Showing papers by "Michael Snyder published in 2000"
TL;DR: A novel protein chip technology is developed that allows the high-throughput analysis of biochemical activities, and this approach is used to analyse nearly all of the protein kinases from Saccharomyces cerevisiae, finding many novel activities and that a large number ofprotein kinases are capable of phosphorylating tyrosine.
Abstract: We have developed a novel protein chip technology that allows the high-throughput analysis of biochemical activities, and used this approach to analyse nearly all of the protein kinases from Saccharomyces cerevisiae. Protein chips are disposable arrays of microwells in silicone elastomer sheets placed on top of microscope slides. The high density and small size of the wells allows for high-throughput batch processing and simultaneous analysis of many individual samples. Only small amounts of protein are required. Of 122 known and predicted yeast protein kinases, 119 were overexpressed and analysed using 17 different substrates and protein chips. We found many novel activities and that a large number of protein kinases are capable of phosphorylating tyrosine. The tyrosine phosphorylating enzymes often share common amino acid residues that lie near the catalytic region. Thus, our study identified a number of novel features of protein kinases and demonstrates that protein chip technology is useful for high-throughput screening of protein biochemical activity.
814 citations
TL;DR: This work found that cells lacking septins at the bud neck failed to maintain the exocytosis and morphogenesis factors Spa2, Sec3, Sec5, and Myo2 in the bud during isotropic growth, and proposed that septin maintain cell polarity by specifying a boundary between cortical domains.
315 citations
TL;DR: It is proposed that apical growth and repolarization at the site of cytokinesis are crucial for establishing spatial cues used by diploid yeast cells to position division planes and how the duration of polarized growth affects bipolar bud site selection is examined.
Abstract: We examined the relationship between polarized growth and division site selection, two fundamental processes important for proper development of eukaryotes. Diploid Saccharomyces cerevisiae cells exhibit an ellipsoidal shape and a specific division pattern (a bipolar budding pattern). We found that the polarity genes SPA2, PEA2, BUD6, and BNI1 participate in a crucial step of bud morphogenesis, apical growth. Deleting these genes results in round cells and diminishes bud elongation in mutants that exhibit pronounced apical growth. Examination of distribution of the polarized secretion marker Sec4 demonstrates that spa2Delta, pea2Delta, bud6Delta, and bni1Delta mutants fail to concentrate Sec4 at the bud tip during apical growth and at the division site during repolarization just prior to cytokinesis. Moreover, cell surface expansion is not confined to the distal tip of the bud in these mutants. In addition, we found that the p21-activated kinase homologue Ste20 is also important for both apical growth and bipolar bud site selection. We further examined how the duration of polarized growth affects bipolar bud site selection by using mutations in cell cycle regulators that control the timing of growth phases. The grr1Delta mutation enhances apical growth by stabilizing G(1) cyclins and increases the distal-pole budding in diploids. Prolonging polarized growth phases by disrupting the G(2)/M cyclin gene CLB2 enhances the accuracy of bud site selection in wild-type, spa2Delta, and ste20Delta cells, whereas shortening the polarized growth phases by deleting SWE1 decreases the fidelity of bipolar budding. This study reports the identification of components required for apical growth and demonstrates the critical role of polarized growth in bipolar bud site selection. We propose that apical growth and repolarization at the site of cytokinesis are crucial for establishing spatial cues used by diploid yeast cells to position division planes.
125 citations
TL;DR: Genetic and biochemical evidence suggest that Elm1 and the three other septin-localised protein kinases work in parallel pathways to regulate septin behaviour and cytokinesis in budding yeast is regulated by Elm1.
Abstract: A Saccharomyces cerevisiae mutant unable to grow in a cdc28-1N background was isolated and shown to be affected in the ELM1 gene. Elm1 is a protein kinase, thought to be a negative regulator of pseudo-hyphal growth. We show that Cdc11, one of the septins, is delocalised in the mutant, indicating that septin localisation is partly controlled by Elm1. Moreover, we show that cytokinesis is delayed in an elm1delta mutant. Elm1 levels peak at the end of the cell cycle and Elm1 is localised at the bud neck in a septin-dependent fashion from bud emergence until the completion of anaphase, at about the time of cell division. Genetic and biochemical evidence suggest that Elm1 and the three other septin-localised protein kinases, Hsl1, Gin4 and Kcc4, work in parallel pathways to regulate septin behaviour and cytokinesis. In addition, the elm1delta;) morphological defects can be suppressed by deletion of the SWE1 gene, but not the cytokinesis defect nor the septin mislocalisation. Our results indicate that cytokinesis in budding yeast is regulated by Elm1.
111 citations
TL;DR: Using a novel multipurpose mini-transposon, a collection of defined mutant alleles for the analysis of disruption phenotypes, protein localization, and gene expression in Saccharomyces cerevisiae are generated and cataloged in TRIPLES, a Web-accessible database of TRansposon-Insertion Phenotypes, Localization and Expression in SacCharomyces.
Abstract: Using a novel multipurpose mini-transposon, we have generated a collection of defined mutant alleles for the analysis of disruption phenotypes, protein localization, and gene expression in Saccharomyces cerevisiae. To catalog this unique data set, we have developed TRIPLES, a Web-accessible database of TRansposon-Insertion Phenotypes, Localization and Expression in Saccharomyces. Encompassing over 250 000 data points, TRIPLES provides convenient access to information from nearly 7800 transposon-mutagenized yeast strains; within TRIPLES, complete data reports of each strain may be viewed in table format, or if desired, downloaded as tab-delimited text files. Each report contains external links to corresponding entries within the Saccharomyces Genome Database and International Nucleic Acid Sequence Data Library (GenBank). Unlike other yeast databases, TRIPLES also provides on-line order forms linked to each clone report; users may immediately request any desired strain free-of-charge by submitting a completed form. In addition to presenting a wealth of information for over 2300 open reading frames, TRIPLES constitutes an important medium for the distribution of useful reagents throughout the yeast scientific community. Maintained by the Yale Genome Analysis Center, TRIPLES may be accessed at http://ycmi.med.yale.edu/ygac/triples.htm
80 citations
TL;DR: Logistic regression showed that increasedmean corpuscular volume and mean corpuscular hemoglobin concentration were the parameters best related to DHS, and specific recombination events consistently suggested that the responsible gene lies between markers D16S402 and D16s3037.
65 citations
TL;DR: Mapping of the interaction domains of the two proteins by two-hybrid analyses indicates that Kar3p and Cik1p associate in a highly specific manner along the lengths of their respective coiled-coil domains, demonstrating that the Kar3-Cik1 complex has a novel heterodimeric structure not observed previously for kinesin complexes.
Abstract: Proteins that physically associate with members of the kinesin superfamily are critical for the functional diversity observed for these microtubule motor proteins. However, quaternary structures of complexes between kinesins and kinesin-associated proteins are poorly defined. We have analyzed the nature of the interaction between the Kar3 motor protein, a minus-end-directed kinesin from yeast, and its associated protein Cik1. Extraction experiments demonstrate that Kar3p and Cik1p are tightly associated. Mapping of the interaction domains of the two proteins by two-hybrid analyses indicates that Kar3p and Cik1p associate in a highly specific manner along the lengths of their respective coiled-coil domains. Sucrose gradient velocity centrifugation and gel filtration experiments were used to determine the size of the Kar3-Cik1 complex from both mating pheromone-treated cells and vegetatively growing cells. These experiments predict a size for this complex that is consistent with that of a heterodimer containing one Kar3p subunit and one Cik1p subunit. Finally, immunoprecipitation of epitope-tagged and untagged proteins confirms that only one subunit of Kar3p and Cik1p are present in the Kar3-Cik1 complex. These findings demonstrate that the Kar3-Cik1 complex has a novel heterodimeric structure not observed previously for kinesin complexes.
61 citations
TL;DR: Known kinesin-binding proteins are discussed, and how they might participate in the activity of their motor partners.
46 citations
TL;DR: A model in which Sbe2p and Sbe22p are involved in the transport of cell wall components from the Golgi apparatus to the cell surface periphery in a pathway independent of Chs5p is suggested.
Abstract: The cell wall of fungal cells is important for cell integrity and cell morphogenesis and protects against harmful environmental conditions. The yeast cell wall is a complex structure consisting mainly of mannoproteins, glucan, and chitin. The molecular mechanisms by which the cell wall components are synthesized and transported to the cell surface are poorly understood. We have identified and characterized two homologous yeast proteins, Sbe2p and Sbe22p, through their suppression of a chs5 spa2 mutant strain defective in chitin synthesis and cell morphogenesis. Although sbe2 and sbe22 null mutants are viable, sbe2 sbe22 cells display several phenotypes indicative of defects in cell integrity and cell wall structure. First, sbe2 sbe22 cells display a sorbitol-remediable lysis defect at 37°C and are hypersensitive to SDS and calcofluor. Second, electron microscopic analysis reveals that sbe2 sbe22 cells have an aberrant cell wall structure with a reduced mannoprotein layer. Finally, immunofluorescence experiments reveal that in small-budded cells, sbe2 sbe22 mutants mislocalize Chs3p, a protein involved in chitin synthesis. In addition, sbe2 sbe22 diploids have a bud-site selection defect, displaying a random budding pattern. A Sbe2p–GFP fusion protein localizes to cytoplasmic patches, and Sbe2p cofractionates with Golgi proteins. Deletion of CHS5, which encodes a Golgi protein involved in the transport of Chs3p to the cell periphery, is lethal in combination with disruption of SBE2 and SBE22. Thus, we suggest a model in which Sbe2p and Sbe22p are involved in the transport of cell wall components from the Golgi apparatus to the cell surface periphery in a pathway independent of Chs5p.
45 citations
TL;DR: It is suggested that M25 is dispensable for viral growth in these organs and the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo.
39 citations
TL;DR: F multifunctional transposon-based mutagenesis systems for the large-scale accumulation of expression, phenotypic, and localization data have been developed and offer the potential to determine gene functions associated with essential genes—a target population previously refractory to large- scale study.
Abstract: Publisher Summary As a discipline, molecular biology has never been static: Newly emerging technologies have always fueled fundamental shifts in the paradigms of biological studies A number of such large-scale functional studies are already in place as a means of studying the budding yeast, Saccharomyces cerevisiae With its fully sequenced genome 4 and straightforward genetics, Saccharomyces has provided the template for large-scale projects analyzing gene expression and protein function Functional studies of the Saccharomyces genome have also encompassed efforts to analyze directly all predicted protein products While these approaches are all positive steps toward developing efficient functional genomic methodologies, each technique in itself is limited To address these drawbacks, multifunctional transposon-based mutagenesis systems for the large-scale accumulation of expression, phenotypic, and localization data have been developed This approach is not biased toward previously annotated genes and offers the additional advantage of generating plasmid-borne alleles of mutagenized genes for convenient analysis within any desired genetic background This system also offers the potential to determine gene functions associated with essential genes—a target population previously refractory to large-scale study
TL;DR: Novel large-scale approaches for generating gene disruptions and analyzing the resulting phenotype are underway in the budding yeast Saccharomyces cerevisiae and other organisms including flies, Mycoplasma, worms, plants and mice.
TL;DR: It is proposed that the carboxy terminus of Tub4p is required for re-organization of the microtubules upon completion of nuclear migration, and facilitates spindle elongation into the bud.
Abstract: The role of gamma-tubulin in microtubule nucleation is well established, however, its function in other aspects of microtubule organization is unknown. The carboxy termini of alpha/beta-tubulins influence the assembly and stability of microtubules. We investigated the role of the carboxy terminus of yeast gamma-tubulin (Tub4p) in microtubule organization. This region consists of a conserved domain (DSYLD), and acidic tail. Cells expressing truncations lacking the DSYLD domain, tail or both regions are temperature sensitive for growth. Growth defects of tub4 mutants lacking either or both carboxy-terminal domains are suppressed by the microtubule destabilizing drug benomyl. tub4 carboxy-terminal mutants arrest as large budded cells with short bipolar spindles positioned at the bud neck. Electron microscopic analysis of wild-type and CTR mutant cells reveals that SPBs are tightly associated with the bud neck/cortex by cytoplasmic microtubules in mutants lacking the tail region (tub4-delta 444, tub4-delta 448). Mutants lacking the DSYLD residues (tub4-delta 444, tub4-delta DSYLD) form many cytoplasmic microtubules. We propose that the carboxy terminus of Tub4p is required for re-organization of the microtubules upon completion of nuclear migration, and facilitates spindle elongation into the bud.
TL;DR: Functional genomics is characterised by methodologies involving high throughput and subsequent computational analysis, which are directed at the gene level or the protein level (proteomics), which has great potential for medical application of functional data in the understanding of disease and the search for remedies.
Abstract: Since its inception less than 15 years ago the field of genomics has expanded enormously, particularly with the concentration of entire genome sequencing. More recently, with the completion of the sequencing of a number genomes, including yeast, worms and fruit flies, and with the end of the Human Genome Project foreseeable in the not too distant future, attention is increasingly being turned to gene-function-oriented studies. The term “functional genomics” has arisen, which refers to the attempts to apply large-scale strategies to analyse gene function. Functional genomics is characterised by methodologies involving high throughput and subsequent computational analysis, which are directed at the gene level (genomics) or the protein level (proteomics). In addition to the academic goals of understanding genome functions, there is great potential for medical application of functional data in the understanding of disease and the search for remedies. Associated with the biomedical potential, there is also a high level of interest being shown by pharmaceutical companies eager to exploit the vast potential of this information in their quest for drug discovery. A new forum for genome function studies
TL;DR: A platform-independent Web front end that integrates a set of programs used in a genomic project analyzing gene function by transposon mutagenesis in Saccharomyces cerevisiae exemplifies a general approach by which independent software programs may be integrated into an efficient protocol for large-scale genomic data processing.
Abstract: Large-scale genome projects require the analysis of large amounts of raw data. This analysis often involves the application of a chain of biology-based programs. Many of these programs are difficult to operate because they are non-integrated, command-line driven, and platform-dependent. The problem is compounded when the number of data files involved is large, making navigation and status-tracking difficult. To demonstrate how this problem can be addressed, we have created a platform-independent Web front end that integrates a set of programs used in a genomic project analyzing gene function by transposon mutagenesis in Saccharomyces cerevisiae. In particular, these programs help define a large number of transposon insertion events within the yeast genome, identifying both the precise site of transposon insertion as well as potential open reading frames disrupted by this insertion event. Our Web interface facilitates this analysis by performing the following tasks. Firstly, it allows each of the analysis programs to be launched against multiple directories of data files. Secondly, it allows the user to view, download, and upload files generated by the programs. Thirdly, it indicates which sets of data directories have been processed by each program. Although designed specifically to aid in this project, our interface exemplifies a general approach by which independent software programs may be integrated into an efficient protocol for large-scale genomic data processing.
Proceedings Article•
01 Jan 2000TL;DR: This work uses TurboGenomics, a software package currently in its alpha-testing phase, to develop a Web-based application that allows integrated access to a set of large-scale sequence data analysis programs used by a transposon-insertion based yeast genome project.
Abstract: Rapid analysis of large amounts of genomic data is of great biological as well as medical interest. This type of analysis will greatly benefit from the ability to rapidly assemble a set of related analysis programs and to exploit the power of parallel computing. TurboGenomics, which is a software package currently in its alpha-testing phase, allows integration of heterogeneous software components to be done graphically. In addition, the tool is capable of making the integrated components run in parallel. To demonstrate these abilities, we use the tool to develop a Web-based application that allows integrated access to a set of large-scale sequence data analysis programs used by a transposon-insertion based yeast genome project. We also contrast the differences in building such an application with and without using the TurboGenomics software.