A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae.

In vivo time-lapse microscopy reveals that the number of peroxisomes in Saccharomyces cerevisiae cells is fairly constant and that a subset of the organelles are targeted and segregated to the bud in a highly ordered, vectorial process. The dynamin-like protein Vps1p controls the number of peroxisomes, since in a vps1Delta mutant only one or two giant peroxisomes remain. Analogous to the function of other dynamin-related proteins, Vps1p may be involved in a membrane fission event that is required for the regulation of peroxisome abundance. We found that efficient segregation of peroxisomes from mother to bud is dependent on the actin cytoskeleton, and active movement of peroxisomes along actin filaments is driven by the class V myosin motor protein, Myo2p: (a) peroxisomal dynamics always paralleled the polarity of the actin cytoskeleton, (b) double labeling of peroxisomes and actin cables revealed a close association between both, (c) depolymerization of the actin cytoskeleton abolished all peroxisomal movements, and (d) in cells containing thermosensitive alleles of MYO2, all peroxisome movement immediately stopped at the nonpermissive temperature. In addition, time-lapse videos showing peroxisome movement in wild-type and vps1Delta cells suggest the existence of various levels of control involved in the partitioning of peroxisomes.

Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast.

The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.

Life with 6000 genes.

The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Many yeast chromosomes lack the telomere-specific Y' sequence.

Chromosomal DNAs of 26 different strains representing Saccharomyces species were analyzed by pulsed-field gel electrophoresis and subsequent hybridization to Y' telomere DNA. Hybridization to Y' was found exclusively in Saccharomyces cerevisiae strains, and among these strains, Y' sequences were found to be lacking in small, middle-sized, and large chromosomes.

Purification of a protein binding to the CDEI subregion of Saccharomyces cerevisiae centromere DNA.

The DNA subregions CDEI and CDEIII of Saccharomyces cerevisiae centromeres are highly conserved, and both are binding sites for proteins. We describe here the purfication of a CDEI-specific binding protein using biotin-labeled synthetic CDEI DNA coupled to streptavidin agarose. The binding properties of this 64-kilodalton (kDa) protein were characterized by competition assays and by methylation interference assays. DNA fragments with single base-pair changes at positions 7 and 8 of CDEI were less efficient competitors than fragments with nonmutated CDEI. Mutations at these positions have previously been shown to decrease centromere activity in vivo. Methylation of guanosines at either side of the 8-base-pair CDEI sequence did not interfere with binding, whereas methylation of any of the four guanosines within CDEI prevented binding. A smaller CDEI-specific binding protein of 37 kDa was also purified and characterized. It is most likely a degradation product of the 64-kDa protein.

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae.

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

Time-lapse video microscopy analysis reveals astral microtubule detachment in the yeast spindle pole mutant cnm67.

Saccharomyces cerevisiae cnm67Delta cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein-labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the gamma-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Delta cells Spc72-gamma-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Delta cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.

Cnm67p is a spacer protein of the Saccharomyces cerevisiae spindle pole body outer plaque.

In Saccharomyces cerevisiae, the spindle pole body (SPB) is the functional homolog of the mammalian centrosome, responsible for the organization of the tubulin cytoskeleton. Cytoplasmic (astral) microtubules essential for the proper segregation of the nucleus into the daughter cell are attached at the outer plaque on the SPB cytoplasmic face. Previously, it has been shown that Cnm67p is an integral component of this structure; cells deleted for CNM67 are lacking the SPB outer plaque and thus experience severe nuclear migration defects. With the use of partial deletion mutants of CNM67, we show that the N- and C-terminal domains of the protein are important for nuclear migration. The C terminus, not the N terminus, is essential for Cnm67p localization to the SPB. On the other hand, only the N terminus is subject to protein phosphorylation of a yet unknown function. Electron microscopy of SPB serial thin sections reveals that deletion of the N- or C-terminal domains disturbs outer plaque formation, whereas mutations in the central coiled-coil domain of Cnm67p change the distance between the SPB core and the outer plaque. We conclude that Cnm67p is the protein that connects the outer plaque to the central plaque embedded in the nuclear envelope, adjusting the space between them by the length of its coiled-coil.

Saccharomyces cerevisiae cells with defective spindle pole body outer plaques accomplish nuclear migration via half-bridge-organized microtubules.

Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Delta1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67Delta1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.

GermOnline, a cross-species community knowledgebase on germ cell differentiation.

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on approximately 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.

Partial degradation of transfer RNAs by different preparations of snake venom exonuclease.

The degradation of yeast tRNASer with eight different exonuclease preparations from four snake venoms was investigated. The reaction products were separated on polyacrylamide gels containing 7 M urea. Patterns of sharp bands were obtained which were more or less similar. Two tRNA fragments were characterized by oligonucleotide analyses, one of which was tRNA degraded by the exonuclease up to the beginning of the T-phi-C-stem. The other one was generated by the additional loss of several nucleotides from the 5'-terminus. The formation of the latter fragment was very probably caused by an endonuclease activity in the exonuclease. The endonuclease contaminant, which was found in all preparations, was further investigated by experiments with modified tRNAs whose 3'-terminus should be resistant to exonuclease (tRNASer-A, tRNASerOX-red). With 3'-AMP as substrate no phosphatase activity was found under the conditions of tRNA degradation. Not only in tRNASer, but also in yeast tRNATyr and tRNAAla as well as in fragments of tRNASer and tRNAPhe, the degradation by exonuclease was inhibited at the beginning of the T-phi-C-stem. The finding of such a retardation site in addition to the general retardation of exonuclease digestion after removal of the C-C-A sequence may indicate that retardation at certain elements of secondary structure is a more general feature of degradations by this enzyme.

Cloning and sequencing of a gene coding for an actin binding protein of Saccharomyces exiguus.

The actin binding protein Abp1p of the yeast Saccharomyces cervisiae is thought to be involved in the spatial organisation of cell surface growth. It contains a potential actin binding domain and an SH-3 region, a common motif of many signal transduction proteins [1]. We have cloned and sequenced an ABP1 homologous gene of Saccharomyces exiguus, a yeast which is only distantly related to S. cerevisiae. The protein encoded by this gene is slightly larger than the respective S. cerevisiae protein (617 versus 592 amino acids). The two genes are 67.4% identical and the deduced amino acid sequences share an overall identity of 59.8%. The most conserved regions are the 148 N-terminal amino acids containing the potential actin binding site and the 58 C-terminal amino acids including the SH3 domain. In addition, both proteins contain a repeated motif of unknown function which is rich in glutamic acids with the sequence EEEEEEEAPAPSLPSR in the S. exiguus Abp1p.

Cell polarity and hyphal morphogenesis are controlled by multiple rho-protein modules in the filamentous ascomycete Ashbya gossypii.

Polarized cell growth requires a polarized organization of the actin cytoskeleton. Small GTP-binding proteins of the Rho-family have been shown to be involved in the regulation of actin polarization as well as other processes. Hyphal growth in filamentous fungi represents an ideal model to investigate mechanisms involved in generating cell polarity and establishing polarized cell growth. Since a potential role of Rho-proteins has not been studied so far in filamentous fungi we isolated and characterized the Ashbya gossypii homologs of the Saccharomyces cerevisiae CDC42, CDC24, RHO1, and RHO3 genes. The AgCDC42 and AgCDC24 genes can both complement conditional mutations in the S. cerevisiae CDC42 and CDC24 genes and both proteins are required for the establishment of actin polarization in A. gossypii germ cells. Agrho1 mutants show a cell lysis phenotype. Null mutant strains of Agrho3 show periodic swelling of hyphal tips that is overcome by repolarization and polar hyphal growth in a manner resembling the germination pattern of spores. Thus different Rho-protein modules are required for distinct steps during polarized hyphal growth of A. gossypii.

Homologous recombination as the main mechanism for DNA integration and cause of rearrangements in the filamentous ascomycete Ashbya gossypii.

A slow and a fast growth phenotype were observed after transformation of the phytopathogenic fungus Ashbya gossypii using a plasmid carrying homologous DNA and as selectable marker the Tn903 aminoglycoside resistance gene expressed from a strong A. gossypii promoter. Transformations with circular plasmids yielded slowly and irregularly growing geneticin-resistant mycelia in which 1% of nuclei contained plasmid sequences. Occasionally, fast growing sectors appeared which were shown to be initiated by homologous integration of the transforming DNA. Transformants obtained with plasmids linearized within the homology region immediately exhibited fast radial growth. In all 28 transformants analyzed plasmid DNA was integrated homologously. Such apparent lack of nonhomologous recombination has so far not been observed in filamentous ascomycetes. In 14 transformants two to four tandemly integrated plasmid copies were found. They underwent several types of genetic changes, mainly in the older mycelium: excision of whole plasmid copies and rearrangements within the integrated DNA (inversions and deletions). These internal rearrangements involved 360-bp inverted repeats, remnants of IS-elements flanking the resistance gene, and 156-bp direct repeats, originating from the strong A. gossypii promoter. Improved vectors lacking sequence repetitions were constructed and used for stable one-step gene replacement in A. gossypii.

Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements.

We have developed a transformation system for the filamentous ascomycete fungus Ashbya gossypii. Mycelial protoplasts were transformed to geneticin-resistance with plasmids containing the Escherichia coli kanamycin-resistance gene as a selectable marker and autonomously replicating sequences (ARS) from Saccharomyces cerevisiae (ARS1, 2 mu ARS). Transformation frequencies of up to 63 transformants per microgram of plasmid DNA were obtained. The transformants were unstable under nonselective conditions. Southern analysis of DNA separated by conventional and pulsed-field-gel electrophoresis showed that the transforming DNA was present as autonomously replicating plasmid. Plasmid integration into chromosomal DNA was not detected. We concluded that the S. cerevisiae ARS elements are functional in A. gossypii, since vectors lacking such elements did not yield transformants.

Construction of LYS2 cartridges for use in genetic manipulations of Saccharomyces cerevisiae.

Linker arrays were added to the 5' and 3' boundaries of the Saccharomyces cerevisiae LYS2 gene, which allow the generation of 18 LYS2 cartridges with different sticky ends. As it was necessary to define the beginning and the end of the approx. 4.5-kb LYS2 gene, we sequenced 1 kb of its 5' and 1.5 kb of its 3' region and mapped the mRNA start point. The open reading frame (ORF) found by this analysis was proven to be the LYS2 ORF by exchanging the sequences upstream from the presumptive ATG with the S. cerevisiae CYC1 promoter and subsequent demonstration of LYS2 expression in vivo. The proper functioning of the LYS2 cartridges was demonstrated by the transformation of lys2 mutant strains to Lys+ prototrophy using plasmids furnished with a LYS2 cartridge.

PCR-based gene targeting in the filamentous fungus Ashbya gossypii.

We have investigated a PCR-based approach for one-step gene targeting in the filamentous fungus Ashbya gossypii. Short guide sequences with 40-46 bp of homology to two sequences of a targeted gene, provided by PCR, were sufficient to mediate homologous recombination. The PCR products used for transformation were generated from the newly constructed chimeric selection marker GEN3. This consists of the open reading frame of the Escherichia coli kanR gene under the control of promoter and terminator sequences of the Saccharomyces cerevisiae TEF2 gene and allows selection of G418/geneticin-resistant transformants. Verification of gene targeting was performed either by PCR or by DNA hybridization analyses, and in all 18 cases tested, correct targeting was confirmed. This approach was used for the complete deletion of the open reading frame of the A. gossypii RHO4 gene for which a double-strand sequence was available as information source for the design of PCR primers. We also demonstrated successful partial deletion of four other ORFs using single-read sequences (SRS) as sole information for the design of targeting primers. A gossypii is the first filamentous fungus in which a PCR-based gene disruption technique has been established. Since short target guide sequences are sufficient to direct homologous integration into the A. gossypii genome it is not necessary to obtain and sequence large DNA fragments from a target locus to provide the long flanking homology regions usually required for efficient targeting of cloned disruption cassettes in filamentous fungi. Thus functional analysis of A. gossypii genes is already possible, based on single-pass sequence information.