Systematic disruption of 456 ORFs in the yeast Saccharomyces cerevisiae.

In the framework of the European Network for Functional Analysis (EUROFAN), five packages of 96 ORFs from chromosomes III, IV, VII, XIII, XIV and XV were subjected to systematic deletions in an isogenic derivative of strain S288c. Deletions were constructed in diploid and haploid strains. Two questionable ORFs overlapping with larger ORFs and seven TY ORFs were discarded. A total of 456 heterozygous and 385 homozygous deletant diploids were obtained. Sixty-nine deletions, 25 of which had never been published before, were lethal in haploid strains and 30 caused slow cellular growth.

Disruption and basic phenotypic analysis of 18 novel genes from the yeast Saccharomyces cerevisiae.

In the frame of the European Network for Functional Analysis (EUROFAN) we have deleted 18 yeast open reading frames (ORFs) from chromosomes II, X and XIV using the short flanking homology-PCR strategy. Two diploid strains were used: FY1679 and CEN.PK2. The deletion kanMX6 cassettes with long flanking homology and the cognate gene clones have also been constructed. Heterozygous diploid deletant strains have been sporulated. Tetrad analysis revealed that all the ORFs studied were non-essential. However, four deletant strains exhibited phenotypes. The YBL025w delta strain showed extremely slow cellular growth under all conditions tested. The YJL204c delta strain grew slower than wild-type at 30 degrees C and 37 degrees C, was cold-sensitive, and the homozygous diploids did not sporulate. The YNL213c delta strain did not grow on glycerol and had lost mitochondrial DNA. The deletion of YNL215w caused slower growth on all media but the defect was more pronounced on glucose-minimal and glycerol-rich media than on glucose-rich medium. All deletion mutants were complemented by the corresponding plasmid borne cognate gene. The YJL204w, YNL213c and YNL215w ORFs do not bear significant homology to proteins of known function. YBL025w has recently been identified as RRN10, a gene that encodes an RNA polymerase I-specific transcription initiator factor. The deletion of the remaining fourteen ORFs did not reveal any mutant phenotype in our basic growth tests.

The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae.

The currently available yeast mitochondrial DNA (mtDNA) sequence is incomplete, contains many errors and is derived from several polymorphic strains. Here, we report that the mtDNA sequence of the strain used for nuclear genome sequencing assembles into a circular map of 85,779 bp which includes 10 kb of new sequence. We give a list of seven small hypothetical open reading frames (ORFs). Hot spots of point mutations are found in exons near the insertion sites of optional mobile group I intron-related sequences. Our data suggest that shuffling of mobile elements plays an important role in the remodelling of the yeast mitochondrial genome.

In-frame recombination between the yeast H(+)-ATPase isogenes PMA1 and PMA2: insights into the mechanism of recombination initiated by a double-strand break.

Chimeric PMA1::PMA2 sequences, placed under the control of the PMA1 promoter, were constructed by in vivo recombination between a gapped linearized plasmid containing the PMA2 gene and four different fragments of the PMA1 gene. Correct in-frame assembly of the PMA sequences was screened by the expression of the lacZ reporter gene fused to the PMA2 coding region. Restriction and sequencing analysis of 35 chimeras showed that in all cases, the hybrid sequences was obtained as fusions between continuous sequences specific to PMA1 and PMA2, separated by a region of identity. In all but three cases, the junction sequences were not located at regions of greatest identity. Strikingly, depending on the PMA1 fragment used, junction distribution fell into two categories. In the first, the junctions were scattered over several hundreds of nucleotides upstream of the extremity of the PMA1 fragment, while in the second, they were concentrated at this extremity. Analysis of the alignment of the PMA1 and PMA2 sequences suggests that the distribution is not related to the size of the region of identity at the PMA1-PMA2 boundary but depends on the degree of identity of the PMA genes upstream of the region of identity, the accumulation of successive mismatches leading to a clustered distribution of the junctions. Moreover, the introduction of seven closely spaced mismatches near the end of a PMA1 segment with an otherwise-high level of identity with PMA2 led to a significantly increased concentration of the junctions near this end. These data show that a low level of identity in the vicinity of the common boundary stretch is a strong barrier to recombination. In contrast, consecutive mismatches or regions of overall moderate identity which are located several hundreds of nucleotides upstream from the PMA1 end do not necessarily block recombination.