CSE1 and CSE2, two new genes required for accurate mitotic chromosome segregation in Saccharomyces cerevisiae

By monitoring the mitotic transmission of a marked chromosome bearing a defective centromere, we have identified conditional alleles of two genes involved in chromosome segregation (cse). Mutations in CSE1 and CSE2 have a greater effect on the segregation of chromosomes carrying mutant centromeres than on the segregation of chromosomes with wild-type centromeres. In addition, the cse mutations cause predominantly nondisjunction rather than loss events but do not cause a detectable increase in mitotic recombination. At the restrictive temperature, cse1 and cse2 mutants accumulate large-budded cells, with a significant fraction exhibiting aberrant binucleate morphologies. We cloned the CSE1 and CSE2 genes by complementation of the cold-sensitive phenotypes. Physical and genetic mapping data indicate that CSE1 is linked to HAP2 on the left arm of chromosome VII and CSE2 is adjacent to PRP2 on chromosome XIV. CSE1 is essential and encodes a novel 109-kDa protein. CSE2 encodes a 17-kDa protein with a putative basic-region leucine zipper motif. Disruption of CSE2 causes chromosome missegregation, conditional lethality, and slow growth at the permissive temperature.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6350-6360.1994 ◽

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.

Download Full-text

Fission yeast genes nda1+ and nda4+, mutations of which lead to S-phase block, chromatin alteration and Ca2+ suppression, are members of the CDC46/MCM2 family.

Molecular Biology of the Cell ◽

10.1091/mbc.4.10.1003 ◽

1993 ◽

Vol 4 (10) ◽

pp. 1003-1015 ◽

Cited By ~ 44

Author(s):

S Miyake ◽

N Okishio ◽

I Samejima ◽

Y Hiraoka ◽

T Toda ◽

...

Keyword(s):

Fission Yeast ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Positive Role ◽

A Cell ◽

Cold Sensitive ◽

Single Nucleus ◽

Yeast Genes ◽

Cell Cycle Block ◽

Mutant Cells

Fission yeast cold-sensitive mutants nda1-376 and nda4-108 display a cell cycle block phenotype at the restrictive temperature (cell elongation with the single nucleus) accompanied by an alteration in the nuclear chromatin region. DNA content analysis shows that the onset of DNA synthesis is blocked or greatly delayed in both mutant cells, the block being reversible in nda4-108. Upon release to the permissive temperature, nda4-108 cells resumed replicating DNA, followed by mitosis and cytokinesis. The nda4 phenotype was partly rescued by the addition of Ca2+ to the medium; Ca2+ plays a positive role in the nda4+ function. The predicted protein sequences of nda1+ and nda4+ isolated by complementation are similar to each other and also, respectively, to those of the budding yeast, MCM2 and CDC46, both of which are members of the gene family required for the initiation of DNA replication. The central domains of these proteins are conserved, whereas the NH2- and COOH- domains are distinct. Results of the disruption of the nda1+ and nda4+ genes demonstrates that they are essential for viability.

Download Full-text

Higher order chromosome structure is affected by cold-sensitive mutations in a Schizosaccharomyces pombe gene crm1+ which encodes a 115-kD protein preferentially localized in the nucleus and its periphery.

The Journal of Cell Biology ◽

10.1083/jcb.108.4.1195 ◽

1989 ◽

Vol 108 (4) ◽

pp. 1195-1207 ◽

Cited By ~ 151

Author(s):

Y Adachi ◽

M Yanagida

Keyword(s):

Schizosaccharomyces Pombe ◽

Kinase Inhibitors ◽

Southern Hybridization ◽

Higher Order ◽

Protein Kinase Inhibitors ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Chromosome Domains ◽

Cold Sensitive ◽

Nuclear Environment

We isolated a novel class of Schizosaccharomyces pombe cold-sensitive mutants with deformed nuclear chromosome domains consisting of thread- or rodlike condensed segments at restrictive temperature. Their mutations were mapped in a novel, identical locus designated crm1 (chromosomal region maintenance). The crm1 mutants also show the following phenotypes. DNA, RNA, and protein syntheses diminish at restrictive temperature. At permissive temperature, the amount of one particular protein, p25, greatly increases. The mutant growth is hypersensitive to Ca2+ and resistant to protein kinase inhibitors. We cloned the 4.1-kb-long crm1+ gene that rescued the above phenotypes by transformation and determined its nucleotide sequence, which predicts a 1,077-residue protein. Affinity-purified antiserum raised against the crm1+ polypeptide expressed in Escherichia coli detected a 115-kD protein in S. pombe extracts. Genomic Southern hybridization and immunoblotting suggested that the crm1+ product might be highly conserved in distant organisms. Through immunofluorescence microscopy, the crm1+ protein appeared to be principally localized within the nucleus and also at its periphery. We speculate that the crm1+ protein might be one of those nuclear components that modify the chromosome structures or regulate the nuclear environment required for maintaining higher order chromosome structures.

Download Full-text

An Extragenic Suppressor of the Mitosis-Defective bimD6 Mutation of Aspergillus nidulans Codes for a Chromosome Scaffold Protein

Genetics ◽

10.1093/genetics/142.3.777 ◽

1996 ◽

Vol 142 (3) ◽

pp. 777-787 ◽

Cited By ~ 2

Author(s):

Cydne L Holt ◽

Gregory S May

Keyword(s):

Aspergillus Nidulans ◽

Chromosome Segregation ◽

Chromosome Structure ◽

Heat Sensitivity ◽

Restrictive Temperature ◽

Mitotic Spindles ◽

Normal Number ◽

Cold Sensitive ◽

Extragenic Suppressors ◽

Chromosome Scaffold

Abstract We previously identified a gene, bimD, that functions in chromosome segregation and contains sequences suggesting that it may be a DNA-binding protein. Two conditionally lethal mutations in bimD arrest with aberrant mitotic spindles at restrictive temperature. These spindles have one-third the normal number of microtubules, and the chromosomes never attach to the remaining microtubules. For this reason, we hypothesized that BIMD functioned in chromosome segregation, possibly as a component of the kinetochore. To identify other components that function with bimD, we conducted a screen for extragenic suppressors of the bimD5 and bimD6 mutations. We have isolated seven cold-sensitive extragenic suppressors of bimD6 heat sensitivity that represent three or possibly four separate sud genes. We have cloned one of the suppressor genes by complementation of the cold-sensitive phenotype of the sudA3 mutation. SUDA belongs to the DA-box protein family. DA-box proteins have been shown to function in chromosome structure and segregation. Thus bimD and the sud genes cooperatively function in chromosome segregation in Aspergillus nidulans.

Download Full-text

ISOLATION OF ANEUPLOID-GENERATING MUTANTS OF ASPERGILLUS NIDULANS, ONE OF WHICH IS DEFECTIVE IN INTERPHASE OF THE CELL CYCLE

Genetics ◽

10.1093/genetics/108.1.107 ◽

1984 ◽

Vol 108 (1) ◽

pp. 107-121

Author(s):

A Upshall ◽

I D Mortimore

Keyword(s):

Cell Cycle ◽

Chromosome Segregation ◽

High Frequency ◽

Mitotic Chromosome ◽

Meiotic Chromosome ◽

Restrictive Temperature ◽

Single Mutation ◽

Chromosome Type ◽

Temperature Exchange ◽

And Behavior

ABSTRACT A method is described for isolating mutants potentially defective in loci involved in mitotic chromosome segregation. Conditional lethal, heat-sensitive (42°) mutants were assayed at a subrestrictive temperature of 37° for an inflated production of colonies displaying phenotypes and behavior patterns of whole chromosome aneuploids. Of 14 mutants, three showed specificity for one disomic phenotype, whereas 11 generated colonies mosaic for different aneuploid phenotypes. This latter group is designated hfa (high frequency of aneuploid). For ten of the 11 mutants temperature sensitivity and aneuploid production cosegregated, indicating a single mutation in each. These mutations were recessive and nonallelic. Analysis was concentrated on the hfaB3 mutation which is mapped to chromosome VI tightly linked to the methB and tsB loci. The disruptive influence of hfaB3 on mitosis at 37° was shown by (1) ploidy and whole chromosome-type segregation of markers in the breakdown sectors of phenotypically aneuploid colonies obtained from multiply marked homozygous hfaB3 disploids; (2) a high frequency of haploid and nondisjunctional diploid segregants among spontaneous yellow-spored parasexual recombinants taken from green-spored homozygous hfaB3 diploids. The mutation had no effect on meiotic chromosome segregation at 37°. The single interphase nucleus in germlings at 42°, coupled with changes in the mitotic index in temperature exchange experiments, showed hfaB3 to arrest the cell cycle in interphase at restrictive temperature. A conclusion drawn is that the hfaB gene product is required both for entry into mitosis and for normal chromosome segregation in dividing nuclei.

Download Full-text

BEHAVIORAL AND BIOCHEMICAL DEFECTS IN TEMPERATURE-SENSITIVE ACETYLCHOLINESTERASE MUTANTS OF DROSOPHILA MELANOGASTER

Genetics ◽

10.1093/genetics/96.4.939 ◽

1980 ◽

Vol 96 (4) ◽

pp. 939-965

Author(s):

Jeffrey C Hall ◽

Stamatis N Alahiotis ◽

David A Strumpf ◽

Kristin White

Keyword(s):

Drosophila Melanogaster ◽

Early Developmental Stage ◽

Substrate Affinity ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Larval Stages ◽

Cold Sensitive ◽

And Function ◽

Catalytic Functions

ABSTRACT Temperature-sensitive (ts) mutants of the Ace gene, which codes for acetylcholinesterase (AChE) in Drosophila melanogaster, were analyzed for defects in viability, behavior and function of the enzyme. The use of heat-sensitive and cold-sensitive mutations permited the function of AChE in the nervous system to be analyzed temporally. All ts mutations were lethal, or nearly so, when animals expressing them were subjected to restrictive temperatures during late embryonic and very early larval stages. Heat treatments to Ace-ts mid- and late larvae had little effect on the behavior of these animals or on the viability or behavior of the eventual adults. Heat-sensitive mutants exposed to nonpermissive temperatures as pupae, by contrast, had severe defects in phototaxis and locomotor activity as adults. AChE extracted from adult ts mutants that had developed at a permissive temperature were abnormally heat labile, and they had reduced substrate affinity when assayed at restrictive temperatures. However, enzyme activity did not decline during exposure of heat-sensitive adults to high temperatures even though such treatments caused decrements in phototaxis (29°) and, eventually, cessation of movement (31°). The cold-sensitive mutant also produced readily detectable levels of AChE when exposed to a restrictive temperature during the early developmental stage when this mutation causes almost complete lethality. We suggest that the relationship among the genetic, biochemical and neurobiological defects in these mutants may involve more than merely temperature-sensitive catalytic functions.

Download Full-text

Cold-sensitive mutant with defective growth at 5 °C from a psychrotrophic bacterium

Canadian Journal of Microbiology ◽

10.1139/m80-001 ◽

1980 ◽

Vol 26 (1) ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

W. D. Murray ◽

W. E. Inniss

Keyword(s):

Atpase Activity ◽

Specific Activity ◽

Carbon Sources ◽

Krebs Cycle ◽

Effect Of Temperature ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Wild Type ◽

Cold Sensitive ◽

Krebs Cycle Intermediates

A cold-sensitive (CS) mutant of the psychrotroph, Bacillus psychrophilus, was obtained by N-methyl-N′-nitro-N-nitrosoguanidine mutagenization and penicillin counterselection. In the presence of citrate, the wild type grew well at both 5 and 20 °C whereas the CS mutant grew well at 20 °C (the permissive temperature) but, at 5 °C (the restrictive temperature), grew at a reduced rate for two to three generations followed by a complete plateau in growth. Upon return of the CS mutant to 20 °C, after a delay of about 40 h, growth resumed at the appropriate rate. The CS mutant exhibited growth rates similar to parental rates on a wide variety of carbon sources at 5 °C, but when Krebs cycle intermediates were used as substrates and in the presence of an equimolar amount of citrate, the typical cold-sensitive growth pattern occurred. Comparison of oxidative phosphorylation in the parent and CS mutant indicated that no phosphorylation occurred at 5 °C in the CS mutant during the plateau in growth. Examination of the effect of temperature on ATPase activity showed that at 5 °C the specific activity of ATPase isolated from the CS mutant grown at 5 °C was 15-fold less than the ATPases isolated from wild-type cells grown at either 5 or 20 °C and 10.5-fold lower than ATPase from CS mutant cells grown at 20 °C. The large reduction in CS mutant ATPase activity at 5 °C appears to be at least partly due to an effect on synthesis since citrate did not inhibit preformed ATPase.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6350 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6350-6360 ◽

Cited By ~ 9

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.

Download Full-text