The PHO80/TUP7 locus in Saccharomyces cerevisiae is on the left arm of chromosome XV: mapping by chromosome engineering.

The PHO80/TUP7 locus in Saccharomyces cerevisiae is reported to be located on the right arm of chromosome XV close to its centromere. In the present study, the locus has been reassigned to the left arm of the same chromosome by reciprocal recombination between chromosomes V and XV at URA3 (on chromosome V) and PHO80/TUP7 loci by using the site-specific recombination system of the yeast plasmid pSR1.     

Mapping of gene controlling thiamine transport in Saccharomyces cerevisiae

A recessive mutation leading to complete loss of thiamine uptake in Saccharomyces cerevisiae was mapped on the left arm of chromosome VII, approximately 56cM centromere-distal to trp5. As the analysed locus is relatively distant from its centromere and from the markers used, its attachment to chromosome VII was confirmed by chromosome loss methods.     

A Ser/Thr-rich multicopy suppressor of a cdc24 bud emergence defect.

MSB2 was identified previously as a multicopy suppressor of a temperature-sensitive mutation in CDC24, a gene required for polarity establishment and bud formation in Saccharomyces cerevisiae. The inferred MSB2 product contains 1306 amino acids, 42% of which are Ser or Thr. Its Ser+Thr-richness and hydrophobicity profile suggest that Msb2p may be an integral membrane protein containing a long, periplasmic, N-terminal domain and a short, cytoplasmic, C-terminal domain. Cells that lack MSB2 display no obvious mutant phenotypes. MSB2 is located between the centromere and KSS1 on the right arm of chromosome VII. Although physical mapping suggests that MSB2 and LEU1 (on the left arm of chromosome VII) are approximately 40 kb apart, the genetic map distance observed between leu1 and an msb2::URA3 marker was only 2.3 cM.     

II. Yeast sequencing reports. The two genes encoding yeast ribosomal protein S8 reside on different chromosomes,and are closely linked to the hsp70 stress protein genes SSA3 and SSA4

A 7·4 kb segment of chromosome II was sequenced and analysed. This segment is part of the 25 kb insert of cosmid clone α1004.10 which is located on the left arm of chromosome II. Sequence analysis revealed four open reading frames (ORFs), of which two had been characterized previously (SSA3, AAR2) and one was not identified. The other ORF was precisely 600 bp long and the deduced protein sequence predicted a very basic protein (pI=11·1; molecular weight=22·5 kDa). Evidence was found that the ORF is the S40 ribosomal protein gene (RPG) S8. Consensus splice signals were found in the 5′ leader sequence and also potential RPG-specific sequences. Chromoblot analysis revealed a second copy of the S8 RPG on chromosome IV or VIII. This copy is also closely linked to an hsp70 protein gene, SSA4. The sequence has been deposited in the EMBL data library under accession number Z26879.     

Chromosome V loss due to centromere knockout or MAD2-deletion is immediately followed by restitution of homozygous diploidy in Saccharomyces cerevisiae

To investigate the possibility of inducing specific chromosome loss by centromere deletion in eukaryotic cells, the yeast diploid strain ZG1, carrying three pairs of heterozygous marker genes (CAN1(S)/can1(R), URA3/Deltaura3, hphMX4/HIS1), widely spread on both arms of chromosome V, was constructed. One of the two centromeres V of ZG1 was replaced by the LEU2 gene via the well-established PCR-mediated knockout technique. After DNA transformation, putative yeast colonies that showed loss of heterozygosity (LOH) for the three markers of chromosome V (CAN1(S) URA3 hphMX4) were identified among the colonies selected for leucine prototrophy. Phenotypic tests, colony-PCR and Southern blot analysis of these cells demonstrated the physical loss of the CAN1(s), URA3, and hphMX4 marker genes from the genome. Further tetrad analysis results were consistent with this conclusion; however, four-spore viability indicated a normal chromosome number of these transformants. To verify the diploidy of the selected chromosome V, the HIS1 gene was deleted with a standard KanMX4 knockout DNA cassette. The resulting heterogeneity of the HIS1/KanMX4 markers, together with quantitative PCR and densitometric analysis on chromosome V, confirmed its diploid complement, thereby indicating that an endoreduplication event had taken place. Restitution of diploidy also occurred in MAD2-deleted strains undergoing higher rates of spontaneous chromosome V loss, indicating a more general phenomenon that is undetectable by phenotypic analysis alone.     

The product of the YCR105 gene located on the chromosome III from Saccharomyces cerevisiae presents homologies to ATP-dependent permeases.

During the systematic sequencing of chromosome III from Saccharomyces cerevisiae, carried out by a network of laboratories sponsored by the Commission of the European Community, we have identified the open reading frame YCR105 located on fragment J11D from the circular derivative of chromosome III in strain XJ24-24a (Palzkill et al., 1986). YCR105 is immediately centromere proximal to the PGK gene (opposite strand) on the right arm of chromosome III about 20 kb from the centromere.     

Sequence of a 12.7 kb segment of yeast chromosome II identifies a PDR-like gene and several new open reading frames.

A 12,684 bp DNA fragment, between FUS3 and the centromere, from the left arm of chromosome II of Saccharomyces cerevisiae was sequenced as part of the European project to sequence the whole chromosome. This segment contains at least five complete new open reading frames (ORFs) and the beginning (191 first 5' codons) of an ORF whose putative translational product is highly similar to the multidrug resistance PDR1 gene previously characterized by Balzi et al. (1987) on chromosome VII.     

Systematic hybrid LOH: a new method to reduce false positives and negatives during screening of yeast gene deletion libraries

We have developed a new method, systematic hybrid loss of heterozygosity, to facilitate genomic screens utilizing the yeast gene deletion library. Screening is performed using hybrid diploid strains produced through mating the library haploids with strains from a different genetic background, to minimize the contribution of unpredicted recessive genetic factors present in the individual library strains. We utilize a set of strains where each contains a conditional centromere construct on one of the 16 yeast chromosomes that allows the destabilization and selectable loss of that chromosome. After mating a library gene deletion haploid to such a conditional centromere strain, which corresponds to the chromosome carrying the gene deletion, loss of heterozygosity (LOH) at the gene deletion locus can be generated in these otherwise hybrid diploids. The use of hybrid diploid strains permits complementation of any spurious recessive mutations in the library strain, facilitating attribution of the observed phenotype to the documented gene deletion and dramatically reducing false positive results commonly obtained in library screens. The systematic hybrid LOH method can be applied to virtually any screen utilizing the yeast non-essential gene deletion library and is particularly useful for screens requiring the introduction of a genetic assay into the library strains.     

Localization of the dominant flocculation genes FLO5 and FLO8 of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae three dominant flocculation genes, FLO1, FLO5 and FLO8 have been described. Until now only the FLO1 gene, which is located at chromosome I, has been cloned and sequenced. FLO5 and FLO8 were previously localized at chromosomes I and VIII respectively (Vezinhet, F., Blondin, B. and Barre, P. (1991). Mapping of the FLO5 gene of Saccharomyces cerevisiae by transfer of a chromosome during cytoduction. Biotechnol. Lett. 13 , 47–52; Yamashita, I. and Fukui, S. (1983). Mating signals control expression of both starch fermentation genes and a novel flocculation gene FLO8 in the yeast Saccharomyces. Agric. Biol. Chem. 47 , 2889–2896). This was not in agreement with our results. Here, we report the location of FLO5 and FLO8 on chromosomes VIII and I respectively. By induced chromosome loss and genetic mapping, the FLO5 gene was localized at the right end of chromosome VIII approximately 34 cM centromere distal of PET3. This is part of the region that is present both at chromosome I and chromosome VIII. The location of FLO5 in this area of chromosome VIII made it necessary to re-evaluate the localization of FLO8, which was previously thought to occur in this region. Both genetic and physical mapping showed that FLO8 is allelic to FLO1. Hence, there are only two known dominant flocculation genes, FLO1 and FLO5. Analysis of the nucleotide sequence of chromosome VIII of a non-flocculent strain revealed an open reading frame encoding a putative protein that is approximately 96% identical to the Flo1 protein. This suggests that both dominant flocculation genes encode similar, cell wall-associated, proteins with the same function in the flocculation mechanism.     

XI. Yeast sequencing reports. The yeast YKL741 gene situated on the left arm of chromosome XI codes for a homologue of the human ALD protein

The yeast gene YKL741 is situated on the left arm of chromosome XI, 12 kb closer to the centromere with respect to the previously localized PAS1 gene. The new yeast gene codes for a homologue of the human ALD protein (ALD: adrenoleukodystrophy). The similarity between the YKL741 protein and the ALD protein is very high in the C-terminal half, which contains an ATP-binding cassette characteristic of the ABC family of transporters. Additionally the YKL741 protein shows some similarity to the ALD protein in the N-terminal half in three putative transmembrane spanning domains. The sequence has been deposited in the EMBL data library under Accession Number X76133 SC YKL.     

Saccharomyces cerevisiae mutants defective in chromosome segregation

We have devised a genetic screen to identify trans-acting factors involved in chromosome transmission in yeast. This approach was designed to potentially identify a subset of genes encoding proteins that interact with centromere DNA. It has been shown that mutations in yeast centromere DNA cause aberrant chromosome segregation during mitosis and meiosis. We reasoned that the function of an altered centromere should be particularly sensitive to changes in factors with which it interacts. We constructed a disomic strain containing one copy of chromosome III with a wild-type centromere and one copy of chromosome III bearing the SUP11 gene and a mutant CEN3. This strain forms white colonies with red sectors due to nondisjunction of the chromosome bearing the mutant centromere. After mutagenesis we picked colonies that exhibited increased nondisjunction of the mutant chromosome as evidenced by increased red-white sectoring. Using this approach, we have isolated three trans-acting chromosome nondisjunction (cnd) mutants that are defective in maintaining chromosomes during mitosis in yeast.     

The sequence of an 8 kb segment on the left arm of chromosome II from Saccharomyces cerevisiae identifies five new open reading frames of unknown functions, two tRNA genes and two transposable elements.

The DNA sequence of an 8079 bp ClaI fragment located at 40 kb from the centromere on the left arm of chromosome II from Saccharomyces cerevisiae has been determined. Sequence analysis reveals five new open reading frames, tRNA(Gly) and tRNA(Leu) genes as well as sigma and truncated delta elements. The disruption of the three larger open reading frames shows that they are not essential for mitotic growth.     

Sequence of the CDC10 region at chromosome III of Saccharomyces cerevisiae

A 4.74 kb DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae, adjacent to the centromere region was sequenced. Four open reading frames with an ATG initiation codon and larger than 200 bp were found in this fragment. The largest open reading frame of 966 bp was identified as the CDC10 gene.     

Physical mapping of a centromere-proximal region of chromosome IV-l defines the placement of genes USO1, MBP1, PSA1 and SLC1

A physical map of a 14·5 kb region close to the centromere on the left arm of chromosome IV of Saccharomyces cerevisiae is presented. This map has been constructed by restriction analysis of a clone from a YCp50 genomic library and by use of pre-existing and new sequence data from this region. The map reveals the following gene order (reading from the most centromere-distal to the most centromere-proximal locus): USO1/INT1–MBP1–PSA1–SLC1–YLA1 and defines the size of the open reading frames and intergenic regions.     

High-Resolution cosmid mapping of the left arm of Saccharomyces cerevisiae chromosome XII; A first step towards an ordered sequencing approach

For the sequencing of the left arm of chromosome XII of Saccharomyces cerevisiae, we fine-mapped the entire 450 kb fragment between the ribosomal DNA (rDNA) and the left telomere. Total yeast DNA in agarose blocks was digested with I-PpoI, which exclusively cuts once in each repeat unit of the rDNA. The resulting fragment was isolated from pulsed-field gels, together with the equally sized chromosome IX. A cosmid library of some 30-fold chromosome coverage was generated from this material, with the cloning efficiency being around 20 000 clones per microgram genomic DNA. The chromosome XII and IX specific clones were identified by complementary hybridizations with the respective chromosomes. For the left arm of chromosome XII, a contiguous cosmid array (contig) with an average map resolution better than 9 kb was generated by clone hybridization procedures. The ordered library serves as a tool for the physical mapping of genetic markers. Also, a minimal set of 15 clones was selected that covers the entire fragment. This subset forms the basis for the generation of a template map of much higher resolution for a directed sequencing of the left arm of chromosome XII.     

The complete sequence of a 10.8kb fragment to the right of the chromosome III centromere of Saccharomyces cerevisiae.

The complete nucleotide sequence of the D10H fragment (10850 bp) was determined. The D10H fragment is located on the right arm of chromosome III near the centromere and contains the SUF2 gene. Six open reading frames (ORFs) larger than 300 bp were found. One of them is the CIT2 gene encoding the cytoplasmic citrate synthase. The others are new putative genes and show no significant similarity with any known gene. In addition two tRNA genes (Asn and Pro) and a solo delta element were identified. Two ORFs were disrupted; no peculiar phenotype was observed.