The MET25 gene of Saccharomyces cerevisiae was cloned by functional complementation after transformation of a yeast met25 mutant. Subcloning of the DNA fragment bearing MET25 located the gene on a 2.3 kb region. The gene was formally identified by integration at the chromosomal MET25 locus. The cloned MET25 gene was used as a probe to measure the MET25 messenger RNA in a wild-type strain grown under conditions which promoted or failed to promote repression of MET25 expression. It was found that, under repression conditions, MET25 messenger RNA was reduced tenfold when compared with non-repression conditions. This suggests that the expression of MET25 is regulated transcriptionally. The direction of transcription, the size of the transcript and the position of the transcribed part of the gene were determined. Deletion mapping of the regulatory region was carried out. Deleted plasmids were introduced back into yeast cells and tested for their ability to complement met25 mutations and to promote regulation of expression of the MET25 gene by exogenous methionine. By this method the regulatory region was found to be confined to a 130 bp region.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Sangsoda S, Cherest H, Surdin-Kerjan Y

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