Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck

1987 ◽  
Vol 7 (10) ◽  
pp. 3678-3687
Author(s):  
B K Haarer ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Gene Product ◽  
Polyclonal Antibodies ◽  
Affinity Purification ◽  
Neck Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Growth ◽  
10 Nm Filaments ◽  
Mutant Cells

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud (B. Byers and L. Goetsch, J. Cell Biol. 69:717-721, 1976). Mutants defective in any of four genes (CDC3, CDC10, CDC11, and CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and cell-wall deposition and an inability to complete cytokinesis. We fused the cloned CDC12 gene to the Escherichia coli lacZ and trpE genes and used the resulting fusion proteins to raise polyclonal antibodies specific for the CDC12 gene product. In immunofluorescence experiments with affinity-purified antibodies, the neck region of wild-type and mutant cells stained in patterns consistent with the hypothesis that the CDC12 gene product is a constituent of the ring of 10-nm filaments. Without careful affinity purification of the CDC12-specific antibodies, these staining patterns were completely obscured by the staining of residual cell wall components in the neck by antibodies present even in the "preimmune" sera of all rabbits tested.

scholarly journals Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck.

1987 ◽  
Vol 7 (10) ◽  
pp. 3678-3687 ◽  
Author(s):  
B K Haarer ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Gene Product ◽  
Polyclonal Antibodies ◽  
Affinity Purification ◽  
Neck Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Growth ◽  
10 Nm Filaments ◽  
Mutant Cells

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud (B. Byers and L. Goetsch, J. Cell Biol. 69:717-721, 1976). Mutants defective in any of four genes (CDC3, CDC10, CDC11, and CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and cell-wall deposition and an inability to complete cytokinesis. We fused the cloned CDC12 gene to the Escherichia coli lacZ and trpE genes and used the resulting fusion proteins to raise polyclonal antibodies specific for the CDC12 gene product. In immunofluorescence experiments with affinity-purified antibodies, the neck region of wild-type and mutant cells stained in patterns consistent with the hypothesis that the CDC12 gene product is a constituent of the ring of 10-nm filaments. Without careful affinity purification of the CDC12-specific antibodies, these staining patterns were completely obscured by the staining of residual cell wall components in the neck by antibodies present even in the "preimmune" sera of all rabbits tested.

scholarly journals Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC3 gene product and the timing of events at the budding site.

1991 ◽  
Vol 112 (4) ◽  
pp. 535-544 ◽  
Author(s):  
H B Kim ◽  
B K Haarer ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Polyclonal Antibodies ◽  
Electron Microscopic ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Biol ◽  
Bud Emergence ◽  
Ring Structures ◽  
Bud Growth ◽  
10 Nm Filaments

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud. Electron microscopic observations suggest that these filaments assemble at the budding site coincident with bud emergence and disassemble shortly before cytokinesis (Byers, B. and L. Goetsch. 1976. J. Cell Biol. 69:717-721). Mutants defective in any of four genes (CDC3, CDC10, CDC11, or CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. We showed previously by immunofluorescence that the CDC12 gene product is probably a constituent of the ring of 10-nm filaments (Haarer, B. and J. Pringle. 1987. Mol. Cell. Biol. 7:3678-3687). We now report the use of fusion proteins to generate polyclonal antibodies specific for the CDC3 gene product. In immunofluorescence experiments, these antibodies decorated the neck regions of wild-type and mutant cells in patterns suggesting that the CDC3 gene product is also a constituent of the ring of 10-nm filaments. We also used the CDC3-specific and CDC12-specific antibodies to investigate the timing of localization of these proteins to the budding site. The results suggest that the CDC3 protein is organized into a ring at the budding site well before bud emergence and remains so organized for some time after cytokinesis. The CDC12 product appears to behave similarly, but may arrive at the budding site closer to the time of bud emergence, and disappear from that site more quickly after cytokinesis, than does the CDC3 product. Examination of mating cells and cells responding to purified mating pheromone revealed novel arrangements of the CDC3 and CDC12 products in the regions of cell wall reorganization. Both proteins were present in normal-looking ring structures at the bases of the first zygotic buds.

scholarly journals The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae.

1993 ◽  
Vol 120 (5) ◽  
pp. 1203-1215 ◽  
Author(s):  
K Kuchler ◽  
H G Dohlman ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Gene Product ◽  
Cell Polarity ◽  
Polyclonal Antibodies ◽  
Apparent Molecular Weight ◽  
Subcellular Fractionation ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Factor Secretion

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.

Nuclear behaviour and cell wall composition in relation to budding in mutants of the yeast Saccharomyces cerevisiae

1986 ◽  
Vol 64 (1) ◽  
pp. 193-200 ◽  
Author(s):  
Mario Lachapelle ◽  
E. Roger Boothroyd
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Spindle Pole ◽  
Minor Role ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Emergence ◽  
Spindle Elongation ◽  
10 Nm Filaments ◽  
A Minor

A temperature-sensitive, cell division cycle mutant (cdc24–1) and karyogamy-deficient (kar1) mutant of Saccharomyces cerevisiae, both of which can produce binucleate or multinucleate cells, were used to study certain aspects of budding, after fluorescent staining for mannan, chitin, and nuclei (DNA). In most binucleate cells the two nuclei lay close together and divided into the same bud. In a few, however, the nuclei were far apart and one or two buds were formed, each proximal to a nucleus. The proximity of daughter nuclei in most blocked cdc24–1 cells suggests a role for the CDC24 gene product in spindle elongation. The relationship between the nuclei and the number and location of buds supports the theory of a preponderant role for the nucleus in budding. Although buds develop preferentially in regions of low chitin content in kar1 heterokaryons, the ability of cdc24–1 cells to bud even with a uniformly high content of chitin and mannan suggests a minor role for these cell wall constituents in determining the sites of bud emergence. The chitin ring is not needed for bud emergence but seems to play a role in normal bud development and in septum formation. Electron microscopy of cdc24–1 cells blocked (37 °C) for 8 h and released (23 °C) for 30 min showed morphologically normal spindle pole bodies, cytoplasmic microtubules, and intranuclear spindles. Although the chitin ring was absent, the ring of 10-nm filaments was present, consistent with its proposed role in bud emergence.

scholarly journals Tightly centromere-linked gene (SPO15) essential for meiosis in the yeast Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (1) ◽  
pp. 158-167 ◽  
Author(s):  
E Yeh ◽  
J Carbon ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Transcriptional Activity ◽  
Intragenic Recombination ◽  
Wild Type ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Linked Gene ◽  
Mutant Cells

We used DNA fragments from the centromere regions of yeast (Saccharomyces cerevisiae) chromosomes III and XI to examine the transcriptional activity within this chromosomal domain. DNA transcripts were found 200 to 300 base pairs from the 250-base-pair centromere core and lie within an ordered chromatin array. No transcripts were detected from the functional centromere region. We examined the cellular function of one of these tightly centromere-linked transcripts. (CEN11)L, by disrupting the coding sequences in vivo and analyzing the phenotype of the mutant yeast cell. Diploids heterozygous for the (CEN11)L disruption sporulated at wild-type levels, and the absence of the (CEN11)L gene product had no effect on the viability or mitotic growth of haploid cells. Diploids homozygous for the (CEN11)L disruption were unable to sporulate when induced by the appropriate nutritional cues. The mutant cells were competent for intragenic recombination and appeared to be blocked at the mononucleate stage. The temporal ordering of (CEN11)L function with respect to the sporulation mutant spo13 suggests that the (CEN11)L gene product may be required at both the first and second meiotic cell divisions. This new sporulation gene has been termed SPO15.

scholarly journals Tightly centromere-linked gene (SPO15) essential for meiosis in the yeast Saccharomyces cerevisiae

1986 ◽  
Vol 6 (1) ◽  
pp. 158-167
Author(s):  
E Yeh ◽  
J Carbon ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Transcriptional Activity ◽  
Intragenic Recombination ◽  
Wild Type ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Linked Gene ◽  
Mutant Cells

We used DNA fragments from the centromere regions of yeast (Saccharomyces cerevisiae) chromosomes III and XI to examine the transcriptional activity within this chromosomal domain. DNA transcripts were found 200 to 300 base pairs from the 250-base-pair centromere core and lie within an ordered chromatin array. No transcripts were detected from the functional centromere region. We examined the cellular function of one of these tightly centromere-linked transcripts. (CEN11)L, by disrupting the coding sequences in vivo and analyzing the phenotype of the mutant yeast cell. Diploids heterozygous for the (CEN11)L disruption sporulated at wild-type levels, and the absence of the (CEN11)L gene product had no effect on the viability or mitotic growth of haploid cells. Diploids homozygous for the (CEN11)L disruption were unable to sporulate when induced by the appropriate nutritional cues. The mutant cells were competent for intragenic recombination and appeared to be blocked at the mononucleate stage. The temporal ordering of (CEN11)L function with respect to the sporulation mutant spo13 suggests that the (CEN11)L gene product may be required at both the first and second meiotic cell divisions. This new sporulation gene has been termed SPO15.

INTERACTION OF THE HIM1 GENE PRODUCT WITH HELICASES Srs2 (RadH) AND Mph1 YEAST SACCHAROMYCES CEREVISIAE

Tsitologiya ◽  
2018 ◽  
Vol 60 (7) ◽  
pp. 555-557 ◽  
Author(s):  
E. A. Alekseeva ◽  
◽  
T. A. Evstyukhina ◽  
V. T. Peshekhonov ◽  
V. G. Korolev ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Yeast Saccharomyces Cerevisiae

scholarly journals SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (5) ◽  
pp. 2154-2164 ◽  
Author(s):  
D J DeMarini ◽  
M Winey ◽  
D Ursic ◽  
F Webb ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Gene Product ◽  
Signal Sequence ◽  
Null Alleles ◽  
Nucleoside Triphosphate ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

scholarly journals The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (6) ◽  
pp. 2719-2727 ◽  
Author(s):  
S Silve ◽  
P Leplatois ◽  
A Josse ◽  
P H Dupuy ◽  
C Lanau ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Proliferation ◽  
Gene Product ◽  
In Vitro Assays ◽  
Gene Products ◽  
Yeast Saccharomyces Cerevisiae ◽  
Isomerase Activity ◽  
Encoding Gene ◽  
Resistant Mutants

SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.

Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein

1992 ◽  
Vol 12 (6) ◽  
pp. 2653-2661
Author(s):  
E Gross ◽  
I Marbach ◽  
D Engelberg ◽  
M Segal ◽  
G Simchen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Adenylyl Cyclase ◽  
Gene Product ◽  
Cyclase Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Terminal Domain ◽  
Yeast Homolog ◽  
Cdc25 Protein ◽  
Beta Galactosidase

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.

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