Several key biotech discoveries have contributed to the growth of biotechnology. This article deals with a) Isolation of RNA polymerase b) Discovery of reverse transcriptase and c) Introduction of DNA into cells by calcium dependent method developed by M. Mandel and A.Higa (Calcium dependent bacteriophage DNA infection)

RNA polymerase isolation

RNA polymerase is an enzyme that catalyzes the synthesis of RNA from either DNA or RNA template. Normally genetic information within DNA is dormant and without the impetus provided by RNA polymerase, DNA cannot be copied into RNA through transcription. Moreover it is RNA polymerase that decides which type of proteins a cell should produce.

Hurwitz, James Bonner and Audrey Stevens working independently in different laboratories are credited with isolation of RNA polymerase. However, Hurwitz and others who isolated RNA polymerase were not the first scientists to study the enzymes involved in RNA synthesis and the mechanisms involved in incorporation of ribonucleotides into RNA. Earlier Marianne Grunberg-Manago and Severo Ochoa had isolated the enzyme polynucleotide phosphorylase and erroneously concluded that it was used for RNA synthesis ( on the contrary it was found to degrade RNA)

Hurwitz used Escherichia coli extracts, and in 1960 established stimulation of RNA synthesis by externally added DNA in his E. coli preparations. In the presence of DNA and Mn2+ or Mg2+, the RNA polymerase isolated by Hurwitz could catalyze the synthesis of RNA from ribonucleoside triphosphates of adenine, uracil, guanine, and cytosine. James Bonner used extracts of peas;and Audrey Stevens used extracts of E. coli for RNA polymerase isolation.

Discovery of reverse transcriptase

Reverse transcriptase is a DNA polymerase that polymerizes DNA precursors. The template used by reverse transciptase for making viral DNA is the single stranded RNA in retroviruses (normally the genome of the retrovirus). So Reverse transcriptase (it is a DNA polymerase as it polymerizes DNA precursors) is a replication enzyme of retroviruses. On the other hand, for making new DNA, --- cellular DNA polymerases use DNA as template.

Temin, together with an American virologist David Baltimore and a Japanese virologist who were conducting research relating to interaction between tumor viruses and cell genetic material are credited with discovering reverse transcriptase in 1970.

How did this come about?

Retroviruses are RNA viruses. These viruses make an enzyme called reverse transcriptase. In order to replicate, these viruses need to make DNA copies of their RNA genomes (and put them into host chromosomes) for which they require the enzyme reverse transcriptase. An example is the HIV-I virus which is a retrovirus that needs to make DNA copies of itself for fusing with host chromosome and replicate---for which reverse transcriptase(RNA-directed DNA polymerase)is required. Reverse transcriptase can synthesize DNA from RNA or one can say that RNA can act as the template for the transcription of DNA. That apart, reverse transcriptase finds use as a molecular mechanism to analyze gene expression and to clone (using c DNA libraries).

The then prevailing popular belief was that genetic information flowed from DNA to RNA to protein (unidirectionally) and the concept of reverse transcription was at odds with this. As shown for other RNA viruses it was then believed that RNA tumor causing viruses were RNA viruses which needed RNA polymerases for replication and certainly not a DNA polymerase. So Temin's hypothesis of flow of genetic information from RNA to DNA seemed improbable at that point in time.
It was only in 1970 that Temin could advance proof for reverse transcription when he discovered the RNA-copying DNA polymerase in purified virions. For this discovery of reverse transciptase Temin shared the Nobel Prize with David Baltimore.


Introduction of DNA into cells by calcium dependent method developed by M. Mandel and A.Higa (Calcium dependent bacteriophage DNA infection)


It was Mandel and Higa that first attempted to transform Escherichia coli by treating Escherichia coli cells with calcium to make them suitable for uptaking exogenous DNA----more specifically for the transfection with DNA of bacteriophage lambda to produce viable phage particles. However, nothing is known of the uptake mechanism of the transforming DNA. Although in S. pneumoniae and B. subtilis DNA is uptaken in single stranded state and in H. influenzae uptake gives a partially single stranded DNA, single strandedness does not always occur from uptake in calcium treated E. coli

M. Mandel and A.Higa's Calcium dependent bacteriophage DNA infection has been used for the following:

a)transfection with different phages.
b) For chromosomal transformation
c) And for transformation with plasmid DNA

Even today the fact that calcium treated cells can be transformed is the basis for other in vitro DNA manipulation techniques for ascertaining biological activity.

Slight variants of M. Mandel and A.Higa's Calcium dependent bacteriophage DNA infection process include
a)Exposure of cells to CaCl2 for elongated periods
b) Instead of Ca2+ ions Rb+ (3), Mn2+, and K+ are used
c) Plate transformation method using selective medium plates containing Ca2+

Synthesis of artificial gene from DNA nucleotides by G.H.Khorana

The question arises what we mean by artificial gene synthesis? It simply means in vitro synthesis of gene (by oligonucleotide synthesis) without using samples of initial template DNA. On the contrary, natural DNA replication requires existing DNA templates for new DNA synthesis.

DNA that exhibited biological functioning was first synthesized by Kornberg in 1961 using a section of viral DNA as primer,and a mix of metal ions, DNA polymerase enzyme and deoxyribonucleoside triphosphates.

Artificial Synthesis of DNA without a Template

However in 1965 using chemical methods Hargobind Khorana a scientist of Indian origin made the first synthetic DNA with the synthesis of gene coding for yeast alanine t RNA--but this didn't work. However a few years later Khorana won the Nobel Prize when he synthesized a yeast tRNA---- by cracking the genetic code of yeast by synthesizing parts of a nucleic acid molecule and which perhaps involved the synthesis of the first complete gene. Once again in 1979 Khorana and his team synthesized the gene coding for tyrosine tRNA of E. coli. It functioned in combination with a phage DNA.

Apart from the first ever totally artificial gene that was synthesized by Har Gobind Khorana, he is also credited with synthesizing acetyl coenzyme A (used for research processes) ----using an inexpensive process.

Why are synthetic genes important?

Synthetic genes are synthesized without using precursor template DNA but with a mix of biology and organic chemistry. Wherever recombinant DNA technology is needed gene synthesis is a vital parameter, as for example in gene therapy and vaccine development. Earlier gene synthesis was reckoned as a key source for cDNA's because at that time there were difficulties in cloning it. But now that sequence verified cloned cDNA are easily available, gene synthesis is not used much as a source for cDNAs.

Herbert Boyer and Alexander Markham are credited with the synthesis of the first peptide- and protein-coding genes

Conclusion

Each of the biotech discoveries enumerated in this article has forced us to radically redefine both life and nature, and mainstream biotechnology as we see today is the culmination of the interplay of each of the discoveries listed in this article.

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