Showing papers by "Michael Snyder published in 2001"

Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF

Abstract: Proteins interact with genomic DNA to bring the genome to life; and these interactions also define many functional features of the genome. SBF and MBF are sequence-specific transcription factors that activate gene expression during the G1/S transition of the cell cycle in yeast. SBF is a heterodimer of Swi4 and Swi6, and MBF is a heterodimer of Mbpl and Swi6 (refs 1, 3). The related Swi4 and Mbp1 proteins are the DNA-binding components of the respective factors, and Swi6 mayhave a regulatory function. A small number of SBF and MBF target genes have been identified. Here we define the genomic binding sites of the SBF and MBF transcription factors in vivo, by using DNA microarrays. In addition to the previously characterized targets, we have identified about 200 new putative targets. Our results support the hypothesis that SBF activated genes are predominantly involved in budding, and in membrane and cell-wall biosynthesis, whereas DNA replication and repair are the dominant functions among MBF activated genes. The functional specialization of these factors may provide a mechanism for independent regulation of distinct molecular processes that normally occur in synchrony during the mitotic cell cycle.

A Genomic Study of the Bipolar Bud Site Selection Pattern in Saccharomyces cerevisiae

Abstract: A genome-wide screen of 4168 homozygous diploid yeast deletion strains has been performed to identify nonessential genes that participate in the bipolar budding pattern. By examining bud scar patte...

The Cbk1p pathway is important for polarized cell growth and cell separation in Saccharomyces cerevisiae.

Abstract: During the early stages of budding, cell wall remodeling and polarized secretion are concentrated at the bud tip (apical growth). The CBK1 gene, encoding a putative serine/threonine protein kinase, was identified in a screen designed to isolate mutations that affect apical growth. Analysis of cbk1Δ cells reveals that Cbk1p is required for efficient apical growth, proper mating projection morphology, bipolar bud site selection in diploid cells, and cell separation. Epitope-tagged Cbk1p localizes to both sides of the bud neck in late anaphase, just prior to cell separation. CBK1 and another gene, HYM1, were previously identified in a screen for genes involved in transcriptional repression and proposed to function in the same pathway. Deletion of HYM1 causes phenotypes similar to those observed in cbk1Δ cells and disrupts the bud neck localization of Cbk1p. Whole-genome transcriptional analysis of cbk1Δ suggests that the kinase regulates the expression of a number of genes with cell wall-related functions, including two genes required for efficient cell separation: the chitinase-encoding gene CTS1 and the glucanase-encoding gene SCW11. The Ace2p transcription factor is required for expression of CTS1 and has been shown to physically interact with Cbk1p. Analysis of ace2Δ cells reveals that Ace2p is required for cell separation but not for polarized growth. Our results suggest that Cbk1p and Hym1p function to regulate two distinct cell morphogenesis pathways: an ACE2-independent pathway that is required for efficient apical growth and mating projection formation and an ACE2-dependent pathway that is required for efficient cell separation following cytokinesis. Cbk1p is most closely related to the Neurospora crassa Cot-1; Schizosaccharomyces pombe Orb6; Caenorhabditis elegans, Drosophila, and human Ndr; and Drosophila and mammalian WARTS/LATS kinases. Many Cbk1-related kinases have been shown to regulate cellular morphology.

Emerging technologies in yeast genomics

Abstract: The genomic revolution is undeniable: in the past year alone, the term 'genomics' was found in nearly 500 research articles, and at least 6 journals are devoted solely to genomic biology. More than just a buzzword, molecular biology has genuinely embraced genomics (the systematic, large-scale study of genomes and their functions). With its facile genetics, the budding yeast Saccharomyces cerevisiae has emerged as an important model organism in the development of many current genomic methodologies. These techniques have greatly influenced the manner in which biology is studied in yeast and in other organisms. In this review, we summarize the most promising technologies in yeast genomics.

A filamentous growth response mediated by the yeast mating pathway.

Abstract: Haploid cells of the budding yeast Saccharomyces cerevisiae respond to mating pheromones by arresting their cell-division cycle in G1 and differentiating into a cell type capable of locating and fusing with mating partners. Yeast cells undergo chemotactic cell surface growth when pheromones are present above a threshold level for morphogenesis; however, the morphogenetic responses of cells to levels of pheromone below this threshold have not been systematically explored. Here we show that MAT a haploid cells exposed to low levels of the α-factor mating pheromone undergo a novel cellular response: cells modulate their division patterns and cell shape, forming colonies composed of filamentous chains of cells. Time-lapse analysis of filament formation shows that its dynamics are distinct from that of pseudohyphal growth; during pheromone-induced filament formation, daughter cells are delayed relative to mother cells with respect to the timing of bud emergence. Filament formation requires the RSR1 ( BUD1 ), BUD8, SLK1/BCK1 , and SPA2 genes and many elements of the STE11/STE7 MAP kinase pathway; this response is also independent of FAR1 , a gene involved in orienting cell polarization during the mating response. We suggest that mating yeast cells undergo a complex response to low levels of pheromone that may enhance the ability of cells to search for mating partners through the modification of cell shape and alteration of cell-division patterns.

Genome-wide transposon mutagenesis in yeast.

Abstract: This unit provides comprehensive protocols for the use of insertional libraries generated by shuttle mutagenesis. From the basic protocol, a small aliquot of insertional library DNA may be used to mutagenize yeast, producing strains containing a single transposon insertion within a transcribed and translated region of the genome. This transposon-mutagenized bank of yeast strains may be screened for any desired mutant phenotype. Alternatively, since the transposon contains a reporter gene lacking its start codon and promoter, transposon-tagged strains may also be screened for specific patterns of gene expression. Strains of interest may be characterized by vectorette PCR (protocol provided) in order to locate the precise genomic site of transposon insertion within each mutant. A method by which Cre/lox recombination may be used to reduce the transposon in yeast to a small insertion element encoding an epitope tag is described. This tag serves as a tool by which transposon-mutagenized gene products may be analyzed further (e.g., localized to a discrete subcellular site).

Control of Cell Polarity and Shape

Abstract: Cell polarity results from asymmetric cell growth (polarized growth) and cell division from a specific plane (polarized division), two phenomena fundamental for the development of organisms ranging from yeasts to humans. Polarized growth leads to the formation of unique cell shapes and subcellular structures required for many cell types to carry out specialized functions. Sensory transduction by the neurites of neurons, nutrient absorption by the microvilli of intestinal epithelial cells and plant fertilization by navigation of pollen tubes are examples of cellular processes that require polarized growth (Mooseker 1985; Bedinger et al. 1994; Eisen 1994). The ability to undergo morphological change is essential for the virulence of many pathogenic fungi (Shepherd 1988; see also Chap. 5, this Vol.). Appropriate division plane selection is important for the equal distribution of essential organelles, such as the nucleus and mitochondria, as well as the generation of differential cell fates.

Making drug addicts out of yeast.

Abstract: Yeast has been engineered so that the binding of small-molecule ligands to a target protein can be simply detected by changes in growth

An XML application for genomic data interoperation

Abstract: As the eXtensible Markup Language (XML) becomes a popular or standard language for exchanging data over the Internet/Web, there are a growing number of genome Web sites that make their data available in XML format. Publishing genomic data in XML format alone would not be that useful if there is a lack of development of software applications that could take advantage of the XML technology to process these XML-formatted data. This paper illustrates the usefulness of XML in representing and interoperating genomic data between two different data sources (Snyder's laboratory at Yale and SGD at Stanford). In particular, we compare the locations of transposon insertions in the yeast DNA sequences that have been identified by BLAST searches with the chromosomal locations of the yeast open reading frames (ORFs) stored in SGD. Such a comparison allows us to characterize the transposon insertions by indicating whether they fall into any ORFs (which may potentially encode proteins that possess essential biological functions). To implement this XML-based interoperation, we used NCBIs "blastall" (which gives an XML output option) and SGD's yeast nucleotide sequence dataset to establish a local blast server. Also, we converted the SGD's ORF location data file (which is available in tab-delimited formal) into an XML document based on the BIOML (BIOpolymer Markup Language) standard.

A small reservoir of disabled ORFs in the Saccharomyces cerevisiae genome and its implications for the dynamics of proteome evolution

Abstract: We surveyed the sequenced Saccharomyces cerevisiae genome (strain S288C) comprehensively for open reading frames (ORF) that could encode full-length proteins but contain obvious mid-sequence disable- ments (frameshifts or premature stop codons). These pseudogenic fea- tures are termed disabled ORFs (dORFs). Using homology to annotated yeast ORFs and non-yeast proteins plus a simple region extension pro- cedure, we have found 183 dORFs. Combined with the 38 existing anno- tations for potential dORFs, we have a total pool of up to 221 dORFs, corresponding to less than3 % of the proteome. Additionally, we found 20 pairs of annotated ORFs for yeast that could be merged into a single ORF (termed a mORF) by read-through of the intervening stop codon, and may comprise a complete ORF in other yeast strains. Focussing on a core pool of 98 dORFs with a verifying protein homology, we find that most dORFs are substantially decayed, with90 % having two or more disablements, and 60 % having four or more. dORFs are much more yeast-proteome specific than live yeast genes (having about half the chance that they are related to a non-yeast protein). They show a dra- matically increased density at the telomeres of chromosomes, relative to genes. A microarray study shows that some dORFs are expressed even though they carry multiple disablements, and thus may be more refrac- tive to nonsense-mediated decay. Many of the dORFs may be involved in responding to environmental stresses, as the largest functional groups include growth inhibition, flocculation, and the SRP/TIP1 family. Our results have important implications for proteome evolution. The charac- teristics of the dORF population suggest the sorts of genes that are likely to fall in and out of usage (and vary in copy number) in a strain-specific way and highlight the role of subtelomeric regions in engendering this diversity. Our results also have important implications for the effects of the (PSIa ) prion. The dORFs disabled by only a single stop and the mORFs (together totalling 35) provide an estimate for the extent of the sequence population that can be resurrected readily through the demon- strated ability of the (PSIa ) prion to cause nonsense-codon read- through. Also, the dORFs and mORFs that we find have properties (e.g. growth inhibition, flocculation, vanadate resistance, stress response) that are potentially related to the ability of (PSIa ) to engender substantial phenotypic variation in yeast strains under different environmental conditions. # 2002 Academic Press

A metadata framework for interoperating heterogeneous genome data using XML.

Abstract: The rapid advances in the Human Genome Project and genomic technologies have produced massive amounts of data populated in a large number of network-accessible databases. These technological advances and the associated data can have a great impact on biomedicine and healthcare. To answer many of the biologically or medically important questions, researchers often need to integrate data from a number of independent but related genome databases. One common practice is to download data sets (text files) from various genome Web sites and process them by some local programs. One main problem with this approach is that these programs are written on a case-by-case basis because the data sets involved are heterogeneous in structure. To address this problem, we define metadata that maps these heterogeneously structured files into a common eXtensible Markup Language (XML) structure to facilitate data interoperation. We illustrate this approach by interoperating two sets of essential yeast genes that are stored in two yeast genome databases (MIPS and YPD).