Chromosome segregation from cell hybrids. VII. Reverse segregation from karyoplast hybrids suggests control by cytoplasmic factors.

Using human and Chinese hamster established lines as cell parents, we constructed hamster-human cell hybrids and human cell - hamster karyoplast hybrids. The cell hybrids retained one or two sets of hamster chromosomes and lost most of the human chromosomes. The karyoplast hybrids, however, retained a full set of human chromosomes and lost most of the Chinese hamster chromosomes. This reverse segregation pattern implies that cytoplasmic factors are major determinants of the direction of chromosome segregation.     

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.     

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.     

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 doubling in vine cacti hybrids

We performed reciprocal crosses between the tetraploid Selenicereus megalanthus and the diploid Hylocereus species, H. undatus and H. polyrhizus. S. megalanthus x H. undatus gave rise to viable hexaploids and 6x-aneuploid hybrids rather than to the expected triploids. No genuine hybrids were obtained in the reciprocal cross. The pollen diameter of the tetraploid S. megalanthus varied widely, indicating the occurrence of unreduced gametes, while that of H. undatus pollen was very uniform, indicating an extremely low frequency of unreduced gametes. This finding suggests that the hexaploids were formed by chromosome doubling after the formation of the hybrid triploid zygote rather than by fusion of unreduced gametes of the two species.     

Chromosome replication in mouse intraspecific hybrids

Hybrid cells (HY SS2 and HY SS6) arising from the fusion of diploid cells of the mouse lymphosarcoma LS/BL and L cells resistant to 8-azaguanine (HGPRT-) showed slower growth and a longer generation time than the parent lines. The inter- and intrachromosomal timing and patterns of early chromosome DNA replication of parent cells was preserved in the hybrid genome and was not influenced by loss of telocentric chromosomes from LS/BL or L (HGPRT-) cells. Thus DNA chromosome replication sequences are not dependent on the presence of a complete set of chromosomes of the parent cells and do not therefore seem to be a result of interaction between chromosomes not segregated in the hybrid genome.     

Chromosome elimination in Hordeum vulgare x H. bulbosum hybrids

Summary The hybrid progeny from a stable amphidiploid of H. vulgare x H. bulbosum involving the cultivar Vada and an unstable amphidiploid involving the cultivar Emir were studied. The genotypes examined contained two genomes from Vada or one from Vada and one from Emir, with one or two genomes from H. bulbosum. Comparisons between the chromosome numbers in root-tips and anthers revealed that there was no chromosome elimination in most plants, whether there was one or two Vada genomes present. The one plant in which chromosome elimination was positively identified had Emir as opposed to Vada cytoplasm. It also had a high incidence of degraded or fragmented chromosomes in the PMCs. Differences in stability between a 27 chromosome plant and other hypotetraploids suggest that Vada contains both elimination genes and elimination suppressor genes. Upon selfing, again irrespective of the number of Vada genomes present, circa triploid hybrids gave rise to diploid H. vulgare offspring while hypotetraploids produced hybrid-like plants. These included diploids, triploids and tetraploids. There was evidence that suggested that H. vulgare as well as H. bulbosum chromosomes had been eliminated.     

Chromosome instability in Hordeum vulgare x H. bulbosum hybrids

Chromosome number in Hordeum vulgare x H. bulbosum hybrids ranged between the haploid and diploid number but with peaks in frequency occurring at the 14 and 7 chromosome level. This was reflected in a gradual change from hybrid morphology to that of haploid H. vulgare. The rate of chromosome elimination differed significantly between hybrids, while within each hybrid, differences in mean chromosome number were recorded between and within individual tillers. An increase in temperature from 25–30° C caused a significant increase in the rate of elimination of H. bulbosum chromosomes.     

Chromosome constitution and cell division in in vitro cultures ofZea mays L. endosperm

Summary Cultures of maize (Zea mays L.) endosperm grown in vitro for over 3 years were examined cytologically. Conditions of aneuploidy and polyploidy were noted. Chromosome numbers ranged from 21 to over 200, with 30 to 60 being observed most often. Although a few extra large cells with polyploid nuclei were scattered throughout the smear preparation, a large proportion of the interphase nuclei appeared similar in volume and probably contained a near normal complement of chromosomes. Anaphase bridges were the most commonly observed chromosome aberration. No cell divisions were observed the first 24 hr after transfer. From 2 to 8 days after transfer the proportion of cells in division was relatively constant with a mitotic index of approximately 5.5%. The proportion of cells in division began to decline 8 days after transfer and in the final sample taken after 13 days only 2.6% of the cells were in division. Examples of localized synchrony were observed and mitotic indices for individual cell clumps ranged from 0 to 17%. Authorized for publication on October 16, 1973 as paper number 4552 in the Journal Series of the Pennsylvania State Agricultural Experiment Station.     

Chromosome pairing in tetraploid hybrids between Lolium perenne and L. multiflorum

Summary Chromosome association at first meiotic metaphase in tetraploid hybrids between Lolium perenne and L. multiflorum was compared with that in autotetraploid L. perenne. The hybrids were found to have significantly higher levels of bivalent frequency, and lower levels of multivalent and chiasma frequency. A significant increase in multivalent frequency with increasing chiasma formation was found in both groups, but the increase was much less in the hybrids. These differences in chromosome associations between the two groups must therefore reflect differences in chiasma distribution and it is suggested that the results indicate a significant degree of preferential bivalent pairing in the hybrids.     

Chromosome control of apomixis in maize-gamagrass hybrids

The results of long-term studies on the transmission of the mode of asexual reproduction through seeds to maize from gamagrass, a closely related wild plant, performed in the Laboratory of Plant Cytology and Apomixis are summarized. The first apomictic hybrids between Zea mays and Tripsacum dactyloides were obtained in this laboratory more than 40 years ago and have been maintained until the present time. Cytogenetic studies on the hybrids have shown that at least nine chromosomes of the wild parent are necessary for the expression of asexual reproduction through seeds. In addition, the genes controlling two elements of apomixis (apomeiosis and parthenogenesis) have been found to be inherited independently from each other.     

Chromosome behavior and cell lineage in triploid and mosaic salivary glands of species hybrids in Sciara

Summary A study of the salivary glands of triploid hybrids between females of Sciara ocellaris and males of Sciara reynoldsi throws light on several problems concerning chromosome behavior, species relationships and embryonic development. The specimens studied possess two sets of ocellaris chromosomes and one set of reynoldsi chromosomes. Three types of conditions are represented; 2X3A; 3X3A; and a mosaic possessing cells of both kinds.Heterozygosity of chromosome band pattern in the hybrids shows that the ocellaris homologs are not true sisters and that the sperm probably united with a diploid fusion nucleus composed of two polar bodies and not with the oötid nucleus. This indicates strongly that in a previously described diploid hybrid mosaic some cells developed parthenogenetically from such a fusion nucleus, while others were biparental in origin. The evidence supports the view that the initial influences or reactions of the foreign reynoldsi sperm differ from those of the ocellaris sperm immediately after entry into the egg, but that then and later the differences are relatively slight, although numerous. This agrees with our earlier evidence from the hybrids in indicating that the two parental species are separated genetically, as well as in chromosome band pattern, by many small differences.Species-specific characteristics of chromosome behavior are described which retain their specificity in the hybrids.The 2X3A cells lack a reynoldsi X. The double ocellaris X in such cells (including those in the mosaic) exhibits the peculiar features manifest by the single X in ordinary diploid XO males in the pure species (and in many other Diptera). It is pale and diffuse in structural appearance, and throughout its entire length is uniformly much greater in diameter than it is in the 3X3A cells. The possible significance of this behavior is discussed in relation to problems of sex determination (e. g., Bridges' ratio theory) and those concerning time of gene action. The structural resemblance to the puffs and bulbs found in salivary gland chromosomes is striking and raises the question as to how far morphological characteristics of chromosomes can be used as criteria of gene activity. The double X in the 2X cells of the mosaic, surrounded by 3X cells, exhibits the same peculiarities as those in a pure 2X3A male-type specimen, indicating that we are dealing with intracellular, not intercellular phenomena.Conditions in the mosaic gland support the evidence from the diploid hybrids (some described here for the first time) in indicating that the salivary gland is formed by differentiation of cells which happen to lie in the proper region, regardless of their ancestry. The triploid mosaic specimen (and also a diploid mosaic) is presumed to be a female in which the normal male-producing type of chromosome elimination occurred in one or more mitotic divisions during cleavage or later.     

Cytoplasmic microtubules in transformed mouse x nontransformed human cell hybrids: correlation with in vitro growth.

Patterns of cytoplasmic microtubules in somatic cell hybrids between transformed mouse cells and nontransformed human skin fibroblasts were examined using antitubulin antibodies as an immunofluorescent probe. Nontransformed cells have been shown to exhibit an extensive cytoplasmic microtubule complex (CMTC), while in transformed cells, this complex is greatly diminished. The hybrid populations contained both types of cells. In addition, they contained cells with previously undescribed intermediate CMTC phenotypes. The percentage of each phenotype present in hybrid populations was determined for sixteen hybrid clones. Seven clones were found which appeared transformed on the basis of their CMTC pattern. The others were comprised of various proportions of all the cell types described. Repeated quantitation of the proportions of these types in the hybrid populations showed them to be stable with time in culture. Growth in vitro of the hybrid clones was assayed by determining their saturation densities, their plating efficiencies on plastic and their colony-forming abilities in soft agar. In vitro growth of a cell population was found to be directly proportional to the percentage of cells in the population which showed the greatly diminished CMTC pattern which has been described for transformed cells. This is strong evidence that a greatly reduced CMTC is associated with transformed behavior, especially the increased capacity of transformed cells for in vitro growth.     

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.