Chromosome segregation from cell hybrids. III. Segregation is independent of spindle constitution

Hamster beta-tubulin (detected as a mutant subunit that confers Colcemid resistance) is either not expressed or is underexpressed in Chinese hamster-mouse somatic cell hybrids. This selectivity of tubulin expression suggests that a uniparental mouse spindle might preferentially engage mouse chromosomes and lead to loss of hamster chromosomes. However, the repression of hamster tubulin was found to have no bearing on the direction of chromosome segregation occurring in eight hybrids studied, some of which segregated predominantly mouse and other hamster chromosomes.     

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.     

Genetic analysis of tumorigenesis: IV. Chromosome reduction and marker segregation in progeny clones from Chinese hamster cell hybrids.

Hybrid cells produced by the fusion of pairs of cells, one a tumorigenic derivative of CHEF/16 and the other a nontumorigenic derivative of CHEF/18, give rise to clones which are largely tetraploid, but rare reduced hybrids with chromosome counts in the diploid range have been recovered from tumors of hybrid origin. This paper describes the recovery in cell culture of reduced hybrids in the diploid range by selection with 5-bromodeoxyuridine (BrdU) or methylcellulose as well as by growth in culture of cells from excised tumors. All selected subclones were tumorigenic and resistant to BrdU, but they segregated for resistance to 6-thioguanine. Unselected subclones were tetraploid, nontumorigenic, and sensitive to both drugs. These data show that chromosome reassortment as well as extensive chromosome reduction both occur in a small fraction of the population during growth of each hybrid clone.     

Chromosome segregation control by Escherichia coli ObgE GTPase

Escherichia coli cells depleted of the conserved GTPase, ObgE, show early chromosome-partitioning defects and accumulate replicated chromosomes in which the terminus regions are colocalized. Cells lacking ObgE continue to initiate replication, with a normal ratio of the origin to terminus. Localization of the SeqA DNA binding protein, normally seen as punctate foci, however, was disturbed. Depletion of ObgE also results in cell filamentation, with polyploid DNA content. Depletion of ObgE did not cause lethality, and cells recovered fully after expression of ObgE was restored. We propose a model in which ObgE is required to license chromosome segregation and subsequent cell cycle events.     

Chromosome segregation: dual control ensures fidelity.

A mitotic checkpoint arrests cell cycle progression in response to spindle damage. It now appears that this checkpoint has two separate arms, one that prevents anaphase and a second that prevents cytokinesis and DNA re-replication.     

Chromosome loss is responsible for segregation at the HPRT locus in Chinese hamster cell hybrids

The phenomenon of segregation of gene expression has been examined in intraspecific somatic cell hybrids. Specifically, segregation at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus has been studied in hybrids of Chinese hamster cell lines. The role of chromosome segregation, or other chromosomal events has been assessed by detailed comparison of karyotypes in the 6-thioguanine resistant segregants with those of the parental hybrid lines. The results clearly demonstrate that loss of an entire X chromosome is the primary event responsible for segregation at the HPRT locus, while deletion of a portion of the short arm of an X chromosome was also a frequent event. The results provide the first direct evidence for the assignment of the mapping of this locus to the distal region of the short arm. Analysis of chromosome number distributions in the hybrids and segregants suggests that in selecting chromosomal segregants one may also select for hybrid lines with reduced chromosome stability.     

Chromosome and cell wall segregation in Streptococcus faecium ATCC 9790.

Segregation was studied by measuring the positions of autoradiographic grain clusters in chains formed from single cells containing on average less than one radiolabeled chromosome strand. The degree to which chromosomal and cell wall material cosegregated was quantified by using the methods of S. Cooper and M. Weinberger, dividing the number of chains labeled at the middle. This analysis indicated that in contrast to chromosomal segregation in Escherichia coli and, in some studies, to that in gram-positive rods, chromosomal segregation in Streptococcus faecium was slightly nonrandom and did not vary with growth rate. Results were not significantly affected by strand exchange. In contrast, labeled cell wall segregated predominantly nonrandomly.     

Chromosome segregation during the prokaryotic cell division cycle

Recent work has dramatically changed our view of chromosome segregation in bacteria. Rather than being a passive process, it involves rapid movement of parts of the circular chromosome. Several genes involved in chromosome segregation have been identified, and the analysis of their functions and intracellular localization are beginning to shed light on the mechanisms that ensure efficient chromosome segregation.     

Chromosome distribution: experiments on cell hybrids and in vitro.

Ostergren (1951) provided a simple explanation for both chromosome distribution in mitosis and chromosome segregation in meiosis, and more recently a molecular extension of his hypothesis has been possible. This report focuses on experimental tests of these ideas. Micromanipulation experiments on cell hybrids containing both meiotic and mitotic spindles demonstrate that differences in meiotic and mitotic chromosome behavior are determined by something intrinsic to the chromosome: meiotic chromosomes transferred to a mitotic spindle (or vice versa) behave just as they normally would. The molecular explanation postulates polarized growth or binding of microtubules at kinetochores. This has just been tested in vitro by McGill & Brinkley (1975) and by Telzer, Moses & Rosenbaum (1975), and their results are reviewed. In addition, a novel method for in vitro studies is described - mechanical demembranation of cells which is compatible with quite normal chromosome movement in anaphase. After addition of microtubule subunits to a demembranated prophase cell, chromosome orientation and movement toward an aster was observed for the first time in vitro. It is concluded that important aspects of chromosome distribution are probably understood at both the cellular and molecular levels, but final tests are still required. The outlook is hopeful indeed because the gaps in our knowledge are well known - the necessity of observations on prophase is a recurrent theme - and the means of filling the gaps are in hand.     

Quantitative analysis of human chromosome segregation in man-mouse somatic cell hybrids.

The presumed random and independent process of human chromosome segregation in man-mouse somatic cell hybrids was studied. The results of chromosome analysis on 196 cells from 15 related hybrid strains have provided the first convincing evidence that segregation of human chromosomes can be nonindependent and often concordant. Different human chromosomes were not retained with equal frequency in these hybrid clones. Some were present in 80% of all the cells, whereas others appeared in less than 10% of the same cells. Linear regression analysis was used to test for correlation of the frequencies of all pair-wise combinations of human chromosomes present in these hybrid clones. Twenty-two of 136 possible correlations were statistically significant, indicating that concordant segregation of particular pairs of human chromosomes is a rather frequent occurrence.     

Chromosome segregation: pulling from the poles

Dual wavelength video microscopy has been used to evaluate how chromatids move poleward upon chromosome separation at anaphase. The data reveal that poleward microtubule flux provides the dominant force for separating chromatids in Drosophila embryos during anaphase A.     

Chromosome rearrangements associated with CAD gene amplification. Experiments with cell hybrids.

Resistance to phosphonacetyl-L-aspartate (PALA) is caused by CAD gene amplification. The marker chromosome of a PALA-resistant cell line containing a homogeneously staining region with amplified CAD gene was introduced into PALA-sensitive Chinese hamster cells by microcell-mediated chromosome transfer. Two monochromosomal hybrids containing the marker chromosome in addition to the normal chromosome complement of sensitive cells and 1 tetraploid hybrid containing the complete genomes of donor (resistant) and recipient (sensitive) cells were studied in detail. It was shown that (i) the presence of the marker chromosome was both a necessary and a sufficient condition for the expression of the PALA-resistant phenotype; (ii) the marker chromosome underwent rearrangements in the monochromosomal hybrids, with preferential loss of non-amplified chromosomal regions, while it was not rearranged in the tetraploid hybrid; (iii) unlike the original PALA-resistant cells obtained after long-term selection in the presence of PALA, the PALA-resistant hybrids did not show chromosomal aberrations of other than the marker chromosome. This result indicates that chromosomal aberrations may be due to the selective procedure and is not an inherent property of cells containing amplified genes.     

Unequal segregation of parental chromosomes in embryonic stem cell hybrids

Chromosome segregation was studied in 14 intra- and 20 inter-specific hybrid clones generated by fusion of Mus musculus embryonic stem (ES) cells with fibroblasts or splenocytes of DD/c mice or Mus caroli. As a control for in vitro evolution of tetraploid karyotype we used a set of hybrid clones obtained by fusion of ES cells (D3) with ES cells (TgTP6.3). Identification of the parental chromosomes in the clones was performed by microsatellite analysis and in situ hybridization with labeled species-specific probes. Both analyses have revealed three types of clones: (i) stable tetraploid, observed only for ES x ES cell hybrids; (ii) bilateral loss of chromosomes of both ES and somatic partners; (iii) unilateral segregation of chromosomes of the somatic partner. Observed unilateral segregation was extensive in ES-splenocyte cell hybrids, but lower in ES-fibroblast hybrid clones. Developmental state of the somatic partner is presumably responsible for directional chromosome loss. Nonrandom segregation implies that initial differences in the parental homologous chromosomes were not immediately equalized implying at least transient persistence of the differentiated epigenotype.     

Genomic instability in interspecific cell hybrids III. Repression of Colcemid resistance in hybrids suggests preferential β-tubulin expression

A Colcemid-resistant Chinese hamster line with an altered form of β-tubulin was used in studies of the expression of spindle proteins in interspecific cell hybrids. Eight hybrids between this line, and a Colcemid-sensitive mouse cell line, were studied. The altered hamster β-tubulin was not expressed as an increased resistance to Colcemid in any hybrid. Since the complete hamster chromosome complement was represented among the hybrids, the absence of altered β-tubulin is not due to segregation of the mutant hamster β-tubulin gene. We suggest either that the hamster β-tubulin gene is repressed in hybrids, or that hamster β-tubulin is excluded from the spindle in hybrid cells. We compare these findings with previous reports of the repression of other highly active, moderately repeated constitutive genes in interspecific hybrids.     

Pattern of segregation of chicken HPRT phenotype in Chinese hamster-chick red blood cell hybrids.

The pattern of segregation of hypoxanthine phosphoribosyltransferase (HPRT, E.C. 2.4.2.8) was determined in synchronized Chinese hamster-chick red blood cell hybrids. Three hybrid lines were synchronized at the G1-S boundary. Bromodeoxyuridine pulses were subsequently applied throughout the S phase, and the frequency of the segregant clones was determined. It was found that the segregation of the chicken-specific HPRT phenotype associated with the loss of a chromosome was potentiated by bromodeoxyuridine administered during the first hour following release of the block.     

Genetic control of tumorigenicity in interspecific mammalian cell hybrids.

The nature of genetic control of cellular malignancy was investigated by examining the tumorigenicity of a series of interspecific mouse-human cell hybrids in the athymic nude mouse. Two highly malignant but genetically distinct mouse cell lines, A9 and PG19, were hybridized with three normal human diploid fibroblast strains, and 19 independently arising hybrid clones were isolated. Each of these clones was capable of forming progressive lethal tumors in the nude mouse, and thus resembled the malignant parental mouse cells rather than the nonmalignant parental human cells. We failed to obtain any evidence for complete suppression of tumorigenicity in these cell hybrids. The absence of suppression was observed regardless of the extent and composition of the human chromosome complements retained in the hybrid clones; the results of detailed cytological and isoenzyme analyses would make it highly improbable that the observed lack of suppression was due to cellular selection in vivo for a more tumorigenic subpopulation in the injected hybrid cells. These data demonstrate that at least for the parental cell combinations used in this study, no human chromosome, when present singly in the mouse-human cell hybrids, can suppress the tumorigenic phenotype of the mouse cells. Our results are consistent with the view that the suppression of cellular malignancy previously demonstrated in intraspecific (mouse × mouse) somatic cell hybrids does not occur in interspecific (mouse-human) cell hybrids, or alternatively, genetic determinants located on two or more human chromosomes are required simultaneously to suppress the malignancy of the mouse cells in cell hybrids derived from malignant mouse cell and nonmalignant human cells.     

Chromosome segregation: Samurai separation of Siamese sisters.

How do cells ensure that sister chromatids are precisely partitioned in mitosis? New studies on budding yeast have revealed that sister chromatid separation at anaphase requires endoproteolytic cleavage of a protein that maintains the association between sister chromatids.