Bio Genetics

How can we support Cultural Genetics?

2012-01-08T05:11:00.000-08:00

Three things are necessary to support a cultural genetics:

1. There must be some process of reproduction and inheritance, in which cultural structures and elements are transmitted from one “generation” to the next.

2. There must be a significant measure of stability in the transmission process, in which the replicators show sufficient copying fidelity to transmit recognizable patterns.

3. There must be some process such as sexuality or mutation that introduces change and variety into the process of inheritance, yet is sufficiently coherent itself to permit scientific analysis.

These conditions are met by religious cults and by at least some other phenomena such as stylistic schools in the various arts. If other parts of the wider culture fail to exhibit these features, an “inorganic chemistry” of culture - if not the full richness of an organic genetics - will still emerge, and the rules of one can illuminate the rules of the other.

Cult is culture writ small. Experts suggest that cults are the Drosophila melanogaster and Escherichia coli that will permit us to develop cultural genetics - in other words, fastreproducing organisms whose evolution can be studied efficiently. In modern society, cults are born out of older cults through two processes biologists call fission and sporulation, and most cults are known to cluster in family lineages. Fission is the common term for reproduction by splitting among microorganisms; the corresponding term in religious studies is schism. Sporulation is the term used in biology to name the process by which organisms of certain species (the mosses, for example) reproduce by throwing off spores (tiny seeds). Similarly, the founders of religious cults almost invariably serve an apprenticeship in earlier successful cults, so they serve as the seeds in this reproduction process. In this way, the beliefs and behaviors of cults and artistic movements are transmitted from one generation to the next, with these characteristics sometimes mutating and often combining from two or more sources.

Tags: Bio Technology, Bio Genetics, Cultural Genetics

Understanding of involvement of polyamines in plants

2011-06-26T09:32:00.000-07:00

Polyamines (PAs) are naturally occurring polycationic aliphatic amines, which due to their ubiquity and versatility are involved in the regulation of various cellular and molecular processes. They are positively charged compounds with their charge distributed along the molecule. The common PAs, spermidine (SPD) and spermine (SPM) and their diamine precursor putrescine (PUT) play a critical role in the normal functioning of all cells. They are involved in the cellular functioning both at the molecular and physiological levels due to their association with various Plant macromolecules (DNA, RNA and proteins) and membranes as well as their high concentration in the cytosol thus behaving as osmolytes. The role of PAs is much better studied in animal systems than plants, though they have been suggested to have a role as new plant growth regulators either by mediating the plant hormone effects or independently signalling other responses.

PAs exist in three forms in the cell, viz. as free cations, covalently bound to low molecular weight phenolic compounds like hydroxycinnamic acids (conjugated form of PAs) and bound to marcomolecules or membranes (bound form of PAs). Though the major form is the free cationic form of PAs, there are instances when the amounts of conjugated form exceed the free form and these are known to be critical in certain physiological processes including seed germination, flower development, defence responses and stress reactions. Besides PUT, SPD and SPM, there are certain unusual PAs found in nature, e.g. thermo SPM which have been detected in bacteria residing in hot springs and they seem to be important in protecting the enzymes from heat denaturation and aminobutylhomo-SPD found in fast growing cells of root nodule bacteria Rhizobium. NorSPD and norSPM are found in thermophilic red algae, brown algae,and Chlamydomonas, Nitella and Chlorella. Similarly, some unusual PAs have been reported in plants, homo-SPD was first detected in sandalwood and also in mosses and ferns. In leguminous plants, other unusual PAs like canavalmine, homoagmatine, aminopropylcanavalmine and aminobutylcanavalmine have been detected. NorSPD and NorSPM have been detected in alfalfa grown under drought conditions and have been postulated to play a protective role under stress conditions. As a matter of fact it has been suggested that PA distribution, especially of SPM, may serve as a phylogenetic marker.

PAs have been demonstrated to be associated with regulation of somatic embryogenesis, root and shoot formation, flower and fruit development , stress responses and senescence. In fact, PAs may serve as ‘biomarkers’ for in vitro morphogenetic potential including plant regeneration via somatic embryogenesis. The multifaceted functions of PAs as well as the variations in their levels in response to changes in the physiological state, point towards their role as possible second messengers, though their high titres do not support the view. Various studies have been conducted to investigate the involvement of PAs in cell functioning, using mutants of PA biosynthetic genes and specific substrate-based inhibitors of PAs. Though much information could be generated regarding the involvement and possible mechanisms of action, no clear picture of their functioning emerged. Hence, transgenic plants expressing PA biosynthetic genes in constitutive and regulated manner were generated, with an aim to answer some of the queries regarding the functioning and role of PAs.

Tags: Bio Technology, Bio Genetics, Polyamines in Plants

Different Mechanisms for Transporting DNA into the Cell

2011-03-12T20:28:00.000-08:00

Following two basic mechanisms are assumed to contribute to the transport of the DNA into the cell.

1) An active, energy dependent uptake of the transfection complexes by a process called endocytosisd

2) “Passive” membrane fusion and release of DNA into the cytoplasm.

The compaction agent used in the first step largely determines which mechanism is more important in a given case. For polycationic molecules, a direct interaction (fusion) with the hydrophobic membranes is not likely. The most likely way for them to enter the cell would be by endocytosis. Cationic lipids, on the other hand, can potentially interact and fuse with the membrane. Experiments with synthetic membranes have demonstrated the fusogenic ability of liposomes formed by cationic lipids,but convincing data that this mechanism is also operative during transfection of living cells are still lacking. Other reports seem to indicate that liposomes also preferably enter the cell by endocytosis.

Endocytosis is a process by which cells take up extracellular molecules such as cholesterol via a receptor-mediated mechanism. Cholesterol, insoluble in aqueous solutions, naturally occurs in association with the so-called low-density lipoproteins (LDL). The uptake of cholesterol by the cells depends on receptors specific for LDL. In a first step the ligands bind to the receptor. Receptors occupied with ligands form clusters and induce the formation of a clathrin-coated pit. Clathrin induces the expansion of the pit. Such pits can subsequently enter the cell as a membrane-bound vesicle containing the ligand/cholesterol-complex. Inside the cell, the vesicle rapidly loses its clathrin coat. Vesicles containing receptor bound ligands undergo further changes. Protons are actively imported into the vesicle leading to a drop in pH from the physiological values of 7 to about 5. Under these mildly acidic pH conditions, receptor and ligand dissociate. Receptors are then recycled back to the membrane with the aid of a sorting vesicle. The ligand/cholesterol-complexes stay within the vesicle and are transported towards the so-called lysosome, an even more acidic vesicle containing digestive enzymes. In the case of the ligand LDL, the ligand/cholesterol complex is digested inside the lysosome into amino acids, cholesterol and fatty acids.

Receptor mediated endocytosis may easily be exploited for DNA transfer into a cell, but, if DNA ends up in a lysozyme, it will be degraded. In order to succeed with gene transfer, the DNA needs to escape the endosome before it is digested by lysosomal nucleases. This is possible, as demonstrated by a number of infectious viruses, which use endocytosis for the efficient transfer of their genetic material into certain target cells. Such viruses have special capsid proteins that allow them to escape the early endosome. The signal for their escape is triggered by the drop in pH. As soon as the pH in the endosome starts to decrease, the capsid proteins undergo a conformational change that enables them to fuse with the membrane of the early endosome. The result is a disruption of the vesicle and the release of the virion into the cytoplasm. A synthetic peptide derived from the capsid of the hepatitis A virus has recently been shown to mimic this endosome escape induced by low pH. Another, less efficient, way to escape the lysosome consists in the utilization of lysosome blocking agents such as chloroquine or - even simpler - in an osmotic shock enforced by exposing the cells to nontoxic and nonionic compounds but osmotically active molecules such as glycerol and DMSO.

How the compacted DNA gets Attached to the Cell Surface

2011-01-11T18:16:00.000-08:00

If a foreign DNA sequence is to be introduced into a cell, it is obviously necessary that the two meet, i.e., that the compacted DNA somehow attaches to the cell surface within and for a reasonable amount of time.

Cell membranes consist of a lipid bilayer into which a number of complex (glyco)protein molecules are inserted or anchored. The dominant mechanism for interaction between the DNA complex and the negatively charged cell surface are electrostatic forces. The negative surface charge is in many cases provided by proteoglycan molecules carrying anionic sulfate groups, which are present on the surface of many cell types. Positively charged complexes may attach themselves to the cell surface via these molecules. The importance of this type of interaction to the success of a transfection has been demonstrated by the following experiment. It has been shown, that DNA charged cationic liposomes fail to transfect so-called Raji cells, which are proteoglycan negative, but transfect genetically modified, proteoglycan positive (syndecan-1), cells of the same cell line with good efficiency.

Electrostatic interaction with the proteoglycans, however, is not the only possibility for interaction between a DNA-carrying transfection agent and a cell surface. Many membrane proteins expose binding sites (receptors) for certain biochemical messenger molecules (ligands).

In general, such receptor proteins control the specific uptake of molecules and make the cell sensitive to hormones and other signal molecules. This natural mechanism can be subverted for DNA transfer. The receptor ligands can be used to increase transfection efficiency in general or they can be used to target the transfection complex to a specific cell or tissue type by evoking an interaction between the transfection complex and a cell-specific receptor. Targeting can, for example, be achieved by introducing ligands such as, insulin, transferrin, lactose, galactose, mannose, folate, poly(acrylic acid) or specific monoclonal antibodies or antibody fragments into the transfection complex. This addition has been shown to dramatically increase the efficiency of transfections with agents such as poly(lysine) or poly(ethyleneimine) for certain cell lines, which otherwise were difficult to transfect. It seems that the improvement is due to the ligand’s ability to subsequently induce receptor-mediated uptake of the DNA into the cell (endocytosis, see below). In addition, receptor mediated transfection can be blocked (controlled) if necessary by complementing the cell culture medium during the transfection with an excess of the free ligand.

Tags: Bio Technology, Bio Genetics, Gene Compaction

Understanding the Process of compaction of DNA

2010-10-22T18:48:00.000-07:00

Pure (“naked”) DNA has little chance to enter a cell. DNA is a huge, negatively charged and hence highly hydrophilic molecule. Cells are surrounded by a hydrophobic plasma membrane and, in addition, bear a negative surface charge. The plasma membrane contains several highly selective transporter units, which allow for the well-controlled introduction and excretion of certain molecules. Foreign DNA is normally not amongst the molecules allowed to enter the cell.

The first and best-understood step of transfection is therefore the necessity for “compaction” of the large, negatively charged DNA molecule. A suitable compacting agent is a positively charged molecule able to interact with the DNA and to neutralize or even overcompensate the negative charges. During compaction, the DNA forms stable complexes with the compaction agent, which either stay in solution or form a precipitate. In a typical transfection experiment, the complexes are formed in a reaction mixture containing the given amounts of purified DNA as well as the compaction agent under defined pH and salt conditions. The complex formation occurs spontaneously upon mixing. Within the next 30 minutes the complexes are added to the target cells. Usually, cells are exposed for several hours to the complexed DNA. Subsequently, the medium is exchanged in order to minimize possible toxic effects.

Two groups of molecules are currently investigated as compaction agents: cationic lipids and cationic polymers. Protonated amino groups provide the required positive charges in both cases. Amino groups are also found in some of the naturally occurring compaction agents such as spermine and spermidine. They are clearly the group of choice, since they allow the generation of a positive charge at physiological (neutral) pH. In addition, eukaryotic cells developed over eons of evolution special proteins (nucleosomes) with a high affinity to DNA, which also can complex DNA. The structure of these nucleosomes may in the future inspire the design of novel compaction agents. Prominent representatives are histones or protamines, naturally occurring ubiquitous DNA binding (compacting) proteins.

Cationic lipids are usually fairly small molecules, which mimic the structure of the cell’s plasma membrane and hence facilitate the passage of DNA into the cell by increasing the solubility of the DNA in the plasma membrane. These molecules consist of a hydrophobic (hydrocarbon) tail and a positively charged head-group. The hydrophobic tail promotes in aqueous solutions self-aggregation into larger structures (micelles, double layers) capable of interaction or even fusion with the cellular membrane.

The cationic polymers (such as polyethyleneimine, polyvinyl pyrrolidone) commonly used for transfection are fairly large molecules (up to 1,000,000 g/mol). They are soluble in water at neutral pH due to their positive charges. Linear as well as branched molecules are employed for transfection. In contrast to the cationic lipids, which usually were developed as dedicated transfection reagents, most cationic polymers have been developed for other applications and purposes. They are therefore available from several suppliers in a wide variety of purity and chemical homogeneity.

Tags: Bio Technology, Bio Genetics, Gene Compaction

What are the barriers to Efficient Gene Transfer?

2010-08-16T20:52:00.000-07:00

DNA, the common carrier of the genetic information for all living entities on this planet, is omnipresent and we are daily exposed to large quantities of foreign DNA (e.g., by food or bacterial infections). Under these circumstances, nature had to provide powerful barriers against the spontaneous insertion of foreign DNA sequences into the genomic DNA of cells. Barriers are the plasma membrane of the cell, the envelope of the cell’s nucleus, but also the possibility for DNA degradation in lysosomes and the cytoplasm. These protective mechanisms work rather well and even under optimized conditions it is by no means easy to genetically modify an eukaryotic cell (the terminus usually employed for this modification is to “transfect” the cell). However, the necessity to transfect cells for research purposes, the discovery of new and efficient reporter systems to verify the success of a transfection experiment (luciferase, green fluorescent protein) as well as the availability of powerful transfection reagents have spurred research in the area for many years. Several methods to transfer genes into cells have been developed during the last 30 years. However, considerable efforts to develop new techniques or to improve the efficiency of old ones are still being made.

Transfection reagents help to overcome the natural barriers to gene transfer by various strategies.

The steps involved in the transfer of a “gene” from the outside into the genome of the cell comprise of the following:

1. Compaction of the DNA,

2. Attachment to the cell surface,

3. Transport into the cytoplasm,

4. Import into the nucleus and

5. Insertion into the chromosomal DNA.

The mechanism by which a certain barrier is overcome is an important feature of the respective transfection reagent. In order to elucidate the difficulties in optimizing the genetic engineering of mammalian cells, the major steps of transfection as well as putative agents for reaching this goal will be discussed in detail in the following sections. The mechanisms for many of the above-mentioned five steps of transfection are still under discussion. This is especially the case for the later steps taking place inside the cell, i.e., transport into the cell and most importantly into the nucleus. The earlier stages of compaction and interaction with the cell surface are better understood. This has important consequences for our current ability to engineer transfection agents and procedures. It should be noted that man-made transfection procedures are still orders of magnitude less efficient than nature’s transfection agents, the viruses are. One to five infectious particles, i.e., viruses, per cell are sufficient in that case, compared to the 105– 106 plasmid molecules needed in most nonviral transfection methods.

Tags: Bio Technology, Bio Genetics, Gene Delivery

Understanding of DNA Molecules

2010-07-08T04:11:00.000-07:00

The structure of the large DNA molecule, which was known to be the main material of the chromosomes, remained a mystery until Watson and Crick proposed the double helix structure in 1953.6 Chromosomal (genomic) DNA consists of two complementary polyanionic chains made up of long sequences of four different nucleic bases. Since the four bases are complementary, the double stranded DNA molecule is capable of exact self-replication from either strand. The diameter of the double helix is about 2 nm, while the length of the DNA polymer can be enormous, i.e., several centimeters in a putatively “stretched out”-state.

In a typical human cell, DNA molecules with a total length of 1 meter have to be packed into a nucleus of about 5 m in diameter. The compaction is mediated by the so-called nucleosomes, which contribute about 50% of the total mass of the chromosomes. Nucleosomes are formed by 4 to 5 different types of histones; small, basic proteins with a high proportion of positively charged amino acids (25% lysine or arginine). Histones, which bind tightly to the sugar-phosphate backbone of DNA, also have important regulatory functions. Compacted DNA is not active, meaning it can be neither replicated nor transcribed into RNA and finally into proteins. The histones control the compaction and the compaction-reversal through a regulated process that is gene or sequence specific. The exact biochemical basis of this regulation strategy is still unknown, but the essential role of histones in life is supported by the fact that their amino acid sequence is among the best conserved throughout evolution. Apparently, even minor changes in the histone structure have dire consequences for the organism in question.

Plasmid DNA is an independent type of DNA, which occurs naturally in many microorganisms in addition to the genomic DNA of the respective organism. Plasmids are comparatively small (typically 5–10 kba), circular DNA molecules that can multiply independently from the genomic (chromosomal) DNA. They occur naturally in the supercoiled (major percentage) and the open circular form. Linearized fragments of plasmid DNA can be obtained by “digestion” of the plasmid with restriction endonucleases, i.e., enzymes that cut the DNA at specific base pair sequences. For various reasons, plasmid molecules are the preferred tools for genetic engineering. Plasmids can easily be amplified in bacteria. They are separated from the larger chromosomal bacterial DNA by a denaturation/renaturation process, where the chromosomal DNA forms an insoluble precipitate, because it renatures more slowly. Purified plasmids can be transferred into eukaryotic cells either in their natural, supercoiled form or as linearized molecules.

Tags: Bio Technology, Bio Genetics, Gene Delivery

Understanding the impact of molecular biology on everyday life

2010-06-15T01:33:00.000-07:00

The impact of molecular biology on everyday life has increased enormously over the last two decades. Medical, pharmaceutical and lately even agricultural applications of “gene technology” have become standard, if sometimes controversially viewed procedures. The feasibility of this “revolution” is based on a few biological facts; most importantly the relationship between DNA, RNA and proteins. DNA carries the information for protein production.

Basic units of information are called genes, which typically are DNA sequences of about 1500 base pairs (bp). Usually, one gene carries the information for one protein. While the proteins are highly specific to a species, the genetic code is universal and shared among all living organisms. Therefore, if a human gene is transferred into a bacterium, the bacterium will be able to translate this DNA sequence into the “correct”, i.e., human, amino acid sequence (protein). The insertion of foreign genes into bacteria has become a routine laboratory procedure1 and genetically modified bacteria have been widely used to produce so-called “recombinant” proteins for the pharmaceutical industry. A well-known example is the production of human insulin in E. coli.

However, there are limitations to the use of bacteria for the production of proteins, especially of complex proteins from higher organisms. While the genetic code is universal, the machinery for protein processing is not and bacteria lack the enzymes and organelles, which, for example, in mammalian cells are responsible for further processing and modification of the proteins (e.g., glycosylation, disulfide bridge formation, cleavage). Especially in the case of larger proteins, bacteria are often not able to fold the amino acid chain into the correct three-dimensional structure required for “biological activity”. Last but not least, the tendency of bacteria to store produced proteins inside the cell in the form of denatured precipitates, so-called inclusion bodies, has been known to considerably reduce the yield of active protein. For this reason, mammalian cells, which have been adapted to propagation in single cell culture, are nowadays used to produce the more complex but also more valuable products of modern biotechnology. Well-known examples are the various CHO cell lines derived from Chinese hamster ovary cells. In order to enable such mammalian cells to produce a desired – human - protein, they too need to be genetically modified. The genetic manipulation of mammalian cells (“transfection”) is much more difficult than that of bacteria. Over the last years a number of transfection strategies have been developed, amongst the methods that utilize (semi-) synthetic polymers. A controllable and successful transfection strategy is not only the basis for the production of recombinant proteins, but even more so for gene therapy. Considerable attention has therefore been paid to the development of synthetic polymers as vehicles for gene delivery. This chapter will focus on the current state of knowledge in regard to the requirements for putative transfection vehicles, but also will summarize and compare the various applications of such systems.

Tags: Bio Technology, Bio Genetics, Organ Rejection

What are the areas needing Efficient Gene Delivery

2010-05-22T06:37:00.000-07:00

Today an enormous amount of genetic information is available from databases, which are continuously fed by worldwide genome sequencing programs. Every day, the human genome-sequencing program alone provides new information about human genes with potential therapeutic value. On a diagnostic level, this will allow detecting “genetic defects” and also a prediction as to which amino acid in a given protein is concerned. However, unless the change in amino acids is meaningless or the malformed protein can be replaced, this information has limited therapeutic impact since curing the DNA defect is at present not possible. Another aspect concerns the large number of genes with unknown function. Since it is not possible to predict the three dimensional structure of a protein, let alone its biological function (interactions with other biological substances), from its amino acid sequence, the only way to “mine” the genetic information consists in a laborious transfection of a mammalian cell with the gene in question to enable said cell to produce the protein. Subsequently this allows studying the activity of the protein either directly within the cell or in vitro once enough of the material has been produced for further characterization.

The problem of quickly producing a certain amount of protein for further characterization and study is a major bottleneck in several areas of the life sciences and the related bioindustry. The list of sequenced genes for which the function of the corresponding protein is poorly understood is long. In addition, it is fairly easy to mutate genes in vitro, so a variety of new proteins can be encoded, some of which might have considerable therapeutic value. In contrast to the quick generation of new genes, the establishing of a stable recombinant production cell line requires at least a year for transfection, screening/amplification and scale up due to the difficulties of inserting the gene stably into a transcriptionally active region of the cell's chromosomal DNA. Recently, a much faster method -transient transfection - has been discussed as a means to produce quickly (within days) milligrams of a given protein. In this case, the foreign DNA is not inserted into the genome of the cell. The method, which until recently was only used for the production of smaller amounts of proteins through-out, had been shown to be compatible with at least the 1 L scale. If transfections could be established at the 100 liter scale or more, gram amounts of any protein could be produced within days. Screening of putative biopharmaceuticals but also basic research would profit enormously. Such large-scale transfections have not yet been achieved.

Gene therapy is another domain where efficient transfer of genes is essential. Many severe human diseases are caused by a genetic defect leading to the mal- or over-/ under-expression of the corresponding protein. Patients could be permanently cured if the missing genes could be transferred in a functional form into the concerned organs. Delivery of genes to specific tissues could become the most efficient medical treatment in the future, but for obvious reasons, the establishment of a very safe and well-controlled method for gene delivery is imperative.

Tags: Bio Technology, Bio Genetics, Gene Delivery

Understanding of Bioartificial Organ Rejection

2010-05-03T03:09:00.000-07:00

The process of rejection may begin with the diffusion of immunogens from the graft across the membrane barrier. There are several possible sources for these antigens, including molecules shed from the cell surface, protein secreted by live cells and cytoplasmic protein liberated from dead cells. Recognition and display of these antigens by antigen presenting cells initiates the cellular and humoral immune response. The former leads to activation of cytotoxic cells, macrophages and other cells of the immune system. These cells must be prevented from contacting grafted tissue, a requirement relatively easy to meet. More difficult is keeping out components of the humoral immune response. These include cytokines, for example, interleukin-1, which can have detrimental effects on beta cells, as well as the antibodies formed as a response to the antigens, which have leaked across the barrier. In addition, there may always be some antibodies already present in the antibody spectrum of the blood serum which correspond to cell surface antigens (e.g., major histocompatibility complexes) on allo- or xenografts. Antibodies produced during preexisting autoimmune disease, such as type I diabetes, might also bind to surface antigens on allogeneic cells. Finally, macrophages and certain other immune cells can secrete low-molecular weight reactive metabolites of oxygen and nitrogen including free radicals, hydrogen peroxide, and nitric oxide that are toxic to cells in a nonspecific manner. These agents can diffuse large distances if their lifetime exceeds 1s.

Any attempts to evaluate biocompatibility in vitro would show some lack of predictability for in vivo experiments. Therefore, implantation experiments are necessary to correlate these phenomena. The majority of experiments have been performed on rodents, and there are only a few reports on systematic experiments in large animal models. The choice of an animal model should reflect the human situation. In diabetes research, the diabetic BB-rat, NOD-mice and STZ-treated mice have generally been accepted to be a representative animal model of autoimmune diabetes.

Tags: Bio Technology, Bio Genetics, Organ Rejection

What is Tissue Sourcing

2010-04-20T00:08:00.000-07:00

Organs and cells of animal origin are being considered as a source of tissue for xenotransplantation. If islet transplantation is to become a widespread treatment for type 1 diabetics, solutions must be found for increasing the availability of insulin-producing tissue and for overcoming the need for continuous immunosuppression. Insulin-producing cells being considered for clinical transplantation include porcine and bovine islets, fish-Brockman bodies, genetically engineered insulin-secreting cell lines and in vitro produced “human” beta-cells.

Both primary tissue and cultured cell lines have been employed in small animal xenotransplantation, including cells that have been genetically modified. Substantial efforts have also been made in the isolation of primary tissue, especially for pancreatic islets, though further improvements are necessary for practical, large-scale processing. The most urgent problem in transplantation is the shortage of donor organs and tissue.

Xenotransplantation could offer some advantages over the use of human organs. Xenotransplantation could be planned in advance, the organ would be transplanted while it was still fresh and undamaged. In addition, a planned transplantation allows the administration of therapeutic regimens that call for the pretreatment of the recipient. Another advantage is the possibility that animal sources could be genetically engineered in order to lower the risk of rejection by expressing specific genes for the benefit of the patient. However, the concern over retroviruses has led to political moratoriums on the clinical use of xenotransplantation. It has yet to be established in nonrodent models as a viable alternative.

Alternative Tissue Sources

The optimal source of xenogeneic islets remains controversial. Islets have been isolated from primates and xenografted into immunosuppressed, diabetic rodents, with short-term reversal of diabetes. However, there are ethical issues surrounding the use of primates for these studies. Other promising islet sources are porcine, bovine and rabbit islets, all of which function remarkably well in diabetic rodents. Long-term human, bovine and porcine islet xenograft survival has been documented in nude mice and rats, suggesting that, in the absence of an immune response, sufficient islet-specific growth factors are present in xenogeneic recipients.

Porcine islets are at present receiving the greatest attention since pigs produce an insulin which is structurally very similar to human insulin and pigs are, on the other hand, the only large animals slaughtered in sufficient quantities to supply the estimated demand from type 1 diabetics. In addition, porcine islets within microcapsules have been reported to correct diabetes in cynomologus monkeys. Elaborate studies are in progress to engineer a “perfect pig”, having adequate levels of complement-inhibiting factors. Thus, porcine sources are

perhaps most likely to provide islets for an inaugural human xeno-islet trial. However, porcine islets are fragile and have poor long-term stability. The in vitro glucose-stimulated insulin secretion rate per unit islet volume appears to be substantially smaller for porcine islets than for other species including human. Lastly, there is significant current concern regarding the potential for transmission of infectious agents from porcine organ sources to human xenograft recipients, and to the population at large. None of these characteristics bode well for their practical large-scale use, and serious consideration and investigation is being given to alternate animal sources. There is also speculation that neonatal porcine islets, which culture better and present minimal infrastructure problems, would be an ultimate substitute. Isolation of bovine islets is technically easier and calf islets are glucose-responsive. However, adult bovine islets are relatively insensitive to glucose. The rabbit pancreas is also an attractive source of islets since rabbit insulin differs from human insulin at only one amino acid and rabbit islets are glucose responsive.

What are the techniques of Interfacial Polymerization & Photo Polymerization

2010-04-02T07:38:00.000-07:00

Interfacial polymerization is a method developed for encapsulation of mammalian cells. Cells are coextruded with a generally hydrophobic polymer solution through a coaxial needle assembly. Shear and mechanical forces due to a coaxial air/liquid stream flowing past the tip of the needle assembly causes the hydrogel to envelop the cells and fall off. The encapsulated cells fall subsequently through a series of oil phases, which cause precipitation of the hydrogel around the cell. This process, based on membrane phase inversion, is used primarily when encapsulating cells with hydrogels from the polyacrylate family. Polyacrylates are well tolerated by the host’s immune system and have exceptional hydrolytic stability. A potential disadvantage of this technique is that organic solvents, which may be harmful to living cells, are used to precipitate the hydrogel. To eliminate the use of organic solvents, complex coacervation was developed using acidic and basic water-soluble polymers. Briefly, a droplet containing one of these polymers and cells is added to the other polymer. A thin membrane encapsulates the droplet due to ionic interactions of the two polymers. The major disadvantage of this method is that the capsules may be unstable due to high water uptake in the capsule wall. Modifications have been made to better control permeability and stability of the hydrogel capsules.

Photopolymerization has also been used to conformally coat hydrogel capsules to:

1) Improve their biocompatibility and

2) Reduce the volume to a minimum in order to reduce implant size, a critical issue if an internal organ is the intended transplantation site.

Photopolymerization permits gelation of the polymer membrane in the presence of dissolved oxygen, which is helpful for cell survival during the encapsulation process. The advantage of this technique is that the membrane is directly in contact with the encapsulated cells. Minimizing diffusion distance for oxygen, nutrients, and cell products is important for eliminating necrosis at the center of the capsule12 and for improving therapeutic efficiency.

Tags: Bio Technology, Bio Genetics, Polymerization

Methods adopted for Micro-capsule Formation

2010-03-25T01:55:00.000-07:00

The most widely used procedure for micro-capsule formation involves the gelation of charged poly-electrolytes around the cell core. The popular alginate-L-polylysine micro-capsules, for example, are obtained in the following sequence:

1) The cells are embedded in alginate droplets with the aid of a droplet generator (air / liquid jet or an electrostatic generator);

2) The droplets are transformed into rigid beads by inducing cross-linking with calcium ions;

3) The beads are coated with polylysine and alginate, thereby forming the semi-permeable capsule; and

4) The alginate core is liquefied with a chelating agent.

Micro-capsules surrounding individual cells or clusters such as islets should be physically durable, smooth and spherical for optimal bio-compatibility. Smoothness is one factor, which, in addition to the interfacial composition, reduces tissue irritation, which decreases the probability of cell overgrowth on the capsule surface if aggregated tissue such as beta-cell clusters (beta cells transform blood glucose concentration stimuli into a regulated, pulsatile, insulin secretion) is employed. The capsules should be as small as possible in relation to the islet size to optimize nutrient ingress and hormone egress.

The poly-electrolyte complexation technique used to make alginate-polylysine capsules is advantageous since the capsules are formed under very mild conditions. A disadvantage, however, is the impurities and batch to batch irreproducibility of the alginate, a naturally derived polysaccharide.The high mannuronic acid content of alginate was shown to be responsible for fibrotic tissue response. Fibrosis was reduced and a more resistant micro-capsule was fabricated by decreasing the mannuronic acid level of the alginate at the expense of the guluronic acid content, although these conclusions have been questioned by some authors. Another disadvantage of alginate-polylysine micro-capsules is that the alginate-polylysine membrane, a weak polyelectrolyte complex, gives the micro-capsules relatively poor mechanical properties.

Local changes in pH or ionic concentration may have influence on the integrity of these microcapsules drastically.

Several different hydrogels have been investigated to determine the efficacy of encapsulation therapy as treatment for multiple diseases in a variety of animal models. For instance, alginate-polylysine-alginate micro-capsules have been employed to encapsulate islets and to reverse the effects of diabetes in rats and mice. The mild encapsulation procedure preserved the integrity of the islet’s secretory function with long term viability maintained. Modified alginate-polylysine micro-capsules, which are smaller and stronger than the previous versions, improved the survival of the xenographic tissue grafts. Coating alginate-polylysine capsules with a poly(ethylene glycol)hydrogel or incorporating monomethoxy poly(ethylene glycol) pendant chains to the polylysine polymer backbone has led to improved biocompatibility compared to unmodified capsules. In an attempt to simultaneously control biocompatibility and permeability, polymer blends have been selected that were optimal with respect to islet cytotoxicity (as measured by in vivo tests or) as well as thermodynamic (swelling / shrinking) and mechanical parameters.

Tags: Bio Technology, Bio Genetics, Bio Artificial organs

Understanding of Bioartificial Organs

2010-03-10T00:45:00.000-08:00

Tissue engineering involves the in vitro or in vivo generation of organoids such as cartilage, skin or nerves. More ambitious projects seek to ameliorate the quality of life of diseased or injured patients and reduce the economic burden of treatment. Bioartificial organs involve an in vitro prepared tissue-material interface fabricated into a durable device. A typical example is the bioartificial pancreas, which will be discussed in the following section as a case study. The extra-corporeal bioartificial liver and more recently the bioartificial kidney14 are examples of the transient replacement of organ functions, the former intended as a bridge to stabilize comatose patients until a whole organ can be procured. As the bioartificial pancreas is often microcapsule based, a specific section will be dedicated to review encapsulation technology prior to the application of this bioartificial organ for in situ insulin production.

Bioartificial organs require the combination of several research areas. The understanding of cellular differentiation and growth and how extracellular matrix components affect cell function comes under the umbrella of cell biology. Immunology and molecular genetics will also be needed to contribute to the design of cells or cell transplant systems that are not rejected by the immune system. Cell source and cell preservation are other important issues. The transplanted cells may come from cell lines or primary tissues—from the patients themselves, other human donors, animal sources or fetal tissue. In choosing the cell source, a balance must be struck between ethical issues, safety issues and efficacy. The sterilization and depyrogenation of the polymers involved in transplants is also critical. The materials used in tissue engineering and polymer processing are other key issues. The development of controlled release systems, which deliver molecules over long time periods, will be important in administering numerous tissue controlling factors, growth factors and angiogenesis stimulators. Finally, it will be useful to develop methods of surface analysis for studying interfaces between cell and materials and mathematical models and in vitro systems that can predict in vivo cellular events.

Tags: Bio Technology, Bio Genetics, Bio Artificial organs

Microencapsulation for Cell Delivery

2010-02-25T20:02:00.000-08:00

Microencapsulation is currently the most widely used form of cell delivery with preparation methods including:

1. Gelation and polyelectrolyte complexation,
2. Interfacial polymerization/phase inversion and
3. Conformal coating.

Microencapsulation involves surrounding a collection of cells with a thin generally micrometer sized, semipermeable membrane. Its primary purpose is to protect the encapsulated cells from the host’s immune system, while allowing the exchange of small molecules and thereby ensuring cell survival and function. There are several requirements for polymer capsules or hydrogels used as components of microcapsules:

# Noncytotoxicity to the encapsulated cells

# Biocompatibility with the surrounding environment where capsules are to be implanted (e.g., minimal fibrotic response)

# Adequate permeability for diffusion of essential nutrients (e.g., oxygen and glucose for islets of Langerhans) and cell secretory products (such as insulin, metabolic waste)

# Impermeability to secreted antibodies of the host’s immune system (e.g., immunoglobulins and glycoproteins after complement activation

# Chemical and mechanical stability

From the technological point of view, the requirements for microencapsulation include:

# Small capsule diameters to ensure sufficient diffusion and internal organ transplantability (depending on application, < 400 μm for bioartificial pancreas), with the cell centering within the microcapsule

# Minimum shrinking/swelling due to changes in osmotic conditions upon transplantation

# Uniform wall thickness for optimum transport of molecules across the membrane and effective immunoprotection.

In addition, the technology used for encapsulation must be nontraumatic to the encapsulated cells. This includes minimizing the mechanical stress during encapsulation and solvent toxicity (if any), as well as optimizing temperature, viscosity, pH and ionic strength. This, in turn, limits the concentration and molecular mass which can be employed. In addition, the ionic content of the polymer backbone (density distribution of charges in the polymer chain), the chemistry and location of functional group attachment, the chain rigidity, aromaticity, conformation and extent of branching were identified as important variables in the type of complex produced. The presence of secondary hydrogen bonding interactions was also found to be significant.

Several problems may prevent wide scale application of microcapsules in the clinic. The capsules can clump together, in which case the cells towards the center may suffer severely from limited diffusion of oxygen and nutrients. A substantial fraction of the capsules may also adhere to tissue. If the capsules degrade, the liberated islet cells, even if nonviable, would greatly increase the antigenic burden on the patient. Semipermeable polymeric membranes have been developed with the aim of permitting the transplantation of xenogenic cells thus removing the need for immunosuppression therapy. However, early clinical implementations is not likely to involve xenografts or genetically modified cells but rather auto- and allografts supplemented by immunosuppression when necessary.

Tags: Bio Technology, Bio Genetics, Cell Encapsulation

Adhesion based Immobilization Techniques

2010-02-15T23:05:00.000-08:00

Each immobilization method has specific properties and advantages. Therefore, the selection of a cell delivery technique depends heavily on the intended application.

Adhesion: Adhesion to a three-dimensional structure is used to immobilize cells for culture or analytical procedures as well as to provide a structural template directing cell growth and differentiation. Adhesion alone does not offer immunoisolation. For in vivo investigations, adhesion-based immobilization must be used in conjunction with either a polymeric membrane or matrix entrapment methods. This method is effective for surface binding, either on top of gel films or within hydrogel foams. Several hydrogels can be engineered with bioadhesive properties by methods which include interfacial polymerization, phase separation, interfacial precipitation and polyelectrolyte complexation. Factors affecting cell affinity and behavior on hydrogels include the general chemistry of the monomers and the crosslinks, hydrophilic and hydrophobic properties, and the surface charge and functionality. One method to enhance cell adhesion is by adding immobilized cell-adhesive proteins or oligopeptides, such as the arginine-glycine-aspartic acid sequence, in the hydrogel. The physical characteristics of the hydrogel also govern the adhesion affinity. Therefore, altering the pore size and network structure can modify cell adhesion as well as morphology and function. For some adhesion applications the mechanical strength is also important with a lower fractional porositygenerally creating stronger networks. Furthermore, closed pore systems make stronger hydrogels than open pore ones. With the adhesion approach, cells are generally plated onto the hydrogel and allowed to attach and migrate. Supplemented culture media provide the cells with essential nutrients for growth and development as well as a means of oxygen and metaboli product transport while in vitro.

Macroporous hydrogel membranes are manufactured by several techniques. One method of constructing pores large enough for cell growth is by phase separation in the polymer and solvent mixture. The “freeze thaw” and the porosigen techniques are two other approaches. The hydrogel is polymerized around a crystalline matrix made from freezing the aqueous solvent (freeze-thaw technique) or around a porosigen of desired size (porosigen technique). With the “freeze-thaw” method, the ice-based crystalline matrix is then thawed after UV polymerization, leaving a macroporous foam. The porosigen technique also requires removal of the crystalline porosigens, in this case usually by leaching or dispersion after polymerizing of the hydrogel with free-radical initiators has taken place. Another method for constructing hydrogel foams uses gas bubbles from sodium bicarbonate to create the macroporous network. Bubbles are trapped during the gelation stage. Thus, the foam morphology is dependent on the polymerization kineics and varies for different hydrogel compositions.

Tags: Bio Technology, Bio Genetics, Cell Encapsulation

Understanding of Immunoisolation

2010-01-28T02:46:00.000-08:00

A variety of systems can be employed for cell or enzyme immobilization. These include, for example, microcarriers, gel entrapment, hollow fibers, encapsulation and conformal coatings. The latter three have been extensively tested in small animal models over the last 20 years, particularly in the area of diabetes therapy. The polymeric materials used in bioartificial endocrine devices (the terms bioartificial and endocrine device are often distinguished from ‘artificial organs’ due to the presence of tissue in the former two) serve two major purposes:

1. As a scaffold and an extracellular matrix they favor the attachment and differentiation of functional cells or cell clusters and keep them separate from one another;

2. As permselective envelopes which provide immunoisolation of the transplant from the host.

The central concept of immunoisolation is the placement of a semipermeable barrier between the host and the transplanted tissue. The properties required for the semipermeable membranes used in cell transplantation depend strongly on the source of cells. An allograft is a transplant between individuals within one species, while a xenograft is a graft between individuals from different species.

Immunoisolation of transplanted cells by artificial barriers that permit crossover of low molecular weight substances, such as nutrients, electrolytes, oxygen, and bioactive secretory products, though not of immune cells and high molecular weight proteins such as antibodies (IgG, IgM), provides great promise for developing new technologies to overcome these problems in a reasonable time frame.

Device Geometry Considerations

The immunoisolation of allogeneic or xenogeneic islets can be achieved via two main classes of technology: macroencapsulation and microencapsulation. Macroencapsulation refers to the reliance on larger, prefabricated “envelopes” in which a slurry of islets or cell clusters is slowly introduced and sealed prior to implantation. An intravascular device usually consists of a tube through which blood flows, on the outside of which is the implanted tissue contained within a housing. The device is then implanted as a shunt in the cardiovascular system. Extravascular devices are implanted directly into tissue in a body space such as the peritoneal cavity, though some have also been vascularized into a major artery such as in Calafiore’s clinical trial. Geometrical alternatives include cylindrical tubular membranes containing tissue within the lumen and planar diffusion chambers comprised of parallel flat sheet membranes between which the implanted tissue is placed.

Microencapsulation refers to the formation of a spherical gel around each group of islets, cell cluster or tissue fragment. Microcapsules based on natural or synthetic polymers have been used for the encapsulation of both mammalian and microbial cells as well as various bioactive substances such as enzymes, proteins and drugs. A review of alternative semipermeable microcapsules prepared from oppositely charged water soluble polyelectrolyte pairs has been presented in recent papers. The main advantage of this approach is that cells, or bioactive agents, are isolated from the body by a microporous semipermeable membrane and the encapsulated material is thus protected against the attack of the immune system. In the case of microencapsulated pancreas islets, a suspension of microcapsules is typically introduced in the peritoneal cavity to deliver insulin to the portal circulation.

Tags: Bio Technology, Bio Genetics, Cell Encapsulation

Future of Plant-cell Tissue Culture

2010-01-21T03:33:00.000-08:00

The commercial potential of plant-cell tissue culture has not yet been fully recognized and is underexploited. Plant-cell tissue culture has two primary products: plant tissue for efficient micropropagation of plants and the use of plant-tissue culture to produce specialty chemicals.

Plant-cell, tissue, and organ cultures can be used in processes analogous to traditional fermentation processes for producing chemicals. Although less than 5% of the world's plants have even been identified taxonomically, from among the known plants over 20,000 chemicals are produced - about 4 times as many as from all microorganisms. Very few of the chemicals in pure or semipure form have been tested for their pharmacological activity for other uses. The enzymatic systems in plants can be used to generate completely new compounds when supplied with analogues of natural substrates; thus, plants contain an underused biochemical diversity. Even the limited use of this vast biochemical potential has had important impacts on mankind; in western countries, about one-fourth of all medicines are derived from compounds extracted from plants. Other plant products are used as flavors, fragrances, or pesticides.

Plant-cell tissue culture to produce chemicals commercially has been exploited in Japan, although regulatory approval for medicinal uses has proved difficult and commercial production is restricted to food uses and pigment production. In Japan, a government-sponsored consortium of universities and corporations was recently developed to establish a foundation for plant-cell culture exploitation (i.e., a precompetitive research thrust). In the US plant-cell tissue is not being exploited for chemical production, although some companies are developing processes for the production of the chemotherapeutic agent taxol.

The major technical barriers to the commercial exploitation of plant-cell tissue culture are low growth rates and relatively low product yields. To mitigate those problems, research is needed in subjects as diverse as bioreactor strategies to maintain high-density cultures and enable large-scale production of chemicals through organ cultures and a mechanistic understanding of the role of elicitors in activating pathways for secondary metabolites that could lead to higher productivities of compounds with therapeutic value.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Role of Bio-Process engineering in agriculture and food

2010-01-05T00:48:00.000-08:00

Bioprocess engineering in agriculture and the food industry involves the application of biocatalysts (living cells or their components) to produce useful and value-added products, and it offers opportunities to design and produce new or improved agricultural and food products and their manufacturing processes. This will likely have a great impact on the food-processing industry. In the increasingly health-conscious society, genetically engineered microorganisms and specialty enzymes will find increased use in improving the nutritional, flavoring, and storage characteristics and safety of food products. Products under development range from genetically improved strains of freeze-resistant yeast used in frozen bakery products to phage-resistant dairy (yogurt) starter cultures. Chymosin, a product of recombinant E. coli, is already used in the milk-clotting step of cheese manufacture, and a recombinant maltogenic amylase is being used as an antistaling agent. Enzyme-based immunoassays could develop into a widely used method for detecting pesticides in foods at parts-per-billion concentrations. Challenges that must be addressed include the economics of production and regulatory issues.

The most important applications of bioprocess-engineering research and development related to agriculture and food involve production of agricultural chemicals for control of animal and plant diseases, growth-stimulating agents for improved yield, and biological insecticides and herbicides; increasing bioprocess efficiencies for fermented foods, natural food additives, food enzymes as processing aids, and separation and purification of the products; use of plant-cell culture systems to produce secondary metabolites or chemical substances of economic importance; and efficient use of renewable biomass resources for production of liquid fuel and chemical feedstocks and efficient treatment and management of agricultural wastes and wastes from food-processing industries.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Gene Based Pharmaceuticals and Gene Therapy

2009-12-18T20:16:00.000-08:00

New classes of products are being tested for use in humans and animals, all sharing genes as common targets. Products based on antisense technology directed toward neutralizing messenger RNA are probably being pursued most vigorously; gene therapy through permanent alteration of chromosomes might hold the greatest potential for treatment of diseases like cancer and for correction of genetic disease. The products depend either on classes of compounds that are related to nucleic acids (oligonucleotides and oligonucleotide analogues), on cells that have been genetically altered, of on viruses that bear appropriate nucleic acids.

For the large-scale production of nucleotides and nucleotide analogues, new molecular techniques must be developed. There are now no procedures for making substantial quantities of these types of materials in high purity and with appropriate chirality. Basic chemical and biochemical techniques must be developed for their preparation; new techniques (probably based on high-pressure chromatography) will be required for large-scale purification, and biological methods might be required for preparation of precursors and perhaps for formation of bonds.

For genetically modified cells and viruses, the usual techniques for mammalian-cell culture and molecular biology will be required, as will additional measures for safety and for economical, patient-specific production.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Understanding the Bio-remediation Process

2009-11-19T21:11:00.000-08:00

Bioremediation refers to the use of entire organisms (mostly soil microorganisms) or selected constituents of microbial cells (mostly enzymes) for chemical transformations. Bioremediation transforms a toxic substance into a harmless or less toxic substance. Ideally, the toxic substance is transformed into carbon dioxide and water. If the toxic substance contains a metal or a halogen, such as chlorine or fluorine, there will be additional side-products (perhaps the free metal atom or its ion or a halide ion). Mineralization is the term used to describe the complete degradation of a chemical substance to water and carbon dioxide. Bioaugmentation, another frequently used term, involves the deliberate addition of microorganisms that have been cultured, adapted, and enhanced for specific contaminants and conditions at the site.

Microorganisms used in bioremediation include aerobic (which use free oxygen) and anaerobic (which live only in the absence of free oxygen). Aerobic microbes have been the organisms of choice for degrading hazardous wastes.

Bioremediation is practiced in two modes - in situ and ex situ. In situ bioremediation involves the use of microorganisms to degrade wastes at the site (both on and below the surface) and avoid excavation of contaminated soil and transfer to different locations. Surface remediation is used to treat the top parts of the soil through aeration by the addition of microorganisms, nutrients, and water. Subsurface bioremediation uses microorganisms already in the soil and groundwater and adds oxygen and nutrients. Ex situ treatment involves the excavation of contaminated soil and its transfer to appropriate treatment sites, i.e., bioreactors. The contaminated soil is aerated and treated with nutrients to provide an active environment for the microorganisms of choice. Treatment continues until the soil is sufficiently clean and can be returned to the site. Ex situ techniques are varied but can involve slurry-phase treatments that combine contaminated soil or sludge in bioreactors or solid-phase treatments that involve placing contaminated soils in lined treatment beds. Bioremediation of water or leachate includes treatment with special bioreactors or filters that contain an active film of microorganisms. The choice of method involves many factors, including the contaminant, the site, and the costs that can be borne. Ex situ treatment is usually very expensive. Most often, the microorganisms are expected to reproduce in situ.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Futuristic use of Transgenic Animals & Transgenic Plants

2009-08-30T06:28:00.000-07:00

Transgenic Animals:Transgenic animals are being developed for a wide variety of applications. In the near future, transgenic animals will be used increasingly in safety evaluation of new pharmaceuticals and accelerating their regulatory approval. The feasibility of producing human pharmaceutical proteins in the milk of transgenic livestock has been established.

As an alternative to cell-culture systems, such livestock appear to be appealing because of high volumetric productivity, low operating costs, capability of posttranslation modification of proteins, and potential for expansion of the producing organism. Bioprocess engineers face numerous technical challenges in converting a transgenic mammary gland system into a commercial prototype for large-scale manufacture of high-market-volume proteins, including the following:

1) Purification techniques for obtaining high-purity proteins that must be recovered and fractionated from a complex mixture of fats, proteins, sugars, and ions, some of which are in colloidal form.

2) Optimization of product stability during recovery.

3) Instrumentation to characterize posttranslation modifications made by the mammary gland "bioreactor."

4) Development of on-line sensors to monitor changes in bioactivity of products during purification.

5) Bioseparations of milk proteins.

In the longer term, transgenic animals might provide a source of tissues and organs for use in transplantation patients. Bioprocess engineering will be needed to design novel equipment to maintain, purify, and store the living tissues without affecting viability or graft response.

The hurdles to be surmounted in developing the necessary genetic tools for systematic pathway engineering are substantial, but basic research at the molecular level will continue to provide improved production strains and novel products, and continued interest in the fundamentals of bioprocessing of milk will help to define separation strategies for this complex biological fluid.

Transgenic Plants:Transgenic plants are capable of generating specialty chemicals or other bioproducts. Special bioprocessing capabilities will then also need to be developed for extracting, concentrating, and purifying such products from plant tissue. This sector of bioprocess engineering might also be important to the prospects of expanding crops or developing new varieties that are rich in fermentable carbohydrates, which are readily used as feedstocks for large-scale manufacturing of specialty and industrial chemicals.

Transgenic tobacco plants have been developed to produce monoclonal antibodies identical in function with the original mouse antibody. Other proteins produced in plants are human serum albumin and enkephalins. Processes to recover and purify proteins from plant-cell extracts will be needed if such systems are commercialized.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Understand the Basics of Metabolic Engineering

2009-08-30T06:27:00.000-07:00

A powerful new approach to product development is the creative application of fermentation technology and molecular biology for "metabolic engineering." Examples of metabolic engineering for heterologous-protein production include deletion of proteases that eliminate product and production of factors that facilitate product maturation and secretion. For protein production on an industrial scale, metabolic engineering could be useful in shifting metabolic flow toward a desired product, creating arrays of enzymatic activities for synthesis of novel structures, and accelerating rate-limiting steps. Metabolic engineering has recently been used to increase the efficiency of nutrient assimilation (increasing the growth rate), improve the efficiency of ATP production (decreasing nutrient demands), and reduce the production of inhibitory end products (increasing final cell densities).

Central to molecular modification of multigene pathways, such as those involved in antibiotic production, is the development of new vectors and transformation procedures and other tools of molecular biology. Another important discovery in metabolic engineering is the isolation of positive-control genes that regulate production of secondary metabolites. Positive regulators have been found in biosynthetic gene clusters for actinorhodin, bialophos, streptomycin, and undecylprodigiosin, all of which are Streptomyces products.

Genes encoding the converting enzymes D-amino acid oxidase and cephalosporin acylase were cloned from Fusarium and Pseudomonas, respectively, into the fungus Acremonium chrysogenum. Expression of this "artificial" antibiotic biosynthetic pathway was confirmed by analysis of transformants that synthesized and secreted detectable amounts of 7- aminocephalosporanic acid.

In addition to classical mutation, new tools have become available for genetic manipulation of important producers of natural products, such as Streptomyces. The ability to clone and manipulate biosynthetic genes for antibiotic production, regulatory genes for improved synthesis, and genes from primary metabolic pathways that contribute to secondary biosynthetic pathways can facilitate construction of strains that have substantially altered metabolic properties. In addition, the cloning of heterologous genes into bacterial hosts has generated strains that can produce compounds that are foreign and even
Deleterious to cell physiology.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Know the Basics of Protein Engineering

2009-08-30T06:25:00.000-07:00

Advances in molecular biology have provided researchers with the opportunity to develop increasingly rational approaches to the design of therapeutic drugs. This technology, when used with computer-assisted molecular modeling, is called protein engineering.

Protein engineering combines many techniques, including gene cloning, site-directed mutagenesis, protein expression, structural characterization of the product, and bioactivity analyses; it can be used to modify the primary sequence of a protein at selected sites to improve stability, pharmacokinetics, bioactivity, and serum half-life. A second application of protein engineering is the design of hybrid proteins that contain regions that aid separation and purification. That is achieved by introducing, next to the structural gene for the desired product, a DNA sequence that encodes for a specific polypeptide "tail."

The tails can be inserted at the N or C terminal of the protein to yield a fusion protein with special properties that facilitate separation. Such genetic modifications can be designed to take advantage of affinity, ion-exchange, hydrophobic, metal chelate, and covalent separations. The special properties of fusion proteins allow crude microbial extracts to be passed over an adsorbent that binds specifically to the tail, so that the desired product is retained and contaminants pass through. After elution and treatment to remove the tail, the product is purified further by standard methods, such as size-exclusion chromatography or high-performance liquid chromatography (HPLC).

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Challenges to Isolation and Purification of Proteins

2009-08-30T06:24:00.000-07:00

Isolation generally denotes the separation of the product from the bulk of the producing organism. The disposition and state of the expressed protein affect the isolation procedure. For mammalian cells and some E. coli, Streptomyces, Bacillus, and yeast products, the protein is released from the cell into the surrounding medium, and isolation is effected by a solid-liquid separation step, usually centrifugation or microfiltration or ultrafiltration. If the product has aggregated either in the cytoplasmic or periplasmic space, isolation is more involved. Generally, the cell is first lysed by mechanical, chemical, or enzymatic treatment (or a combination). In some cases, the more dense aggregate can be separated by centrifugation from most of the soluble and insoluble cell components; in other cases, the aggregate is first solubilized while still in the soluble protein mixture.

Purification of the protein is a critical and often expensive part of the process. It might account for 50% or more of the total production cost. Purification has several objectives: to remove contaminating components from the host organism, i.e., other proteins, DNA, and lipids; to separate the desired protein (or family of proteins) from undesired variants of the desired protein; to remove and avoid the introduction of endotoxin; to inactivate viruses; to obtain required yields at acceptable cost; to avoid chemical or biochemical modification of the protein; and to make the process consistent and reliable. In some cases, the first and additional objective is to fold the protein into its desired conformation.

The most common individual operations are centrifugation, filtration, membrane separation, adsorption separation, and chromatography.

The difficulty of separation can often be decreased by changing the organism or culture conditions to produce a more uniform protein. However, it is still necessary to combine a series of purification steps each of which separates according to a different principle. Ultrafiltration steps are often used between separation steps to concentrate the protein solution or to make the buffer solution compatible with the next separation step. The final steps are designed to place the purified protein in the solution used for the product form.

The complexity of the individual purification steps and the need to be able to integrate them into a manufacturing system translate into a major opportunity for bio-processing engineering as the process moves from the bench to the plant. Research and development in purification, scaleup integration, and system design will continue to have high priority.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Bio-Processing of Renewable Resources & its challenges

2009-08-30T06:21:00.000-07:00

Most of the applications and potential applications of bioprocessing related to renewable and nonrenewable resources involve large-scale operations and products of relatively low value. The most abundant renewable material is lignocellulose. Wood, agricultural residue (corn stover, straw, etc.), plants grown deliberately for biomass (such as hybrid aspen), and recycled pulp fiber are the main sources of lignocellulose. Its largest industrial use is in making pulps for paper and other fiber products; second is the use of wood directly in construction.

Key Technical Challenges in Bioprocessing of Renewable Resources are as under:

1) To develop inexpensive cellulose pretreatment and saccharification processes effective with lignocellulosic materials on large scale with environmentally compatible methods.

2) To develop fermentations capable of converting pentoses to value-added products at yields, rates, and extents similar to those obtained for glucose with yeast and to increase product concentrations achievable in both hexose and pentose fermentations.

3) To develop more efficient separations for recovering fermentation products, sugars,and other dissolved materials from water, i.e., lower cost of separating water from product in fermentation broth.

4) To develop processes for large-scale inoculation, control, and propagation of microorganisms in surface culture (e.g., treatment of wood chips and bioremediation of soils) and solid substrate fermentation.

5) To increase knowledge of combinations of chemical, biochemical, and microbial transformations that result in value-added nonfood products from starch and cellulose.

6) To improve fractionation methods for separating oil, starch, and fiber components during corn milling to obtain higher co-product values with lower capital investment.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Understanding Bio-Process Engineering

2009-08-30T06:17:00.000-07:00

Bioprocess engineering is the subdiscipline within biotechnology that is responsible for translating life-science discoveries into practical products, processes, or systems capable of serving the needs of society. It is critical in moving newly discovered bioproducts into the hands of the consuming public. The bioprocess engineer has many missions. Although the most visible today is the production of biopharmaceuticals, bioprocess engineering also has a major role in the existing fermentation industries responsible for the production of ethanol, amino acids and other organic acids, antibiotics, and other specialty products.

Bioprocessing in space presents unique opportunities, particularly in bioregenerative life support and as a research platform for the study of new types of manufacturing processes.

Bioprocessing for protection and beneficiation of the environment represents another large and important opportunity. Biological processes could offer alternatives to environmentally polluting or fossil-fuel-consuming manufacturing processes and could help to remove toxic pollutants from industrial and municipal wastes. Bioremediation's promise is in its potentially lower cost, compared with other types of technology for cleaning up the environment.

Role of bio-process engineering:
The role of bio-process engineering in the successful commercialization of biotechnology is not fully understood by our national government, industrial, and academic leadership. That is in large measure because first-generation biopharmaceutical products have been successfully produced with only secondary concern for costs of manufacturing. However, products now under development will require novel techniques and more efficient and economical processes. Hence, our participation in the expanding bio-products market will necessitate an expanded role of bio-process engineering. This is all the more important because bio-process engineering could have a profound effect on the existing fermentation industry.

Bio-process development for biopharmaceuticals involves all aspects of generating a safe, effective, and stable product. It begins with the biological system, continues with product isolation and purification, and finishes when the product is placed in a stable, efficacious, and convenient form. The product is initially derived either directly or indirectly from a living organism. Thus, process development starts with the development of the biological system. It is usually a living organism that expresses the desired protein; but it might be an enzyme for protein modification or an antibody for immunoaffinity purification. RDNA and hybridoma technology allow the biological system to be optimized for maximal formation of the product, for facilitation of downstream processing, for high product quality, and for improved interaction with the production equipment. In this phase of bio-process engineering, many disciplines must be applied, including molecular biology, genetics, biochemistry, analytical chemistry, and bio-process engineering. Thus, engineers become full partners with experts trained in the bioscience disciplines in developing and scaling up manufacturing technology for biopharmaceuticals.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Understanding various concerns of Human Genome Project

2009-08-05T01:29:00.000-07:00

The importance of the Human Genome Project has raised many concerns, both biological and ethical. These questions are being addressed as the information generated by the project is being processed and used by people worldwide.

1) Privacy and confidentiality of the genetic information: Who owns the genetic information?

2) Right to use the genetic information by insurance companies, employers, courts, schools, adoption agencies, and so on: Who should have access to individual genetic information and how should it be used?

3) Psychological impact and stigma attached to an individual's genetic differences: How does personal genetic information affect an individual and society's perception of that individual? How does genomic information affect members of minority communities?

4) Reproductive issues, including informed consent for complex and potentially controversial procedures, use of genetic information in reproductive decision making, and reproductive rights: Do health-care personnel properly counsel expectant parents about the risks and limitations of genetic technology? How reliable and useful is fetal genetic testing? What are the larger societal issues raised by new reproductive technologies?

5) Clinical issues, including the education of doctors and other health service providers, patients, and the general public in genetic capabilities, scientific limitations, and social risks, including implementation of standards and quality-control measures in testing procedures: How will genetic tests be evaluated and regulated for accuracy, reliability, and utility? (Currently, there is little regulation at the federal level.) How do we prepare health-care professionals for the new information relating to genetics? How do we prepare the public to make informed choices? How do we as a society balance current scientific limitations and social risk with long-term benefits?

6) Uncertainties associated with genetic tests for susceptibilities and complex conditions (e.g., heart disease) linked to multiple genes and environmental interactions: Should testing be performed when no treatment is available? Should parents have the right to have children tested for adult-onset diseases? Are genetic tests reliable and interpretable by the medical community?

7) Conceptual and philosophical implications regarding human responsibility, free will versus genetic determinism, and concepts of health and disease: Do people's genes make them behave in a particular way? Can people always control their behavior? What is considered acceptable diversity? What is the line between medical treatment and enhancement?

8) Health and environmental issues concerning genetically modified (GM) foods and microbes: Are GM foods and other products safe to humans and the environment? How will these technologies affect developing nations' dependence on the West?

9) Commercialization of products including property rights (patents, copyrights, and trade secrets) and accessibility of data and materials: Who owns genes and other pieces of DNA? Will the patenting of DNA sequences limit their accessibility and development into useful products?

Tags: Bio Technology, Bio Genetics, Genetic Engineering

History of Human Genome Project

2009-08-05T01:28:00.000-07:00

Scientists are still far from identifying and characterizing all the proteins in the human body. However, incredible strides have been made to provide a foundation for protein research. This reaches to the source of proteins and ultimately the source of life. This foundation is laid by deciphering the entire genome sequence, or DNA (gene) sequence of an organism. Beginning with bacteria, microscopic worms, and yeast, scientists and computational biologists have expanded DNA sequence information to include certain animals and plants. The ultimate goal of DNA sequencing is the human genome. This genome sequence would allow the understanding of the basis of human life by identifying the order of DNA nucleotides. To accomplish this goal, many groups have come together to work on the Human Genome Project.

The sequencing of the human genome, which is finding the order of the more than 3 billion nucleotides (A, T, C, and G) in the human chromosomes, is being accomplished by two independent groups of scientists. The two versions of this sequence were published in the magazines Nature and Science in February 2001. One group, formerly led by Craig Venter, is Celera Genomics, of Rockville, Maryland, a company started in 1998. The other research group is the result of a consortium of public agencies with laboratories in several countries.

The sequence of the human genome carried out by the public sector, now led by Francis Collins, has a budget of more than $3 billion. The major sponsors were the U.S. Department of Energy and the National Institutes of Health (NIH), as well as the Wellcome Foundation of England. The current map covers about 95 percent of the human genome and has been found to be 99.96 percent accurate. This work has revealed, in a surprising way, that the human genome only has about 30,000 genes instead of 70,000 to 140,000, according to previous estimates. With a DNA sequence of over 3 billion base pairs (bp) and considering the average gene size of 3,000 bp, it is estimated that only 3 percent of the human genome actually codes for some protein. This means that about 97 percent of the human genome has seemingly no coding function; that is, most of the nucleotide sequences in human DNA do not code for genes. This nonfunctional portion of DNA has, for lack of a more accurate term, been called "junk DNA," and its function and purpose have yet to be understood. More important, the data from the Human Genome Project has also revealed that each human being, independent of apparent differences, is about 99.9 percent identical to any other person.

With so much interest and emphasis on the Human Genome Project, what are the practical applications of the sequence of the human genome? The information will help in the early diagnosis of disease, an understanding of the predisposition to genetic diseases, and in genetic counseling, for example. For instance, the sequence of the human genome allows geneticists to understand why certain people have a predisposition to heart disease, and it will eventually lead to the development of new drugs specifically developed to combat the cause of disease and not the symptoms alone. Sequencing the genome will make available basic scientific knowledge for the development of gene therapies for incurable diseases, such as diabetes, muscular dystrophy, cystic fibrosis, Parkinson's disease, and Alzheimer's disease.

By the beginning of 2002, geneticists had already isolated about 13,000 human genes and learned about their functions, including those that code for eye color, circulatory proteins, and genes that when mutated cause a predisposition for developing breast cancer and prostate cancer. All this complex information is contained in each and every cell of the human body. If it were possible to stretch out the incredible amount of information contained in the DNA of all the chromosomes in a single human cell, it would reach about seven feet. If the DNA of all the cells of the human body were stretched out and aligned, it would be enough to cover the distance from Earth to the moon about 8,000 times. Incredible packaging mechanisms allow this information to be stored within each tiny cell.

Tags: Bio Technology, Bio Genetics, Genetic Engineering

Understanding Engineering Genes

2009-08-05T01:26:00.000-07:00

Once the DNA has been obtained, it is necessary to cut the DNA into pieces to be used for the engineered gene. Restriction enzymes are used for cutting the DNA at specific sites. Most restriction enzymes cut the DNA into diametric fragments, as opposed to symmetric fragments. That cut leaves the DNA double helix with a small sequence of nonpairing bases that overhang on the end. These regions of DNA are generally used for ligation, or joining with other DNA fragments. DNA fragments cleaved with a single restriction enzyme or with complementary enzymes can be ligated to each other because the overhanging regions are complementary and will bind together. The ligation of fragments is facilitated with addition of the enzyme DNA ligases. The true art of genetic engineering is putting together the parts of puzzle, where each DNA fragment must be placed in right order and orientation so the gene is functional. As scientists know the sequences of genes encoding important traits or proteins, the information is used to engineer genes that can be used in a variety of applications.

Genetic engineers are able to manipulate tiny pieces of DNA with enzymes to create new genes and DNA sequences used in biotechnology. Relatively simple tools in a small laboratory are needed for these engineers to practice their craft. The products that result from these methods can then be used in many applications of biotechnology.

Tags: Bio Technology, Bio Genetics, Genetic Engineering

What is Central Dogma of Genetics?

2009-08-05T01:25:00.000-07:00

The genetic material of any organism is the substance that carries the information that determines its life cycle and its characteristics. There is a procedure by which this genetic material is used in living processes; this is the central dogma of genetics. Before the development of modern genetics, it was commonly believed that the substance responsible for heredity was a protein. Once DNA was recognized as the genetic material, the central dogma was established. This states that the information contained in DNA is translated into protein through the processes of transcription and translation. The protein is then used in all life processes, from cell division to electron transport in photosynthesis. For this to occur, DNA is copied (transcribed) into mRNA, and the mRNA is used as template for production of the protein in a process called translation. The message coded by the mRNA sequence gene is translated into a sequence of amino acids, the basic components of protein. Cells cannot produce a protein by simply aligning amino acids; they need to use an RNA template. Additionally, the use of an intermediate mRNA template in protein synthesis reduces the risk of damage to the DNA that can occur from repeated use. Additionally, the central dogma postulates that the intermediate mRNA molecule, a direct copy of DNA, can be used repeatedly in protein synthesis.

Main points of the central dogma are as under:
1) Genes are made of DNA.
2) Genes carry information about structures and biological functions, coded by nucleotides (A, C, G, and T).
3) The genetic information is converted in an mRNA molecule.
4) The mRNA defines the number, type, and order of amino acids in proteins.
5) The protein structure is determined by the linear order of amino acids.
6) The three-dimensional protein structure defines its biological function.

Tags: Bio Technology, Bio Genetics, Genetic Engineering

How do we Manipulate Genes?

2009-08-05T01:21:00.001-07:00

Before beginning any genetic engineering project, it is necessary to obtain a reasonable amount of relatively pure DNA, which is then cut and ligated to build the new gene. Today, several companies make DNA extraction and purification kits, making the genetic engineering process much simpler. The basic procedure requires the releasing of the DNA from the cells and purification of the DNA to be used in the experiments.

In a typical extraction and purification procedure for plasmid DNA, bacterial cells with the desired plasmid are lysed (broken up) under alkaline conditions and the crude lysate (remains of the cells) is purified using either filters or centrifugation. The lysate is then loaded onto an apparatus where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, then plasmid DNA is released in high-salt buffer. The DNA can then be concentrated and desalted for genetic engineering uses.

General Steps for Gene Manipulation are as under:
1) Grow the bacteria in liquid culture.
2) Centrifuge the bacterial suspension to concentrate the bacteria.
3) Discard the supernatant (the liquid part that remains above the pellet).
4) Resuspend the bacteria pellet in a solution with RNAse (enzyme that degrades RNA).
5) Add a buffer to promote an alkaline lyse of the bacteria.
6) Neutralize and adjust the saline conditions of the suspension with the buffer.
7) Centrifuge to separate proteins and other impurities.
8) Adsorb the plasmid DNA onto a membrane by filtration.
9) Rinse the membrane with a solution containing ethanol.
10) Elute plasmid DNA from the membrane with EDTA, a chemical substance that preserves the integrity of DNA.

Tags: Bio Technology, Bio Genetics, Genetic Engineering

What are Viruses?

2009-08-05T01:17:00.000-07:00

Viruses are microorganisms in the gray area of what is living and nonliving. Viruses are made of a protein envelope, which surrounds the genetic material (DNA or RNA). Although viruses have their own genetic material like many other living organisms, they do not possess the capacity to reproduce by themselves. For that, they need to use the machinery of living cells to produce a new virus.

The viruses that live in bacteria are called bacteriophage. They inject their DNA into bacterium, leaving their protein envelope outside. Inside of the bacterium, the virus is a filament of nucleic acid that contains coded information for the synthesis of new virus particles, which can be released with the bacteria lyses. The genetic engineering came about from the observation of how viruses use cells of other organisms or bacteria to express their own genes. In that sense, the viruses can be considered genetic engineers. One of the first experiments of genetic engineering was carried out using a bacteriophage as a true Trojan horse, to introduce DNA from other organisms into the bacterium.

One of the requirements for genetic engineering experiments is the production of DNA fragments that contain the desired information. At the beginning of the molecular biology era, DNA used to be cleaved with vibration by ultrasound waves. One of the difficulties that scientists faced in those experiments was the random fashion in which the DNA fragmented. In 1970, however, Dr. W. Arber discovered that bacteria themselves possess a mechanism to specifically cut DNA at certain sequences. Bacteria produce proteins called restriction enzymes that cleave the DNA at specific recognition sites. It was only after the discovery of the restriction enzymes that genetic engineering became a reality. The restriction enzymes were developed as a defense mechanism of bacteria against viruses.

Viruses inject DNA into bacteria and use their bacteria as a mechanism for reproduction. The bacteria, however, develop a mechanism that fragments the exogenous DNA using restriction enzymes. The restriction enzymes recognize the foreign DNA by means of certain specific nucleotide sequences. Different enzymes recognize and cut the DNA at different sites. Using this knowledge, restriction enzymes became essential tools for the genetic engineer to cut DNA into fragments and build new genes. Hundreds of different restriction enzymes exist, many of which are frequently used in biotechnology.

Tags: Bio Technology, Bio Genetics, Genetic Engineering, Virus

Improving Human Race through the science of Eugenics

2009-06-17T06:14:00.000-07:00

Eugenics seeks the genetic improvement of the human race through selection. This idea became more prominent in the beginning of the 20th century and had another great push during World War II. One of Hitler's main objectives was the purification of the Aryan race.

According to the theory of evolution, developed by Charles Darwin in 1859, more "fit" individuals are capable of leaving a larger number of offspring. However, less fit individuals tend to leave fewer descendants. Therefore, over many generations, genes of less adapted or "inferior" individuals are gradually eliminated from the population. Darwin called this process natural selection.

Due to the use of medicines and improved medical procedures in the last few centuries, man has evaded natural selection. A classic example is the Cesarean section procedure for childbirth. Women that would otherwise die from natural childbirth can now produce offspring. Therefore, today, genes for body shape that prevent natural deliveries are retained in the population.

Natural selection for this trait still exists in indigenous populations that do not have access to modern medicine, but this is the exception and not the rule. In some way, less adapted people with low physical resistance, predisposition for genetic diseases, and so on, continue to leave offspring with the help of modern medical resources. How many of us would not be here if medical resources were not available?

Proponents of eugenics argue that the human species is accumulating bad genes because man has a slow natural selection. Others argue that people need a license for many activities, such as driving, hunting, and fishing, but not to procreate, and therefore the government should also control procreation. In China and India, the government regulates population growth. This is a quantitative and not qualitative control. Some people argue that there is sexual discrimination in those two countries, and that boys tend to be preferred because they can bring larger revenue for their family.

The next 20 years will bring many changes in human behavior, and one can imagine that a revolution could take place that will transform the world. Comparing the world today and that of 50 years ago, no one would think that eugenics could be an issue again. Some of the most despicable human acts were performed in the name of the Aryan race purification. Until 1945, eugenics was taught in many important universities around the world, and the compulsory sterilization of inferior people was relatively common in several countries. There are reports of sterilization of 20,000 people in the United States, 45,000 in England, and 250,000 in Germany during the first half of the 20th century. Eugenics turned public opinion against government intervention in citizens' reproductive choice, and today, compulsory sterilization is only conceivable in the minds of fanatic eugenists. However, with world overpopulation and a growing shortage of resources, many people are afraid that population controls could become a reality again.

Currently, it is difficult to imagine that collective sterilization would be used again, but as genetic tests become routine, it is feared that a new wave of sterilization and abortion could take place, not because of mandatory enforcement, but because of pressure resulting from genetic counseling. Abortion based on genetic counseling is already a reality in countries where it is legal. However, is it right to discriminate against genetic defects or weaknesses, even if it is in the womb? Isn't life just as precious? History has shown that the memory of people is short and that history is cyclical.

With an uncertain future ahead, it seems opportune to recognize that genetic tests and new forms of human reproduction will be part of society from now on. In this scenario, the best alternative seems to be drawing strength from family and moral and religious principles.

Tags: Bio Technology, Bio Genetics, Bioethics

Imagine what hapens when human cloning becomes a reality

2009-06-17T06:13:00.000-07:00

Although human cloning has not been done yet, it is believed that it will happen in a matter of time. Since the Dolly sheep cloning in 1997 by Dr. Ian Wilmut at the Roslin Institute in Scotland, the technique has been advanced with many other mammals (monkeys, cows, cats, pigs, etc.). Many countries, however, are passing laws that forbid human cloning. However, some research groups, mainly in infertility clinics, have indicated their interest in human cloning. Although it is technically possible to clone humans, there are several scientific reasons for not doing so.

Beyond the risks for the pregnant mother and for the clone, a series of ethical issues has been raised in relation to human cloning:

1) Would clones have a soul?
2) How would clones relate in a family setting or in public settings?
3) What would be the limits of paternity and social responsibility to clones?

Some ethicists would argue that cloning violates a child's right to an open future. A cloned child would feel the pressure to become similar to his or her biological donor.

Ethical issues will be raised as society discusses and understands the implications of human cloning. Consider, for example, that human cloning was a reality today.

In this scenario a child could have a variable number of parents, from just one to as many as five:
1) One parent: This would be the case when a woman has been cloned, serving as the egg donor, the donor of somatic cells, and the surrogate mother.
2) Five parents: This would happen when the clone has the following parents:
a) Biological father (somatic cell donor)
b) Biological mother (egg donor)
c) Social father (adoptive)
d) Social mother (adoptive)
e) Surrogate mother

Cloning is a great challenge for society, and moral values certainly will deeply change in the 21st century.

Finally, as humans are not just biological beings, biotechnology should consider its limits on the basis of spiritual values. For example, religious conversions produce profound behavior transformation without any genetic modification. This fact reinforces the idea that human behavior is not just a matter of genes or the environment in which the individual develops. An individual, despite possessing superior genes, can be arrogant and behave irresponsibly in relation to society.

Tags: Bio Technology, Bio Genetics, Bioethics

Can we have our privacy in the era of Genetic advancements

2009-06-17T06:11:00.000-07:00

Privacy and confidentiality are one of our most valued possessions. Soon, it might not be possible to hide from society our weaknesses, limitations, and genetic deficiencies. The individuality of each human being is being unmasked.

The completion of the first version of the Human Genome Project, announced in February 2001, unveiled the genomic sequence of the almost 3.2 billion letters of our chromosomes. This announcement also generated discomfort and fear. The revelations of the deficiencies and predispositions coded by human gene sequences are an unsettling idea for many people. The knowledge of the human genome sequence will make possible the diagnosis of several diseases even before their onset. The association between genes and genetic diseases not only affects patients, but it also raises legal and economic issues for society.

In some countries, like Great Britain, the population of some regions has voluntarily donated DNA samples for establishing a genomic database for criminal use. In that country, the ethical perception is that DNA donation for genomic databases is not an invasion of an individual's privacy. In the United States, the situation is completely the opposite: The great majority of the population has a much stronger sense of individuality and so opposes the idea of genetically exposing themselves. However, some genomic databases have been made mandatory in the United States, such as the one at the FBI. In some states, inmates involved in sexual crimes, murders, and other violent crimes have been required to have their DNA profile included in genomics databases. The fear of many people in relation to the loss of their genetic privacy is that the DNA information could be used for discrimination in employment situations or for health and life insurance.

Some geneticists believe that a DNA profile will not only be used to solve crimes, but also to prevent them. They argue that violence is a genetically transmitted trait. Dutch scientists studied a family in the Netherlands with male individuals with outstanding rates of aggressiveness and rape over five generations. The men were found to carry a genetic defect that causes a deficiency of the enzyme serotonin in their brains. Serotonin is a neurotransmitter related to behavior, humor, and personality in humans. Perhaps this is evidence for the existence of genes associated with violence and other antisocial behavior.

A correlation has also been found between atypical levels of serotonin and dopamine with violent behavior and suicide in different studies. The levels of the neurotransmitter are regulated by genetic and environmental factors as well as the interaction between the two factors. The environment is a broad term referring to nongenetic factors contributing to the trait of interest. This can include upbringing, relationships, lifestyle, and other less tangible influences on behavior or health. Current attempts to link violence to genes are more sophisticated and are applied to individual cases and not to population groups. It is believed that, in the near future, genes for violence or dishonesty will be identified, just like the genes related to cancer and other diseases. In reality, even if all the complex human traits like intelligence, violence, honesty, anxiety, and friendliness are mapped and sequenced, they will remain misunderstood for a long time because the understanding of the human mind is still so limited.

Even if the existence of a gene for violence were confirmed, would it be ethically correct to label a child as prone to violence? Would the simple knowledge of that information alter the isolation patterns of society in relation to a carrier of that gene, inducing him or her to become violent? It should be recognized that the manifestation of most traits results not only from genes, but also environmental influence during an individual's life.

Today there are genetic tests for the detection of genes that predispose an individual to the following diseases: sickle cell anemia, Down's syndrome, Huntington's disease, muscular dystrophy, cystic fibrosis, Tay-Sachs, colon cancer, breast cancer, Alzheimer's disease, and multiple sclerosis. The number of diseases for which genetic tests are available continues to grow.

The inclusion of genetic tests results in information on an individual's medical record that could have serious effects in his or her life. Health or life insurance companies might refuse coverage for medical treatments under the allegation of a pre-existing condition. Today, pre-existing conditions only apply to diseases that have already been manifest in the individual; in the future, this might be extended to include the presence of genetic factors linked to specific medical conditions.

Large corporations routinely request intelligence and personality tests for prospective employees. Some people fear that, in the near future, genetic tests will be routinely requested prior to employment. Today, some companies already use genetic tests to identify employees who are sensitive to chemical products used in the work environment. Those companies have argued that the genetic tests are used only to protect their employees from risks related to work, and obviously, to eliminate the risk of being sued for damages in the future. According to the companies, they do not use genetic tests for selection purposes. In the future, companies could opt to increase the number of genetic tests that are mandatory for recruitment. Such tests might reveal personality patterns and could possibly be used for discriminatory means.

Biotechnology has opened the door to our lives, and questions of genetic privacy still remain to be answered. It is now possible to know more about our genetic makeup, but is that necessarily good? This raises many questions about the use of the enormous amount of information that has been made available.

Tags: Bio Technology, Bio Genetics, Bioethics

Genetic Engineering & Bioethiocs

2009-06-17T06:08:00.000-07:00

Science has proven that DNA is the basis of heredity in nearly all living creatures. It is unusual to think that the same molecules that make a fungus a living creature are also similar for human life. The science of genetics has even found gene sequence coding for specific enzymes and proteins that are virtually identical in humans, plants, and microorganisms. Experiments have shown that genes from one species can be manipulated and expressed in another species.

With the advances from biotechnology in agriculture, medicine, and other areas, genes from highly diverse organisms have been transformed into other species to obtain the expression of a certain trait. An often-cited example is again that of Bt corn. A gene from a soil-borne bacterium was engineered into corn to provide resistance to a devastating insect species. For many, this is not ethically wrong, but others find inherent problems in the use of genes across species. It is a basic issue with the science of biotechnology: whether or not our knowledge of DNA and genetics should allow us to manipulate organisms that are not naturally compatible. Many believe that such genetic manipulation is beyond the realms of responsible and moral science. Does such DNA manipulation change the inherent properties of corn or any other organism, or does biotechnology serve to expand the frontiers of life?

This question is one of the basic ethical arguments behind biotechnology and is actually just the beginning of the many ethical questions that can be directed at this science. Despite arguments about the moral justifications of basic genetic engineering, scientists continue to develop products using advanced methods of gene manipulation. However, many cultures and traditions might be affected by such engineering techniques. For instance, those of the Jewish faith abstain from the use of pork, as it is traditionally considered unclean. What would be the ethical implications of using swine genes in a medicine, plant, or other product? Would it compromise the faith of one who abstains from pork? Such examples can be expanded to include many other scenarios in which this encounter of science with tradition could occur. The issue leads to the questioning of many long-held traditions and beliefs. Perhaps life is simpler than previously thought, and advances in genetics and biotechnology allow us to understand how life is simply contained in the ordered chemistry of DNA. This leads to the importance of public awareness of the applications of biotechnology and must also be included in a debate about ethical implications related to this expanding science.

Finallly where do we move from here?
The fine line between right and wrong, or between ethically acceptable and ethically unacceptable behavior, is a tremendous part of bioethics. If it were possible to define those limits or present a rule of thumb to guide ethically correct decisions, it would certainly be mentioned here. However, it seems that many of the ethical cases related to biotechnology have no clear right or wrong and should be judged on an individual basis. Ethical considerations relating to the many facets of biotechnology should not be something that is only discussed at a company board meeting or within the committee rooms of governing organizations. Many of these new technologies will affect everyone in the near future, and it is important to recognize the many considerations involved in biological sciences. Biotechnology is advancing and making progress on the major factors that limit the lives of billions of people. Solutions to the problems of hunger, disease, pollution, and others are being found using the science of biotechnology, yet many are apprehensive about the technology or fear the technical nature of the science.

Despite the greatest efforts, balanced arguments that will satisfy everyone are impossible to find. The first step for anyone is to become educated in the background, the science, and the applications of biotechnology. It is then important to be informed about how biotechnology affects the risks, benefits, and moral implications associated with superior health care, enhanced crop production, and environmental improvement. This knowledge must be used to make informed and sound judgments, so that opinions are based on fact and study, and not on emotion, hype, or fear. Ultimately, each individual must take the essential steps to understand biotechnology.

Tags: Bio Technology, Bio Genetics, Bioethics

Know the Therapeutic Cloning

2009-06-17T06:06:00.000-07:00

Most people and the scientific media were surprised with the publication of the article "The First Human Cloned Embryo" in the magazine Scientific American in November 2001. Even the most optimistic followers believed that experiences with human cloning would not produce results so early. ACT, a small biotechnology company in Massachusetts, was the first to accomplish the cloning of human cells for therapeutic use. Dr. Michael West, the company's chief executive officer, emphatically stated that his company's objective was research in cloning for exclusively therapeutic treatments and not for reproductive or human cloning purposes. Nevertheless, the public had a strong reaction to this news.

Those who favor the use of embryonic stem cells tend to see the potential to cure genetic problems, and they emphasize the hope of a cure and improved lives for patients with fatal genetic diseases. Opponents recognize that human life begins at conception, and they believe that the price of a cure should not result in the taking of another life, as harvesting embryonic stem cells for this research results in the destruction of embryos. Therefore, embryonic stem cells can be seen as a matter of life by those who can benefit from this technology, or as a matter of death by those who do not agree with the sacrifice of embryos for the production of stem cells.

This is not an easy debate. Imagine a case in which the only hope of cure for a young mother with two small children is the use of embryonic stem cell therapy. Even if this mother's dramatic situation might suggest that it would be ethical to sacrifice a mass of frozen cells stored in liquid nitrogen to obtain the needed stem cells for the therapy, the point that deserves to be addressed is this: Who would have the right to sacrifice a defenseless life (embryo) to save another (adult individual)?

The use of stem cells from bone marrow, umbilical cord, and other parts of the adult human body has not generated as much controversy. The potential benefits from stem cell therapy have been widely discussed. However, the use of embryonic stem cells has raised heated debates in public and scientific arenas. These cells are usually harvested from spare embryos generated through in vitro fertilization that have not been implanted in prospective mothers. Even if the scientist that uses stem cells were not responsible for producing them, he or she would be aiding in this process by creating a demand that results in the destruction of embryos, being an accomplice in the process. This is the same rationale used by the governments that burn ivory confiscated from smugglers, as well as the refusal of the scientific community to use the knowledge generated by the Nazis in the horrific human experiments conducted at the concentration camps during World War II.

This and many other recent discoveries in biotechnology have been occupying the world media. Although the scientific bases for cloning are easy to understand, the greater challenge for society is to address its ethical issues.

The lack of ethical references and the speed of development of new knowledge have exposed the society's lack of readiness to address current ethical issues. Sometimes society fears a technology with great potential benefits; other times it is apathetic about technology with proven negative impacts. Individualism and relativist morale, ideals in fashion in this postmodern society, are fertile ground for justifiable mistakes. These ideologies emphasize that nobody should deny anything to himself or herself that is good unless it is especially harmful to his or her neighbor. The ethical boundaries of society reflect the moral principles that it possesses. Society is dynamic and so are its ethical values. This doesn't mean, however, that the principles within society should develop in a liberal way.

Humans were created with intelligence and this allows them to develop new technologies and expand science. Along with this intelligence they have the freedom to choose between good and bad.

Why should one not be in favor of the evolution of the human race? What are the limits of what is morally acceptable? Any answer that deserves consideration should address the dilemmas of society in light of its principles, morals, and religious beliefs. These are some of the challenges society must deal with.

Tags: Bio Technology, Bio Genetics, Cloning

Problems encountered during Cloning

2009-06-17T06:04:00.000-07:00

There are two major problems or limitations found in the cloning of mammals. First, following the introduction of the donor's nucleus into the egg, it must be reimplanted into a gestating surrogate mother. Most of the implanted eggs abort, forcing scientists to perform several implantations, in the hopes that at least one of the females will have normal gestation. In the case of amphibians (e.g., toads), the development of the embryo occurs outside of the adult's body, thereby facilitating the development of the fetus.

The second major challenge in animal cloning is the size of the fetus at birth. Most of the surrogate mothers have to deliver via Cesarean section. This is especially true in bovines, as the clones tend to be about twice as big as normal newborn calves. The large size of the fetus during gestation can represent a substantial risk for the surrogate mother. Additionally, clones tend to have a high incidence of birth defects, and many clones die in the first hours following birth. Common abnormalities observed in cloned animals include failures of the kidney, heart, circulatory system, liver, and lungs. In addition, the placenta of the surrogate mother does not always function properly during gestation.

The causes of the high abortion rate and abnormalities in clones are still not completely understood, but it is suspected that they are at least partially the result of the complexity of the genetic reprogramming that takes place in the genes from the donor that are inserted into the egg. If a gene is expressed inadequately or it is not expressed at a critical point in development, the result can be a developmental defect. Genetic reprogramming involves the regulation of thousands of genes in a systematic and orderly way. Any asynchrony in the expression of the genes can contribute to defects in the fetus or even result in abortion. Additionally, when cloning is done with nuclei from somatic cells, they bear any preexisting mutations that might have occurred after the cells had differentiated into specialized cells. These mutations would have otherwise been screened out in gametogenesis.

With the current knowledge and technology, mammalian cloning is still a highly unsafe and inefficient procedure. The expectation is that, as new knowledge is generated from more experience, the main limitations in cloning will be at least partially solved. This science is continuing to make progress worldwide, even in developing counties. For example, in Brazil, Embrapa-Cenargen recently pioneered the cloning of the first bovine calf from somatic cells, born in March 2001.

Tags: Bio Technology, Bio Genetics, Cloning

Understanding of Basics of Cloning

2009-06-17T06:01:00.000-07:00

A clone can be defined as an individual or group of individuals that descend, through asexual reproduction, from a single individual. In other words, a clone is an exact copy of the original individual. Humans have practiced cloning of plant species for thousands of years. A leaf, a piece of stem, or root of a certain plant placed in a pot with soil or in a petri dish with tissue culture media can regenerate a new individual, genetically identical (clone) to the plant from which the leaf, stem, or root piece was taken. Today, cloning is a common agricultural practice used in many species that can easily reproduce asexually, such as sugar cane, banana, citrus, potato, strawberry, many grasses, roses, and many tree crops.

Cloning is based on two principles:
1) All cells of any organism contain the complete genetic makeup of the species.
2) Totipotence, the ability of one cell to differentiate and regenerate a completely new individual

Although the regeneration of a complete plant from a somatic tissue (leaf, root, stem, etc.) is an ancient practice, it was only in the 1950s that biologists discovered the principles behind regeneration of whole individuals from a single cell. Unlike animal cells, most plant cells retain their potential to express any of their genes and therefore are able to repeat the developmental processes involved in regenerating complete individuals. Cloning of plants offers the possibility of developing millions of individuals exactly identical to the original source of the regenerated cells. This is a common method of reproduction in asexual plant species.

Most animal cells do not have that same capability of naturally regenerating a complete individual from a cell. In animals, this potential is lost during cell specialization. A specific class of cells called stem cells is the only cell type known to retain their totipotence. Stem cells can be found in marrow tissue, fat tissue, and developing embryos. These types of cells have been the focus of animal cloning efforts. In animals, cloning can be accomplished using the technique called nuclear transplant. The technique has been used for many years in animal cloning using embryonic cells for amphibians such as toads. Animal embryonic cells maintain their totipotence after the first few cellular divisions. As the embryo continues its development, the cells lose their ability to differentiate into other cells and, consequently, the capability for complete regeneration ceases quickly. Contrary to the relative ease of nuclear cell transfer in amphibians, this process is much more complex in mammals. Although cloning of toads was accomplished for the first time in 1952, cloning of mice using the same technique was not accomplished until 1977.

Cloning using nuclear transfer involves the manipulation of two cells. The recipient cell is usually a nonfertilized egg from a female taken soon after ovulation. Harvesting of these eggs is done by laparoscopy or by transvaginal suction. The donor cell, which is the one providing the genetic material for regenerating the clone, is collected from the individual to be copied. Any somatic cell could be used for the purpose, including cells from the skin, mammary glands, or mucous membranes. Under a microscope, the recipient cell (egg) is held, by suction, at the end of a pipette. With an extremely fine micropipette, the chromosomes are removed. At this point the nucleus from the donor cell is then fused with the recipient egg previously deprived of its chromosomes. Some of the cells, if implanted into the uterus of a surrogate mother, start developing into an embryo and eventually a fetus. The procedure involves the removal or destruction of the chromosomes from the recipient egg cell, and the subsequent introduction of the chromosomes from the donor cell. The egg, with the newly introduced genetic material, begins the developmental process in the uterus of a surrogate mother to form a complete individual, genetically identical to the donor that supplied the nucleus. This technique has been used with success for cloning sheep, cattle, mice, monkeys, and other mammals.

The first cloned mammal from somatic cells of an adult donor was the sheep Dolly, born in February 1997. Dolly was cloned using mammary cells from an adult sheep. This widely covered event occurred at the Roslin Institute in Scotland, and the lead scientist was Dr. Ian Wilmut.

Tags: Bio Technology, Bio Genetics, Cloning

Why Human Cloning is Undesirable?

2009-06-17T05:55:00.000-07:00

There is a series of scientific reasons for not cloning human beings. Although many scientists and most of the public share this point of view, it is feared that personal ambition of unscrupulous scientists would make them blind to the scientific reasons for not cloning man. The success in animal cloning is evidence that this technology might be ready to justify its application to humans. In 2000, Dr. Panayiotis Zavos, an Israeli specialist in in vitro fertilization, and Dr. Severino Antinori, an Italian specialist in reproductive physiology, announced their intention to clone humans. In April 2002, Antinori claimed that he had two women carrying cloned babies.

After the birth of Dolly and the successful cloning of mice, cattle, monkeys, goats, and pigs, it is evident that cloning is not a completely safe procedure. Cloning of mammals is considered highly inefficient, and this is unlikely to change in the foreseeable future. Many cloning experiments have resulted in developmental flaws either during gestation or in the neonatal period. Even in the best cases, only a small percentage of cloned embryos survive to birth and, of those, many die shortly after birth. There is no reason to believe this would be any different with humans. This means that to achieve the successful generation of a human clone, many others will have been sacrificed in the developmental phases.

The few animal clones that have survived and been born show abnormal size, a phenomenon called increased offspring syndrome. It is believed that incorrect functioning of the placenta is one of the main causes of embryonic death. The suspected causes of newborn death are respiratory and circulatory problems. Some seemingly healthy survivors might possess immune system dysfunction or kidney and brain malformation. Those problems have been detected in practically all species in which cloning has been accomplished. Therefore, if an attempt to clone a human is made, the concern is not just with the embryos, but also with those that will live to be abnormal children and adults.

The abnormalities in the fetuses and in those few clones that are born alive cannot be easily traced to the nucleus of the donor. The most probable explanations are flaws in the genetic reprogramming or timing and expression of the correct developmental genes. Normal development depends on a necessary sequence of changes in the configuration of DNA and proteins coded by developmental genes. Those developmental changes control the specific genetic expression in the specialized tissues.

Genetic reprogramming of the entire genome is a natural process that happens during spermatogenesis and oogenesis, which can span over months and years in humans. During cloning, reprogramming of the donor's DNA must be done within minutes or, at the most, in a few short hours, during the period of time that nuclear transfer is completed and cell division begins to form the zygote.

Prenatal mortality in clones can occur due to inadequate reprogramming that results in improper gene expression. Some surviving clones have subtle genetic defects that, over time, result in life-threatening conditions. There is no information on genetic regulation in clones, but some evidence seems to indicate errors in gene expression in cloned animals. The expression of marked genes is significantly altered when embryos are cultivated in vitro before they are implanted in the uterus, indicating that even a minimal disturbance of the embryo's environment can have profound effects on gene regulation during development.

All the current evidence now suggests that the experiments on human cloning announced by Zavos and Antinori will have the same failure rates and occurrences of abnormalities that have been detected in animal cloning. Zavos tried to calm the public, informing them their research would use genetically perfect embryos to be implanted as a quality control. However, the public perception of reproductive biotechnology will be seriously damaged if the research fails and defective babies are born from human cloning experimentation. This would likely negatively affect other areas of research, such as the advancements being made with stem cells.

The National Bioethical Advisory Commission in the United States reached the following conclusion six years ago: "At the present, the use of cloning to generate a child would be a premature experiment, and would expose the fetus and the child in development to unacceptable risks." All the data gathered since seems to reinforce this point of view. In many countries, it is unlawful to perform research with human reproductive cells, thereby forbidding embryonic cloning.

Tags: Bio Technology, Bio Genetics, Cloning

Is Human Cloning Possible?

2009-06-16T00:00:00.000-07:00

After the cloning experience of Dolly the sheep, human cloning is theoretically and technically possible. The procedure would consist of taking an egg, removing its chromosomes, and then fusing it with a somatic cell from the individual to be cloned. Some believe that it is inevitable that some scientist will try to clone humans, if it is not already occurring. There seems to be a consensus that within a few years the news of the birth of the first human clone will be the major headline in the media. Scientists in South Korea reported success in creating a cloned human embryo, but it was destroyed instead of being implanted in a surrogate mother. Even if the first human clone is decades from birth, the idea that scientists are secretly trying to do it is a real possibility.

Scientists with an economic interest in this science have been expressing their viewpoint that it would be ethically acceptable to clone human beings. They argue that an embryo up to 10 days after fertilization cannot be considered a life because development of the brain begins at about 14 days after fertilization. It would be interesting to know how those scientists define the ethical limits in relation to their objectives.

It has been assumed by some that human cloning serves only the interests of the narcissists or neo-Nazis, those who would like to create the perfect race. In fact, several scenarios have been created that justify cloning of the Homo sapiens "animal." Some of those scenarios can seem extremely appealing, but an ethical analysis of the dilemmas that clones, their relatives, and society would face during their life indicates that cloning of the most intelligent and rational of the animals is not politically, socially, or religiously acceptable.

Some of the following scenarios show the complexity of the subject:

1) Consider the situation of a homosexual man who feels frustrated with his incapacity to bear children and wants to be cloned.

2) Consider the couple that wants to have a baby, but the husband is sterile. Assuming that cloning is an alternative, the couple could decide to clone the husband, and the wife could contribute as a surrogate mother. Would the child's responses to education differ though he is genetically identical to his father? Would he have the same tastes and preferences as the husband? What if a divorce occurs? How would the mother see her son, who is a copy of the man from whom she is divorced? Would the father have the right to custody of the child because he is genetically related to his father?

3) In another scenario, where a woman gives birth to her own clone, would she be her child's mother or twin sister with a different age?

Obviously, society changes over time. In vitro fertilization was illegal in many countries until about 20 years ago, and the idea of heart transplants was considered immoral in the past. Public opinion on human cloning will probably change in the next few years, but cloning will likely be banned globally before the birth of the first human clone. It would be a terrible mistake to wait until the birth of a baby with genetic defects before that decision is reached. Current experience with animals shows that this technology has too many technical and ethical problems to justify experimentation in humans.

Ethicists are concerned that clones would be considered inferior to human beings, and they would be subject to the limitations and expectations of the knowledge of the copied person. These expectations could be false, as both genetic factors and the environment determine personality. For example, a clone of an extroverted person could be more introverted, depending on his or her upbringing. Clones of athletes, artists, scientists, and politicians could choose different professional careers based on opportunities and the environment in which they are raised.

Predicting the future of human cloning is not an easy task. History shows that society is dynamic, that ethical values change, and moral principles distort over time. In other words, only time will tell. The challenge for bioethicists is to keep science progressing while maintaining the sanctity of life. It is a mistake to think that genetically identical means identical individuals. In the 1978 movie The Boys of Brazil, based on Ira Levin's bestseller, a scientist conspires after World War II to clone Hitler, with the objective of raising a new generation of Nazi leaders. The film shows that without intense indoctrination, the clones can be influenced to pursue other activities than becoming dictators.

Tags: Bio Technology, Bio Genetics, Cloning

What is Transgenic Locus

2009-05-21T02:03:00.000-07:00

Gene constructs used in genetic transformation posses a promoter, coding region, and termination sequence. In the vicillin promoter, specific for expression in seeds, drives the expression of the gene UDP 6-glucose dehydrogenase in the antisense orientation. The construct also possesses the NOS (noplaine synthase) termination sequence, which marks the site for the end of transcription. Besides the transgene of interest, in general, reporter genes are introduced simultaneously to facilitate the identification and selection of transformed individuals.

For the selection of the transformed cells, the gene construct contains a gene sequence that codes for antibiotic or herbicide resistance. Frequently, neomycin or hygromycin antibiotic resistance genes or the phosphinotricim acetyl transferase herbicide tolerance gene is included under a strong constitutive promoter, such as 35SCaMV. The transformed cells would be the only ones possessing the capability to grow in a medium with a selective agent (antibiotic or herbicide), thereby facilitating their selection.

Frequently, a gene reporter is also included in the genetic construction. The function of this reporter is to allow the visual identification of transformed cells. Three genes have been used as reporters in plant transformation: Glucaronidase (GUS), Luciferase (LUC) and Green Fluorescent Protein (GFP). GUS allows the identification of transformed individuals by the expression of a blue color, because they become blue in the presence of the chemicals X-Gal and IPTG (isopropyl beta D-thiogalactoside). Luciferase, a protein in fireflies, turns the transformed individuals phosphorescent, and GFP, isolated from a species of jellyfish, codes for a fluorescent-green color in transformed individuals.

Genetic transformation of individuals is a difficult task. The science behind the methods is understandable on a basic level, but the results from the procedures do not always work out as planned. Specific gene sequences are needed to induce the expression of a transgene, and genes are needed to identify the transformed cells. Still, the use of transformation is being improved to more accurately express desired traits in different organisms. The comprehension of the intricacies of transformation is a key to understanding the broad applications of biotechnology.

Tags: Bio Technology, Bio Genetics, Genetic Transformation

Know the Gene Expression

2009-05-21T01:51:00.000-07:00

All cells possess the typical number of chromosomes of their species. Therefore, root, epidermis, or pod cells of a soybean plant possess all 40 chromosomes typical of this species. However, not all of the genes are expressed in every cell. For instance, genes that code for chlorophyll production are expressed in the leaves and any other green part of the plant. However they are silenced in the roots, which is the reason these cells do not contain chlorophyll. Gene regulation is a complex process that is affected by a series of factors. A common occurrence in genetic engineering is a lack of expression after a gene has been transformed into an organism. Therefore, an understanding of mechanisms involved with gene expression is critical in genetic transformation.

In bacteria, some genes are activated while others are silenced, depending on the conditions in which these microorganisms are grown. For example, the bacteria Escherichia coli can use two different carbohydrates, lactose and glucose, as energy sources. The bacteria needs to synthesize specific enzymes that catalyze the breakdown of the carbohydrates into energy. The enzymes, like all other proteins, are coded by genes. When E. coli is cultivated in a medium with both glucose and lactose (preferably glucose), it metabolizes. The genes coding for the production of the enzymes that metabolize glucose are thus expressed preferentially. The metabolism of lactose requires an additional enzyme that is only synthesized, or activated, after the medium runs out of glucose and lactose is the only energy source available. This phenomenon is called gene regulation.

Gene expression in more complex organisms is still not completely understood. The complexity of gene regulation is a puzzle in the zygote, a cell formed by the union of sperm and egg cells, in which the genes coding for differing functions have to be activated in a precise and orderly manner. The same genetic information present in the zygote is also present in any other cell in the body, from muscles to skin. Obviously, different genes are activated or expressed in each organ in a different way.

Gene expression is not just a function of where the cell is, but also the result of environmental stimuli. Cells of a floral bud of soybeans differentiate into flowers when the plant is grown during long nights. If the soybean plant is grown during short nights, it continues vegetative growth and does not bloom. Another example of gene regulation occurs with animals, including humans. Testicle and ovary cells do not start the production of sexual hormones until the individual reaches puberty.

Another example of the complexity and importance of gene regulation can be observed in the metamorphosis and development of butterflies and moths. These insects take three forms during their lives: caterpillar, pupa, and adult butterfly or moth. The insect possesses the same genes and DNA during these three different developmental phases. Although the caterpillar, pupa, and adult have the same genes, it is interesting to observe that different genes are expressed in the three developmental phases. In the caterpillar phase, the genes for production of several legs and a stronger mouth capable of chewing leaves are expressed, but not the genes for production of wings. However, the genes for the formation of a delicate mouth apparatus, appropriate for nectar feeding, and genes for the formation of wings are active in the insect's adult phase. The gene expression pattern changes during insect development to allow for the correct progression of its life cycle.

The mechanisms regulating gene expression involve regulatory genes. As opposed to the genes discussed up to this point, these DNA sequences do not code for any protein. Their function is to promote the activation or the silencing of genes.

An important part of gene regulation is the promoters. A promoter is a DNA sequence preceding the gene, which contains regulatory sequences to control the rate of RNA transcription. Promoters control when and in which cells a certain gene is expressed. Through the manipulation of promoters it is possible to induce superexpression, underexpression, or even gene silencing.

Some promoters are constitutive—that is, they induce gene expression continually—whereas others are inducible. Among these, there are some that are chemically inducible, and others are activated by heat, light, or hormones. Some promoters are active in certain tissues and organs, but not in others. In this case, they are considered tissue-specific promoters, as in the case of chlorophyll production. The promoters of the chlorophyll genes are not active in roots, but they are active in the leaves and in all green parts of plants.
Some of the promoters frequently used in genetic engineering of plants include the following:
1) Constitutive
a) UBI from corn
b) 35SCaMV from a cauliflower virus

2) Tissue-specific
a) Phaseolina promoter, a seed-specific promoter from field beans
b) Vicillin promoter, a seed-specific promoter from peas
c) Glutamine promoter, an endosperm-specific promoter from wheat

3) Inducible
a) Rubisco 5S promoter, inducible by light

Aside from promoters, other genetic factors are important in proper gene expression. Although the genetic code is universal, it is also considered degenerate, as more than a single codon codes for a certain amino acid. Different organisms have acquired the preferential use of specific codons for certain amino acids during evolution; this can also have an impact in gene expression. That was the case of the Bt gene from Bacillus thuringiensis introduced in corn. Initially, the expression of that bacterial gene in corn was low; however, when a transgene was reengineered to favor the preferential use of certain codons by corn, gene expression occurred at normal levels.

Several other factors can affect the expression of transgenes, such as the presence of a peptide signal, the site of its integration in the genome, the number of copies integrated, and transgene rearrangements during the integration process. Integration of transgenes in the host genome, in general, happens at random; that is, it can occur in any chromosome of the cell and it can land in any part of the chromosome. However, most of the transgenic varieties have the transgene inserted close to the ends of the chromosome. Multiple copies of the transgene are typically introgressed together.

Tags: Bio Technology, Bio Genetics, Genetic Transformation

Problems in Genetic Transformation

2009-05-21T01:46:00.000-07:00

Tissue culture has been identified as one of the largest obstacles in the development of transgenic plant products. It is necessary to develop protocols that allow the regeneration of whole individuals from the transformed cells or tissue. One of the difficulties faced by scientists is that regeneration methodologies work well with some, but not all species or germplasm within a species. This severely limits the spectrum of individuals that can be transformed. In many cases, the procedure has been the transfer of the transgene through classical genetics and breeding methods. An example of this is in the genetic transformation of wheat. Genetic transformation of most wheat varieties is very difficult because of problems in tissue culture. One variety, Bobwhite, is the exception, and protocols have been developed for the transformation of this wheat variety. Once a gene has been successfully transferred into Bobwhite, it can be moved into other varieties through traditional breeding methods.

Another difficulty associated with the use of tissue culture in transformation is somaclonal variations. Plants produced from tissue culture have higher mutation rates and the appearance of abnormal variation. This is due to the delicate environment in which cells are cultured. Many times, the cultured plants have problems associated with the cell cultures and not from the transgene integration.

Transformation methods currently in development promise to revolutionize the introduction of genes in plants. Some of these methods are already being used with the model plant Arabidopsis thaliana, commonly known as mouse ear cress. One of the methods involves the submersion of floral buds in a solution containing plasmids bearing the transgenes. Another alternative technique, still in development, is the transformation of seeds mediated by Agrobacterium tumefaciens. Although the methods have been used with success in Arabidopsis, the literature does not report its use in crop species. The key aspect of these two methods is that transformation is carried out without the need to regenerate plants through tissue culture. These methods are exciting because the transformation procedure works on the seeds that can then be planted to identify transgenic individuals.

Tags: Bio Technology, Bio Genetics, Genetic Transformation

Methods of Genetic Transformation

2009-05-21T01:33:00.000-07:00

Among the several methods of plant transformation, four have yielded the best results: Agrobacterium species-mediated transformation, microprojectile bombardment, microinjection, and direct transformation. Each of these methods has merits and limitations and is used in specific situations. At this time there is no single technique that is suitable for all species.

Agrobacterium Mediated Transformation
Tumors and uncontrolled cellular growth in plants can occur due to genetic factors or bacterial and viral infections. An example is crown gall in plants, where tumors are caused by bacteria that causes uncontrolled growth on the stem of the infected plants. This problem is caused by Agrobacterium tumefaciens, a soil bacterium that infects some plants because of a wound on the plant. Plasmids present in the bacteria are responsible for tumor growth after infection by A. tumefaciens. The bacteria are able to recognize wounds on the plant, and this induces the transfer of the bacterial plasmid into the plant. The plasmids are capable of integrating into the DNA of the host plant, causing uncontrolled plant growth and the formation of tumors. The ability of A. tumefaciens to efficiently transfer plasmid DNA into the host has made it important in early studies in genetic transformation.

Agrobacterium tumefaciens was the first vector used for introduction of foreign DNA in plant cells. Although Agrobacterium has only been used to infect dicot plant species, such as soybean, tomato, pea, and cotton, the protocol has been modified to allow the bacteria to infect some monocot (grass) species as well. Many research groups working with plants have found this to be the preferred transformation approach. Another soil bacteria, Agrobacterium rhizogenes, causes the growth of secondary roots after infection. This bacterial species has also been used for plant transformation.

The basis of this transformation method is the bacterial plasmid, which contains the genetic sequence that is integrated into the host genome. One of the most important parts of a plasmid is the region responsible for the translocation of its DNA into the host plant genome. This is called transfer DNA (T-DNA), and this area of DNA is key to the tumor growth in infected plants. The region is located between the right border and left border (RB and LB) of the plasmid. Plasmids also contain other important DNA sequences; some of them control the production of auxin and cytokinin, two important plant hormones involved in tumor formation. With the use of the restriction enzymes, a transgene can be introduced between the right border and left border of the plasmid, allowing the bacteria to transfer novel genes into the recipient plant.

One of the techniques used for transformation mediated by A. tumefaciens uses leaf disks. Leaf disks of about 6 mm in diameter are cultured on a tissue-culture media containing A. tumefaciens with plasmids containing the transgene. After approximately a month of incubation in the tissue culture medium, seedlings start to develop on the leaf disks. Through selection methods, transgenic seedlings are identified for whole plant regeneration.

Microparticle Bombardment
This technique has also been called microprojectile acceleration or biolistics, but microparticle bombardment is the formal name for the machine called a gene gun. This method, developed at Cornell University, was designated biolistic (biologic + ballistics = biolistic), because high-speed microscopic projectiles (microprojectiles) are accelerated into the cells to be transformed.
This transformation method consists of the acceleration of a macroprojectile loaded with millions of tungsten or gold microspheres about 1 µm in diameter (microparticle). The microspheres are coated with the transgene, or DNA of the gene of interest. Microspheres have a high specific mass, allowing them to acquire the needed momentum to penetrate the target cells. The macroparticle is propelled in the direction of the cells at high speed, but it is retained, after a small distance, on a steel mesh so that the microparticles continue in the direction of the target cells. Helium gas at high pressure is used to propel the macroparticle, and the acceleration chamber operates under a partial vacuum, which allows for improved microsphere movement. Once inside the target cells, the DNA coating the microspheres is released and can be integrated into the plant's genome.

Many of the commercial transgenic crop varieties on the market today were developed using the gene gun. However, due to its cost and the complex integration patterns resulting from this method, several research groups are reducing its use.

Microinjection
This method was developed for animal transformation but has also been extended to plants. Although very difficult and laborious, DNA microinjection has yielded positive results and has been used in several laboratories.

In this technique, microcapillary needles are used to introduce DNA directly into cells. Each cell to be transformed must be manipulated individually. One of the advantages of this method is that the optimum amount of DNA can be injected into the target cells, which helps to ensure optimal integration. Positive results have already been obtained in several crop species such as corn, wheat, soybean, tobacco, and rice, and in animals like salmon, cattle, and swine.

Direct Transformation
Transformation using direct methods was accomplished soon after the first Agrobacterium-mediated transformation. These methods use protoplasts (cells after the removal of the cellular wall) as targets for transformation. This is a simple method that consists of adding great amounts of transgenic plasmids to a protoplast culture, which guarantees that a small proportion of the protoplasts will be taken up (assimilated) by the plasmids. The assimilation rate can be increased with the addition of polyethylene glycol (PEG) or the use of an electric discharge (electroporation). No barrier to direct transformation has been detected, indicating that this method can be used with virtually any species. The problem with this method lies in the difficulty of regenerating a whole plant starting from protoplasts. Therefore, it has not been used as widely as the other methods.

Tags: Bio Technology, Bio Genetics, Genetic Transformation

Understanding Genetic Transformation

2009-05-21T01:26:00.001-07:00

The term genetically modified is frequently used to describe organisms that were genetically transformed or engineered. The science of genetic engineering was developed with the objective of building genes for genetic transformation. Genetic transformation systems possess three main components:
1) A mechanism for introduction of the foreign DNA into the target cell.
2) A cell or tissue suitable for transformation.
3) A method for the identification and selection of transformed cells or individuals.

Success in transformation for any species depends on these three components. Obviously, each one must be optimized and, therefore, as technology develops, transformation should become a more routine activity. The final objective in transformation is the introduction of a new trait in an individual. When the desired trait exists in any other sexually compatible individual, the first alternative should be to transfer the trait through crossing and selection, as has been done in conventional breeding since the 19th century. Modern soybean, corn, cotton, and wheat varieties, as well as swine, cattle, and poultry lines used in agriculture to feed the world, were initially obtained by traditional methods of crossing and selection.

One of the main limitations of conventional genetic improvement is that the breeder is limited to traits among species that are sexually compatible. For instance, the field bean is a species rich in sulfur-containing amino acids. However, beans are naturally deficient in lysine. On the other hand, rice is naturally rich in lysine, but deficient in sulfur-containing amino acids. It is not possible to naturally cross these species, so the conventional plant breeder is unable to develop a new field bean variety with elevated lysine levels or a rice cultivar rich in sulfur-containing amino acids. Genetic transformation allows the exchange of genes between organisms previously limited by sexual incompatibility. With genetic engineering and transformation, it is possible to transfer genes among bacteria, animals, plants, and viruses. In fact, one of the areas of research in biotechnology is the improvement of nutritional profiles in crops. New, more nutritional bean and rice varieties can now be developed through advances in genetic engineering.The basic tools for genetic transformation are restriction enzymes, which are used to cut DNA at specific sites, and ligases, which catalyze the joining of DNA fragments. Using the right restriction enzymes, it is possible to cut the circular bacterial plasmid DNA, causing it to linearize. With a ligase, it is possible to add other DNA fragments containing the gene of interest and join them to the linearized plasmid. Under the right conditions, the ends of the plasmid, now with the added DNA fragments, rejoin to create a new circular plasmid with some DNA modifications. The new plasmid can be introduced into certain bacteria through a process called electroporation, and the bacteria can then be used to transfer the transgene to the target species. If the plasmid DNA is integrated into the genome of the recipient species and the transferred genes are expressed, the individual is considered to be transformed or transgenic.

Tags: Bio Technology, Bio Genetics, Genetic Transformation

Understanding Genetic Transformation

2009-05-21T01:26:00.000-07:00

The term genetically modified is frequently used to describe organisms that were genetically transformed or engineered. The science of genetic engineering was developed with the objective of building genes for genetic transformation. Genetic transformation systems possess three main components:

1) A mechanism for introduction of the foreign DNA into the target cell.

2) A cell or tissue suitable for transformation.

3) A method for the identification and selection of transformed cells or individuals.

Success in transformation for any species depends on these three components. Obviously, each one must be optimized and, therefore, as technology develops, transformation should become a more routine activity. The final objective in transformation is the introduction of a new trait in an individual. When the desired trait exists in any other sexually compatible individual, the first alternative should be to transfer the trait through crossing and selection, as has been done in conventional breeding since the 19th century. Modern soybean, corn, cotton, and wheat varieties, as well as swine, cattle, and poultry lines used in agriculture to feed the world, were initially obtained by traditional methods of crossing and selection.

One of the main limitations of conventional genetic improvement is that the breeder is limited to traits among species that are sexually compatible. For instance, the field bean is a species rich in sulfur-containing amino acids. However, beans are naturally deficient in lysine. On the other hand, rice is naturally rich in lysine, but deficient in sulfur-containing amino acids. It is not possible to naturally cross these species, so the conventional plant breeder is unable to develop a new field bean variety with elevated lysine levels or a rice cultivar rich in sulfur-containing amino acids. Genetic transformation allows the exchange of genes between organisms previously limited by sexual incompatibility. With genetic engineering and transformation, it is possible to transfer genes among bacteria, animals, plants, and viruses. In fact, one of the areas of research in biotechnology is the improvement of nutritional profiles in crops. New, more nutritional bean and rice varieties can now be developed through advances in genetic engineering.The basic tools for genetic transformation are restriction enzymes, which are used to cut DNA at specific sites, and ligases, which catalyze the joining of DNA fragments. Using the right restriction enzymes, it is possible to cut the circular bacterial plasmid DNA, causing it to linearize. With a ligase, it is possible to add other DNA fragments containing the gene of interest and join them to the linearized plasmid. Under the right conditions, the ends of the plasmid, now with the added DNA fragments, rejoin to create a new circular plasmid with some DNA modifications. The new plasmid can be introduced into certain bacteria through a process called electroporation, and the bacteria can then be used to transfer the transgene to the target species. If the plasmid DNA is integrated into the genome of the recipient species and the transferred genes are expressed, the individual is considered to be transformed or transgenic.

Tags: Bio Technology, Bio Genetics, Genetic Transformation

Understanding Stem Cell Gene Therapy

2009-05-08T20:34:00.000-07:00

Stem cell therapy or therapeutic cloning does not involve gene therapy itself. However, in the future it might be used in conjunction with gene therapy for regeneration of tissue and organs after they have been treated with corrective genes. Visually, stem cells are not distinguishable from any other cells of the human body. Under a common microscope (magnification 20 to 40 times), those cells can only be observed using special dyes. Visually there is no significant difference in such cells. The real differences exist at the DNA level, where gene expression is amendable to signals influencing protein expression. The cells can differentiate into any of the 220 cell types of the human body (e.g., kidneys, heart, liver, skin, or retina), a phenomenon called pluripotency. At birth, stem cells can be harvested from an individual's bone marrow, fat tissue, and the umbilical cord. Embryonic stem cells are harvested from embryos up to a few days after fertilization.

Another characteristic of stem cells is their capability to grow indefinitely. Whereas the remaining body cells have a biological programming that limits the number of cell divisions they can go through before dying, stem cells can be maintained indefinitely in a petri dish with nutritive media.

Stem cell therapy provides hope for a cure for patients of incurable afflictions such as Parkinson's disease and Alzheimer's disease, and also for people suffering from paralysis resulting from spinal cord injuries.

At first, some opponents speculated that stem cells would be used in nurseries to produce organs such as livers, hearts, and virtually any other body part. However, most organs possess complex structures with ducts and valves, making it impossible to produce them outside of the organism. Stem cells have opened a new avenue for disease treatment. For example, the injection of stem cells into the liver of a patient with cirrhosis or hepatitis could result in new tissue capable of performing its role. Stem cell therapy also has great potential to cure rheumatoid arthritis and some heart diseases. Recent research has found that spine-injured mice suffering from paralysis were able to move their legs following an injection of stem cells.

Some people believe that if human stem cells are as versatile as those of mice, they might be the long sought after fountain of youth. The combination of stem cells with gene therapy might allow rebuilding of new body parts to substitute for old and defective ones. Right now, different procedures are being tested for curing ADA deficiency. Somatic cell gene therapies have the limitation of lasting for only a few months, which in turn requires repeated applications. With the use of stem cells to regenerate healthy bone marrow cells, a permanent cure is expected, as healthy cells have the capability to grow and divide continuously.

Embryonic stem cells, from embryos about four days old, have been at the center of a heated debate due to ethical issues. The main disagreement is whether or not a four-day-old embryo is already a human life. When would an embryo or a fetus reach the status of life? Those that support the use of embryonic stem cells would argue that human life would not begin until about the 14th day after the fertilization, whereas the opposition argues that life begins at conception (i.e., at the moment of the fertilization of the egg by the sperm). For many, the destruction of embryos for the purpose of treating another human being is wrong. Recently, in the United States, the Bush administration broadened the definition of a child eligible for coverage under the Children's Health Insurance Program by classifying a developing fetus as an "unborn child." Many activists are arguing that the Bush administration's proposal demonstrates its commitment to the strategy of undermining a woman's right to choose abortion by ascribing legal rights to embryos.

Tags: Bio Technology, Bio Genetics, Gene Therapy

What are the Risks associated with Gene Therapy

2009-05-08T20:32:00.000-07:00

The first death associated with gene therapy occurred on September 18, 1999, at the University of Pennsylvania. Jesse Gelsinger was participating in a clinical trial, a biomedical experiment for evaluation of safety and efficiency of a therapy for a disease. Gelsinger, who was 18 years old at the time of the treatment, had a deficiency of ornithine transcarboamylase, an important enzyme in the metabolism of ammonia. Patients with this rare metabolic disorder must maintain a low-protein diet and take a series of medicines to avoid ammonia poisoning in the blood stream. The gene therapy Gelsinger took triggered a chain reaction in his immune system, resulting in hepatic and respiratory failure, and consequently, his death four days after being treated.

Since Gelsinger's death, the University of Pennsylvania has been reevaluating all procedures involved in the vector engineering and in the administration of the therapy. No flaw has been found that would explain such an extreme reaction by his immune defense system. Ever since, the public and the FDA, the agency responsible for oversight of clinical trials in the United States, have been more skeptical and doubtful about whether current scientific knowledge is enough to justify further investigations with humans. The credibility of gene therapy was seriously damaged, resulting in a temporary moratorium on human clinical trials.

Another challenge to gene therapy has been its ephemeral benefits to patients. This has been observed in several clinical trials with cystic fibrosis and ADA deficiency patients, whose cure faded after a few months of therapy, and was followed by a return of the disease symptoms. A possible explanation for that is that the genetically modified somatic cells decreased in amount. Because they are already differentiated and possess only a limited capability to multiply, it is expected that after they are gone, the treated organ could become diseased again.

Future of Gene Therapy

Although the idea of gene therapy has been around for only 20 years, the technique has been drawing a great deal of interest and curiosity through the world. The first trials generated great expectations within the scientific community. Although there have been several disappointments, many believe that it is just a matter of time before the technical and scientific details are mastered and the procedures become routine. This research is being advanced worldwide. In fact, Alain Fischer, a medical doctor in Paris, France, reported the complete cure of two children who had a rare immune deficiency condition.

Another promising result from stem cell research has been reported in type-B hemophilia patients at the Children's Hospital in Philadelphia and at Stanford University, where patients treated with gene therapy presented a reduction in the period for blood coagulation. ADA deficiency, a disease caused by a defective gene for the ADA enzyme present on human chromosome 20 has been a focus for gene therapy in many institutions. In one of the cases, several patients treated with the corrective gene were able to reconstitute their immune systems and are living normal lives out of the isolated bubbles that are needed to maintain an environment free from microbes. The patients started to produce a correct ADA enzyme after receiving the gene therapy.

The potential use of this therapy to cure other more complicated diseases, such as cancer and coronary diseases, also seems promising. Gene therapy is still in its infancy, but it is believed that as it matures, it will become an effective treatment for the myriad of genetic diseases that affect humanity.

Tags: Bio Technology, Bio Genetics, Gene Therapy

What are the pointers to Gene Delivery

2009-05-08T20:31:00.000-07:00

Appropriate methods to deliver DNA used in gene therapy are vital, as the targeted tissues must properly receive the appropriate genes. Gene therapy can be carried out using naked DNA delivered directly into the target cells. However, this procedure of introducing isolated DNA molecules has a very low efficiency rate. To increase the efficiency of DNA uptake by the target cells, special vectors have been engineered for gene transfer. Vectors are plasmids or viruses that are used to move recombinant DNA from one cell to another. A retrovirus is a special class of RNA viruses that can insert its nucleic acid into host cells. The viruses possess a gene for production of the reverse transcriptase, an enzyme that transcribes RNA in DNA in the host cell. Adenovirus, retrotransposons, and liposomes are other vectors used for gene transfer in gene therapy. They are all able to transfer and integrate genes into new cells. Retroviruses used in gene therapy are engineered so that any genes that are harmful to man are removed. Corrective genes are then added to replace the removed genes, and the new, modified retrovirus is then introduced into the patient.

One of the challenges for vectors is to survive the patient's immune system so they can transfer the corrective genes from their genome into the patient's cells. In general, the immune system of the human body contains molecules that immobilize viruses or other microorganisms that could infect the organism. Viruses that escape the immune system need to penetrate the cellular membrane, an additional barrier to infection. Finally, the infecting retrovirus must integrate its genome with that of the host, thereby moving the corrective genes into the genome of the infected cell. This integration happens in a random manner. It should occur in an area of DNA that is not essential to the host genome, or a risk of other complications might occur. Furthermore, the introduced gene must be transcribed and expressed for the production of the correct enzyme. With all these processes at the molecular level, gene therapy becomes a very complex procedure.

Another promising strategy, which has been used for the introduction of therapeutic genes in lung cancer treatment, is the direct injection of the corrective genes into the target area. Using this strategy, scientists have injected a drug containing the normal version of the gene, which suppresses cell tumor growth, directly into the patient's cancerous tumor. This technique bypasses the immune system reaction to the invading vector, a problem frequently associated with gene therapy. Many scientists believe that as gene therapy develops, it will be possible in the near future to easily introduce genes into patients through intramuscular injection, especially for cases of anemia, hemophilia, diabetes, and other diseases related to the circulatory system.

Tags: Bio Technology, Bio Genetics, Gene Therapy

Causes of Genetic Defects

2009-05-08T20:28:00.000-07:00

Each human being carries normal as well as some defective genes. Usually, the individual does not become aware of the presence of a defective gene until a disease associated with the gene is manifested in him or her or in a relative. More than 4,000 medical disorders caused by defective genes have been identified, each with varying degrees of seriousness. About 10 percent of the human population will evidence, sooner or later, some type of disorder. Although genes are responsible for predisposition to disease, the environment, diet, and lifestyle can affect the onset of the illness.

An example of a genetic disease is cystic fibrosis, which frequently becomes evident in the first years of life for the child carrying the defective gene. The mutant gene causes the development of cysts and fibrous tissue in the patient's pancreas and the production of thick and viscous lung mucous. The mucous makes breathing very difficult and, in many cases, is fatal. On average, in Western countries, about 1 child in 2,500 has the disease. If the child receives two defective recessive alleles of the gene named CF (one from each parent), he or she will develop the disease. Patients with cystic fibrosis can reduce the symptoms of the disease with drugs developed through genetic engineering. A cure for cystic fibrosis may come through gene therapy. One possibility is a genetically engineered virus, carrying the corrective gene, which after being introduced into the patient's lung cells would allow the lungs to function properly. The introduced gene would allow the lung cells to produce a protein that eliminates the mucus.
Most people do not manifest genetic diseases because, most of the time, they are carriers of just a single defective copy of the CF gene. As most of the defective genes are recessive, meaning two copies are needed for expression of the disease, most people do not have the disease. This is the reason for the larger incidence of genetic diseases in children from related parents.

If the defective gene, however, is dominant, the disease is expressed in any people that carry the defective gene. Huntington's Disease, a disorder of the nervous system that usually occurs after the age of 45, is an example of a genetic disease caused by a dominant gene.

Having a defective gene does not make disease development a certainty. Besides the large effect from genetics, the environment is also important to the onset of many illnesses. Diseases such as heart disease do have a genetic component, but are largely dependent on diet and lifestyle. Some genetic diseases also have benefits. A classic example of a genetic disease that has a beneficial effect on human survival is sickle cell anemia. There exists in the human population a defective b-hemoglobin gene and individuals carrying two copies of the defective gene develop sickle cell anemia, a blood problem caused by defective hemoglobin and consequently misshapen red blood cells. The genetic mutation in the defective allele of this disease is a single nucleotide change, from an A in normal genes to a T in the mutant. This single nucleotide mutation results in a mutant b-hemoglobin that possesses the amino acid valine instead of glutamine. The mutant b-hemoglobin has less affinity to oxygen, becoming a poor oxygen transporter in the blood.

However, carriers of a single copy of the defective allele do not have the disease, and they are also resistant to malaria. There is an obvious advantage of carrying a single allele of the defective hemoglobin gene, especially in regions where malaria is endemic, as in tropical regions of Africa.
The first case of gene therapy occurred in 1990, at the NIH in Bethesda, Maryland. On that occasion, a four-year-old patient with a severe immune system deficiency (adenosine deaminase enzyme [ADA] deficiency or bubble-boy disease) received an infusion of white blood cells that had been genetically modified to contain the gene that was absent in his genome. Since then, gene therapy has been studied and experimentally tested for several medical conditions.
Diseases caused by the absence of an enzyme or the presence of an inactive enzyme are potential targets for gene therapy. Cystic fibrosis, ADA deficiency, and many other genetic diseases are among the candidates for gene therapy.

Medical Conditions for Which Gene Therapy Is Being Studied are as under
ADA deficiency > Hemophilia
AIDS > Liver cancer
Asthma > Lung cancer
Brain tumor > Melanoma
Breast cancer > Muscular dystrophy
Colon cancer > Neurodegenerative conditions
Diabetes > Ovarian cancer
Heart diseases > Prostate cancer

Tags: Bio Technology, Bio Genetics, Gene Therapy

Understanding Gene Therapy

2009-05-08T20:25:00.000-07:00

Gene therapy has become an increasingly important topic in science-related news. The basic concept of gene therapy is to introduce a gene with the capacity to cure or prevent the progression of a disease. Gene therapy introduces a normal, functional copy of a gene into a cell in which that gene is defective. Cells, tissue, or even whole individuals (when germ-line cell therapy becomes available) modified by gene therapy are considered to be transgenic or genetically modified. Gene therapy could eventually target the correction of genetic defects, eliminate cancerous cells, prevent cardiovascular diseases, block neurological disorders, and even eliminate infectious pathogens. However, gene therapy should be distinguished from the use of genomics to discover new drugs and diagnosis techniques, although the two are related in some respects. The two main types of gene therapy are somatic cell gene therapy and reproductive or germ-line gene therapy. This chapter also discusses therapeutic cloning, which involves stem cell manipulation for tissue and organ production.

Germ-line cell therapy involves the introduction of corrective genes into reproductive cells (sperm and eggs) or zygotes, with the objective of creating a beneficial genetic change that is transmitted to the offspring. When genes are introduced in a reproductive cell, descendant cells can inherit the genes.

Gene therapy of somatic cells, those not directly related to reproduction, results in changes that are not transmitted to offspring. An example of gene therapy in somatic cells is the introduction of genes in an organ or tissue to induce the production of an enzyme. This alteration does not affect the individual's genetic makeup as a whole and it is not transmitted to its descendants. With somatic cell gene therapy, a disabled organ is better able to function normally. This technology has many applications to human health. One variant of somatic cell gene therapy is DNA vaccines, which allow cells of the immune system to fight certain diseases in a method similar to conventional vaccines.

Stem cell therapy involves the use of pluripotent cells, or cells that can differentiate into any other cell type. Stem cells are found in developing embryos and in some tissues of adult individuals. This therapy is similar to a conventional transplant, with the objective of regenerating or repairing a damaged organ or tissue. The procedure has a reduced probability of rejection because it uses the individual's own cells. For instance, stem cells differentiated into nerve cells could be used by patients suffering from paralysis, with the goal of helping them recovering movement; or in cases of heart stroke, muscle cells might be used to rejuvenate the cardiac muscles. Furthermore, the future may bring the growth of stem cells from an individual's body to produce certain tissues or organs in vitro. Stem cell research could eventually blend gene therapy with genetic engineering to create healthy stem cells that can be used to generate healthy organs and tissue.

A fundamental requirement for gene therapy is the correct identification of genes coding for diseases. This can be accomplished at a spectacular speed with the information from the Human Genome Project. Scientific magazines have been announcing, with great frequency, the discovery of genes responsible for several medical conditions, from Alzheimer's disease to baldness. The knowledge of the genes involved in these traits allows unequivocal diagnosis of the disease in the patient, an essential step before treatment can be initiated for the genetic disease. Biotechnology is contributing to the development of the needed genetic tests for detection of defective genes.
The most complex phase in gene therapy is the development of mechanisms to deliver the therapeutic genes to the target organ in an accurate, controlled, and effective way. That step has been developing more slowly and is currently the most limiting factor for gene therapy.

Tags: Bio Technology, Bio Genetics, Gene Therapy

What are the molecule separation techniques based upon size

2009-04-28T08:02:00.000-07:00

The techniques based on size and weight of the molecules are Gel Filtration, Osmotic Pressure & Centrifugation

Gel-filtration Chromatography:
Gel-filtration chromatography is a separation based on size. It is also called molecular exclusion or gel permeation chromatography. In gel filtration chromatography, the stationary phase consists of porous beads with a well-defined range of pore sizes. The stationary phase for gel filtration is said to have a fractionation range, meaning that molecules within that molecular weight range can be separated.

Proteins that are small enough can fit inside all the pores in the beads and are said to be included. These small proteins have access to the mobile phase inside the beads as well as the mobile phase between beads and elute last in a gel-filtration separation. Proteins that are too large to fit inside any of the pores are said to be excluded. They have access only to the mobile phase between the beads and, therefore, elute first. Proteins of intermediate size are partially included-meaning they can fit inside some but not all of the pores in the beads. These proteins will then elute between the large ("excluded") and small ("totally included") proteins.

Consider the separation of a mixture of glutamate dehydrogenase (molecular weight 290,000), lactate dehydrogenase (molecular weight 140,000), serum albumin (MW 67,000), ovalbumin (MW 43,000), and cytochrome c (MW 12,400) on a gel-filtration column packed with Bio-Gel P-150 (fractionation range 15,000 to 150,000). When the protein mixture is applied to the column, glutamate dehydrogenase would elute first because it is above the upper fractionation limit. Therefore, it is totally excluded from the inside of the porous stationary phase and would elute with the void volume (VO). Cytochrome c is below the lower fractionation limit and would be completely included, eluting last. The other proteins would be partially included and elute in order of decreasing molecular weight.

These separations can be described with this equation

Vr, = Vo + KVi

where Vr is the retention volume of the protein, Vo is the volume of mobile phase between the beads (outside the beads) of the stationary phase inside the column (sometimes called the void volume), Vi is the volume of mobile phase inside the porous beads (also called the included volume), and K is the partition coefficient (the extent to which the protein can penetrate the pores in the stationary phase, with values ranging between 0 and 1). In the mixture of proteins listed above, the partition coefficient (K) for glutamate dehydrogenase would be 0 (totally excluded), K = 1 for cytochrome c (totally included), and K would be between 0 and 1 for the other proteins, which are within the fractionation range for the column.

In practice, gel-filtration can be used to separate proteins by molecular weight at any point in purification of a protein. It can also be used for buffer exchange; a protein dissolved in a sodium acetate buffer, pH 4.8, can be applied to a gel-filtration column that has been equilibrated with Tris buffer pH 8.0. Using the Tris buffer as the mobile phase, the protein moves into the Tris mobile phase as it travels down the column, while the much smaller sodium acetate buffer molecules are totally included in the porous beads and travel much more slowly than the protein. Similarly, it can be used for the separation of salts and other small molecules from a protein sample.

Osmotic Pressure:
Molecules always move from the region of their higher concentration to the region of their lower concentration. It is applicable to both solvent and solute molecules in the case of a solution. When a solution is separated from a pure solvent by a membrane that is permeable to the solvent alone, the molecules of the solvent will move into the solution. The flow of the solvent molecules into the solution can be prevented by applying some amount of pressure that is equal to the pressure exerted by the solvent molecules to enter into the solution compartment through the membrane partition. The pressure that is needed to prevent the entry of the solvent molecule into the solution is called osmotic pressure.

The osmotic pressure of a solution depends on the concentration of a solute and the temperature of the solution. It can be used for the calculation of the molecular weight of the solute.

#V=nRT

where #is the osmotic pressure, V is the volume of the solution, n is the number of moles of solute, R is gas constant, and T is the absolute temperature.

#= n/V x RT (n/V= M, molarity of the solution)

Therefore, # = MRT

But in this equation, n-the number of moles = weight of the solute in grams/molecular weight.

i.e.#V = Wt 9 /MW x RT Therefore,

MW = Wt 9 /#V x RT. (Wt 9 /Volume = Concentration, C)
i.e. MW = CRT/#.

Thus, if the osmotic pressure and the concentration of the solution are available the molecular weight of the solute can be determined.

Tags: Bio Technology, Bio Genetics , Biochemical Techniques

What is Density Gradient Centrifugation

2009-04-28T08:00:00.000-07:00

Density gradient centrifugation are of two types:

1) Rate zonal centrifugation

2) Isopycnic centrifugation

Rate zonal centrifugation:
In rate zonal centrifugation, the sample is applied in a thin zone at the top of the centrifuge tube on a density gradient. Under centrifugal force, the particles will begin sedimenting through the gradient in separate zones according to their size, shape, and density or the sedimentation coefficient(s). The run must be terminated before any of the separated particles reach the bottom of the tube. S is the sedimentation coefficient and is usually expressed in Svedbergs (S) units.

Isopycnic centrifugation:
In the isopycnic technique, the density gradient column encompasses the whole range of densities of the sample particles. The sample is uniformly mixed with the gradient material. Each of the particles will sediment only to the position in the centrifuge tube at which the gradient density is equal to its own density, and there it will remain.

The isopycnic technique, therefore, separates particles into zones solely on the basis of their buoyant density differences, independent of time. In many density gradient experiments, particles of both the rate zonal and the isopycnic principles may enter into the final separations. For example, the gradient may be of such a density range that one of the components sediments to its density in the tube and remains there, while another component sediments to the bottom of the tube. The self-generating gradient technique often requires long hours of centrifugation.

Isopynically banding DNA, for example, takes 36 to 48 hours in a self-generating cesium chloride gradient. It is important to note that the run time cannot be shortened by increasing the rotor speed; this only results in changing the position of the zones in the tube since the gradient material will redistribute further down the tube under greater centrifugal force.

Tags: Bio Technology, Bio Genetics , Biochemical Techniques

What is Centrifugation Process for isolation of molecules

2009-04-28T07:57:00.000-07:00

Biomolecules can be isolated and purified by applying different techniques, which are based on various chemical and physical properties. The main physical and chemical properties that can be exploited for their separation and characterization of biomolecules are molecular weight and size, interaction with electromagnetic radiations or spectroscopic properties, solubility, molecular charge, and polarity.

Centrifugation is a technique based on size and weight of the molecules.

A centrifuge is a device for separating particles from a solution according to their sedimentation rate, which depends on factors like size, shape, density, viscosity of the medium, and centrifugal force (rotor speed). This process of separation of particles based on its sedimentation rate is called centrifugation. In biology, the particles are usually cells, sub-cellular organelles, viruses, and large molecules such as proteins and nucleic acids. The rate of sedimentation will be directly proportional to the molecular weight or size, if all other factors are constant. To simplify mathematical terminology we will refer to all biological material as spherical particles.

Following are the ways to classify centrifugation:

1) Analytical and Preparative Centrifugation:

The two most common types of centrifugation are analytical and preparative; the distinction between the two is based on the purpose of centrifugation. Analytical centrifugation involves measuring the physical properties of the sedimenting particles such as sedimentation coefficient or molecular weight. Optimal methods are used in analytical ultracentrifugation. Molecules are observed by an optical system during centrifugation, to allow observation of macromolecules in the solution as they move in the gravitational field. The samples are centrifuged in cells having windows that lie parallel to the plane of rotation of the rotor head. As the rotor turns, the images of the cell (proteins) are projected by an optical system onto film or a computer. The concentration of the solution at various points in the cell is determined by absorption of light of the appropriate wavelength (Beer's law is followed). This can be accomplished either by measuring the degree of blackening of a photographic film or by the pen deflection of the recorder of the scanning system, which is fed into a computer.

The other forms of centrifugations are preparative and the objective is to isolate specific particles, which can be reused. There are many types of preparative centrifugation such as rate zonal, differential, and isopycnic centrifugation.

2) Ultracentrifugation vs Low-speed Centrifugation:

Another system of classification is the rate or speed at which the centrifuge is turning. Ultracentrifugation is carried out at speed faster than 30,000 rpm. High-speed centrifugation is at speeds between 10,000 and 30,000 rpm. Low-speed centrifugation is at speeds below 10,000 rpm (mostly between 3,000 to 9,000 rpm).

3) Moving Boundary vs Zone Centrifugation:

A third method of defining centrifugation is by the way the samples are applied to the centrifuge tube. In moving boundary or differential centrifugation, the entire tube is filled with the sample and centrifuged. Through centrifugation, one obtains a separation of the mixture into two parts-a supernatant and a pellet. But any particle in the mixture may end up in the supernatant or in the pellet or it may be distributed in both fractions, depending upon its size, shape, density, and conditions of centrifugation. The pellet is a mixture of all of the sedimented components, and it is contaminated with whatever unsedimented particles were in the bottom of the tube initially. The only component that is purified is the slowest sedimenting one, but its yield is often very low. The two fractions are recovered by decanting the supernatant solution from the pellet. The supernatant can be recentrifuged at higher speeds to obtain further purification with the formation of a new pellet and supernatant.

Tags: Bio Technology, Bio Genetics , Biochemical Techniques

What is Biopiracy

2009-04-25T22:56:00.000-07:00

Plants constitute a rich source of therapeutically important products. Only 10 percent of plant species have already been tested for pharmaceutical value. About 120 medicines commonly prescribed by doctors are based on plant extracts. Some of the most important medicines used by humans have a history that traces back to medicinal plants collected from wild flora, and others are just now beginning to be discovered.

Aspirin is one example of the health benefits provided by plants. This medicine is based on acetyl-salicylic acid, a very common analgesic. Its history began with the Greek doctor Hypocrites, who in the fifth century B.C. used a bitter powder to treat pain and lower fever. This mystical powder was collected from the cork of Salix a tree of the family Salicaceae. Although the mode of action of aspirin was only unraveled in the 1970s, this medicine has been used as a painkiller and to improve the elasticity of the circulatory system for millions of people. If this species had gone extinct before that discovery, man would have never known the valuable medicine. In fact, Americans annually consume about 80 billion aspirin tablets.

The history of biodiversity collection dates back to 1500 B.C., when Egyptian rulers gathered plant species from their military expeditions. Charles Darwin, the renowned naturalist of the 19th century, accomplished one of the most famous trips for biological collection. During his travels on the ship the HMS Beagle, he collected samples of everything that interested him, from which he elaborated the Theory of the Evolution, the foundation of modern biological research. More recently, Nicolai I. Vavilov, a Russian scientist in the beginning of the 20th century, also collected samples of plant species from five continents, with which he established the Theory of the Center of Origin of the Crop Species.

None of those famous expeditions were legally or morally questioned. Today, the paradigms and laws have changed, and biopiracy is considered a crime. Biopiracy is the unauthorized appropriation of any biological resources. The extraction of aromatic, ornamental, or medicinal plants without the proper authorization is considered biopiracy. Natural resources primarily from Africa and South America are becoming increasingly valued in the international market. In the 1500s, Brazilian wood was prized for making red dyes; today, targeted Brazilian species number about 50,000 plant species, 534 mammals, 3,000 fishes, approximately 1,700 birds, 500 amphibians, and 470 reptiles. The wealth of Brazilian biodiversity makes the country a valuable source of genetic diversity. Every year, thousands of tourists, scientists, environmentalists, and biologists travel around the world under the umbrella of ecological tourism. Although this type of travel has improved research on biodiversity, it has also caused problems relating to biopiracy.

Scientists and pharmaceutical companies are obtaining several patents using plant extracts from different regions around the world. Recently, the English chemist Conrad Gorinsky received a worldwide patent for two pharmaceutical products: Rupununine, extracted from seeds of the Octotea rodioei, for birth control; and Cunaniol, a nervous system stimulant, extracted from Clibadium sylvestre. The use of the plants is part of the traditions of the native Wapixana Indians, who live in the Brazilian state of Roraima. Several other bioprospecting projects are underway in Africa and other places to identify plant extracts, animal toxins, and microorganisms for different purposes such as production of plastics or ore purification and fermentation processes.

When a sample of a species is collected illegally and a new drug or an isolated gene from that sample is patented, the patent can be revoked. If there is proof that the active ingredient used in the new drug was in public use, even if restricted to an indigenous tribe, revocation of the patent is possible. The great dilemma in patenting a natural product is that pharmaceutical companies take advantage of the ethnobiological knowledge of indigenous populations, and later, the companies are the only ones to collect profits from the marketing and production of the drugs.

To prevent such exploitation, regulations are being made worldwide to govern the use of biological diversity. In 1992, the United Nations Conference on Environment and Development (ECO 92) met in Rio de Janeiro, Brazil, with representatives from 120 countries. This conference recognized the national sovereignty of the nations and the genetic resources within their borders. Beyond this international work, the national laws of each country further govern the conservation and development of biodiversity within their respective boundaries. The Brazilian Congress recently recognized the importance of the protection of its biodiversity. This came after an accord with some multinational companies relating to the development of medicines resulting from the exploration of plants and microorganisms from the Atlantic rainforests and the Amazon.

Only 20 years ago, legal aspects related to the collection of samples of plants, microbes, and animals were largely ignored. In most cases, researchers simply made a trip to the place where the species of interest could exist in nature, collected the samples, and returned to their laboratories. Clearly laws didn't exist to regulate that practice. Sometimes, researchers obtained informal authorization from the local authority or from the landlord where the samples were collected. The days of locating, collecting, and returning home are running out, at least legally in most countries. More often it is today considered biopiracy.

Tags: Bio Technology, Bio Genetics, Bio diversity

What is Genetic Erosion

2009-04-25T22:54:00.000-07:00

Until the 1940s, the centers of origin of crop species and animals were considered limitless sources of genetic variability. After World War II, agriculture in developing countries suffered great changes. The expanded use of improved varieties resulted in the reduction of traditional varieties, a process called genetic erosion. The expansion of the agricultural frontiers also contributed to the risk of loss of the wild relatives of crop species.

According to a study carried out by the National Academy of Sciences in the United States, of approximately 3,000 possible plant species, only 20 to 30 constitute the basis of agriculture. For example, amaranth has high economic potential and has been recommended as a species that deserves more attention from plant breeders, with the objective of improving the plant to make it more valuable for commercial use. This requires the removal of undesirable traits and the improvement of other traits to allow for improved production.

The process of genetic erosion also occurs with many other species of flora, fauna, and microorganisms, and it is the first sign indicating possible species extinction. Environmental deterioration initially results in local extinction and later culminates with the global extinction of the species. For instance, well before species vanish, a small number of survivors could result in inbreeding of the population. Inbreeding results from intermating between related individuals that causes the generation of less fit individuals with a greater likelihood of genetic defects.

Biotechnology can help in the diagnosis of genetic erosion before any conventional techniques. This can be achieved by DNA analyses that quantify the remaining genetic diversity. However, as each organism has a different genome, these methods would have to be developed for each species. This technique has been used with success in the study of wolf species, fish, cattle, macaws, whales, and other animals. In many cases, the studies were used to justify the creation of new refuges where such species dwell.

Tags: Bio Technology, Bio Genetics, Bio diversity

Why Preserve Biodiversity

2009-04-25T22:50:00.000-07:00

The preservation of genetic variation has become an important subject for many species. Various species of plants, animals, and microorganisms have been collected and stored, so the immense species variation might not be lost. The culture of cells and tissue, an area within biotechnology, is being used for the maintenance of live collections of the most varied types of plant species of economic importance or others at risk for extinction. For instance, the preservation of the genetic diversity of cassava is accomplished at tissue culture laboratories, where thousands of different varieties and species are maintained in small petri dishes. In the Frozen Zoological Garden in San Diego, California, there are live cellular lineages of species of several families of mammals, many close to extinction. It is expected that in the near future, cloning techniques will be used to regenerate whole animals from the cells. Had tissue culture technologies not been developed, the required space and costs to preserve rare species would be many times larger, limiting the number of species that could be preserved.

The preservation of microbial diversity has also been made possible by biotechnological techniques. If the bacteria, fungi, and viruses had to be maintained in their traditional hosts, only a small fraction of the biodiversity of microorganisms could be preserved. The germplasm banks of bacteria and fungi require a relatively small and rudimentary laboratory for preservation. The main objectives of microorganism gene banks are related to the preservation of species for subsequent laboratory studies.

Legal mechanisms should be developed to protect the biodiversity of the world. The mechanisms should promote the conservation of biodiversity and its use for the well-being of mankind. Developed countries and many others have taken the lead to ensure germplasm preservation for years to come. Extensive efforts are being made to characterize, catalog, and store the germplasm resources collected over many years from all over the world. Further steps are being taken to guarantee the preservation of the biodiversity of ecosystems and centers of origin for future research. Biotechnology will benefit from the world's biodiversity, while creating a means of preservation and continuation of the diversity of life found around the world.

There is an incredible amount of biodiversity worldwide, and much of it is relatively unknown. Despite the actions to explore this diversity, steps are being taken to characterize and preserve this valuable resource.

Tags: Bio Technology, Bio Genetics, Bio diversity

Understanding Biodiversity

2009-04-25T22:40:00.000-07:00

Biodiversity, or biological diversity, refers to every form of life within an area or ecosystem. This includes the genetic variability within the populations and species; the different species of flora, fauna, and microorganisms; the variety of functions and ecological interactions carried out by the organisms in the ecosystems; and the various communities, habitats, and ecosystems formed by the organisms. Biodiversity is the fruit of the great laboratory, which is the planet Earth, with its more than 30 million different species resulting from 4 to 5 billion years of evolution.

The importance of preserving biodiversity is also referred to in sacred books, such as the Bible, which relates that Noah saved domestic and wild animals from the great flood. Biodiversity is one of the fundamental properties of nature responsible for the balance and stability of ecosystems. It is also of great economic value. This diversity is the basis of farming and food production, and it is essential for biotechnology. The ecological functions carried out by various organisms are still poorly understood, but biodiversity is thought to be responsible for the natural processes and products supplied by ecosystems. It accounts for the species that sustain other life forms and also modifies the biosphere, making it suitable and safe for life. Biological diversity possesses, besides an intrinsic worth, a value of ecological, genetic, social, economical, scientific, educational, cultural, recreational, and aesthetic importance.

A reduction in biological diversity is hazardous to sustainable development. Genetic erosion (the loss of species variability) and the extinction of species can influence us to develop strategies that contribute to the preservation of the remaining biodiversity on the planet, at a level that is already smaller than it was a century ago. The preservation of biodiversity is also essential for human well-being. However, recent studies have indicated that extinction rates are 1,000 times faster than those expected naturally, with 50,000 species extinguished every year. Currently, about 34,000 plant species and 5,200 animal species are at risk of becoming extinct.

Biotechnology can be understood as a technology that explores biological systems instead of individual living organisms. Therefore, the preservation of the biological systems with all of their diversity can be considered a priority as well as a challenge to mankind.

Microbes, such as bacteria, are the most diverse of all living organisms. Some estimates indicate that there exist more than 1 million different species of bacteria in the world. Recent reports suggest that an extremely large number of bacteria exist in the biosphere awaiting the development of appropriate techniques needed to grow them, so that they can be characterized.
This is one example of one key part of the greater picture of biodiversity. Plants, animals, and even fungi are also important aspects of the world's biodiversity. This idea of biodiversity is an important part of biotechnology, as useful traits and chemicals are becoming part of important new biotechnology applications. Biotechnology brings, simultaneously, promises of biodiversity preservation and also the fear of genetic erosion and biopiracy.

Tags: Bio Technology, Bio Genetics, Bio diversity

How the Living Organisms interact with the Environment

2009-03-31T23:27:00.000-07:00

Living organisms and the physical environment have a close relationship. They interact with each other. The biosphere is the parts of Earth inhabited by living organisms. The biosphere consists of specific geographical areas known as biomes. A biome is a collection of different types of ecosystems. The ecosystems include grasslands, rain forests, streams, lakes, sea, deserts etc., with various types of organisms starting from bacteria, fungi, algae, and various other types of plants and animals. There are millions of known species of organisms and there are many millions to be discovered. Each organism lives in a specialized regional environment within the ecosystem known as the habitat. An organism can live in a specific habitat because it is adapted to live in that habitat. Deep-sea vent, bottom of sea, arctic rivers, and river banks, etc. are examples of habitat.

Organisms living in a specific environment interact with the environment and also with themselves in very different ways. There are big trees growing along the bank of the stream. Since the trees are very big, they make half of the stream a shady area, and this may make the temperature of the water a bit lower than that in the middle region. It is because the water at the bank side is not directly heated by the sun. Similarly, there are many algae floating in the water freely, and this may reduce the penetration of sunlight. So the light intensity under the water may be decreased. All the organisms living in an environment along with that physical environment form an ecosystem. The organisms living in a fresh water pond, along with the pond, form an aquatic ecosystem. All organisms on land along with their environment form the terrestrial ecosystem.

The energy flow in an ecosystem obeys the laws of thermodynamics. It is an open system. An open System allows the free flow or exchange of energy and matter such as water, carbon dioxide, nitrogen, food materials, and even the movement of organisms from one ecosystem to another. There are producers, consumers, and decomposers in ecosystems. The producers are the photosynthetic organisms or the autotrophs. The producers of the ecosystem take energy from sunlight and convert it into chemical energy. This energy is passed on to consumers and then to decomposers, which cycles back the materials to the environment. But the energy flows only in one direction and is not cycled back. Herbivorous animals consume the organic food synthesized by the producers, which form the primary consumers.

These herbivores form the food of carnivores, which are the secondary consumers. And finally the decomposers act on the dead remains of all these organisms including the producers. decompose the organic materials into inorganic materials, and thus cycle back the materials to the environment. This forms the food chain in the ecosystem. In each step of the food chain energy is also transferred. In each step a portion of the energy is lost in the form of heat. Thus, heat is flowing in one direction and is not cycled back. The energy enters the ecosystem from the sun through producers and leaves the ecosystem in all steps of the food chain in the form of metabolic heat.

The materials in the form of nutrients required for life are cycled between organisms and the environment. The materials are absorbed by the producers for synthesizing the nutrients and are cycled among the consumers and finally returned to the environment by the activity of saprophytes and other decomposers such as fungi and bacteria. Considering the flow of energy and nutrients in the ecosystem and in the biosphere, it can be considered a single living organism.

Tags: Bio Technology, Bio Genetics , Life Forms, organisms

Size & Complexity of Living Organisms

2009-03-31T23:25:00.000-07:00

Living organisms greatly differ in size and complexity of their body. They range from minute unicellular bacteria to very big multicellular organisms such as blue whales and redwood trees. Primitive cell forms such as bacteria and blue-green algae are very simple in organization and function. The cells and organisms are very small and cannot be seen with the naked eye. A microscope is needed for observing these microorganisms. The multicellular organisms and their cells are very complex in organization and function. The body of higher plants and animals consists of billions of various types of specialized, structurally and functionally complex cells. Therefore, their body and its function is highly complicated.

Variations in the body size affect various other body measurements differently. This is because the volume of cells and so the volume of the entire organism increases much faster than the surface area. Entire single-celled organisms and most primitive multicellular organisms use their cell surfaces to acquire nutrients and dispose of wastes. But the amount of nutrients needed, and the quantity of wastes produced, is related to cell volume. Since the surface area to volume ratio of a cell decreases as its size increases, cells have an upper limit on how much volume they can sustain with a given surface area. Large organisms have less surface area relative to mass than do small organisms. This relationship affects the efficient exchange of material between the body and the environment. Allometric relationships describe the effect of body size on biological features. These relationships can reveal general patterns of how organisms function; for example, how much they sleep, their food requirements, and their brain size.

Allometric relationships also have practical applications, as in the proper determination of drug dosages for animals of differing body sizes. For multicellular organisms, increases in overall body size are mostly due to increases in cell number, not cell size. This is also because of surface area to volume ratio limitations on cells.

The evolution of complexity in multicellular organisms is driven by the specialization of cells. Multicellular complexity requires coordination among body cells. Internal communication mechanisms such as hormones and the nervous system help make this possible. Complexity also requires many body cells to give up reproduction in support of a relatively few cells that do reproduce.

Tags: Bio Technology, Bio Genetics , Life Forms, organisms

What are the Hierarchical Levels of Life Forms

2009-03-31T23:23:00.000-07:00

Life on Earth is incredibly extensive and to make it easier to study, biologists have broken living systems up into generalized hierarchical levels as given below:

1) Molecules
2) Organelles
3) Cells
4) Tissues
5) Organs 6) Organisms
7) Populations
8) Communities
9) Ecosystems
10) Biosphere

The lowest level of the biological hierarchy begins with molecules. Examples include proteins, DNA, lipids, etc. Many such specialized molecules are organized into cells, the basic unit of life. There are single-celled organisms such as bacteria, amoeba, yeast, etc., in which the body consists of a single cell. When the body consists of more than one cell it is called multicellular. Multicellular organisms are collection of various types of specialized cells. A group of specialized cells carrying out a specific function is called a tissue. For example, muscle tissue, nervous tissue, connective tissue, etc. When different types of tissues are organized together to perform a common function it is called an organ. Examples include, liver, stomach, heart, etc. When a number of organs function together to accomplish a specific function of the body, it forms an organ system. For example, the stomach, liver, intestine, pancreas, salivary glands, etc. work together to form the digestive system. In an organism there are a number of organ systems that work in an associated way to form the organism and its life activities. Each individual organism is a member of a large population, which exists in a habitat.

A population is a group of organisms belonging to a species. A group of different species that live and interact in a particular area or environment is known as a community. The communities, along with the environment in which they exist, are known as ecosystems. An ecosystem consists of biomes, which are large geographical areas of the world. Each biome is a part of the biosphere, which includes the entire living population on the Earth along with its physical environment.

Tags: Bio Technology, Bio Genetics , Life Forms, organisms

Adaptation the special feature of Organisms

2009-03-31T23:21:00.000-07:00

The existence of an organism in its environment or habitat is closely related to the special features of that organism or the adaptations. Adaptation is the special feature of an organism's morphology, anatomy, and physiology, which improves its interaction with its environment.

Adaptations usually have the following characteristics.
1) Special features are specially suited to a specific habitat.
2) These special features are often complex.
3) These special features help organisms to live in their environment and capture food, regulate the body's physiology, reproduce, disperse, and defend against enemies.

Adaptation is one of the important factors that drives the process of evolution. Adaptations are created through mutation and natural selection. Evolution requires genetic variation. In order for continuing evolution there must be mechanisms to increase or create genetic variation and mechanisms to decrease it. Mutation is a change in a gene. These changes are the source of new genetic variation. Natural selection operates on this variation. If these variations are suited to the changed environment that organism will outperform the others, which leads to the evolution of the population. If these new changes created through mutation are not suitable for existence in that environment, they will lead to extinction.Natural selection:Some types of organisms within a population leave more offspring than other Over time, the frequency of the more prolific type will increase. The difference ii reproductive capability is called natural selection. Natural selection is the only mechanism of adaptive evolution; it is defined as differential reproductive success of pre-existing classes of genetic variants in the gene pool.

The most common action of natural selection is to remove unfit variants as they arise via mutation. In other words, natural selection usually prevents new alleles from increasing in frequency. This led a famous evolutionist, George Williams, to say "Evolution proceeds in spite of natural selection."

Natural selection can maintain or deplete genetic variation depending on how it acts. When selection acts to weed out deleterious alleles, or causes an allele to sweep to fixation, it depletes genetic variation. When heterozygotes are more fit than either of the homozygotes, however, selection causes genetic variation to be maintained. (A heterozygote is an organism that has two different alleles at a locus; a homozygote is an organism that has two identical alleles at a locus.) This is called balancing selection. An example of this is the maintenance of sickle cell alleles in human populations subject to malaria. Variation at a single locus determines whether red blood cells are shaped normally or sickled. If a human has two alleles for sickle cell, he /she develops anemia-the shape of sickle cells precludes them from carrying normal levels of oxygen. However, heterozygotes who have one copy of the sickle cell allele coupled with one normal allele enjoy some resistance to malaria--the shape of sickle cells make it harder for the plasmodia (malariacausing agents) to enter the cell.

Thus, individual homozygous for the normal allele suffer more malaria than heterozygotes. Individual homozygous for the sickle cell are anemic. Heterozygotes have the highest fitness of these three types. Heterozygotes pass on both sickle cell and normal alleles to the next generation. Thus, neither allele can be eliminated from the gene pool. The sickle cell allele is at its highest frequency in regions of Africa, where malaria is most pervasive. Balancing selection involves opposing selection forces. An equilibrium results when two alleles selected in the homozygous state are retained because of the superiority of heterozygotes. Balancing selection is rare in natural populations.

Tags: Bio Technology, Bio Genetics , Adaptation of organisms

Know the Biodiversity of Life forms

2009-03-31T23:19:00.000-07:00

Biodiversity is the occurrence of all life forms in the biosphere. The phenomenon of speciation increases biodiversity. Biodiversity can be for a specific region or geographical area and similarly can be within a species. Within a species there can be varieties or sub-species, strains, and types. This variation within a species constitutes the biodiversity within a species. It is directly linked to the stability of the ecosystem. The magnitude of the biodiversity is not completely studied. The total number of species collected, named, and classified in taxonomic groups is around 1.5 million. This number is only a small fraction, about 10% of all living organisms, in this biosphere. The remaining, more than 90%, remains to be identified and classified.

Out of this 1.5 million known species, 750,000 are insects. The remaining part includes 280,000 animal species and 250,000 numbers of plant species. There are approximately 69,000 fungi, 27,000 algae, 3,000 protozoans, and about 3,000 prokaryotes including eubacteria and archaebacteria. Among these known groups, some have been studied extensively and others have been studied very poorly. Biodiversity, which is created by speciation and evolution, has a direct impact on the stability of the ecosystem and the biosphere. Due to many man-made changes in the environment through deforestation and construction of big dams, there is disturbances in the habitat of the species, slowly leading to their mass extinction and destabilization. This loss of biodiversity is non-reversible unless we take special precautions. The phenomenon of extinction is opposite to that of speciation. Extinction is the ultimate fate of all species.

Reasons for Extinction : The reasons for extinction are numerous. A species can be competitively excluded by a closely related species, the habitat a species lives in can disappear, and/or the organisms that the species exploits could come up with an unbeatable defense. Some species enjoy a long tenure on the planet while others are short-lived. Some biologists believe species are programed to go extinct in a manner analogous to organisms being destined tc die. This is ordinary extinction. The majority, however, believe that if the environment stays fairly constant, a well-adapted species could continue to survive indefinitely.

Mass extinctions: Mass extinctions shape the overall pattern of macroevolution. If you view evolution as a branching tree, it's best to picture it as one that has been severely pruned a few times in its life. The history of life on this earth includes many episodes of mass extinction in which many groups of organisms were wiped off the face of the planet. Mass extinctions are followed by periods of radiation where new species evolve to fill the empty niches left behind. It is probable that surviving a mass extinction is largely a function of luck. Thus, contingency plays a large role in patterns of macroevolution.

Tags: Bio Technology, Bio Genetics , Genetic Variation

Understanding of Speciation

2009-03-31T23:16:00.000-07:00

Speciation is the process of a single species becoming two or more species. Many biologists think Speciation is key to understanding evolution. According to biological species concept, species are groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups. Speciation is thus seen in terms of the evolution of isolating mechanisms and is said to be complete when reproductive barriers are sufficient to prevent gene flow between the two new species. The problem is that the capacity to interbreed cannot always be tested neither the potential for interbreeding. For asexually reproducing organisms and fossils, this concept does not apply.

Modes of Speciation
Biologists recognize two types of speciation: allopatric and sympatric speciation. The two differ in geographical distribution of the populations in question.

Allopatric speciation: is thought to be the most common form of speciation. It occurs when a population is split into two (or more) geographically isolated subdivisions that organisms cannot bridge. Eventually, the two populations gene pools change independently until they cannot interbreed even if they were brought back together. In other words, they have speciated.

Sympatric speciation: occurs when two sub-populations become reproductively isolated without first becoming geographically isolated. Insects that live on a single host plant provide a model for sympatric speciation. If a group of insects switched host plants they would not breed with other members of their species still living on their former host plant. The two sub-populations could diverge and speciate.

Tags: Bio Technology, Bio Genetics , Genetic Variation

Understanding of Genetic Variation

2009-03-31T23:12:00.000-07:00

Evolution requires genetic variation. If there were no dark moths, the population could not have evolved from mostly light to mostly dark. In order for continuing evolution there must be mechanisms to increase or create genetic variation and mechanisms to decrease it. Mutation is a change in a gene. These changes are the source of new genetic variation. Natural selection operates on this variation.

Genetic variation has two components:
allelic diversity and non-random associations of alleles. Alleles are different versions of the same gene. For example, humans can have A, B, or O alleles that determine one aspect of their blood type. Most animals, including humans, are diploid-they contain two alleles for every gene at every locus, one inherited from their mother and one inherited from their father. Locus is the location of a gene on a chromosome. Humans can be AA, AB, AO, BB, 130, or 00 at the blood group locus. If the two alleles at a locus are of the same type (for instance two A alleles) the individual would be called homozygous. An individual with two different alleles at a locus (for example, an AB individual) is called heterozygous. At any locus there can be many different alleles in a population, more alleles than any single organism can possess. For example, no single human can have an A, B, and an 0 allele.

Allele frequency: The number of organisms in a population carrying a particular allele of gene determines the allele frequency. In population genetics the allele frequency is usually expressed as decimals. Thus, a frequency of 99% is represented as 0.99 and the 1% frequency would be 0.01, because the total population represents 100% or 1.0. In population genetics these are represented as:
p+q=1, where p - frequency of dominant allele & q - frequency of recessive allele.

Thus, in the above example total frequency is 0.99 + 0.01 = 1.0.

If we know the frequency of one allele (gene), the frequency of the other allele can be determined.

Non-random breeding: In most of the natural population, mating is nonrandom. But there are many structural and behavioral mechanisms that prevent the random mating. In populations where there is no random mating, fewer heterozygotes (an organism that has two different alleles at a locus) are found than would be predicted under random mating. A decrease in heterozygotes can be the result of mate choice, or simply the result of population sub-division. Most organisms have a limited dispersal capability, so their mate will be chosen from the local population.

Genetic Drift: The variation in allele frequencies can occur only by chance. This is called genetic drift. Drift is a binomial sampling error of the gene pool. What this means is, the alleles that form the next generation's gene pool are a sample of the alleles from the current generation. When sampled from a population, the frequency of alleles differs slightly due to chance alone. Alleles can increase or decrease in frequency due to drift.

Gene Flow: New organisms may enter a population by migration from another population. If they mate within the population, they can bring new alleles to the local gene pool. This is called gene flow. In some closely related species, fertile hybrids can result from interspecific matings. These hybrids can vector genes from species to species. Gene flow between more distantly related species occurs infrequently. This is called horizontal gene transfer.

Mutation: The cellular machinery that copies DNA sometimes makes mistakes. These mistakes alter the sequence of a gene. This is called a mutation. There are many kinds of mutations. A point mutation is a mutation in which one "letter" of the genetic code is changed to another. Lengths of DNA can also be deleted or inserted in a gene; these are also mutations. Finally, genes or parts of genes can become inverted or duplicated. Typical rates of mutation are between 10-1° and 10-1' mutations per base pair of DNA per generation. Most mutations are thought to be neutral with regards to fitness. Only a small portion of the genome of eukaryotes contains coding segments. And, although some non-coding DNA is involved in gene regulation or other cellular functions, it is probable that most base changes would have no fitness consequence.

Most mutations that have any phenotypic effect are deleterious. Mutations that result in amino acid substitutions can change the shape of a protein, potentially changing or eliminating its function. This can lead to inadequacies in biochemical pathways or interfere with the process of development. Organisms are sufficiently integrated that most random changes will not produce a fitness benefit. Only a very small percentage of mutations are beneficial. The ratio of neutral to deleterious to beneficial mutations is unknown and probably varies with respect to details of the locus in question and environment.

Genetic Load: The existence of disadvantageous alleles in heterozygous genotypes within the population is known as genetic load. The disadvantageous alleles when come as homozygous will affect the organism negatively for their phenotype and their existence. Such organisms may be eliminated from the population when these alleles (if they are deleterious in nature) occur in homozygous condition. If the allele is recessive, its effect won't be seen in any individual until a homozygote is formed. The eventual fate of the allele depends on whether it is neutral, deleterious, or beneficial.

Tags: Bio Technology, Bio Genetics , Genetic Variation

How Populations Evolve

2009-03-31T23:09:00.000-07:00

Evolution is a change in the gene pool of a population over time. A gene is a hereditary unit that can be passed on unaltered for many generations. The gene pool is the set of all genes in a species or population. A population is a group of organisms of the same species usually found in a clearly defined geographical area.

The English moth or the peppered moth, biston betularia, is a frequently cited example of observed evolution. In this moth there are two color morphs, light and dark. Dr. Henry Bernard Davis Kettlewell, a British lepidopterist and medical doctor, is notable for his experiments on the peppered moth, most of which were done in Manchester, England. He found that dark moths constituted less than 2°,o of the population prior to 1848. The frequency of the dark morph increased in the years following. By 1898,95% of the moths in Manchester and other highly industrialized areas were of the dark type. Their frequency was less in rural areas. The moth population changed from mostly light colored moths to mostly dark colored moths. The moths' color was primarily determined by a single gene. So, the change in frequency of dark colored moths represented a change in the gene pool. This change was, by definition, evolution.
The increase in relative abundance of the dark type was due to natural selection. The late eighteenth century was the time of England's industrial revolution. Soot from factories darkened the birch trees the moths landed on. Against a sooty background, birds could see the lighter colored moths better and ate more of them. As a result, more dark moths survived until reproductive age and left offspring. The greater number of offspring left by dark moths is what caused their increase in frequency. This is an example of natural selection.

Populations evolve. In order to understand evolution, it is necessary to view populations as a collection of individuals, each harboring a different set of traits. A single organism is never typical of an entire population unless there is no variation within that population. Individual organisms do not evolve; they retain the same genes throughout their life. When a population is evolving, the ratio of different genetic types is changing-each individual organism within a population does not change. For example, in the previous example, the frequency of black moths increased; the moths did not turn from light gray to dark in concert. The process of evolution can be summarized in three sentences: Genes mutate. Individuals are selected. Populations evolve.

The word evolution has a variety of meanings. The fact that all organisms are linked via descent to a common ancestor is often called evolution. The theory of how the first living organisms appeared is often called evolution. This should be called abiogenesis. And frequently, people use the word evolution when they really mean natural selection-one of the many mechanisms of evolution. Phenotype is the morphological, physiological, biochemical, behavioral, and other properties exhibited by a living organism. Genotype is the genetic make up of an organism.
Evolution can occur without morphological change; and morphological change can occur without evolution. Humans are larger now than in the recent past, a result of better diet and medicine. Phenotypic changes like this, induced solely by changes in environment, do not count as evolution because they are not heritable; in other words, the change is not passed on to the organism's offspring. Most changes due to environment are fairly subtle, for example, size differences. Largescale phenotypic changes are obviously due to genetic changes, and therefore are evolution.

Tags: Bio Technology, Bio Genetics , Evolution

Formation of Tissues to become an Organ

2009-03-31T23:08:00.000-07:00

Various types of tissues are associated together to carry out a specific function of the body and such structures are known as organs. In animals, stomach, heart, brain, etc., are specific organs carrying out specific functions due to the interaction of various tissues.

The heart is involved in the pumping of blood, which in turn is circulated to other organs and tissues by a network of arteries. Blood from various organs and tissues is brought back to the heart by another network of tubes called veins. Thus, forms an important system called the circulatory system. Kidneys are another example of organs. They are involved in the excretion of metabolic waste and other toxins produced in the body.

Similarly, there are a large number of organs such as stomach, intestine, liver, pancreas muscles, reproductive organs such as testes, ovaries, external genitalia, etc., that carry out specific functions in association with other organs. There are varieties of glandular organs, both ductless glands such as pituitary, thymus, adrenal, etc., and glands with ducts such as the salivary glands that execute their function primarily by secreting specific enzymes and hormones to carry out various metabolic activities.

Tags: Bio Technology Bio Genetics, Tissues Organs

Know the Plant Tissues

2009-03-31T23:04:00.000-07:00

Vascular plants have distinctive cell types, all of which are surrounded by a cell wall of cellulose fibers and other molecules secreted by the cells. Just as in animals, cells are organized into tissues that perform different functions, but plants do not have organ systems like those of animals. The tissues of plants are grouped into three basic kinds: ground, vascular, and dermal. Meristem is a special embryonic tissue.

Plants differ from animals in that the tips of roots and stems, called apical meristem, remain embryonic and retain the ability to form new structures (e.g., leaves, stems, flowers, and roots). Hormones secreted by meristem cells are transported elsewhere in the plant; meristem is in part analogous to the endocrine system in animals.

Ground Tissues (Simple tissues):
Ground tissues or the simple tissues include parenchyma, collenchyma, and sclerenchyma. Thin-walled parenchyma cells have a variety of functions such as photosynthesis, starch storage, and secretion; they retain the capacity to divide and are important in repair of damage. They form the large part of the bulk of various organs such as stem, root, etc. In some parts they are modified to perform some special functions. For example:

Epidermis:
This is a single-layered tissue that covers the whole plant body. It protects the internal part from infection and loss of water. This layer of cells has a waxy coating on the surface, which is secreted by the cells. This waxy layer is called cuticle, which helps to reduce the water loss.

Mesophylls:
These types of parenchyma cells are found in the leaves between the two epidermal layers. These are specialized for carrying out photosynthesis. Parenchyma cells containing chlorophyll are also known as chlorenchyma. If the cells of the mesophyll tissue are tightly packed without air space, they are known as palisade parenchyma or mesophyll; if a lot of air space is present it is called spongy parenchyma. Endodermis, pericycle, and companion cells, etc. are also an example of modified parenchyma cells.

Collenchyfna:
These cells resemble parenchyma cells but are characterized by the presence of extra cellulose at the corners of the cells. Their walls are thickened and made strong with cellulose and pectin. Collenchyma cells help strengthen the plant parts in which they occur. Celery strings are an example.

Sclerenchyma:
Sclerenchyma cells have very thick secondary walls that are commonly impregnated with lignin, which makes them quite rigid. The lignified sclerenchyma of flax plants is made into linen threads for weaving, sewing, and paper making. Wood is made of lignified xylem cells. The hardness of a coconut shell or a peach pit is caused by lignified cells. Ground tissues are analogous to the supporting connective tissue and skeletal elements in animals. Sclerenchyma cells act as supporting elements in plants. Mature sclerenchyma cells can't elongate. The two types of sclerenchyma cells are fibers and sclereids. Fibers are long, slender, and tapered cells that occur in bundles. Sclereids are shorter than fibers and shaped irregularly. Nutshells and seed coats are composed of sclereids. Sclereids scattered among the soft parenchyma tissue of the pear give it a gritty texture.

Complex Tissues:
Complex tissues consist of more than one type of cells. Vascular tissues of plants include xylem and phloem; this is the plant's circulatory system. Xylem and phloem are the complex tissues.
Xylem consists of four types of cells-trachieds, vessel elements, parenchyma, and fibers. Trachieds are single cells that are elongated and lignified. At maturity, trachieds cells are dead and form interconnected tubes throughout the plant. Vessels are long, tubular structures formed by the fusion of several cells end to end in a row. They conduct water and dissolve nutrients that the plant absorbs from the soil; their thick, sclerified walls allow them to give mechanical support to the plant. Wood is made of xylem cells. Xylem parenchyma has thin cellulose cell walls and living contents similar to the typical parenchyma cells. Xylem fibers are shorter and thinner than trachieds and have much thicker walls.

Phloem consists of tubular cells modified for translocation. These tubular cells have interconnected cytoplasm and they conduct other solutes, chiefly nutrients (e.g., carbohydrates) from areas of food production such as leaves to areas of food storage such as tubers. There are five types of cells in phloem. They are sieve tube elements, companion cells, parenchyma, fibers, and schlerids. Sieve tubes are long tube-like structures formed by the end to end fusion of sieve tube elements. Adjacent to the sieve tube elements lie the companion cells with dense cytoplasm. Phloem parenchyma is similar to the ordinary parenchyma cells and the phloem fibers are like the sclerenchyma fibers.

Dermal tissues include epidermis and cuticle. The epidermis is a continuous layer of tightly packed cells. It is usually coated with a cuticle of waxes embedded in a fatty substance; this is analogous to keratinized outer layer of skin, including your own, in animals that live on land. Leaf epidermis is perforated by stomata for gas exchange between the photosynthetic mesophyll (parenchyma) and the surrounding atmosphere. Thus, leaves function in part like lungs. All these tissue types-both simple and complex tissues-are distributed all over the plant parts, but their position and orientations are different in different organs like stem, roots, leaves flowers, fruits, etc.

Tags: Bio Technology, Bio Genetics , Plant Tissues

Know the Animal Tissues

2009-03-27T03:56:00.000-07:00

In animals, there are four basic types of tissues: epithelial or linings, connective or supporting, muscular, and nervous. An organ of the body may have all the four types of tissues. For example, the stomach, an organ of the digestive system has all the four types of tissues.

Epithelial Tissues:
The cells are arranged in single or multilayered sheets. They basically form the covering on the external and internal surfaces of the organs and body parts. Epithelial cells are not supplied with blood vessels. They protect the internal tissues from physical injury and infection. The free surface of the epithelial tissue may be of different types depending on its special function such as secretory, absorption, or excretory functions. Epithelial cells are basically classified according to their shapes.

There are three basic cell shapes in epithelial tissues: columnar, cubical, and squamous (scale-like).

The deep columnar cells often have a secretory function, and the nucleus is pushed to the bottom by the made and stored secretions near the surface from which they will exit (e.g., the cells lining the stomach, which secrete mucus). Cubical cells form the walls of small ducts as from salivary glands. Squamous cells are very flat, and the nucleus may form a bulge; they look something like a fried egg. The thinness permits diffusion of molecules across membranes (e.g., alveolar walls in lungs). Thick layers of cells (e.g., skin) prevent diffusion. In addition to the above three basic types there are some modified forms of these tissues. They are the following. Ciliated epithelium, the columnar cells with numerous cilia on their free surface, which lines the respiratory passage. Psudostratified epithelium, which forms a single layer of cells but on sectional view appears to be multilayered. The last one is the stratified epithelium, which are multilayered and form a very tough and impervious barrier.

The secretory or glandular cells may be present individually as in the case of goblet cells or in groups forming multicellular glands. An epithelial tissue having many goblet cells that secrete mucus is called mucus membrane. If the glandular cells or glands discharge their secretion on the surface of the cells or through a duct, they are called exocrine glands. But there are glands that discharge their secretion directly into the bloodstream and do not have any ducts. They are called the ductless glands.

Connective Tissues:
This includes the various types of supporting tissues in the body. Connective tissues are cells in a matrix. The matrix may be a fluid, semi-fluid, or a composite structure made up of secretory products of cells such as fibrous proteins. Blood is a connective tissue in which cells are embedded in a fluid matrix. In fibrous connective tissue cells are scattered among the collagen fibers (fibrous protein) they secrete. In bone and cartilage, cells are scattered throughout the hard or pliable matrix. In cartilage, the cells known as chondroblasts deposit in the matrix. The cell, along with the matrix, forms the chondrocytes. The cartilage is hard but flexible because the matrix is compressible and elastic. Bone is a calcified connective tissue. The cells are embedded in a hard matrix. The cells in the bone tissue are called osteoblasts, which are present in lacunae. Lacunae are present throughout the tissue. The main inorganic component of bone is hydroxyapatite.

Muscle Tissues:
These are made up of highly differentiated contractile cells or fibers held together by connective tissues. Muscle tissues are of three types. Striated muscle cells are large, multinucleate, and column-shaped cells; they are chiefly attached to the skeleton and are known as skeletal muscles or voluntary muscles. Voluntary muscles are under the control of the voluntary nervous system. They show powerful rapid contractions. They are attached to the bones in the trunk, limbs, and head. Smooth muscle cells are small and mononucleate; they are found in the walls of tubes such as blood vessels, glandular ducts, and the digestive system. They are also known as unstriated or involuntary muscles. The involuntary muscles are under the control of the autonomic nervous system and show sustained rhythmical contraction and relaxation movements. Cardiac muscle cells of the heart are small, striated, and branched. They are present only in the heart. They show rapid rhythmical contractions and relaxation movements with long refractory periods and do not show any fatigue.

Nervous Tissues:
Nervous tissues consist of nerve cells, the neurons and associated neuroglial cells. Neurons are capable of generating and transmitting electrical impulses. These cells also act as supporting connective tissue in the brain and spinal cord. The neurons transmit the stimuli from receptors such as skin to the effectors such as muscles and glands that then react to the stimuli.

Tags: Bio Technology, Bio Genetics , Animal Tissues

What are the Basic Cell Structure and their components

2009-03-18T20:31:00.000-07:00

Cells are structural and functional units of life. According to the cell theory all living things are composed of one or more cells. One-celled organisms are called unicellular organisms and those with more than one cell are called multi-cellular organisms. Virus particles do not have any cells and therefore, are termed as acellular. No matter what type of cell we are considering, all cells have certain features in common: cell membrane, nucleic acids, cytoplasm, and ribosomes. Cells are small 'sacks' composed mostly of water. The 'sacks' are made from a phospholipid bilayer. The membrane is semi-permeable, allowing some things to pass in or out of the cell and blocking others. Microscopes make it possible to magnify small objects such as cells in order to see the details of their structure. Both light and electron microscopes are used to study cells. Study of cells with a microscope is called cytology. There are some fundamental activities, which are common for most of cell types from bacteria to the nerve cells in humans. The study of these basic cellular processes is called cell biology.

Cells are 90% fluid (cytoplasm), which consists of free amino acids proteins, glucose, and numerous other molecules. The cell environment (i.e., the contents of the cytoplasm, and the nucleus, as well as the way the DNA is packed) affects the gene expression/ regulations, and thus is very important part of inheritance.

Cells basically fall into two groups: prokaryotic cells and eukaryotic cells.

Prokaryotes, Eukaryotes and Viruses: Cells are classified as prokaryotes or eukaryotes based on their basic structure and the way by which they obtain energy. Cells are also classified according to their need for energy. Autotrophs are "self feeders" that use light or chemical energy to make food. Plants are an example of autotrophs. In contrast, heterotrophs ("other feeders") obtain energy from other autotrophs or heterotrophs. Many bacteria an( animals are heterotrophs.

Prokaryotic Cells: Prokaryotes include bacteria and blue-green algae (cyanobacteria). Simply stated, prokaryotes are molecules surrounded by a membrane and cell wall. Prokaryotic cells lack characteristic eukaryotic subcellular membrane-enclosed "organelles," but may contain membrane systems inside a cell wall as an extension or infoldings of the cell membrane. The nucleus is not well-organized and is without any membrane.

Prokaryotic cells may have photosynthetic pigments, such as is found in cyanobacteria ("blue-green algae"). Some prokaryotic cells have external whiplike flagella for locomotion or hair-like pili for adhesion. Prokaryotic cells come in multiple shapes: cocci (round), baccilli (rods), and spirilla or spirochetes (helical cells). All prokaryotes are unicellular organisms and eukaryotes include both unicellular and multicellular organisms.

Eukaryotes : Basic Structure:
The basic eukaryotic cell contains the following: 1) A Plasma membrane2) Nucleus3) Cytoplasm (semifluid)

Viruses : Basic Characteristics of Viruses:
Simply stated, viruses are merely genetic information surrounded by a protein coat. They may contain external structures and a membrane. Viruses are obligate intracellular parasites meaning that they require host cells to reproduce. In the viral life cycle, a virus infects a cell, allowing the viral genetic information to direct the synthesis of new virus particles by the cell. There are many kinds of viruses. Those infecting humans include polio, influenza, herpes, smallpox, chickenpox, and human immunodeficiency virus (HIV) causing AIDS.

Tags: Bio Technology, Bio Genetics , Cell Structure

Understanding of Cell Membranes

2009-03-18T20:28:00.000-07:00

Characteristics of Cell Membranes are as under:
1) Cell membranes are selective barriers that separate individual cells and cellular compartments.

2) Membranes are assemblies of carbohydrates, proteins, and lipids held together by non-covalent forces. They regulate the transport of molecules, control information flow between cells, generate signals to alter cell behavior, contain molecules responsible for cell adhesion in the formation of tissues, and can separate charged molecules for cell signaling and energy generation.

3) Cell membranes are dynamic, constantly being formed and degraded. Membrane vesicles move between cell organelles and the cell surface. Inability to degrade membrane components can lead to lysosomal storage diseases.

4) Lipids of cell membranes include phospholipids composed of glycerol, fatty acids, phosphates, and a hydrophobic organic derivative such as choline or phosphoinositol. Cholesterol is a lipid component of cell membranes that regulates membrane fluidity and is a part of membrane signaling systems. The lipids of membranes create a hydrophobic barrier between aqueous compartments of a cell. The major structure of the lipid portion of the membrane is a lipid bilayer with hydrophobic cores made up predominately of fatty acid chains and hydrophilic surfaces.

5) Membrane proteins determine functions of cell membranes, including serving as pumps, gates, receptors, cell adhesion molecules, energy transducers, and enzymes. Peripheral membrane proteins are associated with the surfaces of membranes while integral membrane proteins are embedded in the membrane and may pass through the lipid bilayer one or more times.

6) Carbohydrates covalently linked to proteins (glycoproteins) or lipids (glycolipids) are also a part of cell membranes, and function as adhesion and address loci for cells.

7) The Fluid Mosaic Model describes membranes as a fluid lipid bilayer with floating proteins and carbohydrates.

8) Cell junctions are a special set of proteins that anchor cells together (desmosomes), occlude water passing between cells (tight junctions), and allow cell-to-cell direct communication (gap junctions).

Tags: Bio Technology, Bio Genetics , Cell Structure

What are the Various Types of Proteomics

2009-03-08T04:48:00.000-07:00

Even though proteomics can be defined as the study of the protein complement of the genome, the dynamic nature of the proteome made it difficult to study and understand. The total protein expression profile always changes with time and micro- and macro-environmental conditions. Only a small percentage of the total gene is expressed at a particular time. Information about the genome and gene structure can predict the structure and function of its protein complement to a limited extent because of the post-transcriptional modifications that the protein undergoes. These modifications are not represented in the respective gene. This is what makes proteomics difficult to study compared to genomics.

The following are the major types of proteomics:

Expression Proteomics:
This is the qualitative and quantitative study of the expression of total proteins under two different conditions. For example, expression proteomics of normal cells and diseased cells can be compared to understand the protein that is responsible for the diseased state or the protein that is expressed due to disease. Using this method disease-specific protein can be identified and characterized by comparing the protein-expression profile of the entire proteome or of the subproteome between the two samples.

For example, tumor tissue samples from a cancer patient and the same type of tissue from a normal person can be analyzed for differential protein expression. Using two-dimensional gel electrophoresis, mass spectrometry combined with chromatography and microarray techniques, additional proteins that are expressed in the cancer tissues or the proteins, which are absent, or those, which are over expressed and under-expressed can be identified and characterized. Identification of these proteins will give valuable information about the molecular biology of tumor formation.

Structural Proteomics:
Structural proteomics, as the name indicates, is about the structural aspects, including the three-dimensional shape and structural complexities, of functional proteins. This includes the structural prediction of a protein when its amino acid sequence is determined directly by sequencing or from the gene with a method called homology modeling. This can be carried out by doing a homology search and computational methods of protein structural studies and predictions.

Apart from this, structural proteomics can map out the structure and function of protein complexes present in a specific cellular organelle. It is possible to identify all the proteins present in a complex system such as ribosomes, membranes, or other cellular organelles and to characterize or predict all the proteins and protein interactions that can be possible between these proteins and protein complexes. Structural proteomics of a specific organelle or protein complex can give information regarding supra-molecular assemblies and their molecular architecture in cells, organelles, and in molecular complexes.

Functional Proteomics:
This is an assembly type of proteomic method to analyze and understand the properties of macromolecular networks involved in the life activities of a cell. With these methods it will be possible to identify specific protein molecules and their role in individual metabolic activities and their contribution to the metabolic network that operates in the system. This forms one of the major objectives of functional proteomics. For example, the recent elucidation of the protein network involved in the functioning of a nuclear pore complex has led to the identification of novel proteins involved in the translocation of macromolecules between the cytoplasm and nucleus through these complex pores.

Functional proteomics is yielding large databases of interacting proteins, and extensive pathway maps of these interactions are being scored and deciphered by novel high-throughput technologies. However, traditional methods of screening have not been very successful in identifying protein-protein interactions and their inhibitors. The identification and measurement of changes in the concentration of specific proteins that cells make as a result of their genetic response to specific toxicants, and how these proteins are related to each other and to the specific biological condition of the cell, also fall under functional proteomics.

Tags: Bio Technology, Bio Genetics , Proteomics

Understanding Genes in Relation to Proteins

2009-03-08T04:46:00.000-07:00

Owing to concerted efforts by numerous state and commercial establishments, the human genome had completely been sequenced in 2000. All 24 human chromosomes are mapped, and the defects of hundreds of genes responsible for the development of hereditary diseases have been revealed.

A new medical concept has been advanced whereby all diseases can be divided into two major groups:

(a) Hereditary diseases : A consequence of transmission of defective genes from parents to their children.

(b) Non-heritable diseases: Or so called socially-significant diseases, which make up more than 95% of all human diseases and result from disturbances in normal genes' expression regulation. Thus, all human diseases, one way or another, are associated with the genome; the only difference is that the diseases of the former group are due to a defect(s) in gene's structure while those of the latter group are caused by disturbances in a gene expression's regulation. The cataloging of human genes is an achievement, which can hardly be overestimated: for many years to come this catalogization will serve as a basis for the development of basic biochemistry, molecular biology, and genome-associated applied sciences. The future of this area of research, called genomics, is even more brilliant. Currently, the emphasis has gone from sequencing the human genome to sequencing the genomes of animals and microorganisms, particularly pathogenic and plant genomes. This research promises enormous achievements in medicine, especially in the struggle against infectious diseases.

How is the informational structure (gene) connected with the actual working molecular machine (protein)?

To answer this question, we have to consider the results of those few works, where the expression map of mRNA was compared with the proteinous map in the same cellular system. It failed to reveal any strict correlation between the two maps. Thus, the informational knowledge cannot be directly converted into the knowledge of actually operating protein molecules. As a result, a new area of research has appeared, called proteomics, which deals with inventory of proteins. At first glance, this is an utterly impossible task. While the human genome map is the same for all human cells (24 chromosomes), in the proteomic map each cell is individual. Although the cell may have only 33,000 functional genes, the numerous modification reactions may increase the number of proteins in it up to several million. There are two definitions for proteomics: a narrow one (the so-called structural proteomics) and a broader one, encompassing both the structural and functional proteomics. In a narrow sense of the word, 'proteomics' is a science dealing with the cataloging of proteins based on a combination of several methods: two-dimensional electrophoresis, mass spectrometric analysis of molecular mass, and sequencing of electrophoretically separated proteinacious biological material with subsequent analysis of the results obtained, with the help of bioinformatical and computational methods.

In a broader sense, the terms 'proteome analysis' or 'proteomics' can be used not only for cataloging proteins of a biological subject but also for the monitoring of reversible post-translational modification of proteins by specific enzymes (i.e., phosphorylation, glycosylation, acylation, phrenylation, su1furization, etc). To date, more than 300 different types of post-translational modification have been characterized with the aid of proteomics. Another aspect of functional proteomics is to clarify the composition of functionally active complexes that constitute different metabolic chains and also, to determine the interactions between various proteins or subunits of oligomeric complexes by a combination of methods to isolate these complexes and subsequent mass-spectrometric analysis. Lately, structural proteomics is often called expressional proteomics while functional proteomics is also designated as cell-mapping proteomics, since it elucidates the interactions of proteins within metabolic pathways. Therefore, in short, the number of proteins and the number of genes are not equal and they are non-linear. The number of proteins easily outnumbers the number of genes.

Tags: Bio Technology, Bio Genetics , Proteins

Understanding Proteomics

2009-03-07T00:50:00.000-08:00

Proteomics permits genome-wide expression profiling providing a vital information required to describe how a cell functions, and gives an insight into the development of diseases.

Out of the two methods of genome-wide analysis, proteomics, the examination of the complete set of proteins synthesized by a cell under a given set of physiological or developmental conditions, should be most informative when making functional assignments. This is mainly due to the fact that the proteome is context-dependent and unlike mRNAs, proteins are functional entities within the cell. The proteome is the proteins expressed by a genome.

Proteomics involve the analysis of a large number of proteins with a combination of two-dimensional gel electrophoresis and mass spectrometry. Today, it includes both the identification of a large number of proteins expressed by a genome as well as the characterization of their functional and structural relationships. It was thought that a single gene would result in the formation of a single protein or polypeptide. But there are different stages in the process of transcription and translation, where the same process can take place but produces a different protein. These processes can take place just after the process of transcription-the post-transcriptional process that can result in different types of mRNAs with the control of splicing mechanisms. The second point where the alteration can occur is after the process of translation or protein synthesis. This is the post-translational modification which can also produce structurally and functionally different proteins from the same gene.

Proteomics also deals with the use of qualitative and quantitative protein-level measurements to characterize biological processes (e.g., diseases). By comparing the protein profiles from normal cells and diseased or metabolically aberrant cells, the reason for the diseased condition can be ascertained. Therefore, diseases can be treated at the protein level or even at the gene level because the proteins are the drug targets.

The Key Technologies in Proteomics
1) Reproducible Two-dimensional Gel Technology
2) Staining and Scanning Technology
3) Mass Spectrometry for Identification
4) Databases (protein and genome)
5) Database Searching Algorithms

The crude protein extracted from the tissue sample or cells or from individuals is separated in gel by two-dimensional electrophoresis comprising of IEF and SDS-PAGE. The gel is suitably stained and compared with that of the standard two-dimensional gel profile of protein from the same organisms grown under normal conditions (or healthy individuals). If the formation of any new protein is observed that spot can be excised from the gel along with the gel matrix and washed with sterile distilled water. This protein spot along with the gel can be digested with trypsin and then introduced into the mass spectrometer and (the MS fingerprint can be taken). The MS fingerprint can be used for the identification of the protein by database search or can proceed further to carry out amino acid sequencing by mass spectrometer. Thus, the protein purification and identification become very easy and simple. But the key challenge in proteome research is the automation and integration of these technologies.

Tags: Bio Technology, Bio Genetics , Proteomics

How to Improve the Nutritional Value of Cereals and legumes

2009-03-07T00:08:00.000-08:00

In human nutrition, cereals and legumes provide a major part of the dietary protein requirement. The storage proteins of these seeds are a good source of essential amino acids. But some of these legumes and cereals create deficiency of some essential amino acids and therefore such cereals and legumes are considered to be poor in quality, even though they are rich sources of dietary protein. They cannot provide a balanced diet. In such cases the diet should be supplemented with other sources of essential amino acids. In some other cases certain rich sources of protein may not be suitable for human consumption because of toxicity, a bad quality such as bad smell, taste, etc. Thus, protein for human consumption should be safe and nutritious.

The major parameters for assessing the general effectiveness of a protein as a dietary protein are the Essential Amino Acids Profile (EAAP), Biological Value (BV), and Protein Efficiency Ratio (PER).

Essential Amino Acids :
Proteins are composed of 20 amino acids. Plants and microbes can synthesize all these amino acids, but animals cannot. Such amino acids have to be supplemented through the diet. Such amino acids are the essential amino acids. The presence of a high percentage of essential amino acids along with other amino acids increases the nutritive quality of protein for human consumption.

The nutritive quality of whey proteins is considered to be higher compared to other types of proteins. They are rich in branched chain amino acids-Ile, leu, val, lys and trp. The branched chain amino acid (BCAA) is needed for rapid energy metabolism for muscular activity. BCAA helps in the bio-availability of complex carbohydrates absorbed by muscle cells for anabolic muscle building activity. BCAA plays an important role in the production of metabolic energy during rapid muscular actions. During exercise BCAAs are released from the muscle cells. The carbon chain part of the molecule is used as fuel and the nitrogen part is used to synthesize alanine. Alanine is transported to the liver where it is turned into glucose to meet the energy requirements. So athletes are advised to take BCAA sources before and after exercises to protect their healthy body. Thus, BCAAs are very important for muscle growth and muscular activity.

Biological Value :
Biological value or BV is a measure of protein nitrogen retained by the body after consuming a particular amount of protein nitrogen. Among various types of dietary proteins whey protein showed the maximum BV compared to rice, soy, egg, and wheat proteins.

Protein Efficiency Ratio:
Protein efficiency ratio or PER is a measure of the efficiency of a protein on the rate of growth. It is the growth performance in terms of weight gain of an adult by consuming 1 gm of dietary protein. The order of PER for various dietary proteins is given below.

Wheat protein :
The quality of protein can be improved by the modem approach of protein engineering through recombinant DNA technology. The storage protein of cereals and legumes can be modified by introducing essential amino acids. These genes can be transferred to food plants to be expressed in the respective storage parts of the plants such as seeds, tubers, or grains. Modifications in proteins can be made by introducing new amino acids or by substituting the existing amino acids with new ones. Efforts are already being made to enhance the nutritive quality of protein genes in maize and other similar food grains. Scientists are on the look out for new types of dietary protein genes that are rich in essential amino acids, with which the nutritive quality of other plants can be increased.

Tags: Bio Technology, Bio Genetics ,Protein Designing

Art of Designing of Proteins

2009-03-07T00:05:00.000-08:00

Protein design is important because it not only allows us to systematically investigate the forces that determine structure and stability but it also affords the possibility of creating proteins through novel and useful activities. The biotechnological industry is more interested in developing new types of enzymes, which are stable and more active under extreme working conditions such as high temperature and extreme pH such as acidic or alkaline pH.

Biochemists can now use a refinement of the genetic modification technique to redesign proteins. Once they have isolated the gene for a particular protein they can alter its code so that a change occurs in the protein's primary structure. They then incorporate the modified gene into a microorganism where it is decoded as before, but this time a new protein appears. Swapping one a-amino add in a protein can have a large effect on how the protein behaves. Biochemists have already used this protein engineering to modify human insulin in a way that makes it become absorbed more quickly after injection. Multiple changes are needed to 'humanize' antibodies.

Computer-modeling techniques allow protein chemists to make predictions about how proposed a-amino acid changes might change the structure and activity of a protein. Genetic and protein modification have enormous potential. Besides insulin, genetic modification can produce human growth hormone and the blood clotting factor VIII. Genetic engineering, thus, has provided a tool to produce designer proteins having special characteristics by changing the amino acid sequence. In addition, it is already possible to introduce into a plant new genes that enable it to produce its own protein insecticide or that make it resistant to disease. Hepatitis B vaccine, oil-digesting bacteria, and bacteria that produce biodegradable plastic are all recently developed products of protein engineering and genetic-modification techniques.

Creation of Novel Proteins: Vaccines are traditionally prepared from heat-inactivated microbes-bacteria or viruses or the surface proteins of these organisms to immunize against various infectious diseases. But in some cases it was frequently observed that some parts or components of the vaccines were creating some deleterious effects on people. It is known that proteins are the main candidates responsible for imparting the stimulus for immunity. Therefore, efforts have been made to design an altered protein molecule, which can produce only very little deleterious effects. There are some amino acid sequences, which are directly involved in the stimulation of immunity and that part of the protein or polypeptide is known as epitopes. A recombinant vaccine can be designed by involving only the essential epitopes in the immune response. Such vaccines will be very simple and will be safe without reducing their effectiveness. Safe vaccines against the hepatitis B virus and anthrax bacteria are under development.

Tags: Bio Technology, Bio Genetics ,Protein Designing

Types of Protein based products based upon their derivation

2009-02-27T03:19:00.000-08:00

Proteins, as biological macromolecules, are very important for the existence of normal activity in a living system. Because of the multifaceted role of proteins, they are also important commercially. Proteins may be classified, according to their derivation as described here:

1) Blood-derived Proteins and Vaccines:
Blood is the fluid connective tissue of humans. It is the most important medium for the transportation of gas and nutrients between tissues and sources. The blood forms the source for a number of therapeutically important proteins. A detailed understanding of the formation of blood cells (hematopoiesis) and detailed studies regarding the coagulation of blood have shown the presence of several useful proteins. In addition to blood-coagulating factors, whole blood and blood plasma have been used traditionally as blood products. These products are obtained from human volunteers who donate blood. But today some of the proteins of blood are produced by a recombinant technique. The blood coagulating factor known as factor VIII used in the treatment of hemophilia A and another factor, the factor IX for treating hemophilia B, etc., and the vaccine for hepatitis B are now synthesized by transgenic bacteria in fermentors.

2) Therapeutic Antibodies and Enzymes :
Antibodies and enzymes that are used for clinical applications are known as therapeutic antibodies. Polyclonal antibodies and monoclonal antibodies are examples. In tissue and organ transplantation, the body will recognize these as foreign objects and will produce antibodies against these tissues and organs leading to the failure of organ transplantation. Certain specific antibodies can be used against these natural antibodies and can revert the rejection process. For example, OKT-3 is used during kidney transplantation to revert the acute organ rejection in patients. Similarly, ReoPro is used to prevent blood clots. A large number of therapeutic antibodies are now produced by recombinant DNA techniques. Tissue plasminogen activator (t-PA) is an example of therapeutic enzyme used for acute myocardial infarction. There are a number of enzymes that are used as drugs. Asparaginase is used against some types of cancers and DNase is used for the treatment of cystic fibrosis.

3) Hormones and Growth Factors as Therapeutic Agents:
There are a number of proteins and peptides that act as hormones and growth factors. In the case of diseases due to metabolic errors such as diabeties, caused by the absence of these peptides, the concerned molecules can be administered to correct the metabolic errors. Insulin is the best example. In earlier times, insulin was prepared from the pancreas of cows and pigs. But now, human insulin is prepared through genetic engineering, by transferring the gene of insulin into bacteria; the transgenic bacteria will produce the active human protein. This insulin is called humulin. The methods of protein engineering have helped to develop altered forms of insulin, which are more active than the normal ones. There are a number of growth factors such as EGF (Epithelial Growth Factor) and plateletderived growth factors that are used for therapeutic purposes. EGF is used for the treatment of burns and injuries, during skin transplantation, for its growth. The platelet-derived growth factors are used for the treatment of diabetic and skin ulcers. A large number of growth factors and hormones are under clinical trials.

Tags: Bio Technology, Bio Genetics , Protein Products

Types of Protein based products based upon their Commercial Importance

2009-02-27T03:11:00.000-08:00

Proteins being biological macromolecules can be classified, according to their commercial importance as described below :

1) Regulatory Factors :
Regulatory factors are also like growth factors, which are closely involved in the process of signal transduction and expression of specific genes. These factors are called cytokines because they are involved in the growth and proliferation of cells. Cytokines include interferones (INF), interleukins, tumor necrosis factors, colonystimulating factors, etc. All these factors find use in therapeutic application for the treatment of different diseases, both infectious and non-infectious. For example, (X-interferon is used in the treatment of hepatitis C, p-interferon is used for the treatment of multiple sclerosis, and y-interferon is used for severe granulomatous disease.

2) Proteins and Enzymes Used in Analytical Applications:
In addition to the use of antibodies and enzymes as therapeutic agents, they are also used in the diagnosis of diseases as the components of some confirmatory tests of certain diagnostic procedures. Hexokinase and glucose oxidase are used in the quantification of glucose in the serum and urine. Glucose-oxidase is used in glucose electrodes. Uricase is used for the estimation of uric acid present in urine. Alkaline phosphatase, horseradish peroxidase, and antibodies are used in ELISA (Enzyme Linked Immunosorbent Assay).

3) Industrial Enzymes and Proteins:
Among commercially useful proteins, industrial enzymes have the first place. Industrially useful enzymes include carbohydrate-hydrolyzing enzymes such as amylases, cellulase, invertases, etc., proteolytic enzymes such as papain, trypsin, chymotrypsin, etc., and other bacterial and fungal-derived proteolytic enzymes and lipases that can hydrolyze various types of lipids and fats. All these enzymes are important in the food and beverage industries, the textile industry, paper industry, and detergent industry. Proteases have a special use in the beverage industry, meat and leather industries, cheese production, detergent industry, bread and confectionary industry, etc. Various types of lipases are used for the modifications of various types of lipids and fats, production of various organic acids including fatty acids, in detergents, production of coco butter, etc. In addition to all these, enzymes are used in chemical industries as reagents in organic synthesis for carrying out stereospecific reactions.

4) Non-catalytic Functional Proteins:
These commercially important proteins are used in the food industry as emulsifiers, for inducing gelation, water binding, foaming, whipping, etc. These non-catalytic functional proteins are classified as whey proteins. The proteins that remain in solution after the removal of casein are by definition called whey proteins.Commercially-available whey protein concentrates contain 35 to 95% protein. If they are added to food on a solid's basis, there will be large differences in functionality due to the differences in protein content. Most food formulations call for a certain protein content and thus whey-protein concentrates are generally utilized as a constant protein base. In this case the differences due to protein content as such should be eliminated. As the protein content increases, the composition of other components in the whey-protein concentrate must also change and these changes in composition have an effect on functionality.

5) Nutraceutical Proteins:
Nutraceutical proteins represent a class of nutritionally-important proteins having therapeutic activity. The whey-protein concentrates and some of the milk proteins of infant foods contain certain pharmaceutical proteins having high nutritive quality. Infants get the required proteins from the mother's milk, which also contains certain therapeutic proteins that protect the baby from infection and other problems. There are other infant foods, which also have more or less the same composition as that of mother's milk, made up of cow's and buffalo's milk. All these food proteins provide the infants the raw building materials in the form of essential amino acids and at the same time protects them from microbial infections and other diseases. Thus, milk is a very good source of nutrition.

Tags: Bio Technology, Bio Genetics , Protein Products

Mass spectrometry for protein identification

2009-02-27T01:59:00.000-08:00

There are various types of mass spectrometry depending on the type of ionization source or the ion analyzers. They are used for specialized studies in proteomics such as identification of proteins by generating mass fingerprints or the ionization pattern of the protein molecule, amino acid sequencing of peptides, and thereby the detailed studies of the three-dimensional structure of the protein and modification of the proteins if any can be detected very easily and efficiently. Some of the common types of mass spectrometry are discussed below.

MALDI-TOF Mass Spectrometry :
Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) is another method of ionization that does not require any heating for the production of ions and can be used for heat-sensitive molecules. In this method, the sample to be analyzed is mixed with a matrix compound and crystallized. The compound of this matrix is usually a weak inorganic acid. This mixture (matrix mixed with sample) is then excited with a laser, which results in the evaporation of the matrix compound. The matrix compound also carries the sample molecules into the vapor phase, resulting in indirect vaporization of the sample. Sample ions are formed by the exchange of electrons and protons with the matrix compound.

MALDI is especially useful for protein and peptide identification using masses alone, since the masses of ions can be determined with great accuracy. Time of flight is the technique used for the sorting and separation of ions generated by laser ionization.

Electrospray Ionization (ESI):
This is a newer method of ionization that does not cause excessive fragmentation. ESI generates ions directly from the solution without heating. Therefore, this method can be used for heat-sensitive molecules, which cannot be ionized by heating. The sample is finely sprayed in the presence of an electrical field. Charge accumulates on the sample droplets, which then explode due to mutual repulsion of charges leading to the formation of ions. Both single and multiple-charged ions can be produced in this manner.

Tandem Mass Spectrometry (MS/MS or Two-dimensional MS):
Tandem MS is a procedure in which multiple rounds of mass spectrometry are performed to obtain greater resolution of the ions generated by the unknown sample molecule. Short stretches of polypeptides can be sequenced with tandem MS. In general a polypeptide is cleaned into a number of fragments enzymatically and the products are taken into the mass spectrometer for analysis one by one to generate the primary structure or the amino acid sequence.

The protein solution is cleaned into short stretches of pep tides preferably by an enzyme such as trypsin or chymotrypsin. The mixture of short pep tides thus generated is injected into the tandem MS for analysis. The tandem MS is an instrument with two MS in a series. They are designated as MS1 and MS2. In the first mass analyser (MS1) the peptide mixture (precursor ions) is sorted resulting in the selection of only one peptide from the several types of pep tides produced by cleavage, which comes out at the outlet of the MSl. The selected peptide (precursor ion) is then transmitted to a collision cell where it undergoes collision-induced dissociation (CIO). The peptide is further fragmented by a high-energy impact with a small amount of inert gas such as helium or argon known as the collision gas. The fragment ions, thus produced, are transmitted to the second mass analyzer (MS2), which measures the m/z ratio of all the charged fragments. During the process of fragmentation the peptide molecule breaks at the peptide bonds resulting in the product ions. This fragmentation does not involve the addition of water as in the case of enzymatic cleavage.

The MS2 scans the product ions and generates one or more sets of peaks. A set of peaks consists of all the charged fragments (product ions) generated by breaking the same type of bond (peptide bond) by CIO. Each peak in a set represents one amino acid less than the previous peak. The difference in mass (m/z) between two adjacent peaks indicates the amino acid that was lost during CID, thus revealing the sequence of amino acids of the peptide. This type of mass spectrometry, also known as 2D MS, is usually used for the analysis of complicated mass spectrum, particularly useful in the amino acid sequencing of pep tides and post-translational modifications.

Tags: Bio Technology, Bio Genetics , Protein Purification

Process of Characterization of Proteins

2009-02-27T01:39:00.000-08:00

The purified protein needs to be identified and characterized. There are various methods to accomplish this. The purity of the protein can be determined by the SDS-PAGE or by isoelectric focusing (IEF) or by two-dimensional gel electrophoresis.

Identification or characterization of protein can be carried out with the help of anyone of the following techniques or by a combination of these:

1) Two-dimensional gel electrophoresis,
2) Peptide fingerprinting,
3) Protein sequencing, and
4) Mass fingerprinting by mass spectrometry. Here we are discussing the technique of Mass Spectrometry.

Mass Spectrometry: It is a chemical-analysis technique that is used to measure the mass of unknown molecules by ionizing, separating, and detecting ions according to their mass-to-charge ratios. Mass spectrometry is also used to determine the structure of molecules.Mass spectrometry helps us in the following areas:

1) To determine the sequence of proteins and peptides.
2) To identify the structure of other biomolecules such as lipids, carbohydrates, oligonucleotides, etc.
3) To detect the presence of banned substances in athletes.
4) To determine the composition of rocks and thus determine their age and origin.
5) To identify isotopes of various elements.
6) To perform forensic analysis to detect the presence of certain substances.

A mass spectrometer creates charged particles (ions) from molecules. It then analyzes those ions to provide information about the molecular weight of the compound and its chemical structure. There are different types of mass spectrometers and sample introduction techniques, which allow a wide range of analyses. This discussion will focus on the principle of mass spectrometry and its use in the study of protein chemistry. The techniques can be used to sequence peptides and proteins and to study their interactions. It is useful in the identification of protein molecules, their accurate molecular weight, the post-translational modifications, and structural functional relationship of proteins.

It is now possible through mass spectrometer to 'see' proteins of molecular weight as high as 100,000 daltons. A big advantage is that a very small sample, as small as picomoles, is required. Thus, it is possible to do two-dimensional gel electrophoresis of proteins of a cell (to separate the few thousand different proteins) and identify them using mass spectrometer. This approach of protein analysis and identification is one of the important techniques of what is called proteomics-the study of the complete protein complement of a cell.

Let us understand as to what is a Mass Spectrum:
A mass spectrum is a plot that shows the relative abundance of ions of various mass-to-charge ratios. The X-axis represents the mass-to-charge ratio of the ions and the Y-axis represents the relative abundance of each ion.

Mass spectrometry can be in association with or linked to one or more separating techniques. For example, in organic chemistry mass spectrometry is always linked to Gas chromatography and therefore, is called GC-MS. Gas chromatography will convert the samples into gaseous phase, which is introduced to the mass spectrometer. In addition to this, GC can be used to separate the components in a mixture and the separated pure compounds can be directly identified and analyzed by a mass spectrometry spectrum. This is the advantage of linking a separating technique to mass spectrometry. Similarly, in the case of protein studies mass spectrometry can be linked to various types of liquid chromatographic systems such as GPC, ion exchange, or affinity chromatography individually or in tandem, or to capillary electrophoretic systems, which can also be operated automatically, like HPLC.

Tags: Bio Technology, Bio Genetics , Protein Characterization

How do we scale up the Protein Purification Process

2009-02-23T20:15:00.000-08:00

When the protein purification protocol forms the part of an industrial process for the production of industrially important or pharmaceutically important protein such as vaccines, hormones, etc., the laboratory scale protocol also has to be scaled up. Actually, it forms the process of downstream processing.

Special care should be taken to bring the cost of production of the protein down by purchasing the chemicals in bulk quantities and using equipment and holding vessels, which are permanent or recyclable. The materials and vessels should be made up of cheap materials and should be inert and resistant to corrosion. Leaching of toxic metals and chemicals into the product should be avoided. The process of manufacturing and downstream processing require the approval of the regulatory authority to produce and market a protein that is useful in food industry or as pharmaceuticals. About 80% of production costs is for downstream processing and quality assurance. Therefore, by controlling the various steps and equipment in downstream processing and product recovery, we can bring down the cost of production without compromising the quality of the product.

Bulk-Protein Production :
The protein-purification procedure that is applicable for the laboratory scale is applicable for the downstream process of the industrial scale also. The process can be used for the purification of enzymes in bulk quantities not only from microbial fermentation systems but also from plant and animal tissues.

Proteins for Therapeutic and Diagnostic Purposes:
Drug proteins such as digestive enzymes, therapeutic proteins such as antibodies or vaccines, hormones, growth factors, etc., and other diagnostic proteins should be purified to a very high degree. Those proteins that are for pericutaneous or intra-venal administration should be highly purified and sterile. Parentral preparations are those that are intended for intra-venal administration or for infusion or for implantation. Such materials should be highly purified and sterile.

Tags: Bio Technology, Bio Genetics, Protein Purification

Extraction & Purification of Proteins

2009-02-16T08:35:00.000-08:00

Biochemists investigate proteins at different levels. At the simplest level they carry out qualitative chemical tests to find out if samples of material contain protein of any sort. At the other extreme they can use the most up-to-date technology to find out the precise arrangement of every atom in each molecule of a particular protein. For structural elucidation, proteins should be very pure homogeneously and in some cases they should be crystallized. There are various methods of extraction and purification of proteins from different sources such as microbes and plant and animal tissues. Extraction and separation techniques for the purification of compounds from microbial cultures and plants and animal tissues are collectively called downstream processing. For industrial purposes, there are some microorganisms identified as non-pathogenic, non-toxic which do not produce any antibiotic. These microorganisms can be used as a source of industrial enzymes and proteins as well as for introducing foreign genes for producing recombinant proteins. Such microorganisms are called generally regarded as safe (GRAS) microbes.

Plants and some animal tissues such as pancrease form the source of some important industrially important proteins and enzymes, which are used in food industry and medicine. Such enzymes should be extracted from non-toxic plant parts and the animal tissues used should be free from infectious diseases. For example, one of the important industrial enzymes, papain, is extracted from the latex of green leaves and fruits of papaya. Papain is used in the meat industry and leather industry for meat tenderization, processing the collagen and other fibrous proteins present in leather, clarification of beverages, and also in medicines as a digestive aid and for cleaning wounds.

Proteins and enzymes of animal origin can be extracted and purified from the respective organs in which the enzyme is present in higher quantities. Slaughterhouses are one of the centers for the supply of tissues and organs necessary for the extraction of certain proteins such as insulin. Traditionally, insulin was obtained from the pancreas of cows and pigs. It requires the slaughtering of about 100 to 150 pigs or 15 to 20 cows to meet the insulin requirement of a single diabetic patient per year. From this we can imagine the number of animals that have to be killed to meet insulin requirements. But modern biotechnology and genetic engineering has come in to help the situation.

Nowadays we don't depend on animals for insulin. There are a number of pharmaceutical companies which manufacture and market human insulin produced by genetic engineering. Similarly, a large number of therapeutic proteins such as vaccines and hormones are now produced by genetic engineering. Today, efforts are there to produce transgenic plants and animals capable of producing these therapeutic antibodies and hormones, and other industrially important proteins in specific organs of plants and animals. For example, the production of edible vaccines (edible plant parts such as fruits and tubers containing the vaccine) and expression of certain proteins such as insulin in the milk of cows and goats, and in eggs. This method of genetic engineering for large-scale production of specialized proteins and other molecules is called molecular farming. The main advantages of molecular farming is that the cost of production can be reduced, costly and time-consuming fermentation procedures and downstream processes can be avoided, large-scale production of the specific compounds is possible, and ease of production and purification procedures is advantageous.

Edible vaccines have the advantage that they can be stored for a long time without refrigeration, can be easily transported, and can be administered by feeding the fruit or the plant parts having the vaccine. While eating the vaccinated fruit, the vaccine molecules will be absorbed into the bloodstream through the mucous membrane lining the mouth and esophagus. Even animals can be vaccinated by this way. Attempts are being made to develop transgenic fodder grass containing the anthrax vaccine. Cattle can be fed with this transgenic fodder grass and be vaccinated effectively against anthrax.

Tags: Bio Technology, Bio Genetics, Proteins

Understanding Two-dimensional Gel Electrophoresis

2009-02-16T08:34:00.000-08:00

Two-dimensional gel electrophoresis is a method for the separation and identification of proteins in a sample by displacement in two-dimensions oriented at right angles to one another. This allows the sample to separate over a larger area, increasing the resolution of each component.

Two-dimensional gel electrophoresis is generally used as a component of proteomics and is the step used for the isolation of proteins for further characterization by mass spectroscopy. In the lab we use this technique for two main purposes. Firstly, for the large-scale identification of all proteins in a sample. This is undertaken when the global protein expression of an organism or a tissue is being investigated and is best carried out on model organisms whose genomes have been fully sequenced. In this way the individual proteins can be more readily identified from mass spectrometry data. The second use of this technique is differential expression; this is when two or more samples are compared to find differences in their protein expression.

Two different protein-separating techniques are combined in sequence to achieve the goal of protein separation and identification-Iso electric Focusing (IEF) and SDS-PAGE. Isoelectric focusing (IEF) is used in the first-dimension. This separates proteins by their charge (pI) and SDS-PAGE (sodium dodecyle sulphate-polyacrylamide gel electrophoresis) is used in the second-dimension. This separates proteins by their size (molecular weight, MW). The procedure is known as ISO-OALT (iso for isoelectric focusing and dalt for molecular weight in dalton).

Isoelectric focusing (IEF). The side chains of amino acid residues of a protein contribute a net charge for protein molecules, which depend on the pH of the medium. In simple electrophoresis the mobility of the protein molecules is dependent on its charge that is controlled by the pH. There is a pH for every protein molecule at which the net charge of the protein becomes zero. This pH is known as isoelectric pH or isoelectric point (PI). At isoelectric pH the protein loses its mobility in the electric field. The technique of protein separation based on the property of isoelectric pH or PI is known as isoelectric focusing (IEF). A pH gradient is generated on an IEF gel and proteins are allowed to move in an electric field and that results in the separation of an individual protein species according to its isoelectric point.

Tags: Bio Technology, Bio Genetics, Proteins

Sickling of Cells & Malaria

2009-02-16T08:32:00.000-08:00

The high representation of the hemoglobin S gene in some populations reflect the protection it provides against malaria. The malaria parasite does not survive as well in the erythrocytes of people with the sickle trait as it does in the cells of normal people. The basis of the toxicity of sickle hemoglobin for the parasite is unknown. One possibility is that the malarial parasite produces extreme hypoxia in the red cells of people with the sickle trait. These cells then sickle and are cleared (along with the parasites they harbor) by the reticuloendothelial system. Another possible mechanism is that low levels of hemichromes are formed in sickle trait erythrocytes. Hemichromes are complexes containing heme moieties that have dissociated from the hemoglobin. Hemichromes catalyze the formation of reactive oxygen species, such as the hydroxyl radical, which can injure or even kill malarial parasites.

The malaria hypothesis maintains that during prehistory, on average, people without the sickle gene died of malaria at a high frequency. On the other hand, people with two genes for sickle hemoglobin died of sickle cell disease. In contrast, the heterozygotes (sickle trait) were more resistant to malaria than normal people and yet suffered none of the ill effects of sickle cell disease. This selection for heterozygotes is called balanced polymorphism. Support for this concept comes from epidemiological studies in malaria-endemic regions of Africa. The frequency of the sickle cell trait is lower in people coming for treatment to malaria clinics than is seen in the general population. The reasonable assumption is that relative protection from malaria is at work in this situation.

Although malaria remains a major health problem in many tropical regions of the world, the disease is not a significant threat to people in temperate zones. Consequently, the protection afforded by the sickle trait no longer has a survival advantage for many groups of people in whom the sickle cell gene is common. This has left sickle cell disease the major health issue in these populations.

Tags: Bio Technology, Bio Genetics, Siclkling of cells

Understanding of Protein Fingerprinting Technique

2009-02-16T08:30:00.000-08:00

Breaking the protein molecules down into shorter fragments called peptides, Pauling subjected these fragments to another technique called protein fingerprinting by paper chromatography.

The technique of protein fingerprinting involves the following steps:

1) Extract and purify hemoglobin from sickle cell RBC and normal RBC separately in a clean test tube.

2) Digest these proteins with a commercial sample of trypsin separately under standard conditions. Trypsin is another type of serine protease that cleaves the peptide bond adjacent to a lysine or arginine residue in a protein molecule.

3) The cleaved peptides are subjected to paper electrophoresis under pH (pH 2.5) and dry the paper.

4) After electrophoresis they are subjected to paper chromatography perpendicular to the direction of electrophoresis using the solvent system water: butanol: acetic acid in the ratio 5:4:1. The peptides will separate depending on their partition coefficient, which further depends on their degree of hydrophobicity. The more hydrophobic peptide will move fast and the less hydrophobic will move slowly.

5) Remove the chromatographic paper and stain with ninhydrin.

6) Examine the peptide spots and compare with the standards.

When this procedure is applied to samples of normal and mutant (sickle) hemoglobin molecules (alpha and beta chains) that had been broken down into specific pep tides, all the spots are the same except for one crucial spot, which represents the difference between sickle cell and normal hemoglobin.

The protein fingerprinting or the peptide mapping developed for the molecular studies of sickle cell hemoglobin became a very powerful technique for the identification of protein samples from different sources. The peptide fingerprint of a protein from new sources can be compared with that of the standard protein and thus,. the variations can be identified or understood. This simple technique of peptide fingerprinting has given rise to another similar and more powerful technique: two-dimensional gel electrophoresis. This is a combination of two electrophoretic techniques - Isoelectric focusing and SDS-PAGE-in a series. First, the protein is subjected to isoelectric focusing, which is followed by 50S-PAGE in a direction perpendicular to the first. This technique was found to be very useful for proteomes studies, expression of protein profiles of cells grown under different conditions (for example, normal cells and diseased cells), and also easy identification of proteins in combination with mass spectrometry. All these technological advancements including amino acid sequencing have provided an enormous quantity of data, and that has given rise to computerized databases and homology searches and protein identification. All these have led to a generation of bioinformatics and computational biology.

Tags: Bio Technology, Bio Genetics, Proteins

Sickle Cell Anemia the cause of oxygen depletion

2009-02-16T08:28:00.000-08:00

Sickle cell anemia, creates serious depletion of oxygen through two mechanisms:
1) Because of molecular changes within the sickled cell, oxygen-carrying capacity of the blood is greatly reduced.

2) Because of their peculiar shape, greater rigidity, and tendency to stick together, sickle cells clog smaller vessels in the circulatory system-the arterioles and capillaries, in particular-preventing the blood from delivering oxygen and nutrients, and removing carbon dioxide and wastes from tissues.

This disease was reported for the first time by a Chicago-based cardiologist James B. Herrick in 1910. It was recognized to be the result of a genetic mutation, inherited according to the Mendelian principle of incomplete dominance. Initially, it was not clear what the actual defect was, that caused the sickling. Various experiments indirectly narrowed down the site of the defect to the hemoglobin molecule. The most direct evidence that mutation affected the hemoglobin molecule came from electrophoretic analysis, a method to separate complex mixtures of large molecules by means of an electric current on a gel. When hemoglobin from people with severe sickle cell anemia, the sickle cell trait, and normal red blood cells was subjected to electrophoresis.

It was clear that hemoglobin molecules of people with sickle cell anemia migrated at a different rate, and thus ended up at a different place on the gel, from the hemoglobin of normal people. What was even more interesting was the observation that individuals with the sickle cell trait had about half normal and half sickle-cell hemoglobin, each type making up 50% of the contents of any red blood cell.

To confirm this latter conclusion, the electrophoretic profile of people with the sickle cell trait could be duplicated simply by mixing sickle cell and normal hemoglobin together and running them independently on an electrophoretic gel. These results fit perfectly with an interpretation of the disease as inherited in a simple Mendelian fashion showing incomplete dominance. Here, then, was the first verified case of a genetic disease that could be localized to a defect in the structure of a specific protein molecule. Sickle cell anemia thus became the first in a long line of what have come to be called molecular diseases. Thousands of such diseases (most of them quite rare), including over 150 mutants of hemoglobin alone, are now known.

Tags: Bio Technology, Bio Genetics, Proteins

Importance of Structure on Function of Proteins

2009-02-16T08:26:00.000-08:00

Proteins are the workhorses of cells and in every activity there is the involvement of one or more proteins in different ways. Now, it is very clear that each protein has a specific three-dimensional shape determined by the amino acid sequence and various other intermolecular interactions. This three-dimensional shape has a great influence on the biological function that it performs in the cells. We consider two proteins, as an example, to understand the importance of a three-dimensional structure on its specific function: a proteolytic enzyme, chymotrypsin, and the oxygen carrying protein, hemoglobin.

Chymotrypsin - A Protein- Digesting Enzyme:
Chymotrypsin is a member of a family of enzymes, all of which cleave peptide bonds through the action of an active site serine (the serine proteases). This family includes the pancreatic enzymes chymotrypsin, trypsin, and elastase as well as a variety of other proteases (e.g., cocoonase, thrombin, acrosomal protease, etc.). Chymotrypsin, trypsin, and elastase show a high degree of similarity in their overall tertiary structure, but have different substrate specificities determined by a specific substrate-binding site on each enzyme.

Chymotrypsin is one of the proteinhydrolyzing enzymes produced by the digestive gland pancreas. The protein present in the food that we eat is digested mainly by two proteases - trypsin and chymotrypsin in the beginning of the small intestine (the duodenum). These two digestive enzymes are produced by pancrease and are released into the duodenum through the pancreatic duct. Thus, the site of production of these enzymes is the pancreas and their site action is the duodenum.

Trypsin and chymotrypsin cut the linear polypeptide chains into short peptides by cutting at specific sites. These short pep tides thus produced are acted upon by other peptidases releasing amino acids. But the pancreas is made up of many proteins. How are these proteins protected from the hydrolytic activity of chymotrypsin? These types of hydrolytic enzymes, particularly proteases are produced in an inactive form called zymogen and are transported to the site of action, the duodenum-where it is converted into an active enzyme by a process known as in situ activation. Because of this process, the protein undergoes a major change in its three-dimensional shape, which is now suitable for its interaction with its substrates. The active chymotrypsin enzyme is known as alpha. chymotrypsin and its inactive form from which it is produced is called chymotrypsinogen.

Know the Chymotrypsinogen:
Chymotrypsinogen, the precursor (zymogen) of active chymotrypsin, consists of 245 amino acid residues. Activation of chymotrypsinogen involves proteolytic cleavage at two sites along the chain and removal of two amino acids at each cleavage site. The resultant three peptide chains are A, B, and C. These three chains are held together by five disulfide bonds and fold into a globular structure. This process of folding brings three distantly placed amino acid residues his 57, asp 102, and ser 195 close together in a particular order to form the active center or the reaction center of the enzyme. The overall chymotrypsin molecule is folded into two domains, each containing six beta strands arranged as antiparallel sheets that form a circular structure known as a beta barrel. The active site residues (ser 195, his 57, and asp 102) are far apart in the primary sequence but are brought together in a crevice formed between the two protein domains

Levels of Complexities of Structure of Proteins

2009-02-08T04:44:00.000-08:00

Proteins and peptides are biopolymers composed of amino acid residues interlinked by amide bonds. Their structures can be discussed in terms of four levels of complexity and are as follows:
1) Primary structure
2) Secondary structure
3) Tertiary structure
4) Quaternary structure

The linear unbranched chain of amino acids linked together by covalent bonds known as peptide bonds is called the primary structure of a protein. In addition to peptide bonds, other types of covalent linkages such as disulphide bonds, if present, are also included in the primary structures. The types and the sequence of amino acids present in the polypeptide chain determine the nature of the secondary structures at different regions of the chain. The secondary structure of a segment of a polypeptide chain is the local spatial arrangement of its main-chain atoms without regard to the conformation of its side chains or to its relationship with other segments.

The major types of secondary structures observed in protein molecules are alpha (a ) helices and beta (b ) pleated sheets in addition to random coils and beta turns. All these secondary structures may be present independently or may be together in the secondary or tertiary structures of a single polypeptide chain. The secondary structure undergoes further folding and reorganization within the molecule resulting in higher order compact structures or the tertiary structure.

There are structural components comprising a few alpha-helices or beta-strands, which are frequently repeated within structures, called supersecondary structures (being intermediate to secondary and tertiary structures). These compact structurally distinct elements are known as motifs. When these structurally distinct regions of protein molecules are associated with a specific function, those structurally and functionally distinct units are called a domain. Structurally-related domains are found in different proteins, which perform similar functions.

The molecular forces, which are responsible for the secondary and tertiary structures, are the non-covalent interactions between the various amino acid side chains within the molecule and with the water molecule surrounding it. The main molecular force responsible for the various secondary structures are the hydrogen bonds and the molecular forces behind the tertiary structures are the ionic bonds, hydrogen bonds, hydrophobic and hydrophilic interactions, and van der Waals force. Secondary and tertiary structures represent the most thermodynamically stable conformations or shapes for the molecule in a solution. The quaternary structure is the assembly of two or more independent polypeptides or proteins at their tertiary stage to form a multimeric protein. The individual component pep tides of the multimeric proteins are known as subunits and are held together via non-covalent forces. The subunits of a multimeric protein may be similar or dissimilar. For example, hemoglobin contains four polypeptide chains (20. chains and 213 chains) held together non-covalently in a specific conformation as required for its function.

The major molecular forces that cause the linear polypeptide chain to undergo a specific type of coiling and folding in space to a characteristic three-dimensional shape are the non-covalent forces. These forces, to a greater extent, lie in the chemical and structural properties of the constituent amino acid residues of the polypeptide chain. There are 20 types of amino acids by which the entire protein of the living system is composed. These amino acids can be broadly classified into three categories-hydrophobic (tryptophan, phenylalanine, leucine, etc.), polar (glutamine, serine, etc.), and charged (aspartic acid, lysine, etc.) amino acids. Therefore, these amino acids are capable of interacting with each other within the protein molecules via various non-covalent interactions leading to a very characteristic shape and biological property for the protein molecule.

Tags: Bio Technology, Bio Genetics, Proteins

An Introduction to Structure of Proteins

2009-02-08T04:42:00.000-08:00

A single cell develops into a multicellular embryo through a large number of complicated biochemical reactions mediated and controlled by various types of proteins expressed during the course of its development. Every function in the living cell depends on proteins. They make us who we are and make our cells operate properly. A cell cannot function without proteins. The shape of a protein determines its biological activity. A single protein may have a varying structure and more than one function. Proteins have many different biological functions. Proteins are even classified according to their biological roles. The key to appreciating how different proteins function in these different ways lies in an understanding of protein structure and their three-dimensional shape. Proteins interact with other molecules such as small molecules, other proteins, nucleic acids, lipids, etc., and these interactions form the basis of their biological roles. Structural complementarity is the means of molecular recognition that allows molecules to interact. The structure of one molecule is complementary to that of its partner(s) in the interaction, like pieces in a puzzle, or a lock and its key. Proteins, by virtue of their architectural diversity, are ideal for such complementary interactions. In short, the structure or the molecular shape of the protein determines its function. Therefore, to understand the function and biological role of a protein it is essential to understand the structure and three-dimensional shape of the protein in detail.

The detailed study of the structure of proteins requires protein extraction and purification to its homogeneity. The purified protein has to be analyzed by various biochemical and instrumental methods to get the details about its chemical composition. The pure protein obtained has to be crystallized to study its three-dimensional shape by x-ray crystallography (an x-ray diffraction technique). Another equally powerful technique to elucidate the three-dimensional structure of protein is NMR spectroscopy. The structural study of protein, thus has two parts-the first part is determination of amino acid sequence and the second part is the elucidation of the three-dimensional shape of the protein formed by the specific folding of the polypeptide chain controlled by a number of molecular forces. The extraction and purification and its crystallization are the preconditions for the detailed structural and functional studies.

The ability to sequence polypeptides was a major step forward in the understanding of the relationship between protein structure and function. It was Dr. Frederick Sanger who developed the basic chemical method for sequencing proteins during the 1940s. He showed for the first time, that proteins are a linear polymer of amino acids, linked in a continuous sequence by peptide bonds. He received the Nobel Prize in 1958 for determining the sequence of the peptide hormone insulin. The peptide bond is formed between the alpha-amino and alpha-carboxyl groups of two adjacent amino acids. Pehr Edman modified this process of amino acid sequencing by introducing a new reagent-phenyl-isothiocyanate for the sequential removal of amino acids and their identification in a protein. This method of sequencing is now automated and called the Edman Degradation Reaction and the instrument is called the Sequenator.

Tags: Bio Technology, Bio Genetics, Proteins

Expression of Proteins in Cells

2009-02-08T04:41:00.000-08:00

The proteins, which are produced, have various life spans or half-life ranging from a few seconds to many months or years.

Each protein can be identified by its unique amino acid sequence, the three-dimensional shape of the protein (that mainly depends on amino acid sequence and other environmental factors), function of the protein, and cellular location.

The proteins expressed in a cell may be intracellular (present within the cell) or extracellular (secreted outside the cells), or circulating in the blood like hormones, immunoglobulins, etc., constantly interacting with other molecules such as proteins, lipids, sugars, DNA, RNA, metal ions, vitamins, etc., or with other cells. The functions of these circulating proteins are also influenced by their interaction with other molecules that are present nearby. Even though the total number of genes estimated is about 35,000, the actual number of proteins is much higher than this. About 17, 000 proteins were identified at the gene level but information regarding their function and biological role is still being investigated in detail.

The type and total number of genes in an organism will be stable (static) and are identical in all somatic cells of an organism. But, the total number of proteins expressed by a cell (protein profile) of an organism is always variable (dynamic). The protein profile of a cell depends on its metabolic state, stage of development, and other micro- and macro-environmental factors, which influence the expression of a set of genes at a particular time. Therefore, the challenge for the future is to determine the actual total number of proteins expressed in each cell type and find out the functions and biological role of these proteins in metabolism, health, and disease. These topics provide an exposure to the three-dimensional structure of proteins and their relationship to proteomics, recombinant DNA technology, genomics, and finally bioinformatics.

Tags: Bio Technology, Bio Genetics, Proteins

Understanding the Broad Compositions of Proteins

2009-02-08T04:39:00.000-08:00

In spite of these diverse biological functions, proteins have relatively homogeneous compositions. All proteins are linear polymers of the same 20 types of amino acids in different combinations. The major difference between proteins is in the sequence in which the amino acids are assembled into polymeric chains. The secret to their functional diversity lies partly in the chemical diversity of the 20 amino acids, but primarily in the diversity of the three-dimensional structures that these amino acid building blocks can form by linking in different sequences. The amazing functional properties of proteins can be understood only in terms of their relationship to the three-dimensional structures of proteins.

Now we know that the amino acid sequence of a protein and thereby its three-dimensional structure is specified by a gene. But, this is not completely true. Even though the gene sequence specifies the amino acid sequence of the protein, the three-dimensional structure is also influenced by a number of other factors. The number of proteins produced in a system always exceeds the number of genes. The Human Genome Project has announced the presence of about 35,000 genes. But the actual number of proteins encoded by these genes exceeds the number of genes.

This is mainly because of the various types of molecular modifications such as deletion of amino acids, chemical modifications of certain amino acids, addition of other macromolecules and groups such as phosphate groups (phosphorylation), acetyl groups (acetylations), sugar and other types of carbohydrates (glycosylations), lipids, etc. All these chemical modifications of proteins just after their formation are collectively called post-translational modifications. Actually, these post-translational modifications are responsible for the diversity in the three-dimensional structures and functions along with the amino acid sequence prescribed by the respective genes. A number of proteins are expressed in all cells irrespective of their functional specialization. Such proteins are called housekeeping proteins, required for the basic life activities of all cells_ But there are certain proteins, which are unique to certain cells. Hemoglobin in erythrocytes (RBC), collagen, myosin, etc., in muscle cells are some examples. This is called cell-specific or organ-specific or tissue-specific gene expression. The expressions of these genes are under the control of very specific regulatory proteins or other types of small molecules called transcription factors.

Tags: Bio Technology, Bio Genetics, Proteins

Effect of Malfunctioning of Proteins in the Body

2009-02-08T04:38:00.000-08:00

The absence or malfunctioning of one or more proteins in the system can cause serious life-threatening diseases. The malfunctioning of proteins can be traced to some type of structural abnormality due to variations in the chemical composition. For example, the absence of one of the subunit, beta chain of the oxygen-carrier protein hemoglobin of RBC, can cause thalassaemia.

This metabolic error due to abnormal hemoglobin affects many children who can only survive on repeated blood transfusion. Another type of abnormal hemoglobin is where the beta chain is mutated and the glutamic acid at position six is replaced with valine and results in deformed RBC and a condition known as sickle cell anemia.

The absence of an enzyme adenosine-deaminase, an important enzyme in nucleotide metabolism, can cause the disease known as SCID (severe combined immunodeficiency) in children. These children cannot survive infancy. There are some types of infectious protein particles known as 'prions', which can turn normal proteins to rogue proteins or incorrectly shaped proteins and can cause diseases such as mad cow disease. To understand more about the relationship between the disease and the structural abnormality of the protein we should know more about the structure and its relationship with biological activity.

Tags: Bio Technology, Bio Genetics, Proteins

Importance of Proteins for Life forms

2009-02-08T04:33:00.000-08:00

Proteins are essential to maintain the structure and function of all life forms. The word 'protein' itself is derived from the Greek word protos, meaning "primary" or "first." Proteins are vital for the growth, repair, and maintenance of muscles, blood, internal organs, skin, hair, and nails, and their functions are endless. Each and every property that characterizes a living organism is affected by proteins, whether it is a bacteria or a human body. Nucleic acids, another major biological macromolecule, are also essential for life; they encode genetic information-mostly specific for the structure of proteins-and the expression of that information depends almost entirely on proteins. The fertilization of an egg with a sperm and the development and differentiation of the resultant zygote into a fully developed organism and its growth and maintenance of life activities up to its death is controlled and programed by a large number of proteins.

In our body, when we breath, oxygen present in lungs will be taken by the hemoglobin present in the RBC of blood to the various cells of the system for the process of cellular respiration. Movements and activities of body parts and systems including lungs, heart, stomach, etc. are happening due to the contractions and relaxations of various types of muscles. Myosin, actin, and collagen are the protein molecules involved in body structure, protection, and muscular contraction and relaxation. The structure of cells, and the extracellular matrix in which they are embedded, is largely made of protein. Plants and many microbes depend on carbohydrates such as cellulose for support. All biological activities of cells are mediated and regulated by a large number of catalytic proteins called enzymes. The function of the human brain and the speed at which the electric impulses are generated and transmitted to coordinate various activities of the systems are meticulously done by a large number of proteins that act as enzymes and receptors.

The receptors and hormones are another class of proteins, which act as signal molecules that are involved in the coordination of different metabolic functions of the system. There are the proteins called transcription factors, which turn the genes on and oft to guide the differentiation of the cells and development, and there are many more activities in which proteins are involved. Thus, proteins are diverse in their functions and are truly the physical basis of life. To understand the diversity in the biological function of proteins, their molecular structure and shape has to be studied in detail, since the function is closely related to the structure.

Tags: Bio Technology, Bio Genetics, Proteins

Understanding the methods of DNA Isolation

2009-02-03T19:35:00.000-08:00

DNA isolation and purification is a technique used in laboratories engaged in molecular biology experiments. There are a number of standardized techniques and variations, which can be adopted according to the type of cells or tissues. The isolation and purification methods used in earlier times were lengthy and tiresome with the use of ultra-centrifugation. But now with the advancement of separation techniques, the procedure is very simple and short.

In any method of extraction and purification, there are three main steps:

1. Breaking of the cells
2. Extraction of DNA
3. Purification

Cells can be broken in different ways. One common method for lysis of bacterial cultures is alkaline lysis. In the case of animals, cells can be lysed by simple detergents or by hypotonic solutions. Plant tissues can be homogenized by strong detergents such as SDS and heating at high temperatures. But there are various types of DNA isolation kits marketed by a number of biotechnology companies, which are very simple, short, and easy to handle.

Isolation of Plasmid DNA by Alkaline Lysis Method:
This method is used for the large-scale isolation of plasmid and cosmid DNA by a modification of alkaline lysis procedure, followed by purification by phenol chloroform extraction. Cells containing the desired plasmid or cosmid are harvested by centrifugation, incubated in a lysozyme buffer (re-suspension buffer), and treated with alkaline detergent. The alkali breaks the cells and the DNA and proteins- are released into the medium. Detergent solubilizes the proteins and DNA. The proteins and membranes are precipitated with sodium acetate. The precipitate is centrifuged out at a higher RPM and the supernatant contains the DNA. Finally, the DNA is precipitated out by adding 95%. ethyl alcohol or propanol. The DNA pellet is resuspended in a Tris EDTA buffer. This DNA sample contains some DNA-binding proteins, which have to be removed. This can be carried out by phenol-chloroform extraction. There are several variations to this protocol, which is suited to the situations- and type of bacterial cultures.

Genomic DNA Isolation from Blood:
Genomic DNA isolation is performed according to the standard protocol suggested by Federal Bureau of Investigation, USA. After the blood samples (stored at -70°C in EDTA vacutainer tubes) are thawed, a standard citrate buffer is added, mixed, and the tubes are centrifuged. The top portion of the supernatant is discarded and additional buffer is added, mixed, and again the tube is centrifuged. After the supernatant is discarded, the pellet is resuspended in a solution of SDS detergent and proteinase K, and the mixture is incubated at 55°C for one hour. The sample then is phenol-extracted once with a phenol/ chloroform/ isoamyl alcohol solution, and after centrifugation the aqueous layer is removed to a fresh microcentrifuge tube. The DNA is ethanol-precipitated, resuspended in buffer, and then ethanol-precipitated a second time. After the pellet is dried, buffer is added and the DNA is resuspended by incubation at 55°C overnight, and the genomic DNA solution is assayed by the polymerase chain reaction.

DNA Isolation from Plant Tissues:
Plant tissues bring up several problems during DNA isolation. Plant cells have a rigid cell wall and the tissue contains a number of toxic metabolites, which can interact with the DNA and change its nature and make it useless for other experimental purposes. Metabolites such as mucilage and other carbohydrates can very easily- form complexes with DNA and it can be damaged. Therefore, the extraction buffer should be supplemented with some compounds that can protect the DNA against these metabolites.

Many DNA-isolation techniques widely employed by plant molecular biologists use a CTAB (Cetyltrimethylammonium bromide) extraction buffer. This compound forms a complex with DNA and thus protects it from other toxic metabolites such as mucilage and phenolics.

The DNA, isolated and purified by any of these methods, can be used for a variety of experimental purposes. It can be used for restriction digestion analysis, cloning, ligation, transformation experiments, in vitro transcription, PCR amplification, RFLP (restriction length polymorphism), fingerprinting, RAPID (random amplification polymorphic DNA), sequencing, nick translation and radiolabeling, preparation of genomic DNA library and cDNA library, etc.

Tags: Bio Technology, Bio Genetics, DNA Isolation

Know the Pedigree Analysis of Humans

2009-02-03T19:28:00.000-08:00

Humans are unique among organisms in many ways. But one way, which is near and dear to a geneticist's heart, is that humans are not susceptible to genetic experimentation. In practice, we humans actually share this characteristic with many long-lived organisms who delay first births. In short, it is not terribly convenient to perform experimental crosses if one has to wait 15 years between generations. However, for humans, one also has to add that our system of morality uniquely does not allow such experimentation on humans. This is an unfortunate state of affairs since there is no other organism for which practical knowledge of their genetics would be more useful, especially in the case of the genetics of heritable diseases. It has been found that human genetics may readily be inferred so long as good records have been kept within large families. This formal mechanism of inference is called pedigree analysis.

Here we are presenting many aspects of human genetics, with particular consideration to strategies of pedigree analysis whereby we are attempting to infer the genetics of human conditions based on knowledge of marriage (mating) and affliction in large extended families.

Procedures for Pedigree Analysis:
Pedigree analysis is one of the central tasks of the human geneticist. It involves the construction of family trees. Family history information is often collected at major family gatherings. A pedigree is used to trace inheritance of a trait over several generations.

Three primary patterns of inheritance in man are the following:

1) Autosomal recessive
2) Autosomal dominant
3) Sex-linked (X-chromosomal)

Autosomal Dominant Inheritance: A dominant condition is transmitted in unbroken descent from each generation to the next. Most matings will be of the form M/m x m/m (i.e., heterozygote to homozygous recessive). We would therefore expect every child of such a mating to have a 50". chance of receiving the mutant gene and thus of being affected.In this pedigree, an affected father passes the trait to half of his six children, including two daughters and a son. One of the daughters passes the same trait to one of her three children.

Autosomal Recessive Pedigree:A recessive trait will only manifest itself when homozygous. If neither parent has the characteristic phenotype (disease) displayed by the child, the trait is recessive. If it is a severe condition it is unlikely that homozygotes will live to reproduce and thus most occurrences of the condition will be in matings between two heterozvgotes (or carriers). An autosomal recessive condition may be transmitted through a long line of carriers before, by the ill chance, two carriers mate. Then there will be a one fourth chance that any child will be affected.

If the parents are related to each other, perhaps by being cousins, there is an increased risk that any gene present in a child may have two alleles identical by descent. The degree of risk that both alleles of a pair in a person are descended from the same recent common ancestor is the degree of inbreeding of the person.

Pedigree of Sex-linked Traits: The transmission of X-linked traits is in a zigzag manner. Females transmit X chromosomes to both sons and daughters. Males transmit the X chromosomes only to daughters and Y chromosomes to sons. The X-linked traits, which are recessive are preferentially seen in males, who are always homozygous for the X chromosomes. Females are heterozygous and form the "carriers" of that trait. Most X-linked traits are recessive.

A color-blind man is the father of "carrier" daughters and normal sons. Carrier daughters have a 50'N, chance to have color-blind sons and a cross between a color-blind male and carrier female can produce color-blind daughters. Hemophilia also has the same type of inheritance. Duchenne muscular dystrophy is another example of X-linked inheritance.Transmission of Y-linked Genes
Men are homozygous for Y-linked genes present on the non-homologous parts. All these genes will be expressed in all conditions. These genes are always transmitted from father to sons and never to daughters. There are no essential genes in the Y chromosomes except the locus for the maleness and fertility.

Pseudoautosomal Inheritance:
There are some homologous regions in the X chromosomes and Y chromosomes. These homologous parts pair during meiosis and may undergo crossing over. Therefore, genes in these homologous regions show inheritance similar to autosomal genes and are called pseudoautosomal inheritance. Such genes or characters are very rare.

Tag: Bio Technology, Bio Genetics, Pedigree Analysis

What are the Breeding Methods in Plants

2009-01-30T19:20:00.000-08:00

Breeding Methods in Plants:

Breeding plants to create new varieties and improve upon old ones is a hobby that nearly everyone can engage in. The crossing techniques are easy to learn and breeders can experiment with many kinds of plants. Generally, amateur plant breeders work with traits that are fairly easy to change-for example, flower color, fruit shape, or plant size. Nevertheless, although experiments may be simple, it is possible to produce unusual or beautiful plants. In order to breed plants successfully it is important to understand the principles of plant reproduction. The purpose of this is to explain the simple techniques that can be used to produce new varieties or strains of plants.

The first step in the plant hybridization procedure is the selection of parent plants with the desired characteristics. Plant characteristics can be changed after many generations by a process of selection. There are two types of selection-natural and artificial. Natural selection is the process that occurs in nature whereby strong and well-adapted plants survive while weak and poorly adapted plants eventually die out. This process has taken place since the beginning of life on earth and it is still occurring in nature. Artificial selection is the process that humans use to obtain more desirable types of plants. Thousands of years ago people learned that saving seed from the kind of plant they wanted to continue growing would increase the chances of getting a plant similar to the original. But our ancestors didn't know what their chances of success were nor did they understand the processes by which traits were changed or maintained. It wasn't until the eighteenth and nineteenth centuries that humans began to understand the laws of heredity and the processes of plant reproduction. Even today, these fundamentals aren't completely understood. But enough is known so that we can select plants for breeding with considerably more assurance of success than our ancestors did.

In our experiment we have to select the parents for the process of hybridization. The plants selected for breeding should be sturdy and healthy. It is usually easier to tell which ones are healthy after a few flowers on the plant have bloomed. Some plants have natural barriers to cross- or self-pollination. It is advisable to check for this before breeding, for although barriers can be overcome, some plants cannot be artificially pollinated. An example of a barrier that cannot be overcome is the selfpollination prohibitor of some orchids; the stigmas of certain orchids produce a substance, which kills the pollen of flowers of the same plant. The mechanism that performs this cannot be removed without destroying the pistil. In choosing a pollen parent (male parent), select one that has a heavy yellow powder on the anther. This powder is the pollen. In choosing a seed parent, examine the stigma. It should have either a glistening substance on it that is sticky to the touch or a "hairy" surface. It is this substance or surface that retains the pollen, thus making fertilization possible. Once the seed parents and pollen have been selected, you are ready to begin pollination.

Tags: Bio Technology, Bio Genetics, Breeding Methods in Plants

How to practically do Conjugation of Bacteria

2009-01-30T19:17:00.000-08:00

Here practical steps are being described to perform conjugation of Bacteria.

Take two strains of bacterial cultures of e.coli. One is the male strain or the P+, which is auxotrophic for biotin and methionine (Bio-, MeC). This bacterium can grow in the minimal medium, only if these two components are supplemented. Similarly, the female bacteria or the P- strain is able to produce both biotin and methionine, but are auxotrophic for threonine and leucine (Thr-, Leu-). These bacteria cannot grow in the minimal medium unless the respective nutrients are supplemented.

But when these two populations are mixed and grown in media with only the salts and the carbon source (minimal media) some of the cells could grow without the supplementation of the additional amino acids and vitamin, biotin. This indicates that when grown together some female cells receive the functional genes of Thr and Leu from the male strains by conjugation. Similarly, some of the male strains receive the functional genes for biotin and methionine from the female strain. These new genetically transformed strains can grow in the minimal medium without any additional nutrient supplementation.

This is a common method of natural recombination in bacteria resulting in the formations of variants. In this context it is very important because conjugation can produce drug resistance among pathogenic bacteria. Therefore, the mechanism of conjugation has to be clearly understood and should be aware that in heterogonous cultures there is the chance of bacterial conjugations and genetic recombinations resulting in new strains with new weapons. Contaminated laboratory cultures, organic factory effluents, and sewage water are good media for bacterial conjugations.

Tags: Bio Technology, Bio Genetics, Bacterial Conjugation

How to practically carry out Bacterial Transformation

2009-01-30T19:15:00.000-08:00

Bacterial transformation is routine work in all molecular biology laboratories as part of recombinant DNA experiment or gene cloning. In rDNA experiments or gene cloning, we prepare recombinant DNA or the gene or plasmid to be cloned, which has to be transferred to a host cell so that the DNA will multiply inside the bacterial cell. Transfer of the plasmid or the rDNA is carried out by bacterial transformation.

The first step is to select a suitable host cell such as a suitable strain of e.coli like DH5 a , a common strain available in all molecular biology laboratories, which can take foreign DNA easily. For this we have to treat the grown bacterial cultures at its log phase of growth, with CaCl2. Centrifuge the cells growing at the log phase under low rpm (3,000-5,000 for 10 minutes) at 4°C and collect the cells. Suspend the cells in chilled CaCl2 of 0.1 M. The cells in calcium chloride are able to accept the small DNA molecules. These cells in CaCl2 can be stored for a long time under low temperatures such as -20 or -70°C.

Sudden exposure of this cell to the room temperature or higher can force the cell to take the DNA from outside. Take the stored competent cells, which are in the frozen condition and add the DNA sample to these cells and expose them to a higher temperature, at 42°C for two to three minutes. Some of these cells take the DNA from outside and will be transformed by intercalating with its genome. These cultures can be plated on a selection agar plate and the transformed colonies can be selected against the untransformed ones.

This transformation is extensively used in genetic engineering experiments. Any gene or DNA, before transferring into an organism, can be tested in a selected host by this transformation method. New promoters can be checked for their strength of expression. Commercially-useful enzymes and therapeutic proteins can be prepared in industrial scales. In short, any genetic engineering or gene cloning cannot be accomplished without bacterial transformation.

Tags: Bio Technology, Bio Genetics, Bacterial Transformation