Isolation of genes coding for known specific proteins

Steps involved in the isolation of a gene coding for a specific protein
Fig. 41.1. Steps involved in the isolation of a gene coding for a specific protein.
Isolation of genes for specific proteins became possible only after the discovery of reverse transcriptase enzyme in 1970. This enzyme can be easily used for the synthesis of copy DNA or complementary DNA (cDNA) from mRNA. This complementary DNA can then be used for the isolation of corresponding gene from genomic DNA. It is therefore obvious that for the isolation of a specific gene, techniques should first be available for the isolation of specific mRNA. For this purpose, antibodies are produced against a specific protein for which the gene is to be isolated. Therefore, isolation of a gene coding for a specific protein involves the following basic steps (Fig. 41.1) : (i) purification of the protein product of the gene; (ii) production of antibodies against this protein product by immunizing animals like rabbit or mouse; (iii) precipitation of polysomes engaged in synthesizing specific protein with the help of the antibody raised in step (ii); (iv) isolation and purification of mRNA from the polysome fraction; (v) using mRNA for synthesizing cDNA with the help of reverse transcriptase; (vi) cloning of cDNA thus synthesized for preparation of a cDNA library; (vii) immunological and electrophoretic analysis of the translation products of cDNA clones to identify specific cDNA clone meant for the specific protein in mind; (viii) use of the specific cDNA probes selected in step (vii) for the identification and isolation of the gene from genomic DNA though screening a complete or partial genomic library (for preparation of cDNA and genomic DNA libraries, consult Genetic Engineering and Biotechnology 1.  Recombinant DNA and PCR (Cloning and Amplification of DNA)).
Steps involved in the isolation of a gene coding for a specific protein
Fig. 41.1. Steps involved in the isolation of a gene coding for a specific protein.

In step (vi) above, for preparation of cDNA library, cDNA may be cloned in expression vectors (vectors in which gene can be expressed due to the presence of promoter sequences for RNA polymerase enzyme) like λgt11, which can accept gene inserts into β galactosidase gene. The chimeric vector will then produce hybrid protein if correctly expressed. The hybrid protein can be identified using antibody as above. Plaques belonging to the chimeric vector carrying the desired gene can also be identified by the reaction of the antibody attached to a radioactive protein. The procedure was used to clone a number of genes including a maize gene regulating zein synthesis.

Steps involved in the isolation of a gene with tissue specific expression using its mRNA
Fig. 41.2. Steps involved in the isolation of a gene with tissue specific expression using its mRNA.
The cDNA clones may also be used directly for gene manipulation and transformation experiments. However, when cDNA clone is used, it should be described as synthesis of gene rather than as isolation of gene, since cDNA has been artificially synthesized, and not isolated from the organism. The above technique for isolation of genes has now been successfully utilized both in plants and animals. However, the first genes isolated were those which existed in multiple copies and were expressed at high level in specific tissues e.g. ovalabumin gene in chicks, globin and immunoglobulin genes in mouse, genes for storage proteins in cereals and legumes, amylase genes in barley, actin genes in some legumes, etc.

Isolation of genes which are tissue specific in expression. It is much easier to isolate genes which are expressed in specific tissues. For instance, genes for storage proteins are expressed only in developing seeds, ovalbumin gene is expressed in oviduct and globin gene is expressed in erythocytes. Such genes can be easily isolated because mRNA extracted from these specific tissues will either exclusively belong to the gene of interest or it will be rich in this species of mRNA. Other mRNA molecules in minor quantities can be eliminated, since these can be identified through their isolation from tissues where this gene is silent. This strategy which is outlined in Figure 41.2, was actually followed for isolation and cloning of carrot genes expressed during the development of the somatic embryos, and also in the isolation of several genes for storage proteins in crop plants.
Steps involved in the isolation of a gene with tissue specific expression using its mRNA
Fig. 41.2. Steps involved in the isolation of a gene with tissue specific expression using its mRNA.

Use of DNA or RNA probes in isolation of genes. Specific molecular probes (whether DNA or RNA probes), if available can be used for isolation of specific genes. These probes may be available either from another plant species for the same gene or may be artificially synthesized using a part of the amino acid sequence of the protein product of the gene in question.

Steps involved in the isolation of a gene using the probe, artificially synthesized on the basis of amino acid sequence of a part of the protein
Fig. 41.3. Steps involved in the isolation of a gene using the probe, artificially synthesized on the basis of amino acid sequence of a part of the protein.
Use of heterologous probes.
The probes obtained from one plant species and used for another plant species are called heterologous probes. These heterologous probes have been found to be effective in identifying gene clones during colony hybridization or plaque hybridization or on Southern blots. For instance, the gene for chalcone synthase (CHS) has been isolated from Antirrhinum majus and Petunia hybrida using heterologous cDNA probe from parsley. Similarly, heterologous Antirrhinum cDNA probe was used for isolation of CHS gene from barley, and heterologous probes from maize were used for isolation of barley genes Wx (waxy gene) and A1 (aleurone gene). Heterologous probes should ordinarily be used with cDNA library and not with the genomic library, since in the latter case, unrelated genes or pseudogenes (which do not express) may be isolated and cloned.

Use of cDNA or synthetic probes. We have already discussed the synthesis of cDNA probes above. However- if protein purified using the technique of two dimensional gel electrophoresis, is used for microsequencing of 5-15 consecutive amino acids, this information can be used for the synthesis of oligonucleotides (using automated DNA synthesizers). These oligonucleotides may then be directly utilized for screening of cDNA or genomic libraries for isolation of specific genes (Fig. 41.3). These can also be used as primers in PCR for the synthesis of cDNA using mRNA isolated from a tissue. A 32 kilodalton (kD) glycoprotein (isolated from style) associated with the S2 incompatibility allele of Nicotiana alata, was used for the synthesis of an oligonucleotide leading to the isolation of a related gene.
Steps involved in the isolation of a gene using the probe, artificially synthesized on the basis of amino acid sequence of a part of the protein
Fig. 41.3. Steps involved in the isolation of a gene using the probe, artificially synthesized on the basis of amino acid sequence of a part of the protein.

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