Bioremediation

Enormous quantities of organic and inorganic compounds are released into the environment each year as a result of human activities. In some cases these releases are deliberate and well regulated (e.g. industrial emissions) while in other cases they are accidental (e.g. chemical or oil spills). Petroleum and its products are one of the most common environmental pollutants. They are a fire hazard, threat to marine life, and a source of air and groundwater pollution. They contaminate land and water bodies by accidental spills like the Alaska Oil spill in 1989 and oil spills during the Gulf War, leakage from pipelines, and other human activities. Detoxification of the contaminated sites is expensive and time consuming by conventional chemical or physical methods.

Bioremediation consists of using naturally occurring or laboratory cultivated micro-organisms to reduce or eliminate toxic pollutants. Petroleum products are a rich source of energy and some organisms are able to take advantage of this and use hydrocarbons as a source of food and energy. This results in the breakdown of these complex compounds into simpler forms such as carbon dioxide and water. Bioremediation thus involves detoxifying hazardous substances instead of merely transferring them from one medium to another. This process is less disruptive and can be carried out at the site which reduces the need of transporting these toxic materials to separate treatment sites.

Using bioremediation techniques, TERI has developed a mixture of bacteria called 'oilzapper' which degrades the pollutants of oil-contaminated sites, leaving behind no harmful residues. This technique is not only environment friendly, but also highly cost-effective.

Biofertilizers

One of the major concerns in today's world is the pollution and contamination of soil. The use of chemical fertilizers and pesticides has caused tremendous harm to the environment. An answer to this is the biofertilizer, an environmentally friendly fertilizer now used in most countries. Biofertilizers are organisms that enrich the nutrient quality of soil. The main sources of biofertilizers are bacteria, fungi, and cynobacteria (blue-green algae). The most striking relationship that these have with plants is symbiosis, in which the partners derive benefits from each other.

Plants have a number of relationships with fungi, bacteria, and algae, the most common of which are with mycorrhiza, rhizobium, and cyanophyceae. These are known to deliver a number of benefits including plant nutrition, disease resistance, and tolerance to adverse soil and climatic conditions. These techniques have proved to be successful biofertilizers that form a health relationship with the roots.

Biofertilizers will help solve such problems as increased salinity of the soil and chemical run-offs from the agricultural fields. Thus, biofertilizers are important if we are to ensure a healthy future for the generations to come.

Mycorrhiza

Mycorrhizae are a group of fungi that include a number of types based on the different structures formed inside or outside the root. These are specific fungi that match with a number of favourable parameters of the the host plant on which it grows. This includes soil type, the presence of particular chemicals in the soil types, and other conditions.

These fungi grow on the roots of these plants. In fact, seedlings that have mycorrhizal fungi growing on their roots survive better after transplantation and grow faster. The fungal symbiont gets shelter and food from the plant which, in turn, acquires an array of benefits such as better uptake of phosphorus, salinity and drought tolerance, maintenance of water balance, and overall increase in plant growth and development.

While selecting fungi, the right fungi have to be matched with the plant. There are specific fungi for vegetables, fodder crops, flowers, trees, etc.

Mycorrhizal fungi can increase the yield of a plot of land by 30%-40%. It can absorb phosphorus from the soil and pass it on to the plant. Mycorrhizal plants show higher tolerance to high soil temperatures, various soil- and root-borne pathogens, and heavy metal toxicity.

Legume-rhizobium relationship

Leguminous plants require high quantities of nitrogen compared to other plants. Nitrogen is
an inert gas and its uptake is possible only in fixed form, which is facilitated by the rhizobium
bacteria present in the nodules of the root system. The bacterium lives in the soil to form root
nodules (i.e. outgrowth on roots) in plants such as beans, gram, groundnut, and soybean.


Blue-green algae

Blue-green algae are considered the simplest, living autotrophic plants, i.e. organisms capable of building up food materials from inorganic matter. They are microscopic. Blue-green algae are widely distributed in the aquatic environment. Some of them are responsible for water blooms in stagnant water. They adapt to extreme weather conditions and are found in snow and in hot springs, where the water is 85 °C.

Certain blue-green algae live intimately with other organisms in a symbiotic relationship. Some are associated with the fungi in form of lichens. The ability of blue-green algae tophotosynthesize food and fix atmospheric nitrogen accounts for their symbiotic associations and also for their presence in paddy fields.

Blue-green algae are of immense economic value as they add organic matter to the soil and increase soil fertility. Barren alkaline lands in India have been reclaimed and made productive by inducing the proper growth of certain blue-green algae.

Bt cotton

Cotton and other monocultured crops require an intensive use of pesticides as various types of pests attack these crops causing extensive damage. Over the past 40 years, many pests have developed resistance to pesticides.

So far, the only successful approach to engineering crops for insect tolerance has been the addition of Bt toxin, a family of toxins originally derived from soil bacteria. The Bt toxin contained by the Bt crops is no different from other chemical pesticides, but causes much less damage to the environment. These toxins are effective against a variety of economically important crop pests but pose no hazard to non-target organisms like mammals and fish. Three Bt crops are now commercially available: corn, cotton, and potato.

As of now, cotton is the most popular of the Bt crops: it was planted on about 1.8 million acres (728437 ha) in 1996 and 1997. The Bt gene was isolated and transferred from a bacterium bacillus thurigiensis to American cotton. The American cotton was subsequently crossed with Indian cotton to introduce the gene into native varieties.

The Bt cotton variety contains a foreign gene obtained from bacillus thuringiensis. This bacterial gene, introduced genetically into the cotton seeds, protects the plants from bollworm (A. lepidoptora), a major pest of cotton. The worm feeding on the leaves of a BT cotton plant becomes lethargic and sleepy, thereby causing less damage to the plant.

Field trials have shown that farmers who grew the Bt variety obtained 25%–75% more cotton than those who grew the normal variety. Also, Bt cotton requires only two sprays of chemical pesticide against eight sprays for normal variety. According to the director general of the Indian Council of Agricultural Research, India uses about half of its pesticides on cotton to fight the bollworm menace.

Use of Bt cotton has led to a 3%–27 increase in cotton yield in countries where it is grown.

Genetic engineering

Genetically modified plants are created by the process of genetic engineering, which allows scientists to move genetic material between organisms with the aim of changing their characteristics. All organisms are composed of cells that contain the DNA molecule. Molecules of DNA form units of genetic information, known as genes. Each organism has a genetic blueprint made up of DNA that determines the regulatory functions of its cells and thus the characteristics that make it unique.

Prior to genetic engineering, the exchange of DNA material was possible only between individual organisms of the same species. With the advent of genetic engineering in 1972, scientists have been able to identify specific genes associated with desirable traits in one organism and transfer those genes across species boundaries into another organism. For example, a gene from bacteria, virus, or animal may be transferred into plants to produce genetically modified plants having changed characteristics. Thus, this method allows mixing of the genetic material among species that cannot otherwise breed naturally. The success of a genetically improved plant depends on the ability to grow single modified cells into whole plants. Some plants like potato and tomato grow easily from single cell or plant tissue. Others such as corn, soy bean, and wheat are more difficult to grow.

After years of research, plant specialists have been able to apply their knowledge of genetics to improve various crops such as corn, potato, and cotton. They have to be careful to ensure that the basic characteristics of these new plants are the same as the traditional ones, except for the addition of the improved traits.

The world of biotechnology has always moved fast, and now it is moving even faster. More traits are emerging; more land than ever before is being planted with genetically modified varieties of an ever-expanding number of crops. Research efforts are being made to genetically modify most plants with a high economic value such as cereals, fruits, vegetables, and floriculture and horticulture species.

Public concern

The potential of biotechnology as a method to enhance agricultural productivity in the future has been accepted globally.

However, because of its revolutionary nature, there is a great degree of risk and uncertainty attached to the process of genetic engineering and the resultant genetically modified products.

Risks are also associated with genetically modified plants that are released into the environment. The nature of interactions with other organisms of the natural ecosystems cannot be anticipated without proper scientific testing. For example, modified plants with enhanced resistance to pests or disease threaten to transfer resistance to the wild relatives. This may have implications for biodiversity and ecosystem integrity. These and other numerous doubts plague the minds of common people and the decision-makers.

Some of the many applications for which Plant Biotechnology is currently being used are

	developing plants that are resistant to diseases, pests, and stress
	keeping fruits and vegetables fresh for longer periods of time, which is extremely important in tropical countries
	producing plants that possess healthy fats and oils
	producing plants that have increased nutritive value
	producing soy beans with a higher expression of the anti-cancer proteins naturally found in soy beans
	producing new substances in plants, including biodegradable plastics, and small proteins or peptides such as prophylactic and therapeutic vaccines.

DNA

Since the time Gregor Mendel began studying about inheritance in garden plants some 150 years back, researchers have worked to learn more about the language of life – how characteristics pass from one generation to another. Researchers began to understand DNA from the 1800s when they stated that all living beings, whether plants, humans, animals, or bacteria, comprised cells that have the same basic components.

Living organism are made up of cells, i.e. cells are the basic units of life. For example, each of us is made up of billions of this basic unit. If one closely inspects the structure of the cell, one is likely to find various smaller bodies or organelles like mitochondria that generates the energy required to perform all life processes (‘the powerhouse’), chloroplast (only in green plants and responsible for their coloration), the central core – ‘the nucleus, to name a few. The nucleus harbours the blueprint of life and the genetic material – DNA or deoxyribonucleic acid – and is the control centre of any cell. The genetic material or the blueprint is contained in all the cells that make up an organism and is transmitted from one generation to another. A child inherits half of the genetic material from each of his/her parents.

The chemical structure of everyone's DNA is the same. Structurally, DNA is a double helix: two strands of genetic material spiraled around each other. Each strand contains a sequence of bases, also called nucleotides. A base is one of four chemicals: adenine, guanine, cytosine, and thymine. The two strands of DNA are connected at each base. Each base will only bond with one other base, as follows: Adenine (A) will only bond with thymine (T), and guanine (G) will only bond with cytosine (C). If one strand of DNA looks like A-A-C-T-G-A-T-A-G-G-T-C-T-A-,the DNA strand bound to it will look like T-T-G-A-C-T-A-T-C-C-A-G-A-T-C.

Together, the section of DNA would be represented as given in Figure

T-T-G-A-C-T-A-T-C-C-A-G-A-T-C

A-A-C-T-G-A-T-A-G-G-T-C-T-A-G

The length of the DNA strand varies from organism to organism but within individuals of a particular species it is nearly constant. For example, a certain virus may have only 50 000 (5 x 10⁴) bases constituting the genetic material whereas a human cell contains nearly 3.2 billion (3.2 x 10⁹) bases in each of the cells (except the germ line cells). The amount and sequence in all the cells of an organism is identical. The DNA is for most part of the time present as condensed body called chromosomes (coloured body) except when it is replicating or dividing. A piece of a chromosome that dictates a particular trait, for example, eye and skin colour in humans, is called a gene. In any cell, the DNA can be classified into two categories – the sequence that codes for traits or genes and the sequence that has no apparent function or the non-coding DNA. The coding sequence (genes) in humans constitutes only five per cent of the total DNA and is identical in all humans. The non-coding sequence, which is nearly 95% in humans, varies from one individual to another, and forms the basis of DNA fingerprinting.

DNA fingerprinting

The only difference between two individuals is the order of the base pairs. Each individual has a different sequence of DNA, specially in the non-coding region. Using these sequences, every person could be identified solely by the sequence of their base pairs. However, because the entire DNA is so huge, the task would be time-consuming and nearly impossible. Instead, scientists are able to use a shorter method.

The steps involved in DNA fingerprinting can be summarized as follows.

	Isolating the DNA in question from the rest of the cellular material in the nucleus.
	Cutting the DNA into several pieces of different sizes.
	Sorting the DNA pieces by size. The process by which the size separation, or ‘size fractionation’, is done is called gel electrophoresis.

This is the basic concept behind fingerprinting technique.

DNA fingerprinting in plants

The concept of DNA fingerprinting can also be extended to plants and many institutions in the country are doing it today. TERI has successfully generated fingerprints of various medicinal plants such as neem, ashwagandha, and amla with the objective of determining their identity. With the help of fingerprints one can find out the genetic diversity in India. This knowledge has profound implications. Based on the extent of genetic diversity, one can establish the centre of origin of a particular plant species. And having done that we are better equipped to prevent bio-piracy or the theft of our genetic resources.

Types of Gene therapy and general strategies

Gene therapy may be classified into two types
1) Germ line gene therapy

2) Somatic cell gene therapy

a) Incase of germ line gene therapy germ cells that is sperms or eggs are modified by the introduction of functional genes, which are ordinarily integrated into their genomes. Therefore the change due to therapy is heritable and passed onto the later generations. This approach, heretically, is highly effective in counteracting the genetic disorders. However this option is not consider, at least for the present for application in human beings for a variety of technical and ethical reasons.

b) In the case of somatic cell gene therapy the gene is introduced only in somatic cells, especially of those tissues in which expression of the concerned gene is critical for health. Expression of the introduced gene relieves symptoms of the disorder, but this effect is not heritable, as it does not involve the germ line. It is the only feasible option, and clinical trials have already started mostly for the treatment of cancer and blood disorders

GENERAL GENE THERAPY STRATEGIES

1) Gene augmentation therapy (GAT): -

It is done by simple addition of functional alleles has been used to treat several inherited disorders caused by genetic deficiency of a gene product. It is also involved in transfer to cells of genes encoding toxic compounds (suicide genes) or prodrugs (reagents which confer sensitivity to subsequent treatment with a drug). It has been particularly applied to autosomal recessive disorders where even modest expression levels of an introduced gene may make a substantial difference.

2) Targeted killing of specific cells: -

Artificial cell killing and immune system assisted cell killing have been popular in the treatment of cancers. It can be done by two ways.

a) Direct cell killing: - it is possible if the inserted genes are expressed to produce a lethal toxin (suicide genes), or a gene encoding a prodrug is inserted, conferring susceptibility to killing by a subsequently administered drug. Alternatively selectively lytic viruses can be used.

b) Indirect cell killing: - It uses immunostimulatory genes to provoke or enhance an immune response against the target cell.

3) Targeted mutation correction: -

The repair of a genetic defect to restore a functional allele, is the exception, technical difficulties have meant that it is not sufficiently reliable to warrant clinical trails.

4) Targeted inhibition of gene expression: -

It is suitable for treating infectious diseases and some cancers. If disease cells display a novel gene product or inappropriate expression of a gene a variety of different systems can be used specifically to block the expression of a single gene at the DNA, RNA or Protein levels.

REFERENCE

1) Tom strachan and Andrew P. Read, Human Molecular Genetics, Second edition.

2) T.A. Brown, Gene Cloning an introduction, Third Edition.

3) S.N. Jogdand, Gene Biotechnology.

4) B.D Singh, Biotechnology.

The human genome project

Although great strides have been made in gene therapy in a relatively short time, its potential usefulness has been limited by lack of scientific data concerning the multitude of functions that genes control in the human body. For instance, it is now known that the vast majority of genetic material does not store information for the creation of proteins, but rather is involved in the control and regulation of gene expression, and is, thus, much more difficult to interpret. Even so, each individual cell in the body carries thousands of genes coding for proteins, with some estimates as high as 150,000 genes. For gene therapy to advance to its full potential, scientists must discover the biological role of each of these individual genes and where the base pairs that make them up are located on DNA.

To address this issue, the National Institutes of Health initiated the Human Genome Project in 1990. Led by James D. Watson (one of the co-discoverers of the chemical makeup of DNA) the project's 15-year goal is to map the entire human genome (a combination of the words gene and chromosomes). A genome map would clearly identify the location of all genes as well as the more than three billion base pairs that make them up. With a precise knowledge of gene locations and functions, scientists may one day be able to conquer or control diseases that have plagued humanity for centuries.

Scientists participating in the Human Genome Project identified an average of one new gene a day, but many expected this rate of discovery to increase. By the year 2005, their goal was to determine the exact location of all the genes on human DNA and the exact sequence of the base pairs that make them up. Some of the genes identified through this project include a gene that predisposes people to obesity, one associated with programmed cell death (apoptosis), a gene that guides HIV viral reproduction, and the genes of inherited disorders like Huntington's disease, Lou Gehrig's disease, and some colon and breast cancers. In April 2003, the finished sequence was announced, with 99% of the human genome's gene-containing regions mapped to an accuracy of 99.9%.

The history of gene therapy

In the early 1970s, scientists proposed "gene surgery" for treating inherited diseases caused by faulty genes. The idea was to take out the disease-causing gene and surgically implant a gene that functioned properly. Although sound in theory, scientists, then and now, lack the biological knowledge or technical expertise needed to perform such a precise surgery in the human body.

However, in 1983, a group of scientists from Baylor College of Medicine in Houston, Texas, proposed that gene therapy could one day be a viable approach for treating Lesch-Nyhan disease, a rare neurological disorder. The scientists conducted experiments in which an enzyme-producing gene (a specific type of protein) for correcting the disease was injected into a group of cells for replication. The scientists theorized the cells could then be injected into people with Lesch-Nyhan disease, thus correcting the genetic defect that caused the disease.

As the science of genetics advanced throughout the 1980s, gene therapy gained an established foothold in the minds of medical scientists as a promising approach to treatments for specific diseases. One of the major reasons for the growth of gene therapy was scientists' increasing ability to identify the specific genetic malfunctions that caused inherited diseases. Interest grew as further studies of DNA and chromosomes (where genes reside) showed that specific genetic abnormalities in one or more genes occurred in successive generations of certain family members who suffered from diseases like intestinal cancer, bipolar disorder, Alzheimer's disease, heart disease, diabetes, and many more. Although the genes may not be the only cause of the disease in all cases, they may make certain individuals more susceptible to developing the disease because of environmental influences, like smoking, pollution, and stress. In fact, some scientists theorize that all diseases may have a genetic component.

On September 14, 1990, a four-year old girl suffering from a genetic disorder that prevented her body from producing a crucial enzyme became the first person to undergo gene therapy in the United States. Because her body could not produce adenosine deaminase (ADA), she had a weakened immune system, making her extremely susceptible to severe, life-threatening infections. W. French Anderson and colleagues at the National Institutes of Health's Clinical Center in Bethesda, Maryland, took white blood cells (which are crucial to proper immune system functioning) from the girl, inserted ADA producing genes into them, and then transfused the cells back into the patient. Although the young girl continued to show an increased ability to produce ADA, debate arose as to whether the improvement resulted from the gene therapy or from an additional drug treatment she received.

Nevertheless, a new era of gene therapy began as more and more scientists sought to conduct clinical trial (testing in humans) research in this area. In that same year, gene therapy was tested on patients suffering from melanoma (skin cancer). The goal was to help them produce antibodies (disease fighting substances in the immune system) to battle the cancer.

These experiments have spawned an ever growing number of attempts at gene therapies designed to perform a variety of functions in the body. For example, a gene therapy for cystic fibrosis aims to supply a gene that alters cells, enabling them to produce a specific protein to battle the disease. Another approach was used for brain cancer patients, in which the inserted gene was designed to make the cancer cells more likely to respond to drug treatment. Another gene therapy approach for patients suffering from artery blockage, which can lead to strokes, induces the growth of new blood vessels near clogged arteries, thus ensuring normal blood circulation.

Currently, there are a host of new gene therapy agents in clinical trials. In the United States, both nucleic acid based (in vivo) treatments and cell-based (ex vivo) treatments are being investigated. Nucleic acid based gene therapy uses vectors (like viruses) to deliver modified genes to target cells. Cell-based gene therapy techniques remove cells from the patient in order to genetically alter them then reintroduce them to the patient's body. Presently, gene therapies for the following diseases are being developed: cystic fibrosis (using adenoviral vector), HIV infection (cell-based), malignant melanoma (cell-based), Duchenne muscular dystrophy (cell-based), hemophilia B (cell-based), kidney cancer (cell-based), Gaucher's Disease (retroviral vector), breast cancer (retroviral vector), and lung cancer (retroviral vector). When a cell or individual is treated using gene therapy and successful incorporation of engineered genes has occurred, the cell or individual is said to be transgenic.

The medical establishment's contribution to transgenic research has been supported by increased government funding. In 1991, the U.S. government provided $58 million for gene therapy research, with increases in funding of $15-40 million dollars a year over the following four years. With fierce competition over the promise of societal benefit in addition to huge profits, large pharmaceutical corporations have moved to the forefront of transgenic research. In an effort to be first in developing new therapies, and armed with billions of dollars of research funds, such corporations are making impressive strides toward making gene therapy a viable reality in the treatment of once elusive diseases.

The future of gene therapy

Gene therapy seems elegantly simple in its concept: supply the human body with a gene that can correct a biological malfunction that causes a disease. However, there are many obstacles and some distinct questions concerning the viability of gene therapy. For example, viral vectors must be carefully controlled lest they infect the patient with a viral disease. Some vectors, like retroviruses, also can enter cells functioning properly and interfere with the natural biological processes, possibly leading to other diseases. Other viral vectors, like the adenoviruses, often are recognized and destroyed by the immune system so their therapeutic effects are short-lived. Maintaining gene expression so it performs its role properly after vector delivery is difficult. As a result, some therapies need to be repeated often to provide long-lasting benefits.

One of the most pressing issues, however, is gene regulation. Genes work in concert to regulate their functioning. In other words, several genes may play a part in turning other genes on and off. For example, certain genes work together to stimulate cell division and growth, but if these are not regulated, the inserted genes could cause tumor formation and cancer. Another difficulty is learning how to make the gene go into action only when needed. For the best and safest therapeutic effort, a specific gene should turn on, for example, when certain levels of a protein or enzyme are low and must be replaced. But the gene also should remain dormant when not needed to ensure it doesn't oversupply a substance and disturb the body's delicate chemical makeup.

One approach to gene regulation is to attach other genes that detect certain biological activities and then react as a type of automatic off-and-on switch that regulates the activity of the other genes according to biological cues. Although still in the rudimentary stages, researchers are making headway in inhibiting some gene functioning by using a synthetic DNA to block gene transcriptions (the copying of genetic information). This approach may have implications for gene therapy.

The ethics of gene therapy

While gene therapy holds promise as a revolutionary approach to treating disease, ethical concerns over its use and ramifications have been expressed by scientists and lay people alike. For example, since much needs to be learned about how these genes actually work and their long-term effect, is it ethical to test these therapies on humans, where they could have a disastrous result? As with most clinical trials concerning new therapies, including many drugs, the patients participating in these studies usually have not responded to more established therapies and often are so ill the novel therapy is their only hope for long-term survival.

Another questionable outgrowth of gene therapy is that scientists could possibly manipulate genes to genetically control traits in human offspring that are not health related. For example, perhaps a gene could be inserted to ensure that a child would not be bald, a seemingly harmless goal. However, what if genetic manipulation was used to alter skin color, prevent homosexuality, or ensure good looks? If a gene is found that can enhance intelligence of children who are not yet born, will everyone in society, the rich and the poor, have access to the technology or will it be so expensive only the elite can afford it?

The Human Genome Project, which plays such an integral role for the future of gene therapy, also has social repercussions. If individual genetic codes can be determined, will such information be used against people? For example, will someone more susceptible to a disease have to pay higher insurance premiums or be denied health insurance altogether? Will employers discriminate between two potential employees, one with a "healthy" genome and the other with genetic abnormalities?

Some of these concerns can be traced back to the eugenics movement popular in the first half of the twentieth century. This genetic "philosophy" was a societal movement that encouraged people with "positive" traits to reproduce while those with less desirable traits were sanctioned from having children. Eugenics was used to pass strict immigration laws in the United States, barring less suitable people from entering the country lest they reduce the quality of the country's collective gene pool. Probably the most notorious example of eugenics in action was the rise of Nazism in Germany, which resulted in the Eugenic Sterilization Law of 1933. The law required sterilization for those suffering from certain disabilities and even for some who were simply deemed "ugly." To ensure that this novel science is not abused, many governments have established organizations specifically for overseeing the development of gene therapy. In the United States, the Food and Drug Administration (FDA) and the National Institutes of Health require scientists to take a precise series of steps and meet stringent requirements before proceeding with clinical trials. As of mid-2004, more than 300 companies were carrying out gene medicine developments and 500 clinical trials were underway. How to deliver the therapy is the key to unlocking many of the researchers discoveries.


In fact, gene therapy has been immersed in more controversy and surrounded by more scrutiny in both the health and ethical arena than most other technologies (except, perhaps, for cloning) that promise to substantially change society. Despite the health and ethical questions surrounding gene therapy, the field will continue to grow and is likely to change medicine faster than any previous medical advancement.

The biological basis of gene therapy

Gene Therapy
Gene therapy is a rapidly growing field of medicine in which genes are introduced into the body to treat diseases. Genes control heredity and provide the basic biological code for determining a cell's specific functions. Gene therapy seeks to provide genes that correct or supplant the disease-controlling functions of cells that are not, in essence, doing their job. Somatic gene therapy introduces therapeutic genes at the tissue or cellular level to treat a specific individual. Germ-line gene therapy inserts genes into reproductive cells or possibly into embryos to correct genetic defects that could be passed on to future generations. Initially conceived as an approach for treating inherited diseases, like cystic fibrosis and Huntington's disease, the scope of potential gene therapies has grown to include treatments for cancers, arthritis, and infectious diseases. Although gene therapy testing in humans has advanced rapidly, many questions surround its use. For example, some scientists are concerned that the therapeutic genes themselves may cause disease. Others fear that germ-line gene therapy may be used to control human development in ways not connected with disease, like intelligence or appearance.

The biological basis of gene therapy

Gene therapy has grown out of the science of genetics or how heredity works. Scientists know that life begins in a cell, the basic building block of all multicellular organisms. Humans, for instance, are made up of trillions of cells, each performing a specific function. Within the cell's nucleus (the center part of a cell that regulates its chemical functions) are pairs of chromosomes. These threadlike structures are made up of a single molecule of DNA (deoxyribonucleic acid), which carries the blueprint of life in the form of codes, or genes, that determine inherited characteristics.

A DNA molecule looks like two ladders with one of the sides taken off both and then twisted around each other. The rungs of these ladders meet (resulting in a spiral staircase-like structure) and are called base pairs. Base pairs are made up of nitrogen molecules and arranged in specific sequences. Millions of these base pairs, or sequences, can make up a single gene, specifically defined as a segment of the chromosome and DNA that contains certain hereditary information. The gene, or combination of genes formed by these base pairs ultimately direct an organism's growth and characteristics through the production of certain chemicals, primarily proteins, which carry out most of the body's chemical functions and biological reactions.

Scientists have long known that alterations in genes present within cells can cause inherited diseases like cystic fibrosis, sickle-cell anemia, and hemophilia. Similarly, errors in the total number of chromosomes can cause conditions such as Down syndrome or Turner's syndrome. As the study of genetics advanced, however, scientists learned that an altered genetic sequence also can make people more susceptible to diseases, like atherosclerosis, cancer, and even schizophrenia. These diseases have a genetic component, but also are influenced by environmental factors (like diet and lifestyle). The objective of gene therapy is to treat diseases by introducing functional genes into the body to alter the cells involved in the disease process by either replacing missing genes or providing copies of functioning genes to replace nonfunctioning ones. The inserted genes can be naturally-occurring genes that produce the desired effect or may be genetically engineered (or altered) genes.

Scientists have known how to manipulate a gene's structure in the laboratory since the early 1970s through a process called gene splicing. The process involves removing a fragment of DNA containing the specific genetic sequence desired, then inserting it into the DNA of another gene. The resultant product is called recombinant DNA and the process is genetic engineering.

There are basically two types of gene therapy. Germ-line gene therapy introduces genes into reproductive cells (sperm and eggs) or someday possibly into embryos in hopes of correcting genetic abnormalities that could be passed on to future generations. Most of the current work in applying gene therapy, however, has been in the realm of somatic gene therapy. In this type of gene therapy, therapeutic genes are inserted into tissue or cells to produce a naturally occurring protein or substance that is lacking or not functioning correctly in an individual patient.

Segration of Cell lines in the Embryo

In all multi cellular organism, the cleavage of the egg gives rise to cells which differ from one another and which, through successive cell divisions, will eventually give rise to homogeneous Cell populations (cell lines) each endowed with its own specific developmental program. This not only implies a process of sorting out of molecules (either pre-existing in the egg before fertilization or being synthesized in the course of development) into the various blastomers; but also of cells recognizing one another and coordinating their movements, their rate of cleavage, their metabolic activities, and the like.

The dichotomy between the two cell lines involves:

a) That in the somatic cell line, the genes which in the unicellular organism code for the surface structures responsible for the recognition of and interaction between cells of the two gametic types, are silenced. The evidence for this is indirect. The formation of mouse chimaeras shows that genetically male and female embryonic cells do not discriminate one another as different. Also, hybrid hystotypic aggregates can be formed in culture from such species as far as apart as chick and mouse. However, the possibility should be taken into consideration that in vitro conditions may alter the organization of the cell surface in such a way that some of its properties such as the species-specificity are lost while the tissue-specificity is retained. These observations are compatible with the view that the structures discriminating between male and female are not expressed at the surface of these cells.

b) The retention of a largely depressed genome by the cells of the germ line. This is inferred from the fact that in the oocyte, the complexity of the transcripts is several-fold greater than in the somatic cells. But there is no such direct evidence in the case of the male germ cells, it has been shown that at least in Drosophila, spermatocytes exhibit lampbrush chromosomes comparable to those of the oocyte.

The emergence of multicellular organism has required the establishment of cell junctions; not only as a means of holding the cells together, but as a vehicle of functional coordination between cells.

A classical example of a very precocious segregation of the somatic from the germ line is that of Ascaris. In this nematode while the lineage cells of the germ line retain their full chromosomes complement, in the cells of the somatic line pieces of chromosomes are lost; the loss amounts to about 27% of the total DNA of the cell. Interestingly, about one-half of the eliminated DNA consists of repetitive sequences and the other half of unique sequences.

RNA Splicing

In the initial stage, RNA transcript introns are synthesized which are removed later on by a process called RNA splicing ( refer picture below). The junctions of intron-exon have a GU sequences at the intron’s 5’-end, and an AG sequence at its 3’OH end. These two sequences are recognized by the special RNA molecule known as small nuclear RNA (snRNA) or snurps (Steitz, 1988).

These together with proteins form small nuclear ribonucleoprotein particles called snRNPs. Some of the snRNPs recognize the splice junction, and splice introns accurately. For example, the UI-snRNP recognizes the 5’-splicing junction, and the U5 snRNP recognizes the 3’ splicing junction. Consequently pre-mRNA is spliced in a large complex called a spliceosome (Guthrie, 1991). The spliceosome consists of pre-mRNA, five types of snRNPs and non-snRNP splicing factors (Rosbash and Seraphin,1991).

Robert and Sharp, the Nobel prize winners in 1993, independently hybridized the mRNA of adenovirus with their progeny of DNA segments of virus. The mRNAs hybridized the ssDNA of virus where the complementary sequences were present. The mRNA-DNA complexes were observed under electon microscope to confirm which part of viral genome had produced the mRNA strand. It was found that mRNA did not hybridize DNA linearly but showed a discontinuous complexes pattern. Huge loops of unpaired DNA between the hybridized complexes clearly revealed the large chunk of DNA strand that carried no genetic information and did not take part in protein synthesis. The adenovirus mRNA contained four different regions of the DNA.

The B-globin genes of mice and rabbits, and tRNA genes of yeast tyrosine-tRNA consists of eight genes. Each genes contains 14 bases (ATTT-AYCAC-TACGA) as intron in the middle. In the same way the pre-tRNA genes contain introns of 18-19 bases. In all the genes introns are present near anticodon. Similarly, a few rRNA genes are also known to contain introns and some of pre-rRNA are self splicing.

Mycorrhizas as biofertilizers

Mycorrhiza (fungus roots) is a distinct morphological structure which develops as a result of mutualistic symbiosis between some specific root – inhabitating fungai and plant roots. Plants which suffer from nutrient scarcity, especially P and N, develop mycorrhiza i.e. the plants belong to all groups e.g. herbs, shrubs, trees, aquatic, xerophytes, epiphytes, hydrophytes or terrestrial ones. In most of the cases plant seedling fails to grow if the soil does not contain inoculum of mycorrhizal fungi.

In recent years, use of artificially produced inoculum of mycorrhizal fungi has increased its significance due to its multifarious role in plant growth and yield, and resistance against climatic and edaphic stresses, pathogens and pests.

Mechanism of symbiosis:
The mechanism of symbiosis is not fully understood. Bjorkman (1949) postulated the carbohydrate theory and explained the development of mycorrhizas in soils deficient in available P and N, and high light intensity. Slankis (1961) found that at high light intensity, surplus carbohydrates are formed which are exuded from roots. This in turn induces the mycorrhizal fungi of soil to infect the roots. At low light intensity, carbohydrates are not produced in surplus, therefore, plant roots fail to develop mycorrhizas.

Types of Mycorrhizas:

By earlier mycologists the mycorrhizas were divided into the following three groups:
i) Ectomycorrhiza: It is found among the gymnosperms and angiosperms. In short roots of higher plants generally root hairs are absent. Therefore, the roots are infected by mycorrhizal fungi which, in turn, replace the root hairs (if present) and form a mantle. The hyphae grow intercellularly and develop Hartig net in cortex. Thus, a bridge is established between the soil and root through the mycelia.
ii) Endomycorrhiza: The morphology if endomycorrhizal roots, after infection and establishment, remain unchanged. Root hairs develop in a normal way. The fungi are present on root surface individually. They also penetrate the cortical cells and get established intracellularly by secreting extracellular enzymes. Endomycorrhizas are found in all groups of plant kingdom.

iii) Ectendomycorrhiza: In the roots of some of the gymnosperms and angiosperms, ectotropic fungal infection occurs. Hyphae are established intracellularly in cortical cells. Thus, symbiotic relation develops similar to the ecto- and endo-mycorrhizas.

Marks (1991) classified the mycorrhizas into seven types on the basis of types of relationships with the host
(i) Vesicular-arbuscular (VA) mycorrhizas (coiled, intracellular hyphae, vesicle and arbuscules present),

(ii) Ectomycorrhizas (sheath and inter-cellular hyphae present),

(iii) Ectendomycorrhizas (sheath optional, inter and intra-cellular hyphae present).

(iv) Arbutoid mycorrhizas (sheath, inter- and intra-cellular hyphae present).

(v) Ericoid mycorrhizas (only coiled intracellular hyphae, long coiled hyphae present)

(vi) Monotropid mycorrhizas (sheath, inter-and intra-cellular hyphae and peg like haustoria present) and

(vii) Orchidaceous mycorrhizas (only coiled intracellular hyphae present).

Type (i) is present in all groups of plant kingdom; Types (ii) and (iii) are found in gymnosperms and angiosperms. Types (iv), (v) and (vi) are restricted to Ericales, Monotropaceae and Ericales respectively. Types (vii) is restricted to Orchidaceous only. Types (iv) and (v) were previously grouped under ericoid mycorrhizas.

Human genetic map - Read your DNA

Article by Bobbie Johnson, San Francisco, October 07 2008

It took hundreds of scientists 13 years and $3bn (£1.7bn) to decode the human genome: now one company says it is ready to slash the cost of reading your DNA to just $5,000. California-based Complete Genomics has announced that it will begin offering the service later this month, after developing new methods that reduce the price of sequencing a human genome.

According to Clifford Reid, the company's chairman and chief executive, the plummeting price tag "will dramatically increase the availability and affordability of human genome sequencing".

"Our sequencing services will be one of the core enablers of the impending revolution in personalised medicine," he said.

Although there are a number of other companies that offer limited genetic testing, Complete Genomics is the first to say it will produce a complete, low-cost reading of any human genome, each of which consists of more than 25,000 genes. The company said it would be able to complete 1,000 sequences in 2009, rising to 20,000 in 2010 – and that it was the result of two years' working "in stealth mode" to create a faster, cheaper system.

Although the announcement could help more members of the public understand their own genetic makeup – and potentially allow them to organise treatment targeted at specific genetic diseases – there may also be benefits for genetic researchers.

Progress in DNA mapping has accelerated enormously in recent years, thanks to advances in technology and high-powered computer systems. It currently costs around $100,000 for most sequences, but experts have suggested the price is falling by an average of 90% every year.

Such rapid progress has helped speed up scientists' understanding of the genome and relationships between different genetic codes – leading to hopes that it can advance treatment for conditions such as cystic fibrosis, Huntington's disease and some cancers.

It has also helped spawn a number of so-called "lifestyle" genetics companies, including Iceland's DeCODE and California's Navigenics, which allow customers to understand some of their genetic predispositions.

Micromeres of the sea urchin embryo

Sixth cleavage sea urchin embryo.

The micromeres are four small blastomers which at the fourth cleavage are segregated at the vegetal pole of the embryo due to the fact that in the macromers the spindle is strongly shifted towards the vegetal pole. The micromeres are committed to the formation of the primary mesenchyme and exert two important roles in morphogenesis:

1. They are responsible for the control of gastrulation.
2. They act as pacemakers of cell divisions during cleavage.

It was discovered by Driesch that the mesenchyme blastula of sphaerechinus there are about 30 primary mesenchyme cells and about 55 in Echinus. Embryos which develop from one of the first two blastomers isolated after the first cleavage contain half the number of the primary mesenchyme cells: about 14 in sphaerechinus and about 27 in Echinus.

Sixth cleavage sea urchin embryo.

It is interesting to note that the micromeres cleave at a lower rate and their division is out of phase with respect to the other cells of the embryo. The cleavage of the first four micromeres give rise to eight cells only four of which, namely the outer ones, continue to divide while the four inner ones appear to have lost the ability to divide any further (atleast through the next two division cycles; later the micromeres become undistinguishable from the other cells of the embryo). Thus within the micromere cell population two sub-populations are soon segregated, each of which again appear to be programmed as to whether of not the inner micromeres are the precursors of a cell line different from that of the other micromeres.

Human Gene Therapy

Human beings suffer from more than 5000 different diseases caused by single gene mutations, e.g., cystic fibrosis acatalasis, hunting tons chorea, tay sachs disease, lisch nyhan syndrome, sickle cell anemia, mitral stenosis, hunter's syndrome, haemophilia, several forms of muscular dystrophy etc. In addition, many common disorders like cancer, hypertension, atherosclerosis and mental illness seem to have genetic components.

The term gene therapy can be defined as introduction of a normal functional gene into cells, which contain the defective allele of concerned gene with the objective of correcting a genetic disorder or an acquired disorder.

The first approach in gene therapy is: -

a) Identification of the gene that plays the key role in the development of a genetic disorder.

b) Determination of the role of its product in health and disease.

c) Isolation and cloning of the gene.

d) Development of an approach for gene therapy.The genetic material may be transferred directly into cells within a patient, which is referred as in vivo gene therapy or else cells may be removed from the patient and the genetic material inserted into them, which is referred as invitro gene therapy. Apart from the two methods mentioned above there is one more method that is ex-vivo gene therapy in which genetic material is inserted into the cells just prior to transplanting the modified cells back into the patient.
Major disease classes under gene therapy include: -

a) Infectious diseases: - infection by a virus or bacterial pathogen

b) Cancers: - uncontrolled and enormous cell division and cell proliferation as a result of activation of an oncogene or inactivation of a tumors suppressor gene or an apoptosis gene.

c) Inherited disorders: - genetic deficiency of an individual gene product or genetically determined in appropriate expression of a gene.

d) Immune system disorders: - includes allergies, inflammation and also autoimmune diseases in which immune system cells appropriately destroy body cells.

GENETIC NEWS

Balding Men May Get Help From Stem Cell, Gene Discoveries
Those with slick domes, thinning tops and receding hairlines may one day be helped by the discovery of genes that put people at risk for baldness and a stem cell that may replenish hair follicles.
Two studies released today in the journal Nature Genetics may help explain why some people lose their hair, and how they may eventually be able to grow it back, scientists from London- based GlaxoSmithKline Plc, the U.K. and Sweden said.

Hair loss affects about one in four Caucasian men before age 30. While drugs such as Johnson & Johnson's Rogaine and Merck & Co.'s Propecia can help hair regrow or prevent loss in some patients, they don't work for everyone. Treatments that target the DNA responsible may be more promising, said Tim Spector, who led the gene study.

``Early prediction before hair loss starts may lead to some interesting therapies that are more effective than treating late-stage hair loss,'' said Spector, a researcher in Kings College London's department of twin research and genetic epidemiology, in a statement.

Spector and colleagues analyzed the genes of 578 men in Switzerland with early-onset hair-loss, and compared them against those of 547 others who were retaining their hair. They then confirmed their findings against groups from the U.K., Iceland and the Netherlands, studying about 5,000 people in all.

Those with hair loss commonly shared the same variations of two genes that together made them seven times more likely to suffer baldness, researchers from Kings College London and GlaxoSmithKline Plc wrote in the journal Nature Genetics.

More Study Needed

The research associates the genes with hair loss, though further studies are needed to prove the connection. The genetic variations were also found in women, though the link wasn't statistically significant and more research is needed, the authors said. The study was partly funded by Glaxo.

In the stem cell study, researchers led by Viljar Jaks of Sweden's Karolinska Institute examined mouse hair follicles for signs of rapid growth. They found a protein, called Lgr5, on the surface of long-lived, active stem cells in hair cells; the same protein has been identified on stem cells in the intestine, they said in the study.

Cells bearing the Lgr5 marker were capable of maintaining hair follicles for as long as 14 months, the researchers said. In mouse studies, just a few of these cells were able to build an entire hair follicle, they said in the study.

The search for a cure for baldness began at least 3,000 years ago. Ancient Egyptians treated hair loss with fats from crocodiles, geese, lions, ibex, snake and hippopotamuses, according to the U.S.-based Coalition of Independent Hair Restoration Physicians.

`Balding Pattern'

Two of three men will be bald or have a ``balding pattern'' of hair loss by 60, according to the U.S. National Institutes of Health. The condition may be hereditary in more than 80 percent of cases, and has also been linked to maladies including heart disease and metabolic syndrome, the authors wrote.

Americans spent more than $115 million on hair transplant therapy last year, the authors said, and Merck's Propecia earned the Whitehouse Station, New Jersey-based drugmaker $405.4 million.

GENETIC NEWS

Testicle Stem Cells Become Bone, Muscle in German Experiments
Stem cells were isolated from the testicles of adult men and turned into bone, muscle, neural and other kinds of cells, German researchers said.
The advance, reported today in the journal Nature, may provide an alternative way to generate powerful stem cells that might be used to repair or replace damaged tissue in male patients with hard-to-treat diseases. Currently, scientists create stem cells by extracting them from embryos or genetically manipulating adult cells to make them pluripotent, or able to become many other cell types.

The use of testicle cells may also represent a new way to make lines of cells from a male with an inherited disease, for the purpose of studying his condition at the cellular level and testing drugs that might be effective in treating it.

The work by Thomas Skutella, director of the Center for Regenerative Biology and Medicine, in Tuebingen, Germany, produced a ``breathtaking result,'' said George Daley, a researcher at the Harvard Stem Cell Institute and at Children's Hospital Boston. While scientists had known that mouse testes give rise to other cells, he said, it wasn't clear that such cells could be isolated from humans.

``These are the only pluripotent cells present in adult human organisms,'' Skutella said. With this method, he said, ``you could take biopsies from people with Parkinson's or any kind of inherited disease'' and study the cells to learn how they function and respond to drugs.

The technique, if confirmed and improved, may allow researchers to sidestep ethical controversies that have dogged the field since University of Wisconsin scientists isolated stem cells from human embryos a decade ago.

Embryo Research Decried

Critics of embryonic research, including the Catholic Church, U.S. President George W. Bush and German lawmakers, view embryo destruction as immoral. Bush banned federal funding of research that uses newly destroyed embryos. German authorities went further, barring researchers there from creating embryonic stem cells, although they can import those derived in other countries, Skutella said.

Another method, developed two years ago by Japanese researchers, uses genes and viruses to reprogram adult skin cells so they become pluripotent and behave like embryonic stem cells. While this method avoids using embryos, the technique may trigger cancer or other unwanted effects. More recently researchers have begun to refine this method by eliminating viruses linked to cancer.

One advantage of Skutella's method is that if a man's own cells were used to make a therapy, they could be used to treat him without fear that his body would reject the cells. While he was able to coax the testicular stem cells to turn into a number of cell types, he wasn't able to make still other types, including heart cells.

Testicles Donated

Skutella and his colleagues obtained testicular tissue from various sources, including organ donors who had died. Others who supplied tissue were being treated for infertility or had their testicles removed in the course of surgery to change their sex to female or to treat prostate cancer.

The scientists used a complex process to identify and isolate a type of precursor cell that normally helps make sperm. They then concocted a chemical cocktail that allowed them to expand the stem cells.

GENETIC NEWS

Researchers Find Breast Cancer Gene, Spur Hope for New Drugs
Researchers discovered a gene involved in the spread of breast cancer, which may lead to new treatments for a disease that kills about 1 in 35 women.

The gene, called metadherin, may be crucial to cancer’s spread because it helps tumor cells stick to blood vessels in distant organs, researchers at Princeton University and the Cancer Institute of New Jersey found. The gene also makes tumors more resistant to drugs used to wipe out deadly cells.

Breast cancer is the most frequently diagnosed malignancy in U.S. women, excluding cancer of the skin, according to the American Cancer Society. Determining the genetic mechanism involved in the disease’s spread, known as metastasis, may help answer one of the biggest mysteries in cancer research.

“Inhibiting this gene in breast cancer patients will simultaneously achieve two important goals -- reduce the chance of recurrence and, at the same time, decrease the risk of metastatic dissemination,” Yibin Kang, an assistant professor of molecular biology at Princeton who led the research, said in a statement on the Cancer Institute’s Web site. “These are the two major reasons why breast cancer patients die from the disease.”

The finding, published today in Cancer Cell, is based on three years of work, using an approach that combines the emerging science of integrative genomics with the classical methods of clinical research and laboratory experiments, the authors said.

Quick Spread

Breast cancer is caused by a malignant tumor that develops from cells in the breast. The most common sign of breast cancer is a new lump or mass in the breast. Scientists once thought that breast cancer spread first to nearby tissue and underarm lymph nodes before reaching other parts of the body. They now believe cancer cells may break away from the primary tumor in the breast even when the disease is in an early stage.

After re-analyzing clinical breast cancer databases and tumor samples collected from patients, the researchers found an area of human chromosome 8 called 8q22 is repeated multiple times in the genomes of potentially lethal breast tumors. Most normal DNA sequences contain only two copies of a given gene, conveyed from the genomes of the male and female parents.

The researchers went on to discover that among a handful of genes in the 8q22 region, metadherin, also known as MTDH, is responsible for the aggressive behavior of some tumors. The scientists found that tumors which over-express MTDH are more likely to spread to the lungs, other vital organs and bones. These tumors were also found to be more resistant to some chemotherapy agents.

“By analyzing 250 breast tumor samples from patients, we found that this gene is amplified and over-expressed in over 30 to 40 percent of breast cancer cases,” Kang said. “This indicates that new drugs against metadherin may potentially benefit a large population of breast cancer patients.”

The work was funded by a Department of Defense Era of Hope Scholar Award and grants from the National Institutes of Health, the American Cancer Society, the Susan G. Komen Foundation and the New Jersey Commission on Cancer Research.

GENETIC CODE AND PROTEIN SYNTHESIS

1. Anticodons are present on:
A. Coding strand of DNA.
B. mRNA
C. tRNA
D. rRNA

Answer: C. tRNA

2. Codons are present on:
A. Non-coding strand of DNA
B. hnRNA
C. tRNA
D. None of the above

Answer: B. hnRNA

3. Nonsense codons are present on:
A. mRNA
B. tRNA
C. rRNA
D. None of the above

Answer: A. mRNA

4. Genetic code is said to be degenerate because:
A. It can undergo mutation
B. A large proportion of DNA is non-coding
C. One codon can code for more than one amino acids
D. More than one codons can code for the same amino acids

Answer: D. More than one codons can code for the same amino acids

5. All the following statements about genetic code are correct except:
A. It is degenerate
B. In is unambiguous
C. It is nearly universal
D. It is overlapping

Answer: D. It is overlapping

6. All the following statements about nonsense codons are true except:
A. The do not code for amino acids
B. They act as chain termination signals
C. The are identical in nuclear and mitochondrial DNA
D. They have no complementary anticodons

Answer: C. They are identical in nuclear and mitochondrial DNA

7. A polycistronic mRNA can be seen in:
A. Prokaryotes
B. Eukaryotes
C. Mitochondria
D. All of the above

Answer: A. Prokaryotes

8. Non-coding sequences are present in the genes of:
A. Bacteria
B. Viruses
C. Eukaryotes
D. All of the above

Answer: C. Eukaryotes

9. Non-coding sequences in a gene are known as:
A. Cistrons
B. Nonsense codons
C. Introns
D. Exons

Answer: C. Introns

10. Splice sites are present in:
A. Prokaryotic mRNA
B. Eukaryotic mRNA
C. Eukaryotic hnRNA
D. All of the above

Answer: C. Eukaryotic hnRNA

Gene Therapy - Viral Vectors

In both types of therapy, scientists need something to transport either the entire gene or a recombinant DNA to the cell's nucleus, where the chromosomes and DNA reside. In essence, vectors are molecular delivery trucks. One of the first and most popular vectors developed were viruses because they invade cells as part of the natural infection process. Viruses have the potential to be excellent vectors because they have a specific relationship with the host in that they colonize certain cell types and tissues in specific organs. As a result, vectors are chosen according to their attraction to certain cells and areas of the body.

One of the first vectors used was retroviruses. Because these viruses are easily cloned (artificially reproduced) in the laboratory, scientists have studied them extensively and learned a great deal about their biological action. They also have learned how to remove the genetic information that governs viral replication, thus reducing the chances of infection.

Retroviruses work best in actively dividing cells, but cells in the body are relatively stable and do not divide often. As a result, these cells are used primarily for ex vivo (outside the body) manipulation. First, the cells are removed from the patient's body, and the virus, or vector, carrying the gene is inserted into them. Next, the cells are placed into a nutrient culture where they grow and replicate. Once enough cells are gathered, they are returned to the body, usually by injection into the blood stream. Theoretically, as long as these cells survive, they will provide the desired therapy.

Another class of viruses, called the adenoviruses, also may prove to be good gene vectors. These viruses can effectively infect nondividing cells in the body, where the desired gene product then is expressed naturally. In addition to being a more efficient approach to gene transportation, these viruses, which cause respiratory infections, are more easily purified and made stable than retroviruses, resulting in less chance of an unwanted viral infection. However, these viruses live for several days in the body, and some concern surrounds the possibility of infecting others with the viruses through sneezing or coughing. Other viral vectors include influenza viruses, Sindbis virus, and a herpes virus that infects nerve cells.

Scientists also have delved into nonviral vectors. These vectors rely on the natural biological process in which cells uptake (or gather) macromolecules. One approach is to use liposomes, globules of fat produced by the body and taken up by cells. Scientists also are investigating the introduction of raw recombinant DNA by injecting it into the bloodstream or placing it on microscopic beads of gold shot into the skin with a "gene-gun." Another possible vector under development is based on dendrimer molecules. A class of polymers (naturally occurring or artificial substances that have a high molecular weight and formed by smaller molecules of the same or similar substances), is "constructed" in the laboratory by combining these smaller molecules. They have been used in manufacturing Styrofoam, polyethylene cartons, and Plexiglass. In the laboratory, dendrimers have shown the ability to transport genetic material into human cells. They also can be designed to form an affinity for particular cell membranes by attaching to certain sugars and protein groups.

First Human Gene Therapy

On September 14, 1990 at the U.S. National Institutes of Health W. French Anderson, M.D., and his colleagues R. Michael Blaese, M.D., C. Bouzaid, M.D., and Kenneth Culver, M.D., performed the first approved gene therapy procedure on four-year old Ashanthi DeSilva. Born with a rare genetic disease called severe combined immunodeficiency (SCID), she lacked a healthy immune system, and was vulnerable to every passing germ or infection. Children with this illness usually develop overwhelming infections and rarely survive to adulthood; a common childhood illness like chickenpox is life-threatening. Ashanthi led a cloistered existence -- avoiding contact with people outside her family, remaining in the sterile environment of her home, and battling frequent illnesses with massive amounts of antibiotics.

In Ashanthi's gene therapy procedure, doctors removed white blood cells from the child's body, let the cells grow in the lab, inserted the missing gene into the cells, and then infused the genetically modified blood cells back into the patient's bloodstream. Laboratory tests have shown that the therapy strengthened Ashanthi's immune system by 40%; she no longer has recurrent colds, she has been allowed to attend school, and she was immunized against whooping cough. This procedure was not a cure; the white blood cells treated genetically only work for a few months, after which the process must be repeated (VII, Thompson [First] 1993). As of early 2007, she was still in good health, and she was attending college. However, there is no consensus on what portion of her improvement should be attributed to gene therapy versus other treatments. Some would state that the case is of great importance despite its indefinite results, if only because it demonstrated that gene therapy could be practically attempted without adverse consequences.

Although this simplified explanation of a gene therapy procedure sounds like a happy ending, it is little more than an optimistic first chapter in a long story; the road to the first approved gene therapy procedure was rocky and fraught with controversy. The biology of human gene therapy is very complex, and there are many techniques that still need to be developed and diseases that need to be understood more fully before gene therapy can be used appropriately. The public policy debate surrounding the possible use of genetically engineered material in human subjects has been equally complex. Major participants in the debate have come from the fields of biology, government, law, medicine, philosophy, politics, and religion, each bringing different views to the discussion.

ENZYMES Few Questions

1. The following is a group-specific enzyme:
A. Pepsin
B. Aminopeptidase
C. Phospholipase D
D. All of the above

Answer: D. All the above

2. The following is a substrate-specific enzyme:
A. Hexokinase
B. Thiokinase
C. Lactase
D. Aminopeptidase

Answer: C. Lactase

3. The following is not a substrate-specific enzyme:
A. Glucokinase
B. Fructokinase
C. Hexokinase
D. Phospofructokinase

Answer: C. Hexokinase

4. Chymotrypsin hydrolyses peptide bonds in which carboxyl group is contributed by:
A. Phenylalanine
B. Tyrosine
C. Tryptophan
D. Any of the above

Answer: D. Any of the above

5. Coenzymes combine with:
A. Proenzymes
B. Apoenzymes
C. Holoenzymes
D. Antienzymes

Answer: B. Apoenzymes

6. Coenzymes are required in the following reactions:
A. Oxidation-reduction
B. Transamination
C. Phosphorylation
D. All of the above

7. The following coenzyme takes part in hydrogen transfer reactions:
A. Tetrahydrofolate
B. Coenzyme A
C. Coenzyme Q
D. Biotin

Answer: C. Coenzyme Q

8. The following coenzyme does not take part in hydrogen transfer reactions:
A. FAD
B. NAD
C. NADP
D. Cobamides

Answer: D. Cobamides

9. The following coenzyme takes part in oxidation reduction reactions:
A. Pyridoxal phospate
B. Lipoic acid
C. Thiamin diphospate
D. None of the above

Answer: B. Lipoic acid

10. In conversion of glucose to glucose-6-phospate, the coenqyme is:
A. Mg++
B. ATP
C. Both of the above
D. Neither of the above

Answer: B. ATP

Diseases targeted for treatment by gene therapy

The potential scope of gene therapy is enormous. More than 4,200 diseases have been identified as resulting directly from abnormal genes, and countless others that may be partially influenced by a person's genetic makeup. Initial research has concentrated on developing gene therapies for diseases whose genetic origins have been established and for other diseases that can be cured or improved by substances genes produce.

The following are examples of potential gene therapies. People suffering from cystic fibrosis lack a gene needed to produce a salt-regulating protein. This protein regulates the flow of chloride into epithelial cells, (the cells that line the inner and outer skin layers) that cover the air passages of the nose and lungs. Without this regulation, patients with cystic fibrosis build up a thick mucus that makes them prone to lung infections. A gene therapy technique to correct this abnormality might employ an adenovirus to transfer a normal copy of what scientists call the cystic fibrosis transmembrane conductance regulator, or CTRF, gene. The gene is introduced into the patient by spraying it into the nose or lungs. Researchers announced in 2004 that they had, for the first time, treated a dominant neurogenerative disease called Spinocerebella ataxia type 1, with gene therapy. This could lead to treating similar diseases such as Huntingtons disease. They also announced a single intravenous injection could deliver therapy to all muscles, perhaps providing hope to people with muscular dystrophy.

Familial hypercholesterolemia (FH) also is an inherited disease, resulting in the inability to process cholesterol properly, which leads to high levels of artery-clogging fat in the blood stream. Patients with FH often suffer heart attacks and strokes because of blocked arteries. A gene therapy approach used to battle FH is much more intricate than most gene therapies because it involves partial surgical removal of patients' livers (ex vivo transgene therapy). Corrected copies of a gene that serve to reduce cholesterol build-up are inserted into the liver sections, which then are transplanted back into the patients.

Gene therapy also has been tested on patients with AIDS. AIDS is caused by the human immunodeficiency virus (HIV), which weakens the body's immune system to the point that sufferers are unable to fight off diseases like pneumonias and cancer. In one approach, genes that produce specific HIV proteins have been altered to stimulate immune system functioning without causing the negative effects that a complete HIV molecule has on the immune system. These genes are then injected in the patient's blood stream. Another approach to treating AIDS is to insert, via white blood cells, genes that have been genetically engineered to produce a receptor that would attract HIV and reduce its chances of replicating. In 2004, researchers reported that had developed a new vaccine concept for HIV, but the details were still in development.

Several cancers also have the potential to be treated with gene therapy. A therapy tested for melanoma, or skin cancer, involves introducing a gene with an anticancer protein called tumor necrosis factor (TNF) into test tube samples of the patient's own cancer cells, which are then reintroduced into the patient. In brain cancer, the approach is to insert a specific gene that increases the cancer cells' susceptibility to a common drug used in fighting the disease. In 2003, researchers reported that they had harnessed the cell killing properties of adenoviruses to treat prostate cancer. A 2004 report said that researchers had developed a new DNA vaccine that targeted the proteins expressed in cervical cancer cells.

Gaucher disease is an inherited disease caused by a mutant gene that inhibits the production of an enzyme called glucocerebrosidase. Patients with Gaucher disease have enlarged livers and spleens and eventually their bones deteriorate. Clinical gene therapy trials focus on inserting the gene for producing this enzyme.

Gene therapy also is being considered as an approach to solving a problem associated with a surgical procedure known as balloon angioplasty. In this procedure, a stent (in this case, a type of tubular scaffolding) is used to open the clogged artery. However, in response to the trauma of the stent insertion, the body initiates a natural healing process that produces too many cells in the artery and results in restenosis, or reclosing of the artery. The gene therapy approach to preventing this unwanted side effect is to cover the outside of the stents with a soluble gel. This gel contains vectors for genes that reduce this overactive healing response.


Regularly throughout the past decade, and no doubt over future years, scientists have and will come up with new possible ways for gene therapy to help treat human disease. Recent advancements include the possibility of reversing hearing loss in humans with experimental growing of new sensory cells in adult guinea pigs, and avoiding amputation in patients with severe circulatory problems in their legs with angiogenic growth factors.

Chromosome elimination in Hemiptera

Chromosome elimination is a frequent occurrence in Hemiptera; one of the most interesting cases is that of Sciara. In Sciara coprophila the zygote carries the x chromosomes, one contributed by the egg and two by the spermatozoon (this results from an equational non dis-junction of the maternally derived x chromosome at the second meiotic division in the male following the selective elimination of paternal homologues at the first spermatocyte division). During early cleavage both paternal x chromosomes are eliminated from the somatic cell line of the males while only one is eliminated in the female. In the germ link one paternal x chromosome is eliminated both in the female and in the male, but not until the germ cells have reached their final destination in the gonad. Chromosome elimination may be thought of as a primitive, and in fact crude mechanism of "gene silencing" to be replaced by more subtle devices in the course of evolution. The eliminated chromosomes, or parts of chromosomes, or parts of chromosomes, contain the sexuality genes.

-> Cross section through the pharynx of Ascaris


Each cell line is committed to a certain number of DNA replication cycles before expressing its specific phenotype: in other words "the program for cell division is a part of the differentiative program of each cell line". We can derive that from a study of development of marine invertebrates. The Ascidian embryo offers unique opportunities to study cell lineage. Indeed, it shows the segregation of the major organ-forming territories occurs before the first cleavage

CHEMISTRY OF CARBOHYDRATES

1. Number of asymmetric carbon atom in glucose is:
A. One
B. Two
C. Three
D. Four

Answer: D. Four

2. Beta-1, 4-Glycosidic bond is present in:
A. Maltose
B. Lactose
C. Sucrose
D. None of the above.

Answer: B. Lactose

3. Number of stereoisomers of glucose is:
A. 4
B. 8
C. 16
D. None of the above

Answer: C.16

4. A homopolysaccharide made up of fructose is:
A. Glycogen
B. Dextrin
C. Cellulose
D. Inulin

Answer: D. Inulin

5. Aglycone portion in methyl glucoside is:
A. Glucose
B. Methanol
C. Both of the above
D. Neither of the above

Answer: B. Methanol

6. Identical osazones are formed by:
A. Glucose and Fructose
B. Glucose and Mannose
C. Mannose and Fructose
D. All the 3 pairs

Answer: D. All the 3 pairs

7. Maltose can be formed by hydrolysis of:
A. Strach
B. Dextrin
C. Glycogen
D. All of the above

Answer: D. All of the above

8. Alpha-1, 6-Glycosidic bond is not present in:
A. Glycogen
B. Dextrin
C. Amylose
D. Amylopectin

Answer: C. Amylose

9. Alpha-D-Glucuronic acid is present in:
A. Hyaluronic acid
B. Chondrointin sulphate
C. Heparin
D. All of the above

Answer: C. Heparin

10. Monosaccharides can be seperated by:
A. Electrophoresis
B. Chromotography
C. Salting out
D. None of the above

Answer: B. Chromotography

BIOFERTILIZERS

Nitrogenous fertilizers produced in industry by Haber-Bosch process consume high energy (about 13,500 K Cal/Kg N fixed). In such industries, fossil fuel is the source of energy. In recent years, due to Gulf crisis, the cost of crude oil increased about three fold within a year. Therefore, fossil fuel (oil and coal) based method of farming has become more expensive accordingly. To combat with this problem, however, it is necessary to develop an alternative method of supplying nutrients to plants.

In recent years, use of microbial inoculants as a source of biofertilizers has become a hope for most of countries, as far as economical and environmental view ponts are concerned. Biologically fixed nitrogen is such a source which can supply an adequate amount of nitrogen to plants and other nutrients to some extent. It is a non-hazardous way of fertilization of field. Moreover, biologically fixed nitrogen consumes about 25 percent to 30 percent less energy than normally done by chemical process.

Therefore, in developing countries like India, it can solve the problem of high cost of fertilizers and help in saving the economy of the country.

The term "Biofertilizers" denote all the "nutrient inputs of biological origin for plant growth" (Subba Rao, 1982). Here biological origin should be referred to as microbiological process synthesizing complex compounds and their further release into outer medium, to the close vicinity of plants roots which are again taken up by plants. Therefore, the appropriate term for biofertilizers should be "microbial inoculants" as suggested by Subba Rao (1982). As bacteria and cyanobacteria (also Frankia) are known to fix atmospheric nitrogen, both bacteria and cyanobacteria are widely used as biofertilizers.