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.

Ty3 transposes in mating populations of yeast: a novel transposition assay for Ty3.

Ty3 is a retrotransposon of Saccharomyces cerevisiae that integrates just upstream of the transcription initiation site of genes transcribed by RNA polymerase III. Ty3 transcription is pheromone-inducible in haploid cells and is mating-type regulated in diploid cells. The specificity of Ty3 integration was exploited in the design of a novel target into which transposition of Ty3 elements could be selected. The target plasmid contains divergently oriented tRNA genes with 19 base pairs separating the two tRNA gene coding sequences. An inactive ochre suppressor tRNA(Tyr) gene with a modified transcription initiation region was used as the selectable marker and a tRNA(Val) (AAC) gene was used to direct Ty3 integration into the transcription initiation region of the suppressor tRNA(Tyr) gene. Integration of Ty3 activated expression of the suppressor tRNA gene, which resulted in suppression of ochre nonsense alleles ade2-101(0) and lys2-1(0) and allowed cell growth on selective medium. Based on the activity of this target, Ty3, under control of a galactose-inducible promoter and present on a high copy-number plasmid, was estimated to transpose into the genome at a rate of 5.6 x 10(-3) per cell division. We show here that induction of Ty3 transcription from its natural promoter results in transposition. Ty3 elements in strains of the a or alpha mating-type transposed efficiently to target plasmids in cells of the opposite mating-type. Thus, natural transposition of Ty3 is regulated temporally to occur in mating populations.

Positive and negative regulatory elements control expression of the yeast retrotransposon Ty3.

We report the results of an analysis of Ty3 transcription and identification of Ty3 regions that mediate pheromone and mating-type regulation to coordinate its expression with the yeast life cycle. A set of strains was constructed which was isogenic except for the number of Ty3 elements, which varied from zero to three. Analysis of Ty3 expression in these strains showed that each of the three elements was transcribed and that each element was regulated. Dissection of the long terminal repeat regulatory region by Northern blot analysis of deletion mutants and reporter gene analysis showed that the upstream junction of Ty3 with flanking chromosomal sequences contained a negative control region. A 19-bp fragment (positions 56-74) containing one consensus copy and one 7 of 8-bp match to the pheromone response element (PRE) consensus was sufficient to mediate pheromone induction in either haploid cell type. Deletion of this region, however, did not abolish expression, indicating that other sequences also activate transcription. A 24-bp block immediately downstream of the PRE region contained a sequence similar to the a1-alpha 2 consensus that conferred mating-type control. A single base pair mutation in the region separating the PRE and a1-alpha 2 sequences blocked pheromone induction, but not mating-type control. Thus, the long terminal repeat of Ty3 is a compact, highly regulated, mobile promoter which is responsive to cell type and mating.

Adjacent pol II and pol III promoters: transcription of the yeast retrotransposon Ty3 and a target tRNA gene.

The Saccharomyces cerevisiae retrotransposon Ty3 integrates 16 to 19 basepairs upstream of tRNA genes in a region where sequences have been shown to affect the expression of tRNA genes in vivo and in vitro. Sigma, the isolated long terminal repeat of Ty3, is also found in this region. The purpose of these experiments was to elucidate the effects of Ty3 and sigma expression on that of an associated SUP2 tRNA(Tyr) gene in vivo. SUP2 pre-tRNA levels were moderately increased when SUP2 was associated with Ty3 or sigma in either orientation. These increases were independent of Ty3 or sigma promoter activity. The presence of Ty3 or sigma also increased the usage of a minor SUP2 transcription initiation site 2 basepairs upstream of the major initiation site and within the 5 basepair direct repeat flanking Ty3 and sigma. Transcription from an isolated sigma directed toward the tRNA gene was observed to extend through the tRNA gene. In contrast to the lack of an effect of sigma induction on pre-tRNA(Tyr) levels, levels of this sigma transcript were increased when the SUP2 promoter was inactivated by a single basepair mutation.