scispace - formally typeset
Search or ask a question

Showing papers by "Michael Snyder published in 1999"


Journal ArticleDOI
06 Aug 1999-Science
TL;DR: 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), finding that 17 percent were essential for viability in rich medium.
Abstract: 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.

4,051 citations


Journal ArticleDOI
25 Nov 1999-Nature
TL;DR: This work has developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and has applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae.
Abstract: Economical methods by which gene function may be analysed on a genomic scale are relatively scarce. To fill this need, we have developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and have applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae. Here we present the largest collection of defined yeast mutants ever generated within a single genetic background--a collection of over 11,000 strains, each carrying a transposon inserted within a region of the genome expressed during vegetative growth and/or sporulation. These insertions affect nearly 2,000 annotated genes, representing about one-third of the 6,200 predicted genes in the yeast genome. We have used this collection to determine disruption phenotypes for nearly 8,000 strains using 20 different growth conditions; the resulting data sets were clustered to identify groups of functionally related genes. We have also identified over 300 previously non-annotated open reading frames and analysed by indirect immunofluorescence over 1,300 transposon-tagged proteins. In total, our study encompasses over 260,000 data points, constituting the largest functional analysis of the yeast genome ever undertaken.

539 citations


Journal ArticleDOI
TL;DR: The results reveal for the first time how cells monitor the organization of their cytoskeleton and demonstrate the existence of a cell cycle checkpoint that responds to defects in the peripheral cytos skeleton.
Abstract: The mechanisms that couple cell cycle progression with the organization of the peripheral cytoskeleton are poorly understood. In Saccharomyces cerevisiae, the Swe1 protein has been shown previously to phosphorylate and inactivate the cyclin-dependent kinase, Cdc28, thereby delaying the onset of mitosis. The nim1-related protein kinase, Hsl1, induces entry into mitosis by negatively regulating Swe1. We have found that Hsl1 physically associates with the septin cytoskeleton in vivo and that Hsl1 kinase activity depends on proper septin function. Genetic analysis indicates that two additional Hsl1-related kinases, Kcc4 and Gin4, act redundantly with Hsl1 to regulate Swe1. Kcc4, like Hsl1 and Gin4, was found to localize to the bud neck in a septin-dependent fashion. Interestingly, hsl1 kcc4 gin4 triple mutants develop a cellular morphology extremely similar to that of septin mutants. Consistent with the idea that Hsl1, Kcc4, and Gin4 link entry into mitosis to proper septin organization, we find that septin mutants incubated at the restrictive temperature trigger a Swe1-dependent mitotic delay that is necessary to maintain cell viability. These results reveal for the first time how cells monitor the organization of their cytoskeleton and demonstrate the existence of a cell cycle checkpoint that responds to defects in the peripheral cytoskeleton. Moreover, Hsl1, Kcc4, and Gin4 have homologs in higher eukaryotes, suggesting that the regulation of Swe1/Wee1 by this class of kinases is highly conserved.

314 citations


Journal ArticleDOI
TL;DR: The results suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22–24-kD domain.
Abstract: Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788–1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22–24-kD domain.

125 citations


Journal ArticleDOI
TL;DR: Results indicate that Kar3p forms functionally distinct complexes with Cik1p and Vik1p to participate in different microtubule-mediated events within the same cell.
Abstract: The mechanisms by which kinesin-related proteins interact with other proteins to carry out specific cellular processes is poorly understood. The kinesin-related protein, Kar3p, has been implicated in many microtubule functions in yeast. Some of these functions require interaction with the Cik1 protein (Page, B.D., L.L. Satterwhite, M.D. Rose, and M. Snyder. 1994. J. Cell Biol. 124:507–519). We have identified a Saccharomyces cerevisiae gene, named VIK1, encoding a protein with sequence and structural similarity to Cik1p. The Vik1 protein is detected in vegetatively growing cells but not in mating pheromone-treated cells. Vik1p physically associates with Kar3p in a complex separate from that of the Kar3p-Cik1p complex. Vik1p localizes to the spindle-pole body region in a Kar3p-dependent manner. Reciprocally, concentration of Kar3p at the spindle poles during vegetative growth requires the presence of Vik1p, but not Cik1p. Phenotypic analysis suggests that Cik1p and Vik1p are involved in different Kar3p functions. Disruption of VIK1 causes increased resistance to the microtubule depolymerizing drug benomyl and partially suppresses growth defects of cik1Δ mutants. The vik1Δ and kar3Δ mutations, but not cik1Δ, partially suppresses the temperature-sensitive growth defect of strains lacking the function of two other yeast kinesin-related proteins, Cin8p and Kip1p. Our results indicate that Kar3p forms functionally distinct complexes with Cik1p and Vik1p to participate in different microtubule-mediated events within the same cell.

124 citations


Book ChapterDOI
TL;DR: A transposon mutagenesis system that produces multipurpose constructs for the monitoring of protein production, localization, and function is described, which provides the basis for a wide variety of studies of gene and protein function.
Abstract: Publisher Summary This chapter describes a transposon mutagenesis system that produces multipurpose constructs for the monitoring of protein production, localization, and function. A single mutagenesis generates a large spectrum of alleles, including null, hypomorphic, and conditional alleles, reporter fusions, and epitope-insertion alleles. The system, therefore, provides the basis for a wide variety of studies of gene and protein function. The chapter provides comprehensive instructions for use of the new transposons to mutagenize a gene of interest, and for use of the transposon insertion libraries to mutagenize the yeast genome. While the application of these specific transposons is limited to organisms in which the Saccharomyces cerevisiae selectable marker URA3 can be used, the approach is generally applicable to mutagenesis of DNA from any organism for which a transformation and selection system exists.

32 citations


Journal ArticleDOI
TL;DR: Yeast cells have an efficient system adapting to large variations in ambient pH and SHC1 is one of the genes required for the growth at alkaline pH, according to the Yeast Genome Directory.

11 citations


Book ChapterDOI
TL;DR: The role of proteins that coassemble with Tub4p in the yeast γ-tubulin complex is discussed, which plays an important role in controlling microtubule assembly and its position at the minus ends of microtubules.
Abstract: Publisher Summary Microtubules perform a variety of essential functions during the yeast cell cycle and life cycle, and consequently microtubule organization varies during these periods. The yeast microtubule organizing center (MTOC)—the spindle pole body (SPB)—is located in the nuclear membrane, which remains intact throughout the entire cell cycle. The organization of microtubules and their roles during the yeast cell cycle are diagrammatically represented in the chapter. This chapter discusses the role of proteins that coassemble with Tub4p in the yeast γ-tubulin complex. The Tub4p complex binds to the nuclear face of the SPB by its association with Nuf1p/Spc110p. The intermolecular interactions within the γ-tubulin complex and between the complex and the SPB are extremely well characterized in yeast. This system is useful in elucidating the mechanisms that regulate Tub4p function during microtubule nucleation. To study the function of the Tub4p-complex under defined conditions, it is necessary to develop an in vitro nucleation assay for the Tub4p complex. Such assays are used with great success in higher eukaryotes. Given its importance in microtubule assembly and its position at the minus ends of microtubules, γ-tubulin and its associated proteins play an important role in controlling these dynamics. Thus, the study of budding yeast presents a unique opportunity to uncover novel aspects of γ-tubulin function.

6 citations


Journal ArticleDOI
TL;DR: A DNA microarray is made that includes not only all the open reading frames (ORFs) and other features in the yeast genome, but also all the intergenic regions, using this as a tool to construct genome-wide maps of DNA-protein interactions for proteins that interact directly or indirectly with DNA or chromatin in vivo.
Abstract: We have made a DNA microarray that includes not only all the open reading frames (ORFs) and other features in the yeast genome, but also all the intergenic regions. We are using this as a tool to construct genome-wide maps of DNA-protein interactions for proteins that interact directly or indirectly with DNA or chromatin in vivo. Proteins are crosslinked to DNA in vivo using formaldehyde and the crosslinked DNA is extracted and sheared. DNA specifically associated with any protein of interest is immunoprecipitated using a specific antibody against the protein or an epitope tag that may be fused to the protein. The selected DNA, representing loci that the protein interacts with in vivo, can be identified by fluorescently labelling it and hybridizing it to the microarray along with an appropriate reference probe. This approach is being used to map the genome-wide interactions of sequence-specific DNA binding proteins, components of the transcription machinery and chromatin components, under a variety of conditions.

2 citations