Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae.

Abstract

We have developed a large-scale screen to identify genes expressed at different times during the life cycle of Saccharomyces cerevisiae and to determine the subcellular locations of many of the encoded gene products. Diploid yeast strains containing random lacZ insertions throughout the genome have been constructed by transformation with a mutagenized genomic library. Twenty-eight hundred transformants containing fusion genes expressed during vegetative growth and 55 transformants containing meiotically induced fusion genes have been identified. Based on the frequency of transformed strains producing 13-galactosidase, we estimate that 80-86% of the yeast genome (excluding the rDNA) contains open reading frames expressed in vegetative cells and that there are 93-135 meiotically induced genes. Indirect immunofluorescence analysis of 2373 strains carrying fusion genes expressed in vegetative cells has identified 245 fusion proteins that localize to discrete locations in the cell, including the nucleus, mitochondria, endoplasmic reticulum, cytoplasmic dots, spindle pole body, and microtubules. The DNA sequence adjacent to the lacZ gene has been determined for 91 vegetative fusion genes whose products have been localized and for 43 meiotically induced fusions. Although most fusions represent genes unidentified previously, many correspond to known genes, including some whose expression has not been studied previously and whose products have not been localized. For example, Sec21-13-gal fusion proteins yield a Golgi-like staining pattern, Tyl-13-gal fusion proteins localize to cytoplasmic dots, and the meiosis-specific Mekl/Mre4-13-gal and Spol 1-13-gal fusion proteins reside in the nucleus. The phenotypes in haploid cells have been analyzed for 59 strains containing chromosomal fusion genes expressed during vegetative growth; 9 strains fail to form colonies indicating that the disrupted genes are essential. Fifteen additional strains display slow growth or are impaired for growth on specific media or in the presence of inhibitors. Of 39 meiotically induced fusion genes examined, 14 disruptions confer defects in spore formation or spore viability in homozygous diploids. Our results will allow researchers who identify a yeast gene to determine immediately whether that gene is expressed at a specific time during the life cycle and whether its gene product localizes to a specific subcellular location.

Vps52p, Vps53p, and Vps54p Form a Novel Multisubunit Complex Required for Protein Sorting at the Yeast Late Golgi

“…Additional mutants were obtained by transforming the same strains with a NotI-digested yeast genomic library containing random insertions of a Tn3-LacZ transposon cassette (Burns et al, 1994). CPY-secreting colonies were identified by an overlay assay (Roberts et al, 1991), and those that exhibited synthetic lethality with end4⌬ (as determined by the inability to grow in the presence of 5-FOA) were chosen for further study.…”

“…Linkage between the transposon insertion and the CPY secretion phenotype was tested by tetrad analysis after sporulating diploids obtained by backcrossing with the parental strains SEY6210 and SEY6211. Yeast genomic DNA containing the Tn3-LacZ cassette was recovered from each backcrossed allele as described previously, except that BamHI/SacI-cut pRSQ303 was used instead of YIp5 for plasmid rescue (Burns et al, 1994;Voos and Stevens, 1998). The insertion point of the transposon was determined by sequencing the recovered genomic DNA.…”

The dosage of chromatin proteins affects transcriptional silencing and DNA repair in Saccharomyces cerevisiae

“…UCC3505 and UCC2225 were mutagenized with transposon-containing genomic DNA plasmid libraries [16], a kind gift from M. Snyder. The libraries were mutagenized by the insertion of the mTn transposon that encodes the lacZ, LEU2, and b-lactamase genes [17].…”

“…In our genetic screen, we mutagenized two telomere-marked strains, the diploid UCC2225 and the haploid UCC3505 (for comparison), with transposon-containing genomic DNA libraries ( [16], Table 1). Because the transposon insertion sites were random, insertions often disrupted genes and created truncation mutations.…”

A Screen for Nigericin-Resistant Yeast Mutants Revealed Genes Controlling Mitochondrial Volume and Mitochondrial Cation Homeostasis

“…Screen for nigericin-resistant mutants: Yeast strain W303-1B was transformed with 0.5 mg of NotI-cut mTn-lacZ LEU2-mutagenized genomic integrative library (Burns et al 1994) using the LiAc/ssDNA/PEG method (Gietz and Woods 2002). Transformants were selected on UTAH plates (SD supplemented with 40 mg/liter uracil, 30 mg/liter tryptophan, 40 mg/liter adenine, and 30 mg/liter histidine).…”