Yeast frameshift suppressor mutations in the genes coding for transcription factor Mbf1p and ribosomal protein S3: evidence for autoregulation of S3 synthesis.

The SUF13 and SUF14 genes were identified among extragenic suppressors of +1 frameshift mutations. SUF13 is synonymous with MBF1, a single-copy nonessential gene coding for a POLII transcription factor. The suf13-1 mutation is a two-nucleotide deletion in the SUF13/MBF1 coding region. A suf13::TRP1 null mutant suppresses +1 frameshift mutations, indicating that suppression is caused by loss of SUF13 function. The suf13-1 suppressor alters sensitivity to aminoglycoside antibiotics and reduces the accumulation of his4-713 mRNA, suggesting that suppression is mediated at the translational level. The SUF14 gene is synonymous with RPS3, a single-copy essential gene that codes for the ribosomal protein S3. The suf14-1 mutation is a missense substitution in the coding region. Increased expression of S3 limits the accumulation of SUF14 mRNA, suggesting that expression is autoregulated. A frameshift mutation in SUF14 that prevents full-length translation eliminated regulation, indicating that S3 is required for regulation. Using CUP1-SUF14 and SUF14-lacZ fusions, run-on transcription assays, and estimates of mRNA half-life, our results show that transcription plays a minor role if any in regulation and that the 5'-UTR is necessary but not sufficient for regulation. A change in mRNA decay rate may be the primary mechanism for regulation.

Quantitative immunofluorescence in single Saccharomyces cerevisiae cells.

We have developed a staining procedure that allows the simultaneous determination of intracellular amounts of DNA and an antigen in Saccharomyces cerevisiae with a single laser flow cytometer. The antigen, beta-galactosidase from a cloned lacZ gene, is inducible and is detected with an indirect immunofluorescent stain. Cell preparation procedures, specifically cell fixation and cell wall removal, have significant effects on measured levels of immunofluorescence and have been optimized to prevent cell loss and maximize immunofluorescence. Average immunofluorescent levels of cell populations expressing different levels of beta-galactosidase show excellent correlation with measurements of average beta-galactosidase activity per cell based on cleavage of o-nitrophenyl-beta-D-galactopyranoside. Experiments with yeast populations containing various numbers of copies of the cloned gene indicate that the relationship between immunofluorescence and antigen content also holds at the single-cell level. Correlated measurements of DNA and beta-galactosidase content on a single-cell level permit the investigation of cellular enzyme content as a function of cell cycle position under various conditions. The procedure can be easily modified to detect other antigens by changing the primary antibody used.