Large-scale phenotypic analysis reveals identical contributions to cell functions of known and unknown yeast genes.

Sequencing of the yeast genome has shown that about one-third of the yeast ORFs code for unknown proteins. Many other have similarity to known genes, but still the cellular functions of the gene products are unknown. The aim of the B1 Consortium of the EUROFAN project was to perform a qualitative phenotypic analysis on yeast strains deleted for functionally orphan genes. To this end we set up a simple approach to detect growth defects of a relatively large number of strains in the presence of osmolytes, ethanol, high temperature, inhibitory compounds or drugs affecting protein biosynthesis, phosphorylation level or nucleic acids biosynthesis. We have now developed this procedure to a semi-quantitative level, we have included new inhibitors, such as hygromycin B, benomyl, metals and additional drugs interfering with synthesis of nucleic acids, and we have performed phenotypic analysis on the deleted strains of 564 genes poorly characterized in respect to their cellular functions. About 30% of the deleted strains showed at least one phenotype: many of them were pleiotropic. For many gene deletions, the linkage between the deletion marker and the observed phenotype(s) was studied by tetrad analysis and their co-segregation was demonstrated. Co-segregation was found in about two-thirds of the analysed strains showing phenotype(s).     

Functional analysis of yeast gene families involved in metabolism of vitamins B1 and B6

In order to clarify their physiological functions, we have undertaken a characterization of the three-membered gene families SNZ1-3 and SNO1-3. In media lacking vitamin B(6), SNZ1 and SNO1 were both required for growth in certain conditions, but neither SNZ2, SNZ3, SNO2 nor SNO3 were required. Copies 2 and 3 of the gene products have, in spite of their extremely close sequence similarity, slightly different functions in the cell. We have also found that copies 2 and 3 are activated by the lack of thiamine and that the Snz proteins physically interact with the thiamine biosynthesis Thi5 protein family. Whereas copy 1 is required for conditions in which B(6) is essential for growth, copies 2 and 3 seem more related with B(1) biosynthesis during the exponential phase.     

A comparative analysis of distinctive features of yeast protein sequences

The recently published sequence of yeast chromosome III (YCIII) provides the longest continuous stretch of a eukaryotic DNA molecule sequenced to date (315 kb). The sequence contains 116 distinct AUG-initiated open reading frames of at least 200 codons in length, more than 50 of which had not been described previously nor bear significant similarity to known proteins. We have analysed the YCIII known and putative-protein sequences with respect to significant statistical features which might reflect on structural and functional characteristics. The YCIII proteins have striking similarities and differences in their sequence attribute distributions compared to the corresponding distributions for all available yeast sequences and other protein collections. Nine examples of YCIII proteins with distinctive sequence features are discussed in detail.     

Quantitative analysis of yeast gene function using competition experiments in continuous culture

One possible route to the evaluation of gene function is a quantitative approach based on the concepts of metabolic control analysis (MCA). An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S. cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, we show that both densitometry and GeneScan^™ analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10–90% of the total cell population. Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments in continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype. © 1998 John Wiley & Sons, Ltd.     

Functional analysis of three adjacent open reading frames from the right arm of yeast chromosome XVI

A 7·24 kb genomic DNA fragment from the yeast Saccharomyces cerevisiae chromosome XVI was isolated by complementation of a new temperature-sensitive mutation tsa1. We determined the nucleotide sequence of this fragment located on the right arm of chromosome XVI. Among the three, complete open reading frames: YPR041w, YPR042c and YPR043w contained within this fragment, the gene YPR041w was shown to complement the tsa1 mutation and to correspond to the TIF5 gene encoding an essential protein synthesis initiation translation factor. The YPR042c gene encodes a hypothetical protein of 1075 amino acids containing four putative transmembrane segments and is non-essential for growth. The gene YPR043c encoding the 10 kDa product, highly similar to the human protein L37a from the 60S ribosomal subunit, was found to be essential and a dominant lethal. We conclude that three tightly linked yeast genes are involved in the translation process. © 1998 John Wiley & Sons, Ltd.     

Simultaneous determination of potassium and rubidium content in yeast

Rubidium is widely used as a potassium analogue in transport studies in yeast and other organisms. As rubidium (potassium) uptake is modulated by the internal potassium concentration, it is often necessary to determine both Rb(+) and K(+) concentrations in the same cell extract. Current methods based on atomic absorption/emission spectroscopy require separate analysis for each cation. Alternatively, unsafe radioactive isotopes can be used. Here we report a convenient, non-radioactive, HPLC/conductivity-based method that allows a complete analysis of both cations with a single injection from a cell extract. The increase in Rb(+) uptake during K(+) starvation in yeast is easily demonstrated with this method.     

Sequence and functional analysis of a 7·2 kb DNA fragment containing four open reading frames located between RPB5 and CDC28 on the right arm of chromosome II

In a coordinated approach, several laboratories sequenced Saccharomyces cerevisiae chromosome II during the European BRIDGE project. Here we report on the sequence and functional analysis of a 7217 bp fragment located on the right arm of chromosome II between RPB5 and CDC28. The fragment contains four open reading frames probably encoding proteins of 79·2 kDa (corresponding gene YBR156c), 12·1 kDa (YBR157c), 62·7 kDa (YBR158w) and 38·7 kDa (YBR159w). All four open reading frames encode new proteins, as concluded from data base searches. The respective genes were destroyed by gene replacement in one allele of diploid cells. After sporulation and tetrad analysis, the resulting mutant haploid strains were investigated. No phenotype with respect to spore germination, viability, carbohydrate utilization, and growth was found for YBR157c, encoding the smallest open reading frame investigated. Gene replacement within the YBR156c gene encoding a highly basic and possibly nuclear located protein was lethal. Ybr158 revealed similarities to the Grr1 (Cat80) protein with respect to the leucine-rich region. Cells harboring a mutation in the YBR158w gene showed strongly reduced growth as compared to the wild-type cells. The protein predicted from YBR159w shared 33% identical amino acid residues with the human estradiol 17-beta-hydroxysterol dehydrogenase 3. Haploid ybr159c mutants were only able to grow at reduced temperatures, but even under these conditions the mutants grew slower than wild-type strains. The DNA sequence was deposited at the EMBL data base with accession numbers Z36025 (YBR156c), Z36026 (YBR157c), Z36027 (YBR158w) and Z36028 (YBR159w).     

Suitability of replacement markers for functional analysis studies inSaccharomyces cerevisiae

The complete yeast sequence contains a large proportion of genes whose biological function is completely unknown. One approach to elucidating the function of these novel genes is by quantitative methods that exploit the concepts of metabolic control analysis. An important first step in such an analysis is to determine the effects of deleting individual genes on the growth rate (or fitness) of Saccharomyces cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, they are most readily determined by competition against a standard strain in chemostat cultures where the true steady state demanded by metabolic control analysis may be achieved. We have constructed two different standard strains in which the HO gene is replaced by either HIS3 or kanMX. We demonstrate that HO is a selectively neutral site for gene replacement. However, there is a significant marker effect associated with HIS3 which, moreover, is dependent on the physiological conditions used for the competition experiments. In contrast, the kanMX marker exhibited only a small effect on specific growth rate (≤±4%). These data suggest that nutritional markers should not be used to generate deletion mutants for the quantitative analysis of gene function in yeast but that kanMX replacements may be used, with confidence, for such studies. © 1997 John Wiley & Sons, Ltd.     

Functional analysis of the Saccharomyces cerevisiaeUBC11 gene

UBC11 is the Saccharomyces cerevisiae gene that is most similar in sequence to E2-C, a ubiquitin carrier protein required for the destruction of mitotic cyclins and proteins that maintain sister chromatid cohesion in animal cells and in Schizosaccharomyces pombe. We have disrupted the UBC11 gene and found it is not essential for yeast cell viability even when combined with deletion of UBC4, a gene that has also been implicated in mitotic cyclin destruction. Ubc11p does not ubiquitinate cyclin B in clam cell-free extracts in vitro and the destruction of Clb2p is not impaired in extracts prepared from Δubc11 or Δubc4Δubc11 cells. These results suggest Ubc4p and Ubc11p together are not essential for mitotic cyclin destruction in S. cerevisiae and we can find no evidence to suggest that Ubc11p is the true functional homologue of E2-C. © 1998 John Wiley & Sons, Ltd.     

Disruption of six Saccharomyces cerevisiae genes from chromosome IV and basic phenotypic analysis of deletion mutants

We report here the construction of six deletion mutants and the analysis of their basic phenotype. Deletion cassettes containing the KanMX4 marker module and long flanking regions homologous to the target locus were constructed for each of the six open reading-frames (ORFs YDL088c, YDL087c, YDL086w, YDL085w, YDL084w and YDL082w) located on chromosome IV. Sporulation and tetrad analysis of heterozygous deletant strains revealed that, in the FY1679 genetic background, ORFs YDL088c, YDL087c and YDL084w are essential genes for vegetative growth whereas YDL086w, YDL085w and YDL082w are non-essential. ydl088cΔ and ydl084wΔ haploid strains are viable in the CEN. PK2 genetic background although ydl084wΔ grows at a slower rate than the wild type. Complementation tests by corresponding cognate genes confirmed that gene inactivation was responsible for these growth defects. © 1998 John Wiley & Sons, Ltd.     

Cloning and identification of HEM14, the yeast gene for mitochondrial protoporphyrinogen oxidase

A respiratory-defective mutant (C54) of Saccharomyces cerevisiae was found to have a phenotype consistent with a mutation in either mitochondrial protoporphyrinogen oxidase or ferrochelatase. The mutant is grossly deficient in hemes, accumulates protoporphyrin and is rescued by exogenous heme. The increased levels of protoporphyrin at the expense of heme is indicative of a block in one of the two last steps of the heme biosynthetic pathway. Complementation of C54 by a known ferrochelatase mutant suggested that the defect was most likely in HEM14 encoding protoporphyrinogen oxidase. A plasmid capable of complementing C54 was obtained by transformation with a yeast genomic plasmid library. A partial sequence of the insert identified the gene as reading frame YER014 of yeast chromosome V (GenBank Accession Number U18778). This reading frame codes for a protein homologous to human protoporphyrinogen oxidase. Disruption of this gene elicits a respiratory defect and accumulation of protoporphyrin. The phenotype of the null mutant together with the homology of YER014p to human protoporphyrinogen oxidase provide compelling evidence that YER014 is HEM14.     

荧光图像分析法进行酿酒酵母生长过程活力分析

应用荧光标记结合显微图像分析技术检测酿酒酵母摇床培养过程中活力的变化规律,分析酿酒酵母在生长过程中不同时期的活力特点及其与活性关系.结果表明,本技术不但可获得酿酒酵母生长的活力水平的变化趋势,而且还可获得酿酒酵母分裂周期中蛋白质的分布变化情况,并通过对酿酒酵母的连续动态观察而获得酵母分裂时相的信息.说明图像分析法能够替代流式细胞仪作为酿酒酵母活力评价的重要手段.     

Genetic analysis of the karyotype instability in natural wine yeast strains.

Yeast strains isolated from the wild may show high rates of changes in their karyotypes during vegetative growth. We analysed over 500 karyotypes from mitotic and meiotic derivatives of strain DC5, which has a chromosome rearrangement rate of 8.2 x 10(-3) changes/generation. About 70% of the meiotic derivatives of DC5 had low rearrangement rates, with an average of 5.8 x 10(-4) changes/generation, suggesting that karyotype instability behaved as a dominant phenotype. Diploid derivatives with low karyotype variability in mitosis also had low rates of chromosomal rearrangement during meiosis, suggesting that the two phenotypes may be linked. DC5 and some of its meiotic derivatives (both with high and low karyotype variability) had chromosome XII hypervariable bands. Their distribution among the meiotic products indicates that they are not indicators for genetic instability. To our knowledge, data in this paper are the first to indicate that karyotypically unstable yeast strains may give stable progeny at high rates. Understanding of the relevant mechanism(s) may allow the design of genetic strategies to stabilize karyotypes from natural and/or industrial wine yeasts with unacceptable karyotype rearrangement rates.     

Nucleotide sequence and analysis of the centromeric region of yeast chromosome IX

We have determined the nucleotide sequence of a cosmid (pIX338) containing the centromere region of yeast (Saccharomyces cerevisiae) chromosome IX. The complete nucleotide sequence of 33·8 kb was obtained by using an efficient directed sequencing strategy in combination with automated DNA sequencing on the A.L.F. DNA sequencer. Sequence analysis revealed the presence of 17 open reading frames (ORFs), four of them previously known yeast genes (sly12, pan1, sts1 and prl1), a tRNA gene and the centromere motif. Exhaustive database searches detected sequence homologues of known function for as many as 14 of the 17 ORFs. These include a mammalian tyrosine kinase substrate; the Escherichia coli cell cycle protein MinD; the human inositol polyphosphate-5-phosphatase (gene OCRL) involved in Lowe's syndrome, a developmental disorder; and helicases, for which the new yeast member defines a distinct DEAD/H-box subfamily. A surprisingly large fraction of the ORFs (at least six out of 17) in the centromeric region are apparently involved in RNA or DNA binding. The nucleotide sequence reported here has been submitted to the EMBL data library under the accession number X79743.     

Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis

In the vacuoles of Saccharomyces cerevisiae yeast cells, vividly moving insoluble polyphosphate complexes (IPCs) <1 µm size, stainable by a fluorescent dye, 4′,6‐diamidino‐2‐phenylindole (DAPI), may appear under some growth conditions. The aim of this study was to quantitatively characterize the movement of the IPCs and to evaluate the viscosity in the vacuoles using the obtained data. Studies were conducted on S. cerevisiae cells stained by DAPI and fluorescein isothyocyanate‐labelled latex microspheres, using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). IPC movement was photorecorded and shown to be Brownian motion. On latex microspheres, a methodology was developed for measuring a fluorescing particle's two‐dimensional (2D) displacements and its size. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated. Apparent viscosity values in the vacuoles of the cells, computed by the Einstein–Smoluchowski equation using the obtained data, were found to be 2.16 ± 0.60, 2.52 ± 0.63, 3.32 ± 0.9 and 11.3 ± 1.7 cP. The first three viscosity values correspond to 30–40% glycerol solutions. The viscosity value of 11.3 ± 1.7 cP was supposed to be an overestimation, caused by the peculiarities of the vacuole structure and/or volume in this particular cell. This conclusion was supported by the particular quality of the Brownian motion trajectories set in this cell as compared to the other three cells. Copyright © 2010 John Wiley & Sons, Ltd.