The multifunctional regulatory proteins ABF1 and CPF1 are involved in the formation of a nuclease-hypersensitive region in the promoter of the QCR8 gene

The abundant DNA-binding proteins ABF1 and CPF1 are members of a family of global regulators with diverse chromosomal functions in the yeast Saccharomyces cerevisiae. Recent evidence suggests that these protein factors may be involved in establishing and maintaining well-defined chromatin structures in promoter regions and other genetic elements. We have investigated the involvement of ABF1 and CPF1 in chromatin organization at the QCR8 gene, encoding subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase. The promoter region of the QCR8 gene contains overlapping binding sites for ABF1 and CPF1. Nucleosome positioning studies indicate that the QCR8 gene is associated with a phased array of nucleosomes under both catabolite-repressed and derepressed growth conditions. Analysis of binding site mutants reveals that both ABF1 and CPF1 are involved in maintaining a nuclease-hypersensitive region in the QCR8 promoter. The chromatin structure at QCR8 during steady-state growth is, however, mainly dependent on binding of ABF1 to the promoter region. Implications of these findings for the role played by ABF1 and CPF1 in the regulation of mitochondrial biogenesis and other processes important for cell growth and division will be discussed.     

Genetic and molecular analysis of hybrids in the genus Saccharomyces involving S. cerevisiae,S. uvarum and a new species,S. douglasii

We have studied the phenomenon of infertility of yeast hybrids obtained with physiological conditions under the control of compatible mating systems. The yeasts investigated are three Saccharomyces species: S. cerevisiae, S. uvarum and a new species, S. douglasii. The diploid hybrids from crosses between these species sporulate well but are essentially infertile. The rare viable spores, one per 10⁴ to 10⁵ asci, that have been examined carry a complete genome comprised of chromosomes contributed by both parents but invariably have extra chromosomes, i.e. they are generally disomic for at least two or three chromosomes. This observation is consistent with a failure, in meiosis I, of the pairing and disjunction of homologous chromosomes which in most cases results in spores with an incomplete set of chromosomes. This apparent lack of pairing of ‘homeologous’ chromosomes in meiosis I was analysed in most detail with S. cerevisiae/S. douglasii hybrids. As a genetic tool we studied frequencies of recombination, taking advantage of an S. douglasii breeding stock of some 50 identified mutations in non-switching haploids. Recombination, although markedly reduced, could be observed at both the chromosomal and allelic levels, implying a sporadic pairing in meiosis to allow genetic exchange. Meiotic recombination frequencies were studied for 14 gene pairs and generally found to be reduced ten-fold. Heteroallelic recombination (gene conversion) frequencies were measured at 22 loci and were judged to be reduced at least two- to 100-fold. DNA hybridization experiments with S. cerevisiae gene probes gave results consistent with low DNA sequence homologies between S. cerevisiae and S. douglasii. Moreover, by chance, our experiments disclosed another Saccharomyces strain (CBS2908, originally classified as S. cerevisiae) with hybridization patterns identical to S. douglasii except for the hybridization with the Ty transposon probes. Crosses between CBS2908 and S. douglasii yielded diploid hybrids with 80–90% spore viability, thus establishing a second member of the S. douglasii species.     

Cloning and characterization of WMSU1, a Williopsis saturnus var. mrakii gene encoding a new yeast SUN protein involved in the cell wall structure

SUN proteins of Saccharomyces cerevisiae have been defined on the basis of high homologies in their C-terminal domain. Recently, two of these four proteins were shown to be involved in cell wall morphogenesis (Mouassite et al., 2000a). In the present study, we have isolated WMSU1 (Accession No. AF418983), a new SUN-related gene, from W. saturnus var. mrakii MUCL 41968. Sequencing of the gene revealed an open reading frame coding for 402 amino acids. The predicted amino acid sequence of WMSU1 is closely related to the S. cerevisiae SUN proteins and to other yeast proteins involved in cell wall metabolism. WMSU1 is proposed to encode a cell wall protein since its predicted product contains a signal sequence, a Kex2p cleavage site and a serine/threonine-rich N-terminal domain. Southern blot analysis of the W. saturnus var. mrakii MUCL 41968 genome using the highly conserved domain of WMSU1 as a probe suggested that the isolated gene belongs to a multigenic family. Expression of WMSU1 in E. coli led to a 45 kDa protein, which appeared to be toxic to this host. Scanning electron microscopy analysis of a recombinant S. cerevisiae producing Wmsu1p showed that this strain exhibited an altered cell wall, thus pointing to a probable role of this protein in the cell wall structure.     

IMP2, a nuclear gene controlling the mitochondrial dependence of galactose, maltose and raffinose utilization in Saccharomyces cerevisiae.

The IMP2 gene of Saccharomyces cerevisiae is involved in the nucleo-mitochondrial control of maltose, galactose and raffinose utilization as shown by the inability of imp2 mutants to grow on these carbon sources in respiratory-deficient conditions or in the presence of ethidium bromide and erythromycin. The negative phenotype cannot be scored in the presence of inhibitors of respiration and oxidative phosphorylation, indicating that the role of the mitochondria in the utilization of the above-mentioned carbon sources in imp2 mutants is not at the energetical level. Mutations in the IMP2 gene also confer many phenotypic alterations in respiratory-sufficient conditions, e.g. leaky phenotype on oxidizable carbon sources, sensitivity to heat shock and sporulation deficiency. The IMP2 gene has been cloned, sequenced and disrupted. The phenotype of null imp2 mutants is indistinguishable from that of the originally isolated mutant.     

PGK1, the gene encoding the glycolitic enzyme phosphoglycerate kinase,acts as a multicopy suppressor of apoptotic phenotypes in S. cerevisiae

In a previous paper we reported the construction of a S. cerevisiae strain lacking the essential gene LSM4, which could survive by the introduction of a truncated form of the orthologous gene from Kluyveromyces lactis. This strain showed apoptotic hallmarks and other phenotypes, including an increased sensitivity to caffeine and acetic acid. The suppression of the latter phenotype by overexpressing yeast genes allowed the isolation of PGK1, the gene encoding the glycolytic enzyme phosphoglycerate kinase. This gene restored normal ageing, oxygen peroxide resistance and nuclear integrity in the mutant. Other phenotypes, such as caffeine sensitivity and glycerol utilization, were also suppressed. Copyright © 2009 John Wiley & Sons, Ltd.     

X–XI. Yeast mapping reports. The use of random-breakage mapping to locate the genes APN1 and YUH1 in the Saccharomyces genome,and to determine gene order near the left end of chromosome XI

We have used the previously described technique of random-breakage mapping to locate the two yeast genes APN1 and YUH1. The APN1 locus is located ～235 kb from the left telomere of chromosome XI, and shows weak (～53 cM) genetic linkage to ura1. The YUH1 locus is located ～140 kb from the right telomere of chromosome X, and genetically maps 3·6 cM distal to cdc11. In addition, we show by random-breakage mapping that TRP3 is located ～45 kb from the left telomere of chromosome XI, whereas FAS1 is ～110 kb from the same telomere. This supports a gene order on the left distal portion of chromosome XI that agrees with other physical reports but is inverted with respect to Edition 11 of the published genetic map. This report confirms that random-breakage mapping is a rapid and convenient method of locating cloned genes.