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Isolation of theTRP2and theTRP3genes ofSaccharomyces cerevisiaeby functional complementation in yeast

✍ Scribed by Markus Aebi; Peter Niederberger; Ralf Hütter

Publisher
Springer-Verlag
Year
1982
Tongue
English
Weight
729 KB
Volume
5
Category
Article
ISSN
0172-8083

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✦ Synopsis

This paper describes the isolation of the TRP2 and the TRP3 genes of Saecharomyces cerevisiae. Two pools of plasmids consisting of BamHI and SalGI yeast DNA inserts into the bifunctional yeast -Escherichia coli vector pLC544 (IOngsman et al. 1979) were constructed in E. eoli and used for the isolation of the two genes by selection for functional complementation of trp2 and trp3 mutations, respectively, in yeast.

The TRP2 gene was isolated on a 6.2 kb BamHI and a 5.8 kb SalGI yeast DNA fragment which shared an identical 4.5 kb BamHI-SalGI fragment. The TRP3 gene was located on a 5.2 kb BamHI fragment.

By physical, genetic and physiological experiments it could be shown that the cloned yeast DNA fragments contained the whole structural sequences as well as the regulatory regions of the TRP2 and the TRP3 genes.

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The structure and function of the TRP3 gene of Saccharomyces cerevisiae were analyzed. Subcloning of an original 4.8 kb BamHI DNA fragment, carrying the yeast TRP3 gene, allowed for a localization of the gene on a 2.5 kb ClaI/BamHI fragment. Transcription was found to proceed from the ClaI site towa