Isolation of Microbial Genes for Cysteine Synthesis and Prospects for their Use in Increasing Wool Growth

Summary

One logical application of recombinant DNA technology is to engineer increased wool productivity by restoring the sheep’s capacity to synthesise essential amino acids. For example, there is substantial evidence that fleece growth in Australian sheep breeds is suboptimal because of an inadequate supply of cysteine or its essential precursor, methionine. With the advent of recombinant DNA techniques it has become feasible to initiate research directed at isolating the minimal requirement of two genes for cysteine synthesis from microbial sources and to express them in sheep via transgenesis. An essential requirement for this particular pathway is to have the two necessary enzymes produced in tissues that have access to sulphide, an obligatory substrate. This means that gene constructs inserted into the sheep genome by transgenesis must express in the gastrointestinal tract.

Our initial efforts were directed towards isolation and characterisation of the genes required. At present we have isolated and sequenced the cysE and cysM genes of Salmonella typhimurium. A major effort was given to identifying the coding sequences of each gene and the products they encode. Using in vitro transcription/ translation methods in conjunction with enzyme assays we have been able to demonstrate that these genes encode the enzymes serine acetyltransferase (SAT) and O-acetylserine sulphydrylaseB respectively. We have isolated the yeast MET17 gene and shown this to be identical to the MET25 gene, isolated previously and shown to encode the bifunctional O-acetylhomoserine, O-acetylserine, sulphydrylase. We have also characterised the yeast CYS1 gene which, when mutated, affects SAT activity. This gene, however, was found not to be the structural gene for SAT but appears to encode another enzyme involved in cysteine/methionine biosynthesis. This finding is indicative of enzyme interactions and suggests that a complex of cysteine biosynthetic enzymes may exist in yeast.

The study has now entered the phase of fusing the coding sequences of the proven microbial genes to selective promoters for expression in cell systems. Our most useful trial system is an SV40-transformed sheep ruminal epithelial cell line which was constructed in our laboratory and will accept and replicate plasmid constructs with an SV40 origin. We have developed sensitive radioactive assays for both enzyme activities and these will be used to monitor the expression of fusion genes in cells and in transgenic animals.

Two promoters are under consideration for controlling the expression of these enzymes in sheep. The well characterised, zinc-controllable sheep metallothionein promoter will be utilised in initial studies, however a keratin-gene derived promoter may be ideal given that the rumen mucosa is a keratinising epithelium. With this in mind we are isolating keratin genes expressed in this tissue.