Light and electron microscopy of rat kangaroo cells in mitosis. II. Kinetochore structure and function.
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Data are reported on the frequency and pattern of Mitomycin C-induced exchanges in lymphocytes of a marsupial hybrid Macropus rufogriseus female x Wallabia bicolor male and in four individuals, two male and two female, of M. rufogriseus. In the hybrid, exchanges are found almost exclusively between the M. rufogriseus chromosomes and the breakpoints are in the C-banded pericentromeric regions. In the four M. rufogriseus animals the data show no evidence for preferential exchanges between homologues and so provide no justification for postulating somatic pairing between homologues. Comparisons of the frequency of exchanges between non-homologous chromosomes in the hybrid and in the four individuals of M. rufogriseus show that there is no difference in the frequency of exchange for any given chromosome but that the specific pattern of exchanges may differ between animals. The data are interpreted as providing evidence for different types of heterochromatin and possible bases for these differences are discussed.
The relationship between progression through anaphase and furrow establishment was investigated in PtK1 cells using the anti-mitotic agent Nocodazole to arrest cells at different points in anaphase. The capacity of cells to furrow was compared to the kinetochore-kinetochore separation attained at the time of arrest. For the stages of anaphase examined, furrowing capacity increased directly with kinetochore-kinetochore separation until complete furrows were formed after kinetochore-kinetochore separations of 14 micron or more were reached. Furrow establishment thus occurs during a definite interval during anaphase in PtK1 cells. Results from electron microscopy of both Nocodazole-treated and control cells suggest that a population of astral microtubules may be important for furrow establishment.
Calcium-containing solutions were microinjected into dividing PtK1 cells to assess the effect of calcium ion concentration on the morphology and physiology of the mitotic spindle. Solutions containing 50 microM or more CaCl2 are immediately and irreversibly toxic to PtK1 cells. Those containing 5-10 microM CaCl2 cause reversible reduction in spindle birefringence followed by normal anaphase and cytokinesis. Microinjection of 5 microM or less CaCl2 into anaphase PtK1 cells has no detectable effect on the rate or extent of chromosome movement. Metaphase cells tend to enter anaphase 4-5 min after injection with 1-10 microM CaCl2, compared with an average of 16 min after injection with calcium-free buffer. Reducing the intracellular calcium concentration by injection of EGTA-CaCl2 buffers increases the lag between injection and anaphase to 20 min or more. Microinjection of calcium solutions does not promote precocious chromatid separation in nocodazole-arrested metaphase cells, indicating that the increase in calcium concentration does not induce centromere separation directly. An increase in the concentration of free calcium ions during metaphase appears to stimulate the onset of anaphase. Such an increase, regulated by the cell itself, may contribute to the initiation of chromosome separation in mammalian cells.
The pairing of the sex chromosomes at pachytene has been examined in twenty-two species of Australian marsupials, including four with complex sex chromosome systems. The axial elements of the sex chromosomes associate in all but one species. However, no synaptonemal complex has been observed between the axes of the X and Y chromosome in any of the examined species. Both the type of association between the sex chromosome axes, and the structural modifications of these axes are conserved within taxonomic groupings. In three species with complex sex chromosome systems, the t(XA), Y, A trivalents do not have a favoured relative orientation of the axes of the Y and A chromosomes, whereas in a fourth species with a t(XA1), t(A2YA1), A2 system the t(XA1) and A2 axes are in a cis arrangement with each other.
Chromosomes from a rat kangaroo (Potorous tridactylus) cell line (PtK2) and from a Chinese hamster (Cricetulus griseus) cell line (CHV79) were isolated by means of fluorescence activated flow cytometric sorting. DAPI (4'-6-diamino-2-phenylindole) was used as the DNA specific fluorescent dye. The karyotype of the PtK2 cells which exhibits 13 chromosomes was separated into 6, and the 22 chromosomes of the CHV79 cells were resolved into 11 fractions. DNA extracted from these chromosomal fractions was used for restriction enzyme digestion and blotting on nitrocellulose filters. The blots were challenged with gene probes corresponding to ribosomal RNA (18S and 28S) and small nuclear RNA (U1-snRNA) genes. The rRNA genes were exclusively assigned to chromosomes containing the nucleolus organizing region (in PtK2: X chromosome; in CHV79: chromosomes 4, 5, 6, and 11). - Solely the largest chromosomes in both cell lines hybridized with U1-snRNA indicating that these gene sequences are located on those chromosomes only. Further possible genetic and biochemical applications of this experimental system are discussed.
Blastocysts and late gestation stages of the marsupial mouse, Antechinus stuartii, were examined cytologically and electrophoretically to investigate X chromosome activity during embryogenesis. A late replicating X chromosome was identified in the protoderm cells of female unilaminar blastocysts and in the cells of embryonic and extra-embryonic regions of older blastocysts. Sex chromatin bodies were also observed in female bilaminar and trilaminar blastocysts. The X linked enzyme alpha-galactosidase showed no evidence of paternal allele expression in the extra-embryonic region of bilaminar blastocysts or in the yolk sac and embryonic tissue of known heterozygotes. It is concluded that the late replicating X chromosome is paternal in origin and that unlike the laboratory mouse, X inactivation is not correlated with cell differentiation in Antechinus.
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The rate of digesta passage was measured in five captive Philippine flying lemurs (Cynocephalus volans). These animals were force fed capsules containing known quantities of either particulate or soluble markers. The volumes of the gastrointestinal tracts of three flying lemurs were determined based on the wet weight of the contents of each section of the gut. The mean rate of digesta passage was 14.37 +/- 3.31 h when determined using the particulate marker and 21.9 +/- 0.03 h when determined using the soluble marker. The values based on the particulate marker are between 2% and 10% of similar values for other arboreal folivores. The morphology of the gastrointestinal system of the Philippine flying lemur is similar to that of other hindgut fermenters. Flying lemurs have a simple stomach and a large caecum. The total gut capacity of the Philippine flying lemur is similar to that of other herbivores, but is slightly smaller than that of either the koala (Phascolarctos cinereus), a hindgut fermenter, or the three-toed sloth (Bradypus variegatus), a foregut fermenter. These data suggest that flying lemurs deal with the problems of a folivorous diet very differently than some other arboreal mammals. Phascolarctos cinereus and Bradypus variegatus may represent one extreme with Cynocephalus volans representing the other extreme along a continuum of foraging strategies that are compatible with the arboreal folivore lifestyle.
Phosphoglycerate kinase (EC 2.7.2.3; PGK) exists in two forms in marsupials. PGK1 is an X-linked house-keeping enzyme, and PGK2 is a mainly testis-specific enzyme under autosomal control. We have used PGK1 probes derived from two closely related species of macropodid marsupials (kangaroos and wallabies) to demonstrate the existence of a large family of pseudogenes in the tammar wallaby (Macropus eugenii). Over 30 fragments are detectable after Taq digestion. We estimate that there are 25-30 copies per genome. Most are autosomally inherited and are apparently not closely linked. Only two restriction fragments that appeared to be sex linked could be detected. Varying degrees of hybridization of fragments to the probes suggest different levels of homology, and hence different ages of origin. The existence of two PGK1 homologous restriction fragments from the X and a large number from the autosomes was also demonstrated by somatic cell hybridization for two other macropodid species, the wallaroo (M. robustus) and the red kangaroo (M. rufus). These results are compared with those from human and mouse, and it is suggested that the propensity of PGK1 to form pseudogenes is an ancient (approximately 130 MYR BP) characteristic of mammals. The high level of polymorphism detected in the tammar makes these PGK1 probes potentially useful for measuring genetic variability in this species and other macropodids.