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REVIEWS ARTICLES
DNA - Retrospect and Prospect
BN Apte
The discovery of the structure of DNA completes 50 years this year. A very large number of explosive development have occurred in life sciences. Every single question on DNA has resulted in opening up Pandora’s box. Questions such as How does the DNA duplicate itself? How is the genetic information stored in this molecule? What is the language used? How is this information retrieved? How is this flow of information regulated? - have given us a fund of invaluable information about various complex molecular reactions that occur in a living system.
What is the optimum size of the DNA molecule which is required for the life processes to go on? There does not seem to be a definitive answer to this question. A virus f2, which grows on E. coli has 3 gene equivalent of genetic material. This virus contains RNA as genetic material. The host E.coli on which it grows has about 3000 gene equivalent of genetic material. E. coli contains 3 x 106 (three million) base pairs of DNA. The length of this DNA is 2 millimeter. Human beings have 3 x 109 base pair equivalent of DNA. The length of DNA per cell is 2 meters. Although we have 1000 times more DNA than E. Coli, the latest work on the human genome project has put the lowest estimate on the total number of genes as about 34000 and the highest estimate as about 94000.
We will now see the historical development of DNA.
In 1745 Maupertuis proposed an adaptation ist account of organic design which presupposes some mechanism for transmitting adaptations from one generation to the next.
Not much happened till 1859 when Darwin published his celebrated theory on the origin of species. He vastly stregthened the adaptionist hypothesis which was proposed by Maupertuis in 1745. The interval between these two events i.e. from 1745 to 1859 was dominated by cytologist doing observations of all kinds of material they laid their hands on under the microscope. Microscope was discovered in late 17th century by Leeuwenhock.
In 1865 Gregor Mendel published evidence for the discreteness and combinatoral rules of inherited traits. Mendel showed that traits are carried by discrete units or genes. These results, however, were not appreciated until early 1900.
In 1869 Miescher discovered nuclein in pus cells. It became known as nucleic acid after 1874 after Miescher was able to separate nuclein into a protein component and the nucleic acid component. He proposed that this material must have a positive role to play in the hereditary processes.
Between 1918 and 1926 Muller formulated major principles of spontaneous gene mutation as point effects of ultramicroscopic physicochemical accidents. He induced such changes using X-rays.
In 1920 Nucleic acid was found to be a major component of the chromosomes. The chemical nature of nucleic acids was investigated in 1930s. It was thought to be a tetranucleotide composed of one unit each of adenylic, guanylic, thymidylic and cytidylic acids. The ubiquitous presence of nucleic acids in the chromosome was generally explained in purely physiological or structural terms. The molecular structure of nucleic acid as then seen was considered to be too simple and was, therefore, not considered to be a good candidate for a carrier of genetic information.
In early 1940s the molecular weight of nucleic acid was found to be much higher than the tetranucleotide hypothesis required, Nucleic acid was, however, still viewed as a uniform polymer, like starch, unaffected by its biological source. Hereditary information was generally believed to be stored in proteins, since they differ across the species, between individuals, and even within an organism.
In 1944 Oswald Avery and his colleagues identified nucleic acids as the active principle in bacterial transformation.
Around the same time bacterial viruses-bacteriophages-were discovered. They provided an ideal material for experimental purposes.
In 1950 Erwin Chargaff showed that the four nucleotides in nucleic acid were not present in stable proportions and that the nucleotide composition differs from one source of nucleic acid to the other. He showed that in any given nucleic acid, the amount of adenine is equal to thyamine and the amount of guanine is equal to cytosine. A+G is always equal to T+C.
Alfred Hershy and Martha Chase published their experimental results in 1952, which conclusively proved that when a bacteriophage infects a bacterial host, only nucleic acid enters the host leaving all the protein coat outside the cell. The nucleic acid then produces a large number of phage particles within the host eventually bursting open the host cell.
Using all this information together with a large amount of X-ray diffraction data Jim Watson and Francis Crick proposed their celebrated structural model of DNA. DNA has two strands which are wound around each other in a helical form. Each nucleotide consists of a deoxyribase sugar molecule to which is attached a phosphate group and one of four nitrogenous bases : two purines (adenine and guanine) and two pyrimidiness (cytosine and thymine). The phosphate sugar forms the back-bone of the molecule. Adenine always pairs with thymine through two hydrogen bonds and guanine always pairs with cytosine through three hydrogen bonds. The base pairs, A=T and G-C, form the rungs of the helix and are arranged perpendicular to the long axis of the molecule.
DNA replication is possible through the complementary nature of the two strands. The chemical complexity of the molecule was thought to be sufficient to store the requisite information.
The discovery of the structure of DNA provided a great impetus to the development of molecular biology which has now developed into the science of biotechnology and genetic engineering with applications in several other fields of science.
In early 1970 genetic regulatory elements were discovered. These regulator genes control the timing and the output of structural genes in living systems in response to their environment.Earlier in 1940, Barbara McClintock had discovered transposable or jumping genes in maize. But her work was not fully recognized and appreciated for a generation till 1980. In her Nobel lecture Barbara said, “The genome is a highly sensitive organ of the cell, that in times of stress could initiate its own restructuring and renovation”. These transposable elements are now a well established entity and have been named transposons
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In 1984 McGinnis discovered homeotic (Hox) regulatory genes which are shown to be responsible for the basic body plan of most animals. In subsequent work, his team demonstrated that a single gene mutation in a Hox gene would suffice to suppress all limb development in the thoracic region of the fruit fly. This discovery may have relevance to human beings as one may be able to relate phocomelia with such an event.
1960 onwards was a very productive period. A large number of plasmids were discovered in various systems - microbes, plants and animals and human beings. The plasmids are being used today as vehicles to engineer and transfer genetic material. A large number of enzymes which form the basic tools in genetic engineering and biotechnology were discovered. This can be considered the golden era of molecular biology. The year 2000 saw the human genome project providing us a complete chemical map of the human genome containing 3.1 billion nucleotide units. However, as Jim Watson puts it “The human genome project has resulted in publishing a book on Human genome. Now somebody has to sit there read it and bring the meaning out of it.”
We will now see a few applications of the knowledge which we have acquired in these years. The applications are in various fields.
1. Agriculture : The term, genetically modified food, has become very common. Pest resistant potato is now in the market. Cotton containing a toxic compound producing gene from a microbe, B. thuringiensis, has now been approved for cultivation in India. An enzyme called beta-glucuronidase is extremely important in human beings. Lack of this enzyme results in a dreaded disease called mucopolysachharidosis (MPS VII) in which the patient suffers from dysmorphic features with debilitating structural abnormalities. This enzyme is also present in tomato. The shelf-life of tomatoes can be substantially increased from few days to three weeks or even more by knocking out this enzyme. Rice with Vitamin A is now available.
2. Medicine : Human insulin gene has been now engineered into microbial systems. Pharmaceutical companies produce insulin - humulin - by growing the microbes in fermentors. Similarly human growth hormone has also been engineered in E. coli. What was previously been obtained from 500000 pituitary bodies from one year old sheep brain can now be obtained by growing 10 liter culture of human growth hormone containing E. coli and that too in about 72 hours.
A factor called Vascular endothelial growth factor (VEGF) develops collateral vessels and is being used to treat patients with acute varicose veins. The use of VEGF in cardiac artery blocks has also been successfully tried.
Experiments on modification of the existing antibiotics and producing entirely new molecules with antibiotic activity are being done by genetically manipulating the antibiotic producing cultures. Oral vaccines will be in the market in the next few years.
3. Diagnosis : Use of polymerase chain reaction using DNA primers for the diagnosis of several microbial diseases has become a routine procedure. DNA based diagnostic methods are also used to diagnose a large number of genetic diseases. Prenatal diagnosis of the disease in the foetus in a family with the previous child having genetic disease is being used to prevent the recurrence of the same disease in the same family.
4. Archeology : The period of a fossil can be ascertained by extracting and studying its DNA. Several such fossils have been studied and their period accurately found.
5. Establishing the Identity : Problems arise when one of the parents disowns the child. The identity of the child can be established by studying the DNA fingerprints of the parents and the child in question. This technique has vast applications in medicolegal cases. The identity of a remnant from this recent disaster of the American space-ship, Columbia, was proved to be that of Kalpana Chawla, the Indian astronaut, by using the same technique of DNA analysis.
6. Computers using DNA Chips : The present day Computers use Silicon chips and a very large number of configuration of silicon chips have been in use. Replacing silicon chips with DNA chips would have several advantages. Silicon chips process the data in sequence. DNA chips have an ability to process data in parallel. DNA chips have much larger memory than the silicon chips. These two factors will make computers based on DNA chips more versatile. Such computers are being used to process data on DNA.
Indeed, there will be many more applications of the basic technologies provided by the research on DNA in future. We have extremely powerful tool in our hands. We must use it judiciously to the betterment of the mankind.
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