8 Results
for term "sites"
- Chromosome Segregation in Budding Yeast: Sister Chromatid Cohesion and Related Mechanisms...and Koshland 2003; Huang et al. 2005). Support for the idea that chromosome-bound cohesin is a ring came with the nding that cohesin subunits remain associated with each other, but not with the chromosomes, after cleavage within the coiled-coil domain of Smc3 or at the separase recognition sites in Scc1 ~~~
- Morphogenesis and the Cell Cycle...of Morphogenesis 52 Polarity establishment in G1 52 Events leading to bud emergence: 53 Bud-site selection: 53 Polarization of Cdc42p: 53 Symmetry breaking: 55 Polarization of the cytoskeleton and growth: 56 CDK-mediated regulation of polarity establishment: 56 Bud-site selection: 58 Polarization of Cdc42p: 59 ~~~
- The Yeast Deletion Collection: A Decade of Functional Genomics...-GATGTCCACGAGGTCTCT-39 or D1: 59-CGGTGTCGGTCTCGTAG-39); a 20-bp unique sequence (the molecular barcode TAG); and 18 and 19 bp of sequence, respectively, homologous to the KanMX4 cassette (U2: 59- CGTACGCTGCAGGTCGAC-39 or D2: 59-ATCGATGAATTCGAGCTCG-39) the other priming site for amplifying the molecular barcodes ~~~
- Topology and Control of the Cell-Cycle-Regulated Transcriptional Circuitry...; Orlando et al. 2008). At the turn of the century, high-throughput chromatin immunoprecipitation-microarray chip (ChIP-chip) technologies enabled researchers to collectively identify the binding sites of all known transcription factors across the budding yeast genome (Ren et al. 2000; Simon et al. 2001 ~~~
Figure 2Polarity establishment. Bud-site selection (purple): prelocalized landmark proteins promote local GTP loading of Rsr1p, which recruits Cdc24p. In establishing polarity (blue), Cdc24p locally activates Cdc42p, employing positive feedback to generate and dynamically maintain a patch of highly concentrated GTP-Cdc42p at the cell cortex. During downstream events, localized GTP-Cdc42p employs various effectors to promote septin ring assembly (green), actin cable polarization (red), local exocytosis (yellow), and possibly also glucan synthesis and actin patch clustering. The downstream events also influence each other (only some of these links are shown) and together promote bud formation.
Figure 5Cytokinesis and cell separation. Sequential panels showing (from left to right): (Left) In late anaphase, actin cables and patches are dispersed and an actomyosin ring (red) forms in the center of the neck, recruited to and maintained at that site by the septin collar (green). (Second from left) Upon CDK inactivation, the septin collar splits to form two rings, and the actomyosin ring constricts, guiding a chitin synthase and its regulators (yellow) to deposit a primary septum (black). At around this time the actin cables and patches reorient toward the neck. (Third from left) Upon completion of the primary septum, mother and bud deposit a secondary septum (gray) on either side. (Right) Daughter cells synthesize and secrete chitinase (purple arrows), which degrades the primary septum (black) and several glucanases, which presumably degrade the edge cell wall connecting mother and bud (gray), allowing cell separation. (Bottom) A cross section through the central plane. The mother cell also contains a ring of chitinous cell wall (black) surrounding the neck, which is synthesized in late G1/S phase as a bud first emerges by a distinct chitin synthase. This ring remains as a bud scar following cell separation.
Figure 7Swe1p degradation pathway. Swe1p shuttles in and out of the nucleus and can be recruited to the mother-bud neck by a hierarchy of interactions involving septins, Hsl1p, and Hsl7p. Hsl1p is activated by Elm1p, another neck-localized kinase. At the neck, Swe1p is phosphorylated at multiple sites by Cdc5p, which is thought to target Swe1p for degradation. Phosphorylation of Swe1p by Clb-Cdc28p (which may occur in the nucleus, in the cytoplasm, or at the neck) primes Swe1p for subsequent phosphorylation by Cdc5p.
Figure 1(A) Saccharomyces cerevisiae Genome Deletion Project overview. The Stanford Genome Technology Center (SGTC) (yellow boxes) served as the resource for: (1) The 20-bp unique molecular barcode or tag sequence (UPTAG and DNTAG) assigned to each ORF (in collaboration with Affymetrix, peach box). (2) Automated primer picking for deletion cassette construction and deletion strain confirmation oligonucleotides (oligos) using sequencing data from the SGD (Cherry et al. 2012). (3) Primer-picking scripts were formatted for use with the automated multiplex oligonucleotide synthesizer (AMOS). (4) Resulting PCR-amplified deletion cassette modules (purple) and five premixed oligonucleotides pairs for the PCR confirmations of each strain (yellow) (A–B, A–kanB, C–D, kanC–D, and A–D) were arrayed into 96-well “6-pks” and sent to consortium members. (5) Successful deletion phenotypes and results of PCR confirmations were logged into the deletion database at the SGTC and directly made available to the yeast community by Research Genetics, SGTC, and ATCC. (6) Strains that failed to be deleted in the first round of strain construction were sent back to the SGTC for primer redesign. (B) Deletion strain strategy. Each deletion “cassette” was constructed using two sequential PCR reactions. In the first amplification, 74-bp UPTAG and 74-bp DNTAG primers amplify the KanMX gene from pFA6-kanMX4 DNA, whose KanMX expression confers dominant selection of geneticin (G418) to yeast (Wach et al. 1994). The primers consist of (5′–3′): 18 bp of genomic sequence that flank either the 5′ or 3′ end of the ORF (directly proximal and distal to the start and stop codons, respectively); 18 and 17 bp of sequence common to all gene disruptions (for amplifying the “molecular barcodes” in a PCR; (U1: 5′-GATGTCCACGAGGTCTCT-3′ or D1: 5′-CGGTGTCGGTCTCGTAG-3′); a 20-bp unique sequence (the molecular barcode TAG); and 18 and 19 bp of sequence, respectively, homologous to the KanMX4 cassette (U2: 5′- CGTACGCTGCAGGTCGAC-3′ or D2: 5′-ATCGATGAATTCGAGCTCG-3′) the other priming site for amplifying the molecular barcodes. In the second PCR reaction, two ORF-specific 45-mer oligonucleotides (UP_45 and DOWN_45) are used to extend the ORF-specific homology to 45 bp, increasing the targeting specificity during mitotic recombination of the gene disruption cassette. The presence of two tags (UPTAG and DNTAG) increases the quality of the hybridization data from the oligonucleotide arrays by adding redundancy (∼3.2% of the strains harbor only one unique UPTAG sequence). Note that in version 2.0 and subsequent collections, the two-step PCR was replaced with a single, longer primer PCR. The original length constraint was due to high error rates in longer primers, a problem that was significantly reduced by the time the V 2.0 strains were constructed. (C) Deletion strain confirmation. The correct genomic replacement of the gene with the KanMX cassette was verified in the mutants by the presence of PCR products of the expected size, using primers that span the left and right junctions of the deletion module within the genome. Four ORF-specific confirmation primers (A, B, C, and D primers) were selected for each ORF disruption. The A and D primers were positioned 200–400 bp from the start and stop codons of the gene, respectively. The B and C primers were located within the coding region of the ORF and, when used with the A or D primers, gave product sizes between 250 and 1000 bp. The KanB and KanC primers are internal to the KanMX4 module. For haploid or homozygous isolates, the junctions of the disruption were verified by amplification of genomic DNA using primers A and KanB and primers KanC and D. Deletion of the ORF was verified by the absence of a PCR product using primers A with B and C with D. In the case of heterozygous strains a successful deletion was indicated by the appearance of an additional, wild-type-sized PCR product in reactions A with B, C with D, and A with D. Each deletion mutant was checked for a PCR product of the proper size using the primers flanking the gene. In addition, each strain background was checked for the appropriate auxotrophic markers and mating type. The rigorous strain verification used in the deletion project is unfortunately not the norm. Formally, the five confirmations were required for confirmation; this was reduced to three when the long A–D PCR product proved problematic, with many groups verifying only the upstream and downstream KanMX-genomic junctions, omitting the A and D reactions that verify both the presence of the deletion and, equally importantly, that confirm the absence of the wild-type allele.