Centromeric Regions Control Autonomous Segregation Tendencies in Single-Division Meiosis of Saccharomyces Cerevisiae

We have previously shown that yeast cdc5 or cdc14 homozygotes can be led through a single-division meiosis in which some of the chromosomes segregate reductionally whereas others, within the same cell, segregate equationally. Chromosomes XI tend to segregate reductionally, whereas chromosomes IV tend to segregate equationally. In this report we present experiments with cdc5 homozygous strains, in which the centromeres of one or both chromosomes XI was replaced by the centromeric region from chromosome IV. Analysis of the products of single-division meioses in these strains demonstrates that the choice between reductional or equational segregation is directed by sequences in the vicinity of the centromeres. Although the choice is made separately for each individual chromosome, the analysis also reveals the existence of a system responsible for coordinated segregation of the two chromosomes of a given pair.     

The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination

When diploid cells of Saccharomyces cerevisiae homozygous for the temperature-sensitive cell division cycle mutation cdc6-1 are grown at a semipermissive temperature they exhibit elevated genomic instability, as indicated by enhanced mitotic gene conversion, mitotic intergenic recombination, chromosomal loss, chromosomal gain, and chromosomal rearrangements. Employing quantitative Southern analysis of chromosomes separated by transverse alternating field gel electrophoresis (TAFE), we have demonstrated that 2N-1 cells monosomic for chromosome VII, owing to the cdc6-1 defect, show slow growth and subsequently yield 2N variants that grow at a normal rate in association with restitution of disomy for chromosome VII. Analysis of TAFE gels also demonstrates that cdc6-1/cdc6-1 diploids give rise to aberrant chromosomes of novel lengths. We propose an explanation for the genomic instability induced by the cdc6-1 mutation, which suggests that hyper-recombination, chromosomal loss, chromosomal gain and chromosomal rearrangements reflect aberrant mitotic division by cdc6-1/cdc6-1 cells containing chromosomes that have not replicated fully.     

The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination

When diploid cells of Saccharomyces cerevisiae homozygous for the temperature-sensitive cell division cycle mutation cdc6-1 are grown at a semipermissive temperature they exhibit elevated genomic instability, as indicated by enhanced mitotic gene conversion, mitotic intergenic recombination, chromosomal loss, chromosomal gain, and chromosomal rearrangements. Employing quantitative Southern analysis of chromosomes separated by transverse alternating field gel electrophoresis (TAFE), we have demonstrated that 2N-1 cells monosomic for chromosome VII, owing to the cdc6-1 defect, show slow growth and subsequently yield 2N variants that grow at a normal rate in association with restitution of disomy for chromosome VII. Analysis of TAFE gels also demonstrates that cdc6-1/cdc6-1 diploids give rise to aberrant chromosomes of novel lengths. We propose an explanation for the genomic instability induced by the cdc6-1 mutation, which suggests that hyper-recombination, chromosomal loss, chromosomal gain and chromosomal rearrangements reflect aberrant mitotic division by cdc6-1/cdc6-1 cells containing chromosomes that have not replicated fully.     

Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system

Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications.     

Effects of Homology, Size and Exchange on the Meiotic Segregation of Model Chromosomes in Saccharomyces Cerevisiae

In most eukaryotic organisms, chiasmata, the connections formed between homologous chromosomes as a consequence of crossing over, are important for ensuring that the homologues move away from each other at meiosis I. Some organisms have the capacity to partition the rare homologues that have failed to experience reciprocal recombination. The yeast Saccharomyces cerevisiae is able to correctly partition achiasmate homologues with low fidelity by a mechanism that is largely unknown. It is possible to test which parameters affect the ability of achiasmate chromosomes to segregate by constructing strains that will have three achiasmate chromosomes at the time of meiosis. The meiotic partitioning of these chromosomes can be monitored to determine which ones segregate away from each other at meiosis I. This approach was used to test the influence of homologous yeast DNA sequences, recombination intiation sites, chromosome size and crossing over on the meiotic segregation of the model chromosomes. Chromosome size had no effect on achiasmate segregation. The influence of homologous yeast sequences on the segregation of noncrossover model chromosomes was negligible. In meioses in which two of the three model chromosomes experienced a crossover, they nearly always disjoined at meiosis I.     

Mitotic Transmission of Artificial Chromosomes in Cdc Mutants of the Yeast, Saccharomyces Cerevisiae

In the yeast, Saccharomyces cerevisiae, cell division cycle (CDC) genes have been identified whose products are required for the execution of different steps in the cell cycle. In this study, the fidelity of transmission of a 14-kb circular minichromosome and a 155-kb linear chromosome fragment was examined in cell divisions where specific CDC products were temporarily inactivated with either inhibitors, or temperature sensitive mutations in the appropriate CDC gene. All of the cdc mutants previously shown to induce loss of endogenous linear chromosomes also induced loss of a circular minichromosome and a large linear chromosome fragment in our study (either 1:0 or 2:0 loss events). Therefore, the efficient transmission of these artificial chromosomes depends upon the same trans factors that are required for the efficient transmission of endogenous chromosomes. In a subset of cdc mutants (cdc6, cdc7 and cdc16), the rate of minichromosome loss was significantly greater than the rate of loss of the linear chromosome fragment, suggesting that a structural feature of the minichromosome (nucleotide content, length or topology) makes the minichromosome hypersensitive to the level of function of these CDC gene products. In another subset of cdc mutants (cdc7 and cdc17), the relative rate of 1:0 events to 2:0 events differed for the minichromosome and chromosome fragment, suggesting that the type of chromosome loss event observed in these mutants was dependent upon chromosome structure. Finally, we show that 2:0 events for the minichromosome can occur by both a RAD52 dependent and RAD52 independent mechanism. These results are discussed in the context of the molecular functions of the CDC products.     

Mixed Segregation and Recombination of Chromosomes and Yacs during Single-Division Meiosis in Spo13 Strains of Saccharomyces Cerevisiae

Diploid yeast strains, homozygous for the mutation spo13, undergo a single-division meiosis and form dyads (two spores held together in one ascus). Dyad analysis of spo13/spo13 strains with centromere-linked markers on five different chromosomes and on a pair of human DNA YACs shows that: (a) in spo13 meiosis, chromosomes undergo mixed segregation, namely some chromosomes segregate reductionally whereas others, in the same cell, segregate equationally; (b) different chromosomes exhibit different segregation tendencies; (c) recombination between homologous chromosomes might not determine that a bivalent undergoes reductional rather than equational segregation.     

Assignment of the human gene for hexosaminidase B to chromosome 5

Mouse-human somatic cell hybrids between different mouse and human cells were studied for the expression of human hexosaminidases A and B activities. The expression of human hexosaminidase B in the hybrids was found to segregate concordantly with the presence of the human chromosome 5. Mouse-human hybrid clones containing either the human chromosomes 5 and 7 only or the human chromosome 7 only were also included in this study. Expression of human hexosaminidase B activity was detected only in those clones containing human chromosome 5. These results indicate that the gene(s) for human hexosaminidase B is located on chromosome 5. No hexosaminidase A activity was detected in clones which contained either human chromosomes 5 and 7 or chromosome 7.     

Evidence for the lack of mismatch-repair directed antirecombination during mouse meiosis

Meiotic recombination was studied in DNA mismatch repair (MMR)-deficient mice using a strain carrying a Pms2 knockout mutation. Using single-sperm typing, recombination was analyzed over five intervals on four chromosomes in four Pms2 -/- animals. A total of 1936 meioses were studied and compared to 1848 meioses from three Pms2 +/+ controls. A smaller study was carried out on a single interval in each of two chromosomes in an MMR-deficient mouse homozygous for the Msh2 knockout mutation. A total of 792 meioses were examined in the Msh2 -/- and 880 meioses in the Msh2 +/+ animal. Recombination fractions were not significantly different in either of the MMR-deficient mouse strains when compared to MMR-proficient controls. Our results appear to conflict with mouse embryonic stem (ES) cell gene-targeting experiments where MMR plays a major role in determining the efficiency of homologous recombination between nonidentical sequences. A number of possibilities could explain the apparent lack of a significant effect on meiosis.     

The nucleolar phosphatase Cdc14B is dispensable for chromosome segregation and mitotic exit in human cells

In yeast, the protein phosphatase Cdc14 promotes chromosome segregation, mitotic exit, and cytokinesis by reversing M-phase phosphorylations catalyzed by Cdk1. A key feature of Cdc14 regulation is its sequestration within the nucleolus, which restricts its access to potential substrates for much of the cell cycle. Mammals also possess a nucleolar Cdc14 homolog, termed Cdc14B, but its roles during mitosis and cell division remain speculative. Here we analyze Cdc14B’s subcellular dynamics during mitosis and rigorously test its functional contributions to cell division through homozygous disruption of the Cdc14B locus in human somatic cells. While Cdc14B is initially released from nucleoli at the start of mitosis, the phosphatase quickly redistributes onto segregating sister chromatids during anaphase. This relocalization is mainly driven by Cdk1 inactivation, as pharmacologic inhibition of Cdk1 in prometaphase cells redirects Cdc14B onto chromosomes. However, in sharp contrast to yeast cdc14 mutants, human Cdc14BΔ/Δ cells were viable and lacked defects in spindle assembly, anaphase progression, mitotic exit, and cytokinesis, and continued to segregate ribosomal DNA repeats with near-normal proficiency. Our findings reveal substantial divergence in mitotic regulation between yeast and mammalian cells, as the latter possess efficient mechanisms for completing late M-phase events in the absence of a nucleolar Cdc14-related phosphatase.     

Identification of mouse chromosomes required for murine leukemia virus replication.

The replication patterns of five ecotropic and two amphotropic strains of murine leukemia virus (MuLV) were studied by infecting 41 Chinese hamster x mounse hybrid primary clones segregating mouse (Mus musculus) chromosomes. Ecotropic and amphotropic strains replicated in mouse and some hybrid cells, but not in hamster cells, indicating that replication of exogenous virus requires dominantly expressed mouse cellular genes. The patterns of replication of the five ecotropic strains in hybrid clones were similar; the patterns of replication of the two amphotropic strains were also similar. When compared to each other, however, the replication patterns of ecotropic and amphotropic viruses were dissimilar, indicating that these two classes of MuLV require different mouse chromosomes for replication. Chromosome and isozyme analyses assigned a gene, Rec-1 (replication of ecotropic virus), to mouse chromosome 5 that is necessary and may be sufficient for ecotropic virus replication. Because of preferential retention of mouse chromosomes 15 and 17 in the hybrid clones, however, the possibility that these chromosomes carry genes that are necessary but not sufficient for ecotropic virus replication cannot be excluded. Similarly, the data indicate that mouse chromosome 8 (or possibly 19) carried a gene we have designated Ram-1 (replication of amphotropic virus) which is necessary and may be sufficient for amphotropic virus replication. Because chromosomes 8 and 19 tended to segregate together and two of the three clones excluding 19 have chromosome reaggrangements, we cannot exclude 19 as being independent of amphotropic virus replication. In addition, because of preferential retention, chromosomes 7, 12, 15, 16 and 17 cannot be excluded as being necessary but not sufficient. Hybrid cell genetic studies confirm the assignment of the Fv-1 locus to chromosome 4 previously made by sexual genetics. In addition, our results demonstrate that hybrid cells which have segregated mouse chromosome 4 but have retained 5 become permissive for replication of both N and B tropic strains of MuLV.     

The segregation of the Escherichia coli origin and terminus of replication

Escherichia coli chromosome replication forks are tethered to the cell centre. Two opposing models describe how the chromosomes segregate. In the extrusion-capture model, newly replicated DNA is fed bi-directionally from the forks toward the cell poles, forming new chromosomes in each cell half. Starting with the origins, chromosomal regions segregate away from their sisters progressively as they are replicated. The termini segregate last. In the sister chromosome cohesion model, replication produces sister chromosomes that are paired along much of their length. The origins and most other chromosomal regions remain paired until late in the replication cycle, and all segregate together. We use a combination of microscopy and flow cytometry to determine the relationship of origin and terminus segregation to the cell cycle. Origin segregation frequently follows closely after initiation, in strong support of the extrusion-capture model. The spatial disposition of the origin and terminus sequences also fits this model. Terminus segregation occurs extremely late in the cell cycle as the daughter cells separate. As the septum begins to invaginate, the termini of the completed sister chromosomes are transiently held apart at the cell centre, on opposite sides of the cell. This may facilitate the resolution of topological linkages between the chromosomes.     

Genetic characterization of parental cell lines and biochemical analysis of somatic cell hybrids between mouse (RAG) cells and fibroblasts of Ateles paniscus chamek (primates,platyrrhini)

Mouse (RAG) cells, (deficient in hypoxanthine-phosphoribosyl-transferase), and Ateles paniscus chamek primary fibroblasts were used in fusion experiments to generate somatic cell hybrids. Both parental cell lines were genetically characterized by karyological and biochemical analyses with 27 isozyme systems. These procedures were useful for monitoring primate chromosome segregation in somatic cell hybrids, for detecting chromosome rearrangements of primate chromosomes, and for identifying individual primate chromosomes. These characterizations are necessary to distinguish between different hybrid cell lines and to generate a panel for gene mapping studies. This is achieved by selecting cell lines that segregate different sets of relatively few primate isozymes and chromosomes. Conversely, we eliminated hybrid cell lines either showing: (1) rearrangements between primate and mouse chromosomes, (2) extensive rearrangements of primate chromosomes, or (3) a large number of primate biochemical markers. © 1993 Wiley-Liss, Inc.     

Meiotic Disjunction of Homologs in Saccharomyces Cerevisiae Is Directed by Pairing and Recombination of the Chromosome Arms but Not by Pairing of the Centromeres

We explored the behavior of meiotic chromosomes in Saccharomyces cerevisiae by examining the effects of chromosomal rearrangements on the pattern of disjunction and recombination of chromosome III during meiosis. The segregation of deletion chromosomes lacking part or all (telocentric) of one arm was analyzed in the presence of one or two copies of a normal chromosome III. In strains containing one normal and any one deletion chromosome, the two chromosomes disjoined in most meioses. In strains with one normal chromosome and both a left and right arm telocentric chromosome, the two telocentrics preferentially disjoined from the normal chromosome. Homology on one arm was sufficient to direct chromosome disjunction, and two chromosomes could be directed to disjoin from a third. In strains containing one deletion chromosome and two normal chromosomes, the two normal chromosomes preferentially disjoined, but in 4-7% of the tetrads the normal chromosomes cosegregated, disjoining from the deletion chromosome. Recombination between the two normal chromosomes or between the deletion chromosome and a normal chromosome increased the probability that these chromosomes would disjoin, although cosegregation of recombinants was observed. Finally, we observed that a derivative of chromosome III in which the centromeric region was deleted and CEN5 was integrated at another site on the chromosome disjoined from a normal chromosome III with fidelity. These studies demonstrate that it is not pairing of the centromeres, but pairing and recombination along the arms of the homologs, that directs meiotic chromosome segregation.     

Chromosome strand segregation during sporulation in Bacillus subtilis

After the initiation of spore formation in Bacillus subtilis, the products of the final round of DNA replication segregate into two cells, i.e. the prespore and the mother cell. The prespore, which is known to contain a single completed chromosome, develops into a mature endospore which can be readily separated from mother cells and non-sporulating cells on the basis of its resistance properties. We have used a procedure originally developed to label the terminus region of the B. subtilis chromosome to specifically label the newly synthesized strands of DNA during the final round of DNA replication before sporulation. We have purified prespore DNA and used strand-specific probes to measure the radioactivity incorporated. The results show that the sister chromosomes segregate at random into the prespore. This result has implications for the segregation of chromosomes during vegetative growth and for the generation of cellular asymmetry during sporulation.     

Characterization of pig-mink cell hybrids: assignment of the TK1 and UMPH2 genes to pig chromosome 12

By fusion of thymidine kinase-deficient mink cells with pig leukocytes, a new type of cell hybrid was produced. It was demonstrated that pig chromosomes segregate in pig-mink hybrids and that hybrid cells contain no cytologically visible rearrangements between the chromosomes of parental species, or chromosome fragmentation. With a set of subclones of two primary hybrid clones, the genes for thymidine kinase-1 (TK1) and uridine 5-monophosphate hydrolase-2 (UMPH2) were assigned to pig Chromosome (Chr) 12. A cell line with a single pig Chr 8 on the background of mink chromosomes was established. This clone could serve as a source of DNA for building a chromosome-specific library of pig Chr 8. The data obtained suggest that pig-mink cell hybrids can be used for mapping of pig chromosomes.     

Genes controlling receptors for ecotropic and xenotropic type C virus in Mus cervicolor and Mus musculus.

Gene loci controlling cell surface receptors for murine leukemia virus were studied by using murine X Chinese hamster hybrid cells. Hybrids which exclusively segregate murine chromosomes were made by fusing Mus cervicolor and Mus musculus lymphocytes to hamster fibroblasts. Sensitivity to Moloney murine leukemia virus infecotion and specific binding of the envelope glycoprotein of Rauscher murine leukemia virus (gp70) cosegregate and isozyme analysis show an association with chromosome 5 in both species. With the possible exception of one clone, no evidence was found for a proviral integration site independent of chromosome 5. Evidence is presented for additional unlinked ectropic and xenotropic receptors independent of chromosome 5.     

Arabidopsis Cell Division Cycle 20.1 Is Required for Normal Meiotic Spindle Assembly and Chromosome Segregation

Cell division requires proper spindle assembly; a surveillance pathway, the spindle assembly checkpoint (SAC), monitors whether the spindle is normal and correctly attached to kinetochores. The SAC proteins regulate mitotic chromosome segregation by affecting CDC20 (Cell Division Cycle 20) function. However, it is unclear whether CDC20 regulates meiotic spindle assembly and proper homolog segregation. Here, we show that the Arabidopsis thaliana CDC20.1 gene is indispensable for meiosis and male fertility. We demonstrate that cdc20.1 meiotic chromosomes align asynchronously and segregate unequally and the metaphase I spindle has aberrant morphology. Comparison of the distribution of meiotic stages at different time points between the wild type and cdc20.1 reveals a delay of meiotic progression from diakinesis to anaphase I. Furthermore, cdc20.1 meiocytes exhibit an abnormal distribution of a histone H3 phosphorylation mark mediated by the Aurora kinase, providing evidence that CDC20.1 regulates Aurora localization for meiotic chromosome segregation. Further evidence that CDC20.1 and Aurora are functionally related was provided by meiosis-specific knockdown of At-Aurora1 expression, resulting in meiotic chromosome segregation defects similar to those of cdc20.1. Taken together, these results suggest a critical role for CDC20.1 in SAC-dependent meiotic chromosome segregation.     

Segregation of the replication terminus of the two Vibrio cholerae chromosomes

Genome duplication and segregation normally are completed before cell division in all organisms. The temporal relation of duplication and segregation, however, can vary in bacteria. Chromosomal regions can segregate towards opposite poles as they are replicated or can stay cohered for a considerable period before segregation. The bacterium Vibrio cholerae has two differently sized circular chromosomes, chromosome I (chrI) and chrII, of about 3 and 1 Mbp, respectively. The two chromosomes initiate replication synchronously, and the shorter chrII is expected to complete replication earlier than the longer chrI. A question arises as to whether the segregation of chrII also is completed before that of chrI. We fluorescently labeled the terminus regions of chrI and chrII and followed their movements during the bacterial cell cycle. The chrI terminus behaved similarly to that of the Escherichia coli chromosome in that it segregated at the very end of the cell division cycle: cells showed a single fluorescent focus even when the division septum was nearly complete. In contrast, the single focus representing the chrII terminus could divide at the midcell position well before cell septation was conspicuous. There were also cells where the single focus for chrII lingered at midcell until the end of a division cycle, like the terminus of chrI. The single focus in these cells overlapped with the terminus focus for chrI in all cases. It appears that there could be coordination between the two chromosomes through the replication and/or segregation of the terminus region to ensure their segregation to daughter cells.     

Distributive Disjunction of Authentic Chromosomes in Saccharomyces Cerevisiae

Distributive disjunction is defined as the first division meiotic segregation of either nonhomologous chromosomes that lack homologs or homologous chromosomes that have not recombined. To determine if chromosomes from the yeast Saccharomyces cerevisiae were capable of distributive disjunction, we constructed a strain that was monosomic for both chromosome I and chromosome III and analyzed the meiotic segregation of the two monosomic chromosomes. In addition, we bisected chromosome I into two functional chromosome fragments, constructed strains that were monosomic for both chromosome fragments and examined meiotic segregation of the chromosome fragments in the monosomic strains. The two nonhomologous chromosomes or chromosome fragments appeared to segregate from each other in approximately 90% of the asci analyzed, indicating that yeast chromosomes were capable of distributive disjunction. We also examined the ability of a small nonhomologous centromere containing plasmid to participate in distributive disjunction with the two nonhomologous monosomic chromosomes. The plasmid appeared to efficiently participate with the two full length chromosomes suggesting that distributive disjunction in yeast is not dependent on chromosome size. Thus, distributive disjunction in S. cerevisiae appears to be different from Drosophila melanogaster where a different sized chromosome is excluded from distributive disjunction when two similar size nonhomologous chromosomes are present.