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Chromosome segregation in Escherichia coli B/r at various growth rates.

Chromosome segregation was analyzed in three substrains of Escherichia coli B/r growing at various rates. The cultures were pulse labeled with [14C]thymidine and bound to the bottom surface of a nitrocellulose membrane filter, and the radioactivity in newborn cells released from the surface during continuous elution with growth medium was measured. Since there was a fixed orientation in the release of newborn cells, the time course of the change in radioactivity per effluent cell could be used to investigate the orientation of chromosome segregation. If the radioactive deoxyribonucleic acid strands were partitioned at random between the progenies remaining attached to the membrane filter and those released into the effluent, the radioactivity per cell would decrease twofold after each generation of elution. The decrease in radioactivity was less than twofold at C + D min of elution and larger than twofold one generation later, indicating that chromosome segregation was nonrandom.

Cell Division

Extent and rate of chromosome segregation in two intraspecific mouse cell hybrids: A9 x diploid foetal erythrocyte and A9 x B82.

Patterns of chromosome segregation were studied in 2 different intraspecific mouse cell hybrids: (1) A9 x B82, formed by fusing 2 cell lines of heteroploid fibroblasts, and (2) UWE, originating from the fusion of A9 cells with euploid foetal erythrocytes. Detailed analyses of Giemsa (G)-banded chromosomes and chromosome arms of both parental and hybrid cells were made for each hybrid type, in order to determine the specificity of the losses and to assess the influence of ploidy and cell differentiation. Unlike the A9 x B82 hybrids, which revealed a significant chromosome loss under selective tissue culture pressures only after 9 months, the UWE hybrids showed a sharp reduction in the total chromosome number during the initial 2 months under similar pressures. However, with no additional cloning, UWE remained karyotypically stable after that time. This rapid chromosomal segregation in UWE hybrids may be caused by properties of the parental foetal erythrocytes. In UWE cells, the majority of the chromosome arms were retained or duplicated. Less than a quarter of the total number of chromosome arms were segregated or lost, and these were all chromosome arms with abnormal mouse G-banding patterns, present only in the heteroploid A9 parental cells. In two of the four A9 x B82 hybrid lines, there was marked segregation of chromosome arms whose banding patterns were identical to those of wild type mouse telocentric chromosomes. For both types of intraspecific cell hybrids, two thirds or more of the chromosome arms had banding patterns which were the same as those of the wild type genome.

Animals

Maternal CENP-C restores centromere symmetry in mammalian zygotes to ensure proper chromosome segregation.

Across metazoan species, the centromere-specific histone variant CENP-A is essential for accurate chromosome segregation, yet its regulation during the mammalian parental-to-zygote transition is poorly understood. To address this, we generated a CENP-A-mScarlet mouse model that revealed sex-specific dynamics: mature sperm retain 10% of the CENP-A levels present in MII oocytes. However, this difference is resolved in zygotes prior to the first mitosis, using maternally inherited cytoplasmic CENP-A. Notably, the increase in CENP-A at paternal centromeres is independent of sensing CENP-A asymmetry or the presence of maternal chromosomes. Instead, CENP-A equalization relies on the asymmetric recruitment of maternal CENP-C to paternal centromeres. Depletion of maternal CENP-A decreases total CENP-A in both pronuclei without disrupting equalization. In contrast, reducing maternal CENP-C or disruption of its dimerization function impairs CENP-A equalization and chromosome segregation. Therefore, maternal CENP-C acts as a key epigenetic regulator that resets centromeric symmetry at fertilization to preserve genome integrity.

Animals

Kinetochore targeting of fission yeast Mad and Bub proteins is essential for spindle checkpoint function but not for all chromosome segregation roles of Bub1p.

Several lines of evidence suggest that kinetochores are organizing centers for the spindle checkpoint response and the synthesis of a "wait anaphase" signal in cases of incomplete or improper kinetochore-microtubule attachment. Here we characterize Schizosaccharomyces pombe Bub3p and study the recruitment of spindle checkpoint components to kinetochores. We demonstrate by chromatin immunoprecipitation that they all interact with the central domain of centromeres, consistent with their role in monitoring kinetochore-microtubule interactions. Bub1p and Bub3p are dependent upon one another, but independent of the Mad proteins, for their kinetochore localization. We demonstrate a clear role for the highly conserved N-terminal domain of Bub1p in the robust targeting of Bub1p, Bub3p, and Mad3p to kinetochores and show that this is crucial for an efficient checkpoint response. Surprisingly, neither this domain nor kinetochore localization is required for other functions of Bub1p in chromosome segregation.

Chromosomes, Fungal

Suppression of production of mouse 28S ribosomal RNA in mouse-human hybrids segregating mouse chromosomes.

Mouse-human somatic cell hybrids that lose (segregate) human chromosomes produce only mouse 28S ribosomal RNA even when they retain copies of the human chromosomes that contain the genes for 28S ribosomal RNA. In contrast, mouse-human hybrid cells that segregate mouse chromosomes produce only human 28S ribosomal RNA even when they have retained copies of mouse chromosomes that contain the 28S ribosomal RNA genes.

Animals

Radiation-induced nondisjunction.

The methodology and results of epidemiological studies of the effects of preconception diagnostic x-rays of the abdomen on chromosome segregation in humans are described. Many studies have been conducted in a number of different countries. The vast majority show the same positive, though not significant, trend to increased nondisjunction among the offspring of irradiated women. The results of the various studies, however, cannot be pooled because of differing methodologies used. A worldwide co-operative project with standardized methodology is recommended. Such a study should identify the parental origin of the nondisjunctional event before etiological factors are investigated. Abnormal chromosome segregation during mitotic division has been inducted experimentally by the in vitro exposure of human lymphocytes to a low dose of 50 R gamma irradiation. First meiotic nondisjunction has been successfully induced by whole body exposure of female mice to a low dose of radiation. Further experiments are being conducted to try to induce abnormal segregation during second meiotic division. Because of difficulties encountered in trying to estimate total gonad doses resulting from differing techniques employed by radiologists and other health personnel, no attempt has been made to estimate the doubling dose nor minimum safe dose regarding the effects of radiation on chromosome segregation in humans. The question of time-related repair of the mechanism involved in chromosome segregation is raised.

Abnormalities, Radiation-Induced

Inactivation of the pre-mRNA cleavage and polyadenylation factor Pfs2 in fission yeast causes lethal cell cycle defects.

Faithful chromosome segregation is fundamentally important for the maintenance of genome integrity and ploidy. By isolating conditional mutants defective in chromosome segregation in the fission yeast Schizosaccharomyces pombe, we identified a role for the essential gene pfs2 in chromosome dynamics. In the absence of functional Pfs2, chromosomal attachment to the mitotic spindle was defective, with consequent chromosome missegregation. Under these circumstances, multiple intracellular foci of spindle checkpoint proteins Bub1 and Mad2 were seen, and deletion of bub1 exacerbated the mitotic defects and the loss of cell viability that resulted from the loss of pfs2 function. Progression from G1 into S phase following release from nitrogen starvation also required pfs2+ function. The product of the orthologous Saccharomyces cerevisiae gene PFS2 is a component of a multiprotein complex required for 3'-end cleavage and polyadenylation of pre-mRNAs and, in keeping with the conservation of this essential function, an S. pombe pfs2 mutant was defective in mRNA 3'-end processing. Mutations in pfs2 were suppressed by overexpression of the putative mRNA 3'-end cleavage factor Cft1. These data suggest unexpected links between mRNA 3'-end processing and chromosome replication and segregation.

Carrier Proteins

Meiosis in male Drosophila melanogaster. II. Nonrandom segregation of compound-second chromosomes.

The segregation of compound-second chromosomes in males from two different stocks has been examined. Segregation is random in males from the C(2L)RM4, dp; C(2R)RM4, px stock. Gametes containing only one of the two compound chromosomes comprise 50% of the gametes, and gametes containing either both elements or neither element make up the other 50% of the gametes. In males from the C(2L)RM, b; C(2R)RM, cn stock, gametes containing either C(2L)RM, b or C(2R)RM, cn make up the majority of the gametes. Gametes containing both chromosomes or neither chromosome account for only 2-3% of the gametes. The nonrandom segregation is due to the C(2R)RM, cn chromosome. Viability is reduced in flies carrying the C(2R)RM, cn chromosome. This includes larval lethality, delayed development and premature adult lethality. Cytologically, this chromosome contains a large duplication of 2L material, which includes material proximal to region 38 or 39. It is suggested that the viability and segregational properties associated with this chromosome are due to the duplicated 2L material.

Animals

Native yeast kinetochore structures identify an essential inner kinetochore interaction.

Kinetochores must accurately assemble on centromeres for faithful chromosome segregation. Although a conserved centromeric nucleosome is essential for kinetochore assembly, budding yeast centromeric DNA is a poor template for nucleosome formation in vitro, perhaps due to its intrinsic rigidity. To better understand yeast inner kinetochore assembly, we develop a one-step protocol to purify native inner kinetochore subcomplexes for structural studies. We perform cryoelectron microscopy on the purifications and generate density maps of four separate inner kinetochore complexes, two of which have not been previously visualized and may represent intermediate assemblage states. We identify an Ndc10 trimerization domain that engages centromeric DNA and a pair of CBF3 complexes and is associated with substantial bending of centromeric DNA. Ndc10 trimerization is essential for kinetochore assembly and chromosome segregation. We propose that Ndc10 trimerization facilitates centromeric DNA bending to stabilize the centromeric nucleosome and inner kinetochore.

Kinetochores

RNAseq analysis of oocyte maturation from the germinal vesicle stage to metaphase II in pig and human.

During maturation oocytes at the germinal vesicle (GV) stage progress to metaphase II (MII). However, during in vitro maturation a proportion often fail to progress. To understand these processes, we employed RNA sequencing to examine the transcriptome profile of these three groups of oocytes from the pig. We compared our findings with similar public oocyte data from humans. The transcriptomes in oocytes that failed to progress was similar to those that did. We found in both species, the most upregulated genes in MII oocytes were associated with chromosome segregation and cell cycle processes, while the most down regulated genes were relevant to ribosomal and mitochondrial pathways. Moreover, those genes involved in chromosome segregation during GV to MII transition were conserved in pig and human. We also compared MII and GV oocyte transcriptomes at the isoform transcript level in both species. Several thousands of genes (including DTNBP1, MAPK1, RAB35, GOLGA7, ATP1A1 and ATP2B1) identified as not different in expression at a gene transcript level were found to have differences in isoform transcript levels. Many of these genes were involved in ATPase-dependent or GTPase-dependent intracellular transport in pig and human, respectively. In conclusion, our study suggests the failure to progress to MII in vitro may not be regulated at the level of the genome and that many genes are differentially regulated at the isoform level, particular those involved ATPase- or GTPase-dependent intracellular transport.

Humans

Identification of a putative RocS homolog through phenotypic profiling of uncharacterized essential genes in Streptococcus mutans.

Genome-wide viability catalogs produced by transposon sequencing (Tn-seq) and CRISPR interference (CRISPRi) have successfully mapped the essential genome of Streptococcus mutans . In this study, we combined predictive bioinformatics, conditional CRISPRi transcriptional silencing, transmission electron microscopy, transcriptomics, and genetic suppressor screens to investigate nine poorly characterized essential genes in S. mutans . From this screen, phenotypic and genetic analyses identified SMU_393 as a functional homolog of the pneumococcal chromosome segregation factor, RocS. Depletion of SMU_393 resulted in abnormal cell widening, hypersensitivity to DNA damage, and a significant subpopulation of anucleate cells. These phenotypes were bypassed by a spontaneous surface-exposed missense mutation ( dnaA Q197E ) within the AAA+ ATPase domain of the replication initiator. Together, this study refines annotations within the S. mutans essential genome and provides genetic insights into streptococcal chromosome segregation and cell cycle control.

Journal Article

Phosphorylation as a regulatory mechanism of HP1 protein multifunctionality.

The Heterochromatin Protein 1 (HP1) family proteins are key regulators of chromatin structure and genome function, acting as "reader" proteins that recognize and bind to histone H3 lysine 9 methylation (H3K9me). Beyond their canonical role in heterochromatin formation and transcriptional repression, HP1 proteins exhibit functional versatility, participating in transcriptional activation, RNA processing, DNA repair, and chromosome segregation. This multifunctionality is mediated partially by post-translational modifications (PTMs), with phosphorylation emerging as a central regulatory mechanism. This review explores the diverse effects of HP1 phosphorylation on protein function and chromatin interactions, focusing on Drosophila melanogaster HP1a and its orthologs, mammalian HP1α and S. pombe Swi6. Phosphorylation in the N-terminal tail enhances HP1's affinity for H3K9me, promoting transcriptional silencing. Mitotic phosphorylation of serine residues in the hinge region, regulated by kinases such as AURKB and NDR1/2, leads to chromatin release and relocalization to the kinetochore, enabling proper chromosome segregation. Additionally, phosphorylation modulates HP1 phase separation dynamics, influencing nuclear compartmentalization and chromatin condensation. These findings highlight phosphorylation as a versatile molecular switch that enables HP1 proteins to transition between structural and regulatory roles, contributing to their evolutionary conserved multifunctionality in genome regulation and cell division. Further investigation into HP1 phosphorylation across species and contexts is essential to fully understand its contributions to chromatin biology.

Phosphorylation

Chromosome age and segregation during sporulation of Bacillus megaterium.

The effect of chromosome age on segregation during sporulation was investigated. Vegetative cells of Bacillus megaterium were labeled with [Me-3H]thymine and then were grown at 30 degrees C in nonradioactive medium for various times before being allowed to sporulate. The ratio of the amount of label in sporal DNA to that in sporangial DNA, obtained after minor correction for the sporulation frequency, remained essentially constant as the postlabeling growth period was increased from one to seven generations. The spores were preferentially located at the older poles of sporangia, i.e. the poles formed by divisions occurring prior to those forming the sporangia. Therefore, it seems that old (labeled) chromosomes segregate randomly with respect to both the morphological and genealogical polarities of sporangia. Examination of total cell lysates by dye-buoyant density gradient centrifugation revealed the presence of covalently closed circular DNA from cells grown at 37 degrees C, but none was obtained from cells grown at 30 degrees C. Thus, possible interference by large amounts of extrachromosomal DNA in the determination of the chromosomal segregation pattern is unlikely.

Bacillus megaterium

Cytogenetics of South American akodont rodents (Cricetidae). V. Segregation of chromosome No. 1 polymorphism in Akodon molinae.

Akodon molinae is polymorphic with 2n=42, 43, 44, where the metacentric autosome No. 1 is homologous to 2 acrocentrics 1a and 1b. Matings between 2n=43 heterozygotes 1/1a, 1b gave a surplus of 1/1 offspring, a moderate reduction of heterozygous and a strong reduction of homozygous 1a, 1b/1a, 1b offspring. The latter type also has a highly reduced fertility.

Animals

[Partial disorganization of the anaphasic segregation of chromosomes in plant cells: combined actions of griseofulvin, producer of pluripolar anaphases and 2 ipecac alkaloids, producers of floating pole anaphases].

Anaphasis may be slightly checked by various treatments which however result in a normal chromosomic separation. Griseofulvin exerts a direct though partial influence on the mitotic apparatus, which entails "pluripolar anaphasis"; on the other hand Ipecac alkaloïds act indirectly and produce "floating poles anaphases". Treatments combining griseofulvin with cepheline or tubulosine show that there is never any synergy between the two processes. These results support our hypothesis that floating poles anaphases are not a sign of slight C-mitotic action but only come from a lag between the appearance/disappearance of microtubules and that of chromosomes during anaphasis.

Anaphase

Genetic studies on heterochromatin in Drosophila melanogaster and their implications for the functions of satellite DNA.

In Drosophila melanogaster the centromeric heterochromatin of all chromosomes consists almost entirely of several different satellite DNA sequences. In view of this we have examined by genetic means the meiotic consequences of X chromosomes with partial deletions of their heterochromatin, and have found that the amount and position of recombination on each heterochromatically deleted X is substantially different from that of a normal X. It appears that the amount of heterochromatin is important in modifying the "centromere effect" on recombination.--In all the deleted Xs tested, chromosome segregation is not appreciably altered from that of a nondeleted control chromosome. Thus satellite DNA does not appear to be an important factor in determining the regular segregation of sex chromosomes in Drosophila. Additionally, since X chromosomes with massive satellite DNA deficiencies are able to participate in a chromocenter within salivary gland nuclei, a major role of satellite DNA in chromocenter formation in this tissue is also quite unlikely.--In order to examine the mechanisms by which the amount of satellite DNA is increased or decreased in vivo, we have measured cytologically the frequency of spontaneous sister chromatid exchanges in a ring Y chromosome which is entirely heterochromatic and consists almost exclusively of satellite DNA. In larval neuroblast cells the frequency of spontaneous SCE in this Y is approximately 0.3% per cell division. Since there is no meiotic recombination in D. melanogaster males and since meiotic recombination in the female does not occur in heterochromatin, our results provide a minimum estimate of the in vivo frequency of SCE in C-banded heterochromatin (which is predominantly simple sequence DNA), without the usual complications of substituted base analogs, incorporated radioactive label or substantial genetic content.--We emphasise that: (a) satellite DNA is not implicated in any major way in recognition processes such as meiotic homologue recognition or chromocenter formation in salivaries, (b) there is likely to be continuous variation in the amount of satellite DNA between individuals of a species; and (c) the amount of satellite DNA can have a crucial functional role in the meiotic recombination system.

Animals