Synthesis of gene for a true precursor tRNA

Different parts of the complete E. coli tyrosine suppressor tRNA gene
Fig. 41.12. Different parts of the complete E. coli tyrosine suppressor tRNA gene (redrawn from Science, Vol. 203, 1979).
Before Khorana could complete the synthesis of gene for yeast alanyl tRNA in 1970, as outlined above, it became obvious that tRNA was not the direct product of transcription. Instead a precursor molecule is first synthesized which subsequently after losing segments of RNA by cleavage, gives rise to tRNA.
Obviously, therefore, the actual gene for yeast alanyl tRNA will be longer than the DNA duplex synthesized by Khorana. In view of this, Khorana subsequently initiated synthesis of a gene for E. coli tryosine suppressor tRNA precursor. DNA duplex which will give rise to this tRNA precursor, was synthesized in the form of 26 small oligonucleotide segments. These were then arranged into six DNA duplex fragments having single stranded ends. These six fragments gave rise to presumed gene for E. coli tyrosine suppressor tRNA precursor. This gene, however, still lacked promoter region and other sequences essential for processing.

Later, in 1979, Khorana reported completion of the total synthesis of a biologically functional tyrosine suppressor transfer RNA gene carrying all regulatory sequences. This gene was 207 base pairs long and included the following : (i) a 51 base pairs long DNA promoter region, (ii) a 126 base pairs long DNA corresponding to the precursor tRNA and (iii) a 25 base pairs long duplex DNA corresponding to 16 base pairs adjoining CCA end of tRNA and the remainder, a modified sequence including EcoRIendonuclease specific sequence. The details of the complete gene are shown in Figure 41.12. The complete synthetic gene was cloned in the vector bacteriophage (lambda virus) by gene cloning method discussed in Genetic Engineering and Biotechnology 1.  Recombinant DNA and PCR (Cloning and Amplification of DNA). On transformation of E. coli, the phage could multiply with the cloned gene.
Different parts of the complete E. coli tyrosine suppressor tRNA gene
Fig. 41.12. Different parts of the complete E. coli tyrosine suppressor tRNA gene (redrawn from Science, Vol. 203, 1979).

Total synthesis of a human leukocyte interferon gene.
Interferon is a protein, which has a property of inhibiting viral infection and cell proliferation and thus can be used for treatment of viral infection and malignancies (cancer). These properties have generated great interest in human interferons (IFNs). Atleast three kinds of interferon genes are known in human genome : (i) leukocyte interferon genes (IFN-α genes), (ii) fibroblast interferon gene (IFN-β genes) and (iii) immune interferon gene (IFN-γ genes). Genes for different kinds of interferons differ and details of base sequences are not known for all of them.

In 1980, Weismann and coworkers published the nucleotide sequence of first IFN-a gene derived from cloned IFN-α cDNA. On the basis of this information, total synthesis of this human leukocyte interferon gene, 514 base pairs long, was achieved and published in 1981 (Edge etal., 1981). Since there are 514 base pairs in the gene, it involved synthesis of DNA strands with 1028 nucleotides in predetermined way. This was the longest gene synthesized till 1982. The synthesis involved coupling of initial nucleotide on to a polyacrylamide resin to which further nucleotides could be added in pairs. Sixty six (66) oligonucleotide segments varying in size from 14 to 21 were first synthesized which were then arranged in a predetermined way and joined. This gene has been incorporated into a plasmid and is capable of synthesizing a-interferon. Further details about synthesis of this α-IFN gene are available in the original article published in Nature (Vol. 292, pages 756-761, 1981). The 514 base pairs coded for 166 amino acids = 498 base pairs, and include the initiation and termination signals. There are no intron sequences present in this gene.

The plasmid with this synthesized gene was transferred into bacteria (E. coli)which are being utilized now for synthesis of interferon in industry. This has greatly reduced the cost of production of this drug (interferon) which was earlier sold at the rate of Rs. 16 million per 50 mg.