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.     

The diversity of retrotransposons in the yeast Cryptococcus neoformans.

We have undertaken an analysis of the retrotransposons in the medically important basidiomycetous fungus Cryptococcus neoformans. Using the data generated by a C. neoformans genome sequencing project at the Stanford Genome Technology Center, 15 distinct families of LTR retrotransposons and several families of non-LTR retrotransposons were identified. Members of at least seven families have transposed recently and are probably still active. For several families, only partial elements could be identified and these are quite diverse in sequence, suggesting that they are ancient components of the C. neoformans genome. Most C. neoformans elements are not closely related to previously identified fungal retrotransposons, suggesting that the diversity of fungal retrotransposons has been only sparsely sampled to date. C. neoformans has fewer distinct retrotransposon families than Candida albicans (37 or more), in particular fewer families represented solely by ancient and inactive elements, but it has considerably more families than either Saccharomyces cerevisiae (five) or Schizosaccharomyces pombe (two). The findings suggest that elimination of retrotransposons is faster in C. neoformans than in C. albicans, but perhaps not as rapid as in S. cerevisiae or Sz. pombe. The identification of the retrotransposons of C. neoformans should assist in the molecular characterization of this important pathogen, and also further our understanding of the role played by retroelements in genome evolution.     

Characterization of a disulphide-bound Pir-cell wall protein (Pir-CWP) of Yarrowia lipolytica

In this work we have studied the disulphide-bound group of cell wall mannoproteins of Yarrowia lipolytica and Candida albicans. In the case of Y. lipolytica, SDS-PAGE analysis of the beta-mercaptoethanol-extracted material from the purified cell walls of the yeast form, showed the presence of a main polypeptide of 45 kDa and some minor bands in the 100-200 kDa range. This pattern of bands is similar to that obtained in identical extracts in Saccharomyces cerevisiae (Moukadiri et al., 1999), and besides, all these bands cross-react with an antibody raised against beta-mercaptoethanol-extracted material from the purified cell walls of S. cerevisiae, suggesting that the 45 kDa band could be the homologue of Pir4 of S. cerevisiae in Y. lipolytica. To confirm this possibility, the amino-terminal sequences of two internal regions of the 45 kDa protein were determined, and degenerate oligonucleotides were used to clone the gene. The gene isolated in this way codes a 286 amino acid polypeptide that shows homology with the Pir family of proteins of S. cerevisiae (Russo et al., 1992; Toh-e et al., 1993), accordingly we have named this gene YlPIR1. Disruption of YlPIR1 led to a slight increase in the resistance of the cells to calcofluor white, Congo red and zymolyase, but did not cause changes in cell morphology, growth rate or morphological transition.     

The complete sequence of a 6146 bp fragment of Saccharomyces cerevisiae chromosome III contains two new open reading frames.

As part of the EEC project to sequence the entire chromosome III of Saccharomyces cerevisiae we have sequenced a total of 11,040 bp from near the right end of the chromosome. A new protein kinase gene was found at one extremity of the sequenced region (Wilson et al., 1992), while the previously sequenced actin binding protein gene, ABP1, (Drubin et al., 1990) was found at the other extremity. We present here the sequence of the region between these two genes which has the potential to code for two new open reading frames (ORFs).     

RHO1 (YlRHO1) is a non-essential gene in Yarrowia lipolytica and complements rho1Delta lethality in Saccharomyces cerevisiae

The synthesis of beta-1,3-glucan, the structural component of the yeast cell wall that gives shape to the cell, occurs at the plasma membrane and is the result of the activity of at least a two-component complex. Fks1p is the catalytic subunit directly responsible for the synthesis of beta-1,3-glucan, whilst the second subunit, Rho1p, has a GTP-dependent regulatory role (Yamochi et al., 1994). RHO1 has been characterized in Saccharomyces cerevisiae (Yamochi et al., 1994), and in several other fungal species. In this work, we have used degenerate oligonucleotides derived from the conserved regions of Rho1ps to isolate the RHO1 gene of Yarrowia lipolytica. The gene isolated in this way, which we have named YlRHO1, encodes a 204 amino acid protein that shows a high degree of homology with other Rho1ps. However, unlike S. cerevisiae, the ylrho1Delta disruptant strain in Y. lipolytica is viable, although it exhibits an increased sensitivity to Calcofluor white and Congo red. Also, YlRHO1 complements rho1 lethality in S. cerevisiae at both 28 degrees C and 37 degrees C. The complete sequence of YlRHO1 can be obtained from GenBank under Accession No. AF279915.     

An 11.4 kb DNA segment on the left arm of yeast chromosome II carries the carboxypeptidase Y sorting gene PEP1, as well as ACH1, FUS3 and a putative ARS.

We report the nucleotide sequence of an 11.4 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome II. This sequence contains a typical structure of a functional ARS as well as five open reading frames (ORFs) longer than 300 bp. One is PEP1, a gene encoding a transmembrane protein of 1579 amino acids which transits through the secretory pathway and is involved in vacuolar protein sorting. Two genes were previously sequenced: ACH1 (Lee et al., 1990) and FUS3 (Elion et al., 1990), which encode an acetyl-CoA hydrolase and a protein kinase involved in the cell division cycle, respectively. The last two ORFs localized on the complementary strand of ACH1 are not likely to be expressed.     

Regulation of genes encoding proteolytic enzymes during mammary gland development

The mammary gland undergoes extensive tissue remodelling during each lactation cycle. During pregnancy, the epithelial compartment of the gland is vastly expanded (Benaud et al. 1998). At the end of lactation the epithelial cells undergo apoptosis and adipocyte differentiation is induced (Lilla et al. 2002). Ductal and alveolar growth during puberty and pregnancy, and the involution process require the action of proteolytic enzymes (including matrix metalloproteinases, plasminogen and membrane-peptidases) and the corresponding genes are activated during these periods (Benaud et al. 1998; Alexander et al. 2001). Matrix metalloproteinases (MMP) are expressed in several cell types of the mammary gland including stromal fibroblasts (e.g., MMP3, MMP2), epithelial cells (e.g., MMP7 or MMP9), adipocytes (e.g., MMP2) and lymphoid cells (e.g., MMP9) (Crawford et al. 1996; Lund et al. 1996; Wiseman et al. 2003). A number of knock-out mice, which are deficient for individual MMP genes (e.g., MMP2, MMP3) or plasminogen, display alterations to mammary gland structure and impairment of lactation (Lund et al. 1999; Wiseman et al. 2003).     

TDH2 is linked to MET3 on chromosome X of Saccharomyces cerevisiae.

The MET3 gene of Saccharomyces cerevisiae was cloned and its restriction map was found to differ in the upstream region from an earlier published map (Cherest et al. Gene 34, 269-281, 1985) and nucleotide sequence (Cherest et al. Mol. Gen. Genet. 210, 307-313, 1987). Southern blot analysis of genomic DNA from strains S288C and FL100 (the genetic backgrounds from which these different copies of the gene had been cloned) showed that our clone from a S288C-based library had the same restriction map as the chromosomal DNA from both of the strains. Comparison of the nucleotide sequence of the two clones indicated that the earlier published clone probably represented a cloning artifact. In our clone, we found upstream of MET3, the nucleotide sequence of the TDH2 gene (Holland and Holland, J. Biol. Chem. 255, 2596-2605, 1980). The chromosomal orientation of the two genes was determined to be MET3-TDH2-CEN10.     

副溶血弧菌菌株229基因组测序及其致病因子的注释

本文对从冰冻海鱼中分离到的副溶血弧菌菌株229进行基因组测序,并对注释的基因进行功能聚类分析,以分析其致病因子,为研究其致病机制提供基因组全局水平的数据支持。完成了副溶血弧菌菌株229的基因组测序及组装,所得基因组4.97 Mb,GC含量为43.90%。基因注释获得4940个蛋白质编码基因(CDS),其中1304个CDS具有GO功能注释。WEGO注释显示大量CDS对于副溶血弧菌入侵宿主及随后的代谢过程发挥重要作用。获得了与副溶血弧菌致病性相关的致病因子,包括溶血素(hemolysin)、Ⅲ型因子(TypeⅢsecretion system,TTSS),这些因子位于不同的染色体位点,形成独立的致病因子簇。副溶血弧菌菌株229基因组组装和功能注释为研究副溶血弧菌致病性的遗传学基础提供了有价值的数据。     

Intercomparison of thermal and optical measurement methods for elemental carbon and black carbon at an urban location

Despite intensive efforts during the past 20 years, no generally accepted standard method exists to measure black carbon (BC) or elemental carbon (EC). Data on BC and EC concentrations are method specific and can differ widely (e.g. Schmid et al., 2001, ten Brink et al., 2004). In this study, a comprehensive set of methods (both optical and thermal) is compared. Measurements were performed under urban background conditions in Vienna, Austria, a city heavily impacted by diesel emissions. Filter and impactor samples were taken during 3 weeks in summer 2002 and analyzed for EC with thermal methods: a modified Cachier method (Cachier et al., 1989), a thermal-optical method (Schmid et al., 2001), and the VDI method (VDI, 1996); for BC with optical methods: a filter transmission method and the integrating sphere method (Hitzenberger et al., 1996); and for total carbon (TC) with a combustion method (Puxbaum and Rendl, 1983). The online methods aethalometer (Hansen et al., 1984) and the multiangle absorption photometer MAAP (Petzold et al., 2002) to measure BC were also used. The average values of BC and EC obtained with the methods agreed within their standard deviations. A conversion table was set up to allow comparisons between data measured elsewhere under urban background conditions (with similar source characteristics) with different instruments. An approach to estimate the absorption coefficient from attenuation data is derived so that existing records of aethalometer data in urban environments may be used to obtain also the absorption coefficients.     

Two pathways of DNA double-strand break repair in G1 cells of Saccharomyces cerevisiae

G1 cells of the diploid yeast Saccharomyces cerevisiae are known to be capable of a slow repair of DNA double-strand breaks (DSB) during holding the cells in a non-nutrient medium (Luchnik et al., 1977; Frankenberg-Schwager et al., 1980). In the present paper, S. cerevisiae cells gamma-irradiated in the G1 phase of the cell cycle are shown to be capable of fast repair of DNA DSB; this process is completed within 30-40 min of holding the cells in water at 28 degrees C. For this reason, the kinetics of DNA DSB repair during holding the cells in a non-nutrient medium are biphasic, i.e., the first, 'fast' phase is completed within 30-40 min, whereas the second, 'slow' phase is completed within 48 h. Mutations rad51, rad52, rad54 and rad55 inhibit the fast repair of DNA DSB, whereas mutations rad50, rad53 and rad57 do not significantly influence this process. It has been shown that the observed fast and slow repair of DNA DSB in the G1 diploid cells of S. cerevisiae are separate pathways of DNA DSB repair in yeast.     

New drug-resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe

We describe new heterologous modules for PCR-based gene targeting in the fission yeast Schizosaccharomyces pombe. Two bacterial genes, hph and nat, which display dominant drug-resistance phenotypes, are used as new selectable markers in these modules. Both genes have been used successfully in the budding yeast Saccharomyces cerevisiae, in which hph confers resistance to hygromycin B, while nat confers nourseothricin resistance (Goldstein and McCusker, 1999). Vector modules for gene disruption and C-terminal tagging with 3HA, 13Myc and GFP(S65T) are constructed using previously constructed pFA6a-MX6-derived plasmids (B?hler et al., 1998; Wach et al., 1997). In combination with the existing systems that are based upon the G418-resistance gene (kan), triple gene deletions or tags could be constructed. In addition a vector for one-step integration of a monomeric RFP (mRFP) to the C-terminus of proteins of interest is developed. Finally, oligonucleotides that allow a simple marker switch from kan to hph or nat, and vice versa, are described. The new constructs developed here should facilitate post-genomic molecular analysis of protein functions in fission yeast.     

A single nucleotide change in the MNR1 (VCX1/HUM1) gene determines resistance to manganese in Saccharomyces cerevisiae

Several spontaneous Mn2+-resistant mutants were isolated from Saccharomyces cerevisiae strain W303-1b. All displayed an identical semidominant resistance phenotype. The gene responsible for this phenotype from one of these mutants, as well as that from the wild-type, was cloned and sequenced, which allowed the identification of a single nucleotide change in the former. Further sequencing work with the remaining mutants, as well as with others on the MNAR1 gene (Bianchi et al., 1981), indicated that all displayed the same mutation (mnr1 allele). In addition, MNR1 was shown to correspond to VCX1 and HUM1, which determine a vacuolar membrane protein. The nucleotide sequence has been deposited at EMBL, with Accession No. AJ001272.     

Cytosolic redox metabolism in aerobic chemostat cultures of Saccharomyces cerevisiae

Cytosolic redox balance has to be maintained in order to allow an enduring cellular metabolism. In other words, NADH generated in the cytosol has to be re-oxidized back to NAD(+). Aerobically this can be done by respiratory oxidation of cytosolic NADH. However, NADH is unable to cross the mitochondrial inner membrane and mechanisms are required for conveying cytosolic NADH to the mitochondrial electron transport chain. At least two such systems have proved to be functional in S. cerevisiae, the external NADH dehydrogenase (Luttik et al., 1998; Small and McAlister-Henn, 1998) and the G3P shuttle (Larsson et al., 1998). The aim of this investigation was to study the regulation and performance of these two systems in a wild-type strain of S. cerevisiae using aerobic glucose- and nitrogen-limited chemostat cultures. The rate of cytosolic NADH formation was calculated and as expected there was a continuous increase with increasing dilution rate. However, measurements of enzyme activities and respiratory activity on isolated mitochondria revealed a diminishing capacity at elevated dilution rates for both the external NADH dehydrogenase and the G3P shuttle. This suggests that adjustment of in vivo activities of these systems to proper levels is not achieved by changes in amount of protein but rather by, for example, activation/inhibition of existing enzymes. Adenine nucleotides are well-known allosteric regulators and both the external NADH and the G3P shuttle were sensitive to inhibition by ATP. The most severe inhibition was probably on the G3P shuttle, since one of its member proteins, Gpdp, turned out to be exceptionally sensitive to ATP. The external NADH dehydrogenase is suggested as the main system employed for oxidation of cytosolic NADH. The G3P shuttle is proposed to be of some importance at low growth rates and perhaps its real significance is only expressed during starvation conditions.     

不同培养条件下酿酒酵母菌的转录组差异分析

从组学水平分析富硒条件下酿酒酵母菌(Saccharomyces cerevisiae)内在分子机制,为酿酒酵母菌富硒研究及富硒基因的挖掘利用提供理论依据。该研究以不加硒培养的酿酒酵母菌作为对照组Kb,以加20μg/mL硒培养的酿酒酵母菌为实验组Se,利用Illumina高通量测序平台对两组进行转录组测序,通过生物信息学方法对数据进行分析处理。结果表明,转录组测序共获得6 445个Unigenes,分别有1 401个(21.74%)、3 665个(56.87%)、5 630个(87.35%)、6 112个(94.83%)、6 077个(94.29%)、5 059个(78.49%) Unigenes被注释到GO、KEGG、COG、NR、Swiss Prot和Pfam数据库,共有6 150个(95.42%) Unigenes得到注释。在GO功能注释中,共得到41个GO功能小类,在KEGG代谢通路分析时,获得了113条KEGG通路。该转录组测序数据质量高,结果覆盖面广,为酿酒酵母菌富硒基因挖掘和研究提供了一定的理论参考。     

Limitations of codon adaptation index and other coding DNA-based features for prediction of protein expression in Saccharomyces cerevisiae

The relationship between codon usage and protein/mRNA expression in S. cerevisiae has been extensively studied. Recently, protein expression data for the whole yeast genome was published. We investigate which properties of coding DNA sequences can be used to predict expression levels. The new algorithm by Carbone et al. for computing dominating codon bias in a genome is evaluated. It is concluded that it works at least as well as existing methods, and eliminates the need to arbitrarily choose a set of highly expressed genes. Also, the hypothesis that information on codon pair frequencies can be used to predict expression is investigated. Our conclusion is that codon pairs do not contribute more information than do single codon frequencies. Overall correlation between predicted and actual expression data using properties of coding DNA sequences is around 0.65. Hence, while being a useful source of information, the expression levels predicted by these methods should only be used as a rule of thumb.     

The HTL1 gene (YCR020W-b) of Saccharomyces cerevisiae is necessary for growth at 37 degrees C, and for the conservation of chromosome stability and fertility

A small 78 codon ORF, named HTL1 (Chen et al., unpublished results), situated between loci MAK31 and HSP30 on chromosome III of Saccharomyces cerevisiae, is required for growth at 37 degrees C. In this communication, we characterize the ORF and show that disruption of HTL1, besides preventing growth at 37 degrees C, causes genetic and/or epigenetic instability at 26 degrees C: ploidy increases in about 10% of cells grown from individual disruptants and a fraction of disruptant clones are predestined to a rapid and progressive loss of fertility during growth at 26 degrees C.     

Genetic, genomic, and molecular tools for studying the protoploid yeast, L. waltii

Sequencing of the yeast Kluyveromyces waltii (recently renamed Lachancea waltii) provided evidence of a whole genome duplication event in the lineage leading to the well-studied Saccharomyces cerevisiae. While comparative genomic analyses of these yeasts have proven to be extremely instructive in modeling the loss or maintenance of gene duplicates, experimental tests of the ramifications following such genome alterations remain difficult. To transform L. waltii from an organism of the computational comparative genomic literature into an organism of the functional comparative genomic literature, we have developed genetic, molecular and genomic tools for working with L. waltii. In particular, we have characterized basic properties of L. waltii (growth, ploidy, molecular karyotype, mating type and the sexual cycle), developed transformation, cell cycle arrest and synchronization protocols, and have created centromeric and non-centromeric vectors as well as a genome browser for L. waltii. We hope that these tools will be used by the community to follow up on the ideas generated by sequence data and lead to a greater understanding of eukaryotic biology and genome evolution.     

A novel aspartyl protease allowing KEX2-independent MF alpha propheromone processing in yeast

Mutants of Saccharomyces cerevisiae which lack the KEX2-encoded endopeptidase are unable to process proteolytically the mating factor alpha (MF alpha) propheromone produced from the chromosomal MF alpha 1 and MF alpha 2 genes (Julius et al., 1983). Overproduction of pheromone precursor from multiple, plasmid-borne MF alpha genes did, however, lead to the production of active MF alpha peptides in the absence of the KEX2 gene product. S. cerevisiae therefore must possess an alternative processing enzyme. The cleavage site of this enzyme appeared identical to that of the KEX2-encoded endopeptidase. To identify the gene responsible for the alternative processing, we have isolated clones which allowed production of mature MF alpha in a kex2-disrupted strain even from the chromosomal MF alpha genes. The gene isolated in this way was shown also to be essential for the KEX2-independent processing of propheromone overproduced from plasmid-borne MF alpha 1. The amino acid sequence deduced from the gene shows extensive homology to a number of aspartyl proteases including the PEP4 and BAR1 gene products from S. cerevisiae. In contrast to the BAR1 gene product, the novel aspartyl protease (YAP3 for Yeast Aspartyl Protease 3) contains a C-terminal serine/threonine-rich sequence and potential transmembrane domain similar to those found in the KEX2 gene product. The corresponding gene YAP3 was located to chromosome XII. The normal physiological role of the YAP3 gene product is not known. Strains disrupted in YAP3 are both viable and able to process the mating factor a precursor.