Several great scientists have contributed to the stupendous growth of biotechnology as we know today. Gregor Mendel's work on pea plants in 1866 is probably one of the earliest published works in genetics. Thereafter Muller showed as early as 1927 that damage to cells could cause mutations as for example the inducement of mutations in Drosophila melanogaster due to effect of X-rays. Muller's findings took place a quarter of a century before Hershey and Chase showed in 1952 that genes live within the DNA and 26 years before DNA was made threadbare by Watson and Crick in 1953. What does this mean? This means that we had knowledge of mutagenesis and genetic toxicology before resolution of DNA structure itself. However the molecular basis on which mutations and cytotoxicity occurred came to be known only since 1953 ---because by then we came to know how DNA replicated, how toxic agents could interact with DNA and the role of DNA polymerase and recombination processes---and how these could affect DNA sequence changes.

This article evaluates as far as possible chronologically certain path- braking biotech discoveries that have gradually contributed to the progression of modern transgenic science as we see today starting from the amino acid sequencing by Fred Sanger in 1951.

The first of this series of articles will deal with a) F Sanger and H.Tuppy's work on elucidating the first amino acid sequence b) JD Watson and FHC Crick's DNA double helix model and c) Kornberg's discovery of DNA polymerase

F Sanger and H.Tuppy's work on elucidating the first amino acid sequence

By 1943, it was pretty much known that proteins were made of amino acids held together by peptide bonds. Furthermore, at that point in time it was possible to accurately ascertain how many of the twenty or so then known amino acid residues were there in a given protein. But the way in which the amino acids were arranged in the protein was then unknown. This arrangement was thought to contribute to the variations in the biological properties of different proteins (and genes had a role in controlling them) although proteins had the same type of amino acids. In other words, the amino acid sequence in different proteins was not known.

Fred Sanger and H.Tuppy changed all that. They started working on the insulin molecule because of its simpler structure as it did not have tryptophan and methionine two of the most common amino acids found in most proteins. Sanger used partial acid hydrolysis (peptide bonds are split randomly) to study insulin structure and had tryptophan been there it would have degraded on acid hydrolysis.

Fred's work resulted in showing that insulin had only two peptide chains---a A chain with 20 or so amino acids, and B chain with 30 or so amino acids and it was possible to ascertain the structure of each peptide chain. So the first amino acid sequences of insulin came from partial acid hydrolysis followed by fractionation of the products and then end group analysis.

For this work, Fred was awarded the Nobel Prize in 1958. He was to get yet another Nobel Prize in 1980 for work relating to sequencing nucleic acids.

Perhaps this epoch making earlier discovery is the cornerstone of the study in the nature of changes in proteins when a disease condition exists. The growth of biotechnology as a dominant science would not have been possible without the sequencing of proteins and nucleic acids.

JD Watson and FHC Crick's DNA double helix structure

Before J. D. Watson and F. H. C. Crick announced their theory of the structure of DNA, Pauling and Corey proposed a model of three intertwined chains with phosphates in close proximity to the fibre axis with the bases outside. Another theory propounded by Fraser was also a three chain structure with bases within and phosphates outside apparently connected by hydrogen bonds

J. D. Watson and F. H. C. Crick's DNA double helix structure theory ---- entails two helical chains intertwined around the same axis with both chains following right handed helices---and with the two chains held together by the purine and pyrimidine bases.

In any diagram depicing the DNA you will see two intertwining ribbons that depict the two phosphate-sugar chains, and the horizonal rods indicate the pairs of bases holding both the chains together. An important finding was that, only specific pairs of bases could bond together---for example, adenine (purine) could bond with thymine (pyrimidine), and guanine (purine) could bond with cytosine (pyrimidine).The vertical line denotes the fibre axis in the diagrammatic depiction of DNA. And furthermore in deoxyribose nucleic acid, the ratio of the amounts of adenine to thymine, and the ratio of guanine to cytosine, is always in the proximity of 1

Watson and Crick's research had more to do with the complimentary nature of the bases within the DNA double helix and it showered light on the mechanism of DNA replication. However at that point in time their work could not tell how this molecule could ultimately dictate the functioning of all proteins within the cell. In other words, although it suggested how DNA might replicate, how the molecule could control the nature of proteins present in the cell was then not known. Even the replication of DNA was proven by experiment by Meselson and Stahl only in 1958 that was in consonance with Watson and Crick's earlier prediction. Furthermore, it was not until 1966 that the genetic code was finally cracked, but in the interim period the mechanisms of interaction between DNA, mRNA, tRNA, and ribosomes to produce genetically important proteins became known.

Further to Watson and Crick hypothesis in 1953 and insights into DNA replication and how DNA makes RNA, certain things came to be understood about how these things could go wrong. For example it came to be understood how changes in DNA sequence could produce missense and other mutations---missense means messenger RNA strand section that contains a codon altered through mutation and which codes for a different amino acid. In other words damage to DNA could cause permanent sequence changes resulting in different mutations. Human beings have several genes that produce certain proteins whose main job is to make proteins that could repair damage to DNA and protect against mutation. Extrapolation of this field has led us to the domain of genetic toxicology.

Kornberg's discovery of DNA polymerase

DNA polymerase is a key factor in assembling the building blocks of DNA and is a template directed enzyme. How did the discovery of the first DNA polymerase, DNA polymerase I of Escherichia coli, come about?

Kornberg is credited with finding this enzyme in 1955. He discovered this enzyme system that catalyzed the deoxyribonucleotides into DNA in Escherichia coli extracts and concluded that polymerized DNA was the template that facilitated the making of the new DNA. In other words, DNA polymerase catalyzes the template-directed synthesis of DNA. Using DNA polymerase Kornberg was able to cobble together a 5000 nucleotide DNA chain that had the same genetic activity as compared to a DNA from a natural virus. So in 1967 the Ï•X174 virus became the first biologically active virus ever produced in a lab.

The research approach they took was once a method of quantitative assay in a cellular extract was established, the enzymes could be identified by purification and furthermore lead to the understanding the reactions they catalized.

The discovery of DNA polymerase helps us understand how DNA replication occurs, how it is repaired and how it is transcribed. That apart, it is also responsible for the development of PCR and DNA sequencing which is critically important in biotechnology. For the elucidation of the mechanisms involved in the biosynthesis of ribonucleic acid and deoxyribonucleic acid Kornberg was awarded the Nobel Prize.

Kornberg is also credited with research on
a) DNA polymerase's proofreading and editing functions
b) DNA polymerase III holoenzyme.
c) Discovery of the intermediate 5′-phosphoribosyl-1-pyrophospate (PRPP) and the steps entailing pyrimidine and purine nucleotide synthesis.



Conclusion

Now when we look to the sequencing of the human genome that's mostly been completed---one could easily recognize the importance of each of the discoveries cited above.

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