Nature and distribution of large sequence polymorphisms in Saccharomyces cerevisiae

To obtain a better understanding of the genome-wide distribution and the nature of large sequence polymorphisms (LSPs) in Saccharomyces cerevisiae, we hybridized genomic DNA of 88 haploid or homozygous diploid S. cerevisiae strains of diverse geographic origins and source substrates onto high-density tiling arrays. On the basis of loss of hybridization, we identified 384 LSPs larger than 500 bp that were located in 188 non-overlapping regions of the genome. Validation by polymerase chain reaction-amplification and/or DNA sequencing revealed that 39 LSPs were due to deletions, whereas 74 LSPs involved sequences diverged far enough from the S288c reference genome sequence as to prevent hybridization to the microarray features. The LSP locations were biased toward the subtelomeric regions of chromosomes, where high genetic variation in genes involved in transport or fermentation is thought to facilitate rapid adaptation of S. cerevisiae to new environments. The diverged LSP sequences appear to have different allelic ancestries and were in many cases identified as Saccharomyces paradoxus introgressions.     

Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.     

Nature and timing of some sporulation-specific protein changes in Saccharomyces cerevisiae.

During meiosis and spore formulation in Saccharomyces cerevisiae, changes that occur in a/alpha diploids, but not in isogenic nonsporulating a/a diploids, have been detected in cellular polypeptides. These were found by the technique of prelabeling growing cells with 35SO4(2-) and suspending them in sulfur-free sporulation medium. Under the conditions used, about 400 polypeptides were detected by two-dimensional gel electrophoresis, and 45 were altered during sporulation; of these, 21 changes were specific to a/alpha strains. These alterations were mainly due to the appearance of new polypeptides or to marked increases in the concentrations of a few polypeptides produced during vegetative growth. They could have been due either to modifications of existing polypeptides present in growing cells or to de novo synthesis of new gene products. They occurred at characteristic times during sporulation; whereas the majority of changes took place early (within the first 6 h in sporulation conditions), there were several changes characterizing the later stages of sporulation. Ten of the 35SO4(2-)-labeled polypeptides were also labeled with 32P in the presence of [32P]orthophosphate; of these, three were previously found to be sporulation specific. One of these was phosphorylated at all stages of sporulation and was labeled when [32P]orthophosphate was added either during growth of the culture of 1 h after transfer to sporulation medium. Another was labeled in the same way by adding 32P at either time, so that by 7 h in sporulation medium it was phosphorylated, but was dephosphorylated by 24 h. The third sporulation-specific peptide was labeled in extracts prepared at 7 h in sporulation medium (but not at 24 h) when [32P]-orthophosphate was added during presporulation growth, but not when [32P]-orthophosphate was added 1 h after transfer of the culture to sporulation medium. This polypeptide appeared early during sporulation; it is probably phosphorylated as it appears and is dephosphorylated at some time between 7 h and 24 h of sporulation.     

Stable denaturation of chromosomal DNA from Saccharomyces cerevisiae during meiosis.

Partial denaturation of Saccharomyces cerevisiae chromosomal DNA was found to occur spontaneously during meiosis. Short regions of strand separation (300 base pairs long) were seen in DNA molecules prepared for electron microscopy by the aqueous spreading technique. These regions were clustered along the DNA. The time course of their appearance indicated that the denatured regions were present during the periods of premeiotic DNA replication and recombination. A similar pattern of denaturation was also detected in the DNA from vegetatively grown cells of a conditional cdc8 mutant, which is defective in DNA replication.     

Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae

We describe a novel chromosome engineering technique for shuffling selected regions of chromosomes from two strains in Saccharomyces cerevisiae: The technique starts with the construction of MAT a and MATα strains in which a particular chromosome is split at exactly the same site in both strains such that the split chromosomes generated are marked with different markers. The two strains are then crossed, and the resultant diploid is cultivated in nutrient medium to induce loss of the split chromosome originating from either of the strains. We predicted that some of these clones that are hemizygous for the split chromosome would spontaneously restore a homozygous configuration of the split chromosome during cultivation. We verified this prediction by tetrad analysis and quantitative Southern hybridization analysis, indicating that it is possible to create diploid hybrids in which a selected region of a chromosome from one strain is replaced by the corresponding chromosomal region from another strain. We also found that some chromosomal segments maintain a hemizygous state. This novel technique, which we call ‘chromosome shuffling’, could provide a new tool to analyze phenotypic alterations caused by the replacement or hemizygosity of a selected chromosomal region in not only laboratory but also industrial strains of S. cerevisiae.     

Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.

We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication Enhancer (RE) domain, has no inherent replication competence but is essential for ensuring maximum function of the RS domain. The nucleotide sequence of these domains reveals several conserved sequences one of which is strikingly similar to the yeast ARS consensus sequence.     

DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae.

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.     

Genetic selection for reciprocal translocation at chosen chromosomal sites in Saccharomyces cerevisiae.

We have constructed viable Saccharomyces cerevisiae strains containing a reciprocal translocation between the URA2 site of chromosome X and the HIS3 site of chromosome XV. Our methodology is an extension of the method originally developed to introduce an altered cloned sequence at the chromosomal location from which the parent sequence was derived (S. Scherer and R.W. Davis, Proc. Natl. Acad. Sci. U.S.A. 76:4951-4955, 1979). It comprises three essential steps. First, a nonreverting ura2- strain was constructed by deleting a 3.7-kilobase fragment from the coding sequence of the wild-type URA2 gene. Second, part of the coding sequence of the wild-type URA2 gene (without promotor) was inserted at the HIS3 locus of the ura2- strain. Third, after several generations of growth on uracil-supplemented medium, ura2+ colonies were selected which resulted from mitotic recombination between the nonoverlapping deletions of URA2 located on chromosomes X and XV.     

The ARS consensus sequence is required for chromosomal origin function in Saccharomyces cerevisiae.

Replication origins have been mapped to positions that coincide, within experimental error (several hundred base pairs), with ARS elements. To determine whether the DNA sequences required for ARS function on plasmids are required for chromosomal origin function, the chromosomal copy of ARS306 was deleted and the chromosomal copy of ARS307 was replaced with mutant derivatives of ARS307 containing single point mutations in domain A within the ARS core consensus sequence. The chromosomal origin function of these derivatives was assayed by two-dimensional agarose gel electrophoresis. Deletion of ARS306 deleted the associated replication origin. The effects on chromosomal origin function of mutations in domain A paralleled their effects on ARS function, as measured by plasmid stability. These results demonstrate that chromosomal origin function is a property of the ARS element itself.     

Regulation of gross chromosomal rearrangements by ubiquitin and SUMO ligases in Saccharomyces cerevisiae

Gross chromosomal rearrangements (GCRs) are frequently observed in many cancers. Previously, we showed that inactivation of Rad5 or Rad18, ubiquitin ligases (E3) targeting for proliferating cell nuclear antigen (PCNA), increases the de novo telomere addition type of GCR (S. Smith, J. Y. Hwang, S. Banerjee, A. Majeed, A. Gupta, and K. Myung, Proc. Natl. Acad. Sci. USA 101:9039-9044, 2004). GCR suppression by Rad5 and Rad18 appears to be exerted by the RAD5-dependent error-free mode of bypass DNA repair. In contrast, Siz1 SUMO ligase and another ubiquitin ligase, Bre1, which target for PCNA and histone H2B, respectively, have GCR-supporting activities. Inactivation of homologous recombination (HR) proteins or the helicase Srs2 reduces GCR rates elevated by the rad5 or rad18 mutation. GCRs are therefore likely to be produced through the restrained recruitment of an HR pathway to stalled DNA replication forks. Since this HR pathway is compatible with Srs2, it is not a conventional form of recombinational pathway. Lastly, we demonstrate that selection of proper DNA repair pathways to stalled DNA replication forks is controlled by the Mec1-dependent checkpoint and is executed by cooperative functions of Siz1 and Srs2. We propose a mechanism for how defects in these proteins could lead to diverse outcomes (proper repair or GCR formation) through different regulation of DNA repair machinery.     

Expression of human chromosomal proteins HMG-14 and HMG-17 in Saccharomyces cerevisiae

The cDNAs coding for human chromosomal proteins HMG-14 and HMG-17 were cloned into yeast expression vector pBM150, under the control of the Gal10 promoter. Northern analysis of transformed yeast cells revealed that both cDNAs were efficiently transcribed. Western analysis indicated that the mRNAs were translated into authentic proteins. Expression of human HMG proteins in yeast cell did not produce detectable phenotypic changes, as measured by the growth rate of the yeast cells under a variety of conditions. The antibiotic resistance of the transfected cells was similar to that of control cells, suggesting that the presence of HMG did not affect the expression of actively transcribed genes. However, examination of the protein profile on two-dimensional polyacrylamide gel electrophoresis revealed differences between control and HMG-transfected cells.     

Enhanced production of anticoagulant hirudin in recombinant Saccharomyces cerevisiae by chromosomal delta-integration

Recombinant Saccharomyces cerevisiae strains were developed to overproduce an anticoagulant hirudin. The delta-sequences of the yeast retrotransposon Ty1 and URA3 were used as target sites for a hirudin expression cassette. High copy-number transformants were successfully selected using a dominant selection antibiotic, G418. The copy numbers of the hirudin expression cassette integrated into delta-sequences of the yeast chromosome ranged from five to ten copies per cell. Production of hirudin in the delta-integrated recombinant S. cerevisiae system increased over two-fold compared with the YEp-based episomal hirudin expression system. A linear relationship between the copy number of the hirudin expression cassette and hirudin expression level was observed up to 10 copies. The hirudin expression cassettes integrated into the yeast chromosome were stably maintained in non-selective culture conditions.     

Reidentification of chromosomal CUP1 translocations in the wine yeasts Saccharomyces cerevisiae

Reciprocal translocations between chromosomes XVI and VIII were revealed in eight Saccharomyces cerevisiae strains (mostly wine ones) using pulse-field electrophoresis of native chromosomal DNAs and their hybridizations with the CUP1 and GAL4 probes. New and reciprocal translocations of at least the gene CUP1 occur at the expense of crossing-over in the hybrids of such strains with the genetic lines of normal karyotype during meiosis. Relationship between these reciprocal translocations and the sulfite (Na₂SO₃) resistance gene SSU1-R is discussed.     

Effects of reciprocal chromosomal translocations on the fitness of Saccharomyces cerevisiae

Yeast species have undergone extensive genome reorganization in their evolutionary history, including variations in chromosome number and large chromosomal rearrangements, such as translocations. To determine directly the contribution of chromosomal translocations to the whole organism's fitness, we devised a strategy to construct in Saccharomyces cerevisiae collinear "evolutionary mimics" of other species originally differing by the presence of reciprocal translocations in their genome. A modification of the Cre/loxP system was used to create in S. cerevisiae the translocations detected in the sibling species Saccharomyces mikatae IFO 1815 and 1816. Competition experiments under different physiological conditions showed that the translocated strains of S. cerevisiae consistently outcompeted the reference S. cerevisiae strain with no translocation, both in batch and chemostat culture, especially under glucose limitation. These results indicate that chromosomal translocations in Saccharomyces may have an adaptive significance, and lend support to a model of fixation by natural selection of reciprocal translocations in Saccharomyces species.     

Condensin binding at distinct and specific chromosomal sites in the Saccharomyces cerevisiae genome

Mitotic chromosome condensation is chiefly driven by the condensin complex. The specific recognition (targeting) of chromosomal sites by condensin is an important component of its in vivo activity. We previously identified the rRNA gene cluster in Saccharomyces cerevisiae as an important condensin-binding site, but both genetic and cell biology data suggested that condensin also acts elsewhere. In order to characterize the genomic distribution of condensin-binding sites and to assess the specificity of condensin targeting, we analyzed condensin-bound sites using chromatin immunoprecipitation and hybridization to whole-genome microarrays. The genomic condensin-binding map shows preferential binding sites over the length of every chromosome. This analysis and quantitative PCR validation confirmed condensin-occupied sites across the genome and in the specialized chromatin regions: near centromeres and telomeres and in heterochromatic regions. Condensin sites were also enriched in the zones of converging DNA replication. Comparison of condensin binding in cells arrested in G(1) and mitosis revealed a cell cycle dependence of condensin binding at some sites. In mitotic cells, condensin was depleted at some sites while enriched at rRNA gene cluster, subtelomeric, and pericentromeric regions.     

Nature of Ribonucleic Acid Synthesis During Early Sporulation in Saccharomyces cerevisiae

Phosphate uptake in sporulating cultures of Saccharomyces cerevisiae has been found to occur approximately 2 h after the transfer to sporulation medium. Early ribonucleic acid synthesis begins at approximately 4 h and continues to 8 h. Incorporation of phosphate into acid-extractable precursor pools parallels phosphate uptake. In triple-labeling experiments it was observed that the breakdown of vegetatively synthesized ribonucleic acid is not a significant source of precursors for ribonucleic acid synthesis during sporulation. The majority of the ribonucleic acid made in a 10-min period during sporulation does not migrate on gels with precursor or mature ribosomal ribonucleic acid.