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Plasmid and chromosome segregation in prokaryotes.

Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic-like apparatus in prokaryotes. The identification of chromosomal homologues of the well-characterized plasmid partitioning genes indicates that there could be a general mechanism of bacterial DNA partitioning.

Bacteria↗

Ribosomal DNA transcription-dependent processes interfere with chromosome segregation.

The ribosomal DNA (rDNA) is a specialized genomic region not only owing to its function as the nucleolar organizing region (NOR) but also because it is repetitive in nature and, at least in budding yeast, silenced for polymerase II (Pol II)-mediated transcription. Furthermore, cohesin-independent linkages hold the sister chromatids together at the rDNA loci, and their resolution requires the activity of the conserved protein phosphatase Cdc14. Here we show that rRNA transcription-dependent processes establish linkages at the rDNA, which affect segregation of this locus. Inactivation of Cfi1/Net1, a protein required for efficient rRNA transcription, or elimination of Pol I activity, which drives rRNA transcription, diminishes the need for CDC14 in rDNA segregation. Our results identify Pol I transcription-dependent processes as a novel means of establishing linkages between chromosomes.

Cell Cycle Proteins↗

Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage.

The rad2 mutant of Schizosaccharomyces pombe is sensitive to UV irradiation and deficient in the repair of UV damage. In addition, it has a very high degree of chromosome loss and/or nondisjunction. We have cloned the rad2 gene and have shown it to be a member of the Saccharomyces cerevisiae RAD2/S. pombe rad13/human XPG family. Using degenerate PCR, we have cloned the human homolog of the rad2 gene. Human cDNA has 55% amino acid sequence identity to the rad2 gene and is able to complement the UV sensitivity of the rad2 null mutant. We have thus isolated a novel human gene which is likely to be involved both in controlling the fidelity of chromosome segregation and in the repair of UV-induced DNA damage. Its involvement in two fundamental processes for maintaining chromosomal integrity suggests that it is likely to be an important component of cancer avoidance mechanisms.

Amino Acid Sequence↗

Chromosome segregation and cancer: cutting through the mystery.

Mitosis is the most dramatic--and potentially dangerous--event in the cell cycle, as sister chromatids are irreversibly segregated to daughter cells. Defects in the checkpoints that normally maintain the fidelity of this process can lead to chromosomal instability (CIN) and cancer. However, CIN--a driving force of tumorigenesis--could be the cancer cell's ultimate vulnerability. An important goal is to identify novel anticancer compounds that directly target the mitotic errors at the heart of CIN.

Aneuploidy↗

Antisense inhibition of CAS, the human homologue of the yeast chromosome segregation gene CSE1, interferes with mitosis in HeLa cells.

We have analyzed the effects on HeLa cells of reduction of the CAS protein, the human homologue to yeast chromosome segregation protein CSE1. Expression of CAS antisense cDNA decreases the amount of CAS protein in HeLa cells and perturbs progression from G2 (retards transition from G2) to G1 in the cell cycle. Increased levels of cyclin B in CAS antisense transfected cells correlated with an arrest in G2 phase or mitosis. This arrest upon CAS attenuation is consistent with observations that yeast with CSE1 mutations are defective in mitosis and cyclin B degradation.

Cellular Apoptosis Susceptibility Protein↗

Two new X-autosome Robertsonian translocations in the mouse. I. Meiotic chromosome segregation in male hemizygotes and female heterozygotes.

Two new X-autosome Robertsonian (Rb) translocations, Rb(X.9)6H and Rb(X.12)7H, were found during the course of breeding the Rb(X.2)2Ad rearrangement at Harwell. The influence of these new Rbs on meiotic chromosome segregation was investigated in hemizygous males and heterozygous females and compared to that of Rb(X.2)2Ad. Screening of metaphase II spermatocytes gave incidences of sex chromosome aneuploidy of 9.2% in Rb(X.2)6H/Y and 9.6% in Rb(X.9)2Ad/Y males; no metaphase II cells were present in the testes of the Rb(X.12)7H/Y males examined and no males with this karyotype have so far proved fertile. In breeding tests, 5% of the progeny of Rb(X.2)2Ad/Y males were sex chromosome aneuploids compared to 10% of the Rb(X.9)6H/Y offspring. The difference was not significant, however. Cytogenetic analyses of metaphase II stage oocytes showed elevated rates of hyperhaploidy (n + 1) in Rb heterozygous females over chromosomally normal mice: 4.2% for Rb(X.2)2Ad/+; 2.1% for Rb(X.9)6H/+; 2.2% for Rb(X.12)7H/+ and 1.1% for normal females. There was, however, no statistically significant difference in the rates of hyperhaploidy between the three different Rb types, nor overall between Rb/+ and normal females. Karyotypic analyses of liveborn offspring of Rb heterozygous females revealed low incidences of X0 animals but no other type of sex chromosome aneuploidy. Intercrosses of heterozygous females and hemizygous males yielded 5.5% aneuploidy for Rb(X.2)2Ad and 5.4% for Rb(X.9)6H. In heterozygous females, there was evidence from the metaphase II and breeding test data for all three rearrangements, of preferential segregation of the Rb metacentric to the polar body resulting in a deficiency of cells and progeny carrying a translocation chromosome.

Aneuploidy↗

The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation.

We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

Amino Acid Sequence↗

Chlamydia trachomatis causes centrosomal defects resulting in chromosomal segregation abnormalities.

Chlamydiae traffic along microtubules to the microtubule organizing center (MTOC) to establish an intracellular niche within the host cell. Trafficking to the MTOC is dynein dependent although the activating and cargo-linking function of the dynactin complex is supplanted by unknown chlamydial protein(s). We demonstrate that once localized to the MTOC, the chlamydial inclusion maintains a tight association with cellular centrosomes. This association is sustained through mitosis and leads to a significant increase in supernumerary centrosomes, abnormal spindle poles, and chromosomal segregation defects. Chlamydial infection thus can lead to chromosome instability in cells that recover from infection.

Centrosome↗

The human CAS protein which is homologous to the CSE1 yeast chromosome segregation gene product is associated with microtubules and mitotic spindle.

Human CAS cDNA contains a 971-aa open reading frame that is homologous to the essential yeast gene CSE1. CSE1 is involved in chromosome segregation and is necessary for B-type cyclin degradation in mitosis. Using antibodies to CAS, it was shown that CAS levels are high in proliferating and low in nonproliferating cells. Here we describe the distribution of CAS in cells and tissues analyzed with antibodies against CAS. CAS is an approximately 100-kDa protein present in the cytoplasm of proliferating cells at levels between 2 x 10(5) and 1 x 10(6) molecules per cell. The intracellular distribution of CAS resembles that of tubulin. In interphase cells, anti-CAS antibody shows microtubule-like patterns and in mitotic cells it labels the mitotic spindle. CAS is removed from microtubules by mild detergent treatment (cytoskeleton preparations) and in vincristine- or taxol-treated cells. CAS is diffusely distributed in the cytoplasm with only traces present in tubulin paracrystals or bundles. Thus, CAS appears to be associated with but not to be an integral part of microtubules. Immunohistochemical staining of frozen tissues shows elevated amounts of CAS in proliferating cells such as testicular spermatogonia and cells in the basal layer cells of the colon. CAS was also concentrated in the respiratory epithelium of the trachea and in axons and Purkinje cells in the cerebellum. These cells contain many microtubules. The cellular location of CAS is consistent with an important role in cell division as well as in ciliary movement and vesicular transport.

Antibodies↗

A chromatin-associated kinesin-related protein required for normal mitotic chromosome segregation in Drosophila.

The tiovivo (tio) gene of Drosophila encodes a kinesin-related protein, KLP38B, that colocalizes with condensed chromatin during cell division. Wild-type function of the tio gene product KLP38B is required for normal chromosome segregation during mitosis. Mitotic cells in tio larval brains displayed circular mitotic figures, increased ploidy, and abnormal anaphase figures. KLP38B mRNA is maternally provided and expressed in cells about to undergo division. We propose that KLP38B, perhaps redundantly with other chromosome-associated microtubule motor proteins, contributes to interactions between chromosome arms and microtubules important for establishing bipolar attachment of chromosomes and assembly of stable bipolar spindles.

Amino Acid Sequence↗

Histone H2B repression causes cell-cycle-specific arrest in yeast: effects on chromosomal segregation, replication, and transcription.

To determine which cellular processes are dependent on histones, we blocked histone H2B mRNA synthesis in asynchronously growing yeast after fusing the H2B gene to a repressible GAL10 promoter. Chromosomal segregation, replication, and transcription were then examined. We found that the cells arrested in mitosis, with a cell division cycle (cdc) phenotype. Chromatin structure and nuclear segregation were disrupted. A full round of DNA replication took place after the repression of histone H2B mRNA synthesis. Active transcription and the induction of new transcripts also continued in the arrested cells.

Cell Cycle↗

Cell cycle-dependent localization of two novel prokaryotic chromosome segregation and condensation proteins in Bacillus subtilis that interact with SMC protein.

Disruption of ypuG and ypuH open reading frames in Bacillus subtilis leads to temperature-sensitive slow growth, a defect in chromosome structure and formation of anucleate cells. The genes, which were named scpA and scpB, were found to be epistatic to the smc gene. Fusions of ScpA and ScpB to the fluorescent proteins YFP or CFP showed that both proteins co-localize to two or four discrete foci that were present at mid-cell in young cells, and within both cell halves, generally adjacent to chromosomal origin regions, in older cells. ScpA and ScpB foci are associated with DNA and depend on the presence of SMC and both Scps. ScpA and ScpB are associated with each other and with SMC in vivo, as determined using the FRET technique and immunoprecipitation assays. Genes similar to scpA and scpB are present in many bacteria and archaea, which suggests that their gene products form a condensation complex with SMC in most prokaryotes. The observed foci could constitute condensation factories that pull DNA away from mid-cell into both cell halves.

Bacillus subtilis↗

Chromosome segregation from cell hybrids. VI. Centromeres of both parental chromosome sets stain with antikinetochore antibody.

Antikinetochore antibodies obtained from serum of patients with the CREST syndrome of scleroderma were used to test the hypothesis that there are differences in protein binding to retained- and segregant-set centromeres in Chinese hamster--human hybrids. This hypothesis is not supported since identical staining of the two types of kinetochores was observed with CREST antibody.

Animals↗

Double or nothing: a Drosophila mutation affecting meiotic chromosome segregation in both females and males.

We describe a Drosophila mutation, Double or nothing (Dub), that causes meiotic nondisjunction in a conditional, dominant manner. Previously isolated mutations in Drosophila specifically affect meiosis either in females or males, with the exception of the mei-S332 and ord genes which are required for proper sister-chromatid cohesion. Dub is unusual in that it causes aberrant chromosome segregation almost exclusively in meiosis I in both sexes. In Dub mutant females both nonexchange and exchange chromosomes undergo nondisjunction, but the effect of Dub on nonexchange chromosomes is more pronounced. Dub reduces recombination levels slightly. Multiple nondisjoined chromosomes frequently cosegregate to the same pole. Dub results in nondisjunction of all chromosomes in meiosis I of males, although the levels are lower than in females. When homozygous, Dub is a conditional lethal allele and exhibits phenotypes consistent with cell death.

Alleles↗