Rescuing yeast mutants with human genes.

The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae have, in addition to being extensively studied themselves, both been utilized for the last quarter century as experimental systems for the isolation of genes from other organisms. Mutations conferring growth defects in either of the two yeast strains have frequently been complemented by expression of cDNA libraries from heterologous species, often human. Many successful experiments have utilized available yeast mutations to allow successful complementation by a human gene, which can thus be deduced to have the same, or an overlapping function as the mutated yeast gene. However complementation in yeast has also been used with success to study two fields, apoptosis and steroid receptor signalling, which, at first glance, seem to be foreign to the yeast life cycle.     

Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line

Hybrid myeloma cell lines secreting monoclonal antibodies to tubulin have been prepared using rat myelomas and spleen cells from rats immunized with yeast tubulin. A comparison between the results obtained with the rat myeloma Y3-Ag 1.2.3., which secretes a light chain, and a new line, YB2/O, which does not, shows that they are both excellent parental lines and that the second produces hybrids with no myeloma chain components. The antitubulin antibodies in the serum of rats bearing two of the hybrid myeloma tumors gave titers of up to 1:10(6) from which large amounts of monoclonal antibodies could be easily purified. They recognized tubulin from yeast as well as from birds and mammals. The two antibodies gave clear immunofluorescent staining of yeast mitotic spindles as well as the interphase microtubule network of tissue culture cells. Some difference in the pattern of immunofluorescence staining of yeast cells and nuclei was observed between the two antibodies. The purified antibodies could be conjugated to colloidal gold particles and used for direct labeling of yeast microtubules for electron microscopy.     

Two autonomously replicating sequences near oli-1 gene of yeast mitochondrial DNA

We cloned two autonomously replicating sequences from a short segment of mtDNA of an oligomycin-resistant petite yeast, O-111, into a vector pYleu 12 constructed from yeast LEU 2 gene and pBR 322. These plasmids, pYmit 4 and pYmit 1, had frequencies of transformation of yeast as high as that of YEp 13, having a replicator of 2 mu DNA. They were maintained as plasmids in yeast under selective conditions and shuttled from yeast to E. coli. No evidence was obtained that these plasmids were incompatible with the wild-type mitochondrial genome. These sequences were located in intergenic regions.     

Yeast calmodulin: structural and functional elements essential for the cell cycle.

The budding yeast Saccharomyces cerevisiae is a suitable organism for studying calmodulin function in cell proliferation. Genetic studies in yeast demonstrate that vertebrate calmodulin can functionally replace yeast calmodulin. In addition, expression of half of the yeast calmodulin molecule is found to be sufficient for cell growth. Characterization of conditional-lethal mutants of yeast calmodulin as well as the intracellular distribution of calmodulin have suggested that at least two cell cycle steps require calmodulin function. One is nuclear division and the other is the maintenance of cell polarity. A current focus is to understand which kinds of target proteins are involved in mediating the essential functions of yeast calmodulin in these processes. Thus far, three yeast enzymes whose activity is regulated by calmodulin have been identified.     

Transcriptional mapping of two yeast genes coding for glyceraldehyde 3-phosphate dehydrogenase isolated by sequence homology with the chicken gene

Homology between the coding regions of the chicken and yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes was directly demonstrated by the hybridization of a cDNA clone coding for GAPDH in the chicken with EcoRI-digested yeast DNA. A yeast EcoRI fragment library in bacteriophage lambda was screened using the chicken cDNA plasmid as probe, and two recombinant phages were isolated, each one containing a different GAPDH gene. The initiation and termination sites for the GAPDH mRNA were localized for the two different GAPDH genes and compared to those of other yeast genes. Measurements of the relative mRNA levels for the two genes show that both genes are transcribed at about the same level when yeasts are grown on glucose media.     

Automation of yeast two-hybrid screening

We have developed an automated format for screening yeast two-hybrid libraries for protein-protein interactions. The format consists of a liquid array in which pooled library subsets of yeast, expressing up to 1000 different cDNAs, are mated to a yeast strain of the opposite mating type, expressing a protein of interest. Interactors are detected by a liquid assay for beta-galacsidase following prototrophic selection. The method is demonstrated by the detection of interactions between two encoded yeast RNA polymerase subunits in simulated libraries of varied complexity. To demonstrate its utility for large scale screening of complex cDNA libraries, two nuclear receptor ligand-binding domains were screened through two cDNA libraries arrayed in pooled subsets. Screening these libraries yielded clones which had previously been identified in traditional yeast two hybrid screens, as well as several new putative interacting proteins. The formatting of the cDNA library into pooled subsets lends itself to functional subtraction of the promiscuous positive class of interactor from the library. Also, the liquid arrayed format enables electronic handling of the data derived from interaction screening, which, together with the automated handling of samples, should promote large-scale proteome analysis.     

抑制浓度下锰铁对啤酒废酵母产LYCD活力影响

目的:探讨锰铁离子对啤酒废酵母产生活性酵母细胞衍生物(LYCD)的活力影响.方法:以啤涌废酵母为研究对象、以废液为培养基,在生长抑制浓度下,分别研究了锰、铁离子的作用浓度与时间对啤酒废酵母产生LYCD的活力影响.结果:废酵母在废液中培养28h可达到对数生长期;当废液中Mn~(2+)、Fe~(3+)浓度分别达到200mg/L、50mg/L时可对废酵母产生生长抑制;当Mn~(2+)、Fe~(3+)的作用浓度分别为220mg/L、60mg/L、应激时间分别是25、45min时,啤酒废酵母产的LYCD活性强.结论:应激培养基中金属离子Mn~(2+)、Fe~(3+)达到一定浓度,在一定时间下可使废酵母产生强活力的LYCD.     

Functional adaptation between yeast actin and its cognate myosin motors

We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins.     

Monitoring mRNA decapping activity

mRNA decapping is a common step shared between two important mRNA decay pathways in yeast, Saccharomyces cerevisiae. To investigate how mRNAs are decapped, we have developed an assay that can be easily used to measure the decapping activity. This assay has been used to isolate yeast strains with altered decapping activities. The results demonstrated that decreased decapping activity in vitro corresponds well with the decapping-deficient phenotype in vivo. This assay has been applied to the purified yeast decapping enzyme Dcp1 protein as well as crude yeast extracts and Xenopus oocyte extracts.     

Effects of accumulated S-adenosylmethionine on growth of yeast cells

S-adenosylmethionine (SAM) accumulated in cultured yeast cells and affected growth in two ways. High levels of intracellular SAM in yeast inhibited early growth, but increased growth in medium without sources of nitrogen and sulfur. Accumulated SAM in the yeast cells was recycled as a nutritional source depending on the sulfur and nitrogen contents of the medium.     

DNA polymerase I and DNA primase complex in yeast

Chromatographic analysis of poly(dT) replication activity in fresh yeast extracts showed that the activities required co-fractionate with the yeast DNA polymerase I. Since poly(dT) replication requires both a primase and a DNA polymerase, the results of the fractionation studies suggest that these two enzymes might exist as a complex in the yeast extract. Sucrose gradient analysis of concentrated purified yeast DNA polymerase I preparations demonstrates that the yeast DNA polymerase I does sediment as a complex with DNA primase activity. Two DNA polymerase I peptides estimated at 78,000 and 140,000 Da were found in the complex that were absent from the primase-free DNA polymerase fraction. Rabbit anti-yeast DNA polymerase I antibody inhibits DNA polymerase I but not DNA primase although rabbit antibodies are shown to remove DNA primase activity from solution by binding to the complex. Mouse monoclonal antibody to yeast DNA polymerase I binds to free yeast DNA polymerase I as well as the complex, but not to the free DNA primase activity. These results suggest that these two activities exist as a complex and reside on the different polypeptides. Replication of poly(dT) and single-stranded circular phage DNA by yeast DNA polymerase I and primase requires ATP and dNTPs. The size of the primer produced is 8 to 9 nucleotides in the presence of dNTPs and somewhat larger in the absence of dNTPs. Aphidicolin, an inhibitor of yeast DNA polymerase I, is not inhibitory to the yeast DNA primase activity. The primase activity is inhibited by adenosine 5'-(3-thio)tri-phosphate but not by alpha-amanitin. The association of yeast DNA polymerase I and yeast DNA primase can be demonstrated directly by isolation of the complex on a column containing yeast DNA polymerase I mouse monoclonal antibody covalently linked to Protein A-Sepharose. Both DNA polymerase I and DNA primase activities are retained by the column and can be eluted with 3.5 M MgCl2. Part of the primase activity can be dissociated from DNA polymerase on the column with 1 M MgCl2 and this free primase activity can be detected as poly(dT) replication activity in the presence of Escherichia coli polymerase I.     

Recombinant plasmids carrying multiple markers: isolation during yeast co-transformation

Cotransformants of yeast cells by two partially homologous plasmids, one of which is incapable of autonomous replication, has been used to construct multiply marked recombinant plasmids. Only simultaneous elimination of three yeast markers was registered when episomal plasmid, carrying Ade2 gene, and integrative plasmid, carrying yeast genes LEU2 and URA3, were cotransformed. Transformants, in which yeast genes LEU2, URA3 and HIS3 are linked, have been isolated by analogous technique. The genetic analysis has confirmed existence of plasmid cointegrates in the transformant cells, which carry three yeast genes, bacterial DNA fragment and 2 micrometers DNA fragment, coding for replicative functions. Recombination in the region of bacterial plasmid pBR322 might have resulted in formation of such plasmids. Plasmid recombination in cotransformants has been used to construct multiply marked circular chromosomes, having included yeast genes LEU2, URA3 and TRP1, centromere of the IV yeast chromosome and the sequence coding for their replication in yeast as well as in E. coli cells.     

The yeast homolog of the U1 snRNP protein 70K is encoded by the SNP1 gene.

The product of the yeast SNP1 gene has high homology to two domains of the metazoan U1 snRNP protein 70K, which binds to stem/loop I of the U1 RNA. However, the absence of other domains conserved in metazoan 70K and the minimal effect of yeast U1 RNA stem/loop I deletion make the assignment of SNP1 as yeast 70K less clear. To address this question, we have expressed the SNP1 gene as a fusion protein in E. coli and developed a gel shift assay for U1 RNA binding. We show here that the product of the yeast SNP1 gene binds directly and specifically to the first 47 nucleotides of yeast U1 RNA, which include the stem/loop 1 structure. We therefore conclude that the SNP1 gene product is the yeast 70K homolog. This is the first yeast protein to be identified as a homolog of a metazoan snRNP protein.     

酵母表达体系及其在抗体工程中的应用

酵母是最简单的真核生物,以其为宿主表达异源蛋白具有很大的优越性。近十几年来,各种各样的酵母表达体系陆续发展起来。本文介绍了两种酵母表达系统,并对现有的主要表达体系进行了比较。另外,本文还涉及到酵母在表达抗体或其片段方面的成功应用。     

Origin of changes in electrical impedance during the growth and fermentation process of yeast in batch culture

The electrical impedance of the culture medium shows complex changes during the growth and fermentation process of yeast, and this prevents its possible application for the monitoring of certain yeast activities. Clarification of the mechanism of such changes is thus essential for practical use. As a first step toward this aim, the impedance, yeast concentration, and pH of a batch culture medium were measured using special cells with two compartments and also the usual type of cell with one compartment. In the special cells, the yeast was cultured in one compartment only. Conducting ions and nonconducting substances diffused through an intermediate porous membrane sandwiched between the two compartments. The impedances of the two compartments were measured simultaneously by the four-electrode method. The main mechanism responsible for increasing the impedance was the conducting ions produced by the yeast extract added as a nutrient to the culture broth by certain nonconducting substances during the process of growth. The increase in the yeast concentration was also a minor factor increasing the impedance. These increases surpassed the impedance decrease caused by the increase of H(+) ions produced by some accumulated acidic substances, and the impedance thus increased.     

The interaction of yeast citrate synthase with yeast mitochondrial inner membranes

The specific interaction of yeast citrate synthase with yeast mitochondrial inner membranes was characterized with respect to saturability of binding, pH optimum, effect of ionic strength, temperature response, and inhibition by oxalacetate. The binding ability of the inner membranes is inhibited by proteolysis and heat treatment, which implies that the membrane component(s) responsible for binding is a protein. A protein fraction from inner membranes when added to liposomes will bind citrate synthase. In addition, the binding of yeast fumarase, mitochondrial malate dehydrogenase, and cytosolic malate dehydrogenase to yeast inner membranes was examined. For these studies the yeast mitochondrial matrix enzymes, citrate synthase (from two types of yeast), malate dehydrogenase, and fumarase, as well as cytosolic malate dehydrogenase, were purified using rapid new techniques.     

A conserved family of Saccharomyces cerevisiae synthases effects dihydrouridine modification of tRNA

Dihydrouridine modification of tRNA is widely observed in prokaryotes and eukaryotes, as well as in some archaea. In Saccharomyces cerevisiae every sequenced tRNA has at least one such modification, and all but one have two or more. We have used a biochemical genomics approach to identify the gene encoding dihydrouridine synthase 1 (Dus1, ORF YML080w), using yeast pre-tRNA(Phe) as a substrate. Dus1 is a member of a widespread family of conserved proteins, three other members of which are found in yeast: YNR015w, YLR405w, and YLR401c. We show that one of these proteins, Dus2, encoded by ORF YNR015w, has activity with two other substrates: yeast pre-tRNA(Tyr) and pre-tRNA(Leu). Both Dus1 and Dus2 are active as a single subunit protein expressed and purified from Escherichia coli, and the activity of both is stimulated in the presence of flavin adenine dinucleotide. Dus1 modifies yeast pre-tRNA(Phe) in vitro at U17, one of the two positions that are known to bear this modification in vivo. Yeast extract from a dus1-A strain is completely defective in modification of yeast pre-tRNAPhe, and RNA isolated from dus1-delta and dus2-delta strains is significantly depleted in dihydrouridine content.     

The production of extracellular polysaccharides by a hydrocarbon assimilating yeast.

Investigations were made on the extracellular polysaccharides production by a hydrocarbon assimilating yeast. The yeast Candida lipolytica was grown on two different media containing n-hexadecane as the sole carbon source. Polymeric materials precipitated from the culture medium with ethanol were determined gravimetrically at various growth periods. The hydrolysis of the precipitated material and their chromatographic analysis revealed the presence of mannose, glucose and galactose in the yeast extract-containing medium. The proportions of these sugars differed at various growth periods. The hydrolysates of the polymers of the yeast extract-free medium contained more xylose than those of the yeast extract containing medium.