Artificial Synthesis of Gene

For the first time in 1955, Michelson chemically synthesized a dinucleotide in laboratory. Later on in 1970, Har Govind Khorana and K.L. Agarwal for the first time chemically synthesized gene coding for tyrosine tRNA of yeast. For the synthesis of tRNA and rRNA there are specific genes. However, genes oftRNA are the smallest genes containing about 80 nucleotides. In 1965, Rober W. Holley and coworkers worked out first the molecular structure of yeast alanine tRNA. This structure lent support to Khorana in deduction of structure of the gene. A gene is responsible for encoding mRNA, and mRNA for polypeptide chain. If the structure of a polypeptide chain is known, the structure of mRNA from genetic code dictionary and in turn the structure of gene can easily be worked out. There are two approaches for artificial synthesis of the gene, by using chemicals and through mRNAs.

Synthesis of a Gene for Yeast alanine tRNA
As mentioned earlier that the molecular structure of yeast alanine tRNA was worked out by R.W. Holley and coworkers in 1965 which helped Khorana to deduce the structure of alanine tRNA. They found out that yeast alanine tRNA contains 77 base pairs. It was very difficult to assemble 77 base pairs of nucleotides in ordered form. Therefore, they synthesized chemically the short deoxynucleotide sequences which was joined by hydrogen bonding to form a long complementary strand. By using polynucleotide ligase the double stranded pieces were produced. The complete procedure of synthesizing gene for yeast alanine tRNA is discussed in the following steps.

(i) Synthesis of Oligonucleotides. In the first approach, fifteen oligonucleotides ranging from pentanucleotide (i.e. oligodeoxynucleotide of five bases) to an icosanucleotide (i.e. oligodeoxynucleotide of twenty bases) were synthesized. The chemical synthesis was brought about through condensation between the - OH group at 3' position of one deoxynucleotide and the - PO₄ group at 5' position of the second deoxynucleotide. All other functional groups of deoxyribonucleotides not taking part in condensation processes were protected so that the condensation could be brought about. The deoxynucleotides were protected as below:

(a) The amino group of deoxyadenosine was protected by benzyl group, and the amino group of deoxycytidine was protected by anisoyl group, the amino group of deoxyguanosine was protected by isobutyl group. These protective groups were removed by treating with ammonia when synthesis was over.

(b) The hydroxyl group (-OH) at the 5' position of receiving deoxynucleotide was protected by cyanoethyl group (HN-CH₂-CH₂-).

(c) The -OH group at the 3' position of second or incoming deoxynucleotide was protected by acetyl group.

The different protecting groups used and treatment required to remove the protecting groups are given in Table 2.4. When the groups of deoxynueleotides were protected, the products reacted to form deoxyoligonucleotide. When deoxynueleotides were condensed into oligonucleotides, different protecting groups were removed by treating with ammonia, acid or alkali (Table 2.5). For example, both the cyanoethyl group at the 5' position and the acetyl group at the 3' position were removed by alkali treatment.

Table 2.5. Different protective groups of nucleotides and their removal
Finally, condensation between the groups of two, three or four nucleotides was brought about. The receiving segment had a free 3'-OH group and a protected 5'-OH group, whereas the incoming segment had a free 5'-OH group and a protected 3'-OH group. After each addition, the protective group at the 3' end had removed so that free 3'-OH group could receive another segment.

(ii) Synthesis of three duplex fragments of a gene. By using 15 single stranded oligonucleotides, three large double stranded DNA fragments were synthesized. These three fragments contained (i) segment of A having the first 20 nucleotides with the nucleotides 17-20 as the single stranded, (ii) segment B consisting of nucleotides 17-50 with the nucleotides 17-20 and 46-50 as the single stranded, and (iii) segment C containing the nucleotides 46-77 with the single stranded region 46-50.(iii) Synthesis of a gene from three duplex fragments of DNA. The three segments (A,B,C) synthesized is above were joined by using the enzyme polynucleotide ligase to produce the complete gene for alanine tRNA (Fig. 2.12).

The joining of the three fragments was done by any of two following methods:
(a) In one approach, fragment A was joined to B by taking advantage of overlapping in nucleotide residues 17-20. Then, the fragment C was added with the overlap in nucleotides 46- 50. Thus, a complete double stranded DNA with 77 base pairs was prepared.
(b) In the second approach, the fragment B was joined to C. At the end the fragment A was added to nucleotide residues 17-20 to obtain the complete gene for alanine tRNA.
Khorana et. al. (1970) prepared this gene in vitro which was used for future work. They found that alanine tRNA gene replicated and transcribed into tRNA just like the natural gene. It is not known whether tRNA prepared from artificially synthesized gene had the molecular organization similar to alanine tRNA or not.

Artificial Synthesis of a Gene for Bacterial tyrosine tRNA
In 1975, Khorana and co-workers completed the synthesis of a gene for E. coli tyrosine tRNA precursor. E. coli tRNA precursors are formed from the larger precursors. The tyrosine tRNA precursor has 126 nucleotides. They synthesized the complete sequence of DNA duplex coding for tyrosine tRNA precursor of E. coli, and promoters are terminator genes. Though these segments are not the proper structural gene yet are the regions involved in its regulation.

Twenty six small oligonucleotide DNA segments giving rise to tRNA precursor were synthesized which were arranged into six double stranded fragments each containing single stranded ends. These six fragments were joined to give rise complete gene of 126 base pairs for tyrosine tRNA precursor of E. coli.

Khorana (1979) completely synthesized a biologically functional tyrosine tRNA suppressor gene of E. coli which was 207 base pairs long and contained (i) a 51 base pairs long DNA corresponding to promoter region, (ii) a 126 base pair long DNA corresponding to precursor region of tRNA, (iii) a 25 base pair long DNA including 16 base pairs contained restriction site for EcoRI. This complete synthetic gene was joined in phage lambda vector which in turn was allowed to transfect E. coli cells. After transfection phage containing synthetic gene was successfully multiplied in E. coli.

Khorana (1979) made the phosphodiester approach for synthesizing the oligonucleotides of the biologically active tRNA. The demerits of this approach are: (i) the completion of reaction in long time, (ii) rapidly decrease in yield with the increase in chain length, and (iii) time taking procedure of purification.

Artificial Synthesis of a Human Leukocyte Interferon Gene
Interferons are proteinaceous in nature produced in human to inhibit viral infection. These are of three types secreted by three genes i.e. (i) leukocyte interferon gene (IFN-oc gene), (ii) fibroblast interferon gene (IFN-p gene), and (iii) immune interferon gene (IFN-γ gene). In 1980, Weismann and co-workers published the nucleotide sequence of IFN-a gene. Taking advantage of this information Edge et al. (1981) successfully synthesized the total human interferon gene of 514 base pairs long.

Edge et al. (1981) made the phosphotriester approach in artificially synthesizing 67 oligonucleotides of 10-20 nucleotide residue long segment. The phosphotriester approach overcomes some of the demerits of phosphodiester approach by blocking the function of each internucleotide phosphodiester during the process of synthesis. A completely protected mononucleotide containing a fully masked 3' phosphate triester group is used in this method.

Coupling of initial nucleotide onto a polyacrylamide resin was done to which further nucleotides in pairs were added. In this way 66 oligonucleotides of 14-21 nucleotide residues were first synthesized. These were arranged in predetermined ways and joined chemically. The 514 base" pairs long IFN-a gene contained the initiation and termination signals.

Edge et. al. (1981) incorporated the artificially synthesized gene into a plasmid through biotechnological technique (see Tools of Genetic Engineering and Techniques of Genetic Engineering). The recombinant plasmid was transferred into E.coli cells which expressed oc-interferon. This technique now-a-days is being adopted to produce interferon commercially.