scholarly journals MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae.

1993 ◽  
Vol 123 (2) ◽  
pp. 387-403 ◽  
Author(s):  
M T Brown ◽  
L Goetsch ◽  
L H Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Structural Integrity ◽  
Spindle Pole ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
In Vitro Mutagenesis ◽  
Spindle Elongation

The function of the essential MIF2 gene in the Saccharomyces cerevisiae cell cycle was examined by overepressing or creating a deficit of MIF2 gene product. When MIF2 was overexpressed, chromosomes missegregated during mitosis and cells accumulated in the G2 and M phases of the cell cycle. Temperature sensitive mutants isolated by in vitro mutagenesis delayed cell cycle progression when grown at the restrictive temperature, accumulated as large budded cells that had completed DNA replication but not chromosome segregation, and lost viability as they passed through mitosis. Mutant cells also showed increased levels of mitotic chromosome loss, supersensitivity to the microtubule destabilizing drug MBC, and morphologically aberrant spindles. mif2 mutant spindles arrested development immediately before anaphase spindle elongation, and then frequently broke apart into two disconnected short half spindles with misoriented spindle pole bodies. These findings indicate that MIF2 is required for structural integrity of the spindle during anaphase spindle elongation. The deduced Mif2 protein sequence shared no extensive homologies with previously identified proteins but did contain a short region of homology to a motif involved in binding AT rich DNA by the Drosophila D1 and mammalian HMGI chromosomal proteins.

DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (9) ◽  
pp. 6350-6360
Author(s):  
F Houman ◽  
C Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Essential Gene ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Nonsense Mutations ◽  
Carboxy Terminal

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.

scholarly journals RPC82 encodes the highly conserved, third-largest subunit of RNA polymerase C (III) from Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (10) ◽  
pp. 4433-4440 ◽  
Author(s):  
N Chiannilkulchai ◽  
R Stalder ◽  
M Riva ◽  
C Carles ◽  
M Werner ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Stop Codon ◽  
Sequence Similarity ◽  
Start Codon ◽  
Rrna Synthesis ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Multicopy Plasmid

RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes. In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa. Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82. It maps as a single-copy gene on chromosome XVI. The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon. The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins. Disruption of the RPC82 gene was lethal. An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis. Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits. A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.

DIPLOID SPORE FORMATION AND OTHER MEIOTIC EFFECTS OF TWO CELL-DIVISION-CYCLE MUTATIONS OF SACCHAROMYCES CEREVISIAE

Genetics ◽  
1980 ◽  
Vol 96 (4) ◽  
pp. 859-876 ◽  
Author(s):  
David Schild ◽  
Breck Byers
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cell Division Cycle ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Single Spore ◽  
Diploid Cells ◽  
Meiosis I

ABSTRACT The meiotic effects of two cell-division-cycle mutations of Saccharomyces cerevisiae (cdc5 and cdc14) have been examined. These mutations were isolated by L. H. Hartwell and his colleagues and characterized as defective in mitosis, causing a temperature-sensitive arrest in late nuclear division. When subjected to the restrictive temperature in meiosis, diploid cells homozygous for either of these mutations generally proceeded through premeiotic DNA synthesis and commitment to meiotic levels of recombination, but then arrested at a stage following spindle pole body (SPB) duplication and separation. The two SPBs lacked the interconnection by spindle microtubules typical of the complete meiosis I spindle. Challenge of these homozygotes by a semi-restrictive temperature often caused the production of asci containing two diploid spores. Genetic analysis of the viable pairs of spores revealed that each spore had become homozygous for centromere-linked markers significantly more frequently than for distal markers, indicating that the two spores each contained pairs of sister centromeres that had co-segregated in the reductional division of meiosis I. Ultrastructural analysis of the cdc5 homozygote demonstrated that these cells had completed meiosis I and formed two meiosis II spindles, but that the latter remained unusually short. This resulted in the encapsulation of both poles of each spindle within a single spore wall. These mutations therefore are defective in both meiotic divisions, as well as in the mitotic division described originally.

scholarly journals Stu1p Is Physically Associated with β-Tubulin and Is Required for Structural Integrity of the Mitotic Spindle

2002 ◽  
Vol 13 (6) ◽  
pp. 1881-1892 ◽  
Author(s):  
Hongwei Yin ◽  
Liru You ◽  
Danielle Pasqualone ◽  
Kristen M. Kopski ◽  
Tim C. Huffaker
Keyword(s):  
Mitotic Spindle ◽  
Structural Integrity ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Poles ◽  
Balance Of Forces ◽  
Related Proteins ◽  
Β Tubulin

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1–5mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1–5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin–related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with β-tubulin and identify the domains required for this interaction on both Stu1p and β-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.

scholarly journals Stably denatured regions in chromosomal DNA from the cdc2 Saccharomyces cerevisiae cell cycle mutant.

1983 ◽  
Vol 3 (9) ◽  
pp. 1665-1669 ◽  
Author(s):  
M N Conrad ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Early Stage ◽  
Dna Binding Protein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
Cdc2 Gene ◽  
Initiation Of Dna Replication

DNA isolated from Saccharomyces cerevisiae strains carrying temperature-sensitive mutations in the CDC2 gene after incubation at the restrictive temperature contains multiple stably denatured regions 200 to 700 base pairs long. These regions are probably stabilized by a DNA-binding protein. They are found in both replicated and unreplicated portions of DNA molecules, suggesting that they are not an early stage in the initiation of DNA replication.

scholarly journals A Temperature-Sensitive dcw1 Mutant of Saccharomyces cerevisiae Is Cell Cycle Arrested with Small Buds Which Have Aberrant Cell Walls

2004 ◽  
Vol 3 (5) ◽  
pp. 1297-1306 ◽  
Author(s):  
Hiroshi Kitagaki ◽  
Kiyoshi Ito ◽  
Hitoshi Shimoi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Wall ◽  
Cell Walls ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Mrna Levels ◽  
Homologous Proteins ◽  
Aberrant Cell ◽  
Cell Wall Biogenesis

ABSTRACT Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a Δdcw1 Δdfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3 Δdfg5). When DC61 cells were incubated at 37°C, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37°C. At 37°C, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37°C, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37°C, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (Δdcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.

Isolation and characterization of the RNA2, RNA3, and RNA11 genes of Saccharomyces cerevisiae

1984 ◽  
Vol 4 (11) ◽  
pp. 2396-2405
Author(s):  
R L Last ◽  
J B Stavenhagen ◽  
J L Woolford
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Copy ◽  
Yeast Genome ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Mrna Accumulation ◽  
Isolation And Characterization ◽  
Polyadenylated Rna

Temperature-sensitive mutations in the genes RNA2 through RNA11 cause accumulation of intervening sequence containing precursor mRNAs in Saccharomyces cerevisiae. Three different plasmids have been isolated which complement both the temperature-sensitive lethality and precursor mRNA accumulation when introduced into rna2, rna3, and rna11 mutant strains. The yeast sequences on these plasmids have been shown by Southern transfer hybridization and genetic mapping to be derived from the RNA2, RNA3, and RNA11 genomic loci. Part of the RNA2 gene is homologous to more than one region of the yeast genome, whereas the RNA3 and RNA11 genes are single copy. RNAs homologous to these loci have been identified by RNA transfer hybridization, and the specific RNAs which are associated with the Rna+ phenotype have been mapped. This was done by a combination of transcript mapping, subcloning, and in vitro mutagenesis. The transcripts are found to be enriched in polyadenylated RNA and are of very low abundance (0.01-0.001% polyadenylated RNA).

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 In vitro reactivation of spindle elongation in fission yeast nuc2 mutant cells.

1990 ◽  
Vol 110 (2) ◽  
pp. 417-425 ◽  
Author(s):  
H Masuda ◽  
T Hirano ◽  
M Yanagida ◽  
W Z Cande
Keyword(s):  
Fission Yeast ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Sensitive Mutant ◽  
Vitro Assay ◽  
Chromosome Separation ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Mutant Cells

To investigate the mechanisms of spindle elongation and chromosome separation in the fission yeast Schizosaccharomyces pombe, we have developed an in vitro assay using a temperature-sensitive mutant strain, nuc2. At the restrictive temperature, nuc2 cells are arrested at a metaphase-like stage with short spindles and condensed chromosomes. After permeabilization of spheroplasts of the arrested cells, spindle elongation was reactivated by addition of ATP and neurotubulin both at the restrictive and the permissive temperatures, but chromosome separation was not. This suggests that the nuc2 cells are impaired in function at a stage before sister chromatid disjunction. Spindle elongation required both ATP and exogenous tubulin and was inhibited by adenylyl imidodiphosphate (AMPPNP) or vanadate. The ends of yeast half-spindle microtubules pulse-labeled with biotinylated tubulin moved past each other during spindle elongation and a gap formed between the original half-spindles. These results suggest that the primary mechanochemical event responsible for spindle elongation is the sliding apart of antiparallel microtubules of the two half-spindles.

scholarly journals Mutations which block the binding of calmodulin to Spc110p cause multiple mitotic defects

1996 ◽  
Vol 109 (6) ◽  
pp. 1297-1310 ◽  
Author(s):  
D.A. Stirling ◽  
T.F. Rayner ◽  
A.R. Prescott ◽  
M.J. Stark
Keyword(s):  
Nuclear Dna ◽  
Spindle Pole Body ◽  
Point Mutations ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Primary Defect ◽  
Mutant Proteins ◽  
Synchronous Cultures

We have generated three temperature-sensitive alleles of SPC110, which encodes the 110 kDa component of the yeast spindle pole body (SPB). Each of these alleles carries point mutations within the calmodulin (CaM) binding site of Spc110p which affect CaM binding in vitro; two of the mutant proteins fail to bind CaM detectably (spc110-111, spc110-118) while binding to the third (spc110-124) is temperature-sensitive. All three alleles are suppressed to a greater or lesser extent by elevated dosage of the CaM gene (CMD1), suggesting that disruption of CaM binding is the primary defect in each instance. To determine the consequences on Spc110p function of loss of effective CaM binding, we have therefore examined in detail the progression of synchronous cultures through the cell division cycle at the restrictive temperature. In each case, cells replicate their DNA but then lose viability. In spc110-124, most cells duplicate and partially separate the SPBs but fail to generate a functional mitotic spindle, a phenotype which we term ‘abnormal metaphase’. Conversely, spc110-111 cells initially produce nuclear microtubules which appear well-organised but on entry into mitosis accumulate cells with ‘broken spindles’, where one SPB has become completely detached from the nuclear DNA. In both cases, the bulk of the cells suffer a lethal failure to segregate the DNA.

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