Joe Horecka

Cloning and characterization of the Saccharomyces cerevisiae LYS7 gene: evidence for function outside of lysine biosynthesis

The Saccharomyces cerevisiae LYS7 gene has been cloned based on its genetic map position and complementation of a lys7 mutant. The 1453-bp sequence contains an open reading frame (ORF) that predicts a unique 249 amino acid (aa) protein. A Northern blot experiment demonstrated that LYS7 transcription was not regulated by lysine-specific or general aa control mechanisms. To investigate the effects of total loss of LYS7 function, we created a complete deletion of the gene and introduced this allele into wild-type yeast. The lys7 delta mutant requires lysine and simultaneously displays an array of phenotypes that include pH and temperature sensitivity. The pleiotropic phenotypes of the lys7 delta mutant and the constitutive transcription pattern are at odds with the hypothesis that Lys7p functions solely in the lysine biosynthesis pathway.

[7] Use of imidazoleglycerolphosphate dehydratase (His3) as a biological reporter in yeast

Publisher Summary The use of imidazoleglycerolphosphate dehydratase ( HIS3 ) reporters for growth selection has been incorporated into the yeast two-hybrid system and its variations for detecting protein–protein and protein–nucleic acid interactions. HIS3 reporters can also be used in screens to identify mutants that decrease reporter expression, and thereby render cells histidine auxotrophs. This chapter presents materials and methods for the construction and use of HIS3 reporters in yeast. Subclone transcriptional regulatory sequences from your favorite gene ( YFG1 ), upstream of the HIS3 -coding region in a reporter plasmid, stably integrate the YFG1–HIS3 reporter at an innocuous locus in the yeast genome, and then assay reporter activity. The YFG1–HIS3 reporter should be the strains only source of HIS3 expression, and the chapter describes materials and methods to alter the native HIS3 locus. The chapter also highlights potential pitfalls associated with HIS3 reporters and the ways to avoid them. The interests lie in the use of HIS3 reporters to probe signal transduction pathways and trans-acting factors that regulate gene expression in yeast.