Insect sex chromosomes

A presumptive mechanism of X inactivation has been investigated by using tritiated uridine-induced chromosome aberrations to distinguish active from inactive X chromosome arms in the insect Gryllotalpa fossor. Previous work on therian mammals has shown that constitutive and facultative heterochromatin are less susceptible to breakage by ³H-Urd than euchromatin (active). The present study indicates that, irrespective of the presence of two X chromosomes in females, only one of these is affected as in males and that the total number of aberrations induced by ³H-Urd in both male and female Gryllotalpa is the same. This suggests that in the female only one arm of one X chromosome is active and that a facultative heterochromatinization of the homologous arm of the other X is operative coupled with the presence of constitutive heterochromatin in the second arm of both X chromosomes.     

Insect sex chromosomes

The two X chromosomes in tetraploid spermatogonial cells from Gryllotalpa fossor respond differentially to the production of chromatid aberrations by ³H-uridine (³H-U). As in diploid female somatic cells, only the euchromatic arm of one X shows such aberrations. The equivalent arm of the other X and the constitutive arms of both Xs are not affected. This differential response of the homologous arms of the two Xs appears to be due to a facultative heterochromatinization of one of them. It is suggested that an imprinting process, which has been assumed to occur during fertilization in other cases of X-inactivation, may not be necessary for the differential regulation of two X chromosomes in this case.     

Clastogenic effects of methylnitrosourea and ethylnitrosourea on chromosomes from human fibroblast cell lines.

Human fibroblast cell lines were pulse-treated for 1 h with either methylnitrosourea (MNU) or ethylnitrosourea (ENU) at various time intervals before harvesting for chromosome analysis. Cells treated with 1 X 10(-3) M, 5 X 10(-4) M, and 1 X 10(-4) M final concentrations of MNU and ENU during the G2 or M phases of the cell cycle showed a significant increase in chromatid-type abnormalities over controls. Cells exposed to MNU or ENU 23 h before harvest showed some chromosome-type abnormalities, reflecting probable damage induced during the G1 phase of the cell cycle or derived from chromatid damage induced during the previous cell cycle. The mitotic indices and incidences of abnormalities suggested a dose response effect when cells were treated with the two higher concentrations and the three concentrations, respectively, of MNU or ENU. Chromatid abnormalities were observed in MUN and ENU-treated cells from each of four cell lines. From this investigation, it was concluded that MNU and ENU treatment of human diploid cell lines in vitro induced both chromatid and chromosome aberrations. MNU and ENU, both of which had previously been shown to be mutagenic in experimental animals, are, therefore, also considered to be mutagenic at the chromosome level in human fibroblasts grown and treated in cell culture.     

Insect sex chromosomes. VIII. Identification of active/inactive X-chromosomes in Gryllotalpa fossor by 5-BrdU/AO fluorescence

By employing 5-BrdU/AO fluorescence technique to distinguish active from inactive X-chromosomes, we have, for the first time, provided evidence in support of the validity of this technique for a non-mammalian system Gryllotalpa fossor (Orthoptera). In the female somatic cells of Gryllotalpa, it is only in the active (euchromatic) arm of X-chromosome that the fluorescence is bright, whereas it is dull in the facultative and constitutive heterochromatic arms. In tetraploid spermatogonial cells one of the two X-chromosomes is inactive, suggesting that this phenomenon is probably a random one and that the inactivation process is independent of the imprinting mechanism.     

Human X chromosomes: synchrony of DNA replication in diploid and triploid fibroblasts with multiple active or inactive X chromosomes

We have analyzed patterns of DNA replication in X chromosomes from diploid cultured human fibroblasts and from three triploid 69,XXY fibroblast strains, using BrdU--33258 Hoechst--Giemsa techniques. Both X chromosomes in each of these Barr body-negative triploid strains were early-replicating. The results of gene dosage studies using (1) a histochemical stain to measure X-linked glucose-6-phosphate dehydrogenase (G6PD) activity in single cells and (2) cellulose acetate electrophoresis of G6PD activity in cell extracts also indicated that both Xs in these strains were genetically active. When we compared the synchrony of X chromosome DNA replication kinetics both between cells and within cells containing multiple inactive Xs, a marked variability and asynchrony was observed for late-replicating X chromosomes. In a culture of 47,XXX fibroblasts administered an 8-h terminal pulse of dT after growth in BrdU-containing medium, asynchrony was detected between the two late-replicating Xs in approximately 70% of cells examined. No such asynchrony was observed between the two early-replicating Xs in similarly cultured 69,XXY cells; in the triploid strains, the two Xs were distinguished by asynchronous replication in only approximately 15% of cells. The striking variability in late X chromosome replication kinetics appears, then, to be a property unique to inactive Xs and is not inherent to all X chromosomes.     

Origin and evolution of mammalian sex chromosomes

Mammals present an XX/XY system of chromosomal sex determination, males being the heterogametic sex. Comparative studies of the gene content of sex chromosomes from the major groups of mammals reveal that most Y genes have X-linked homologues and that X and Y share homologous pseudoautosomal regions. These observations, together with the presence of the two homologous regions (pseudoautosomal regions) at the tips of the sex chromosomes, suggest that these chromosomes began as an ordinary pair of homologous autosomes. Birds present a ZW/ZZ system of chromosomal sex determination where females are the heterogametic sex. In this case, avian sex chromosomes are derived from different pairs of autosomes than mammals. The evolutionary pathway from the autosomal homomorphic departure to the present-day heteromorphic sex chromosomes in mammals includes suppression of X-Y recombination, differentiation of the nascent non-recombining regions, and progressive autosomal addition and attrition of the sex chromosomes. Recent results indicate that the event marking the beginning of the differentiation between the extant X and Y chromosomes occurred about 300 million years ago.     

Cell cycle-dependent nucleosome occupancy at cohesin binding sites in yeast chromosomes

In the budding yeast, cohesin is loaded onto the chromosome during the late G1 phase, establishes sister chromatid cohesion concomitant with DNA replication, and dissociates by the telophase. Here, using oligonucleotide tiling arrays, we show that, at the anaphase, nearly all of the cohesin binding sites contain nucleosome-free regions. The majority of these sites remain nucleosome-free throughout the cell cycle, consistent with the suggestion of a DNA-binding anchoring protein present at these sites, although such a region could also serve as part of a marker for the binding of cohesin in the next cell cycle. However, a third of these sites are remodeled in the G1 phase, being reoccupied by nucleosomes by the G1/S boundary, though their subsequent removal in the S phase appears to be independent of DNA replication. Whether this difference is a result of other functions of cohesin or of the chromatin remains to be elucidated.     

Evolution of mammalian sex chromosomes: cooperation of genetic and epigenetic factors

The X and Y chromosomes of mammals, which significantly differ in structure and genetic composition, are thought to originate from a pair of autosomes. During evolution of sex chromosomes in placental mammals, the degradation of the Y chromosome and inactivation spreading along the X chromosome occurred gradually and in concert. Thus, at the molecular level, the genetic and epigenetic factors interacted toward greater differentiation of the X/Y pair. In this review, in context of a comparison permitting to trace this evolutionary pathway, we consider the structural features of mammalian sex chromosomes focusing on the X-chromosomal genes and the unique epigenetic mechanism of their regulation. Possible causes and consequences of the genes skipping inactivation and aspects of molecular mechanism of X-chromosome inactivation are discussed. A number of hypotheses are considered on evolutionary relationships of X-chromosome inactivation and other molecular processes in mammals.     

X chromosomes alternate between two states prior to random X-inactivation

Early in the development of female mammals, one of the two X chromosomes is silenced in half of cells and the other X chromosome is silenced in the remaining half. The basis of this apparent randomness is not understood. We show that before X-inactivation, the two X chromosomes appear to exist in distinct states that correspond to their fates as the active and inactive X chromosomes. Xist and Tsix, noncoding RNAs that control X chromosome fates upon X-inactivation, also determine the states of the X chromosomes prior to X-inactivation. In wild-type ES cells, X chromosomes switch between states; among the progeny of a single cell, a given X chromosome exhibits each state with equal frequency. We propose a model in which the concerted switching of homologous X chromosomes between mutually exclusive future active and future inactive states provides the basis for the apparently random silencing of one X chromosome in female cells.     

New species of mole crickets of the Gryllotalpa gryllotalpa group (Orthoptera: Gryllotalpidae) from Israel, based on morphology, song recordings, chromosomes and cuticular hydrocarbons, with comments on the distribution of the group in Europe and the Mediterranean region

Two sibling species of mole crickets of the Gryllotalpa gryllotalpa group inhabit Israel. Both are described as new species based on differences in their morphology, acoustic behaviour, chromosome number, cuticular hydrocarbon pattern and habitats. The species with males having 2n = 23 chromosomes, described in the past as the 'Dead Sea race', is designated as G. marismortui sp.n. It is an endangered species. In morphology and chromosome number it is similar to G. cossyrensis Bacetti & Capra from Italy, but differs in the composition of cuticular hydrocarbons. Gryllotalpa marismortui occurs in a limited geographical area along the shore of the Dead Sea. Another species, with 2n = 19 chromosomes, is described as G. tali sp.n. and is found throughout Israel. The calling songs of the males of the new species differ markedly. Females of the respective chromosomal numbers discriminate in favour of the homospecific call. Their habitats are strikingly different: G. marismortui lives in hypersaline soil along the Dead Sea shore (total chloride 187.25 meq %), G. tali inhabits freshwater soil (9.31 meq %). The distribution of the twelve known sibling species and one chromosomal race (not yet described) of the G. gryllotalpa group from Europe and the eastern Mediterranean is summarized. The zoogeography and phylogeny of the group are discussed.     

X inactivation in a mammal species with three sex chromosomes

X inactivation is a fundamental mechanism in eutherian mammals to restore a balance of X-linked gene products between XY males and XX females. However, it has never been extensively studied in a eutherian species with a sex determination system that deviates from the ubiquitous XX/XY. In this study, we explore the X inactivation process in the African pygmy mouse Mus minutoides, that harbours a polygenic sex determination with three sex chromosomes: Y, X, and a feminizing mutant X, named X*; females can thus be XX, XX*, or X*Y, and all males are XY. Using immunofluorescence, we investigated histone modification patterns between the two X chromosome types. We found that the X and X* chromosomes are randomly inactivated in XX* females, while no histone modifications were detected in X*Y females. Furthermore, in M. minutoides, X and X* chromosomes are fused to different autosomes, and we were able to show that the X inactivation never spreads into the autosomal segments. Evaluation of X inactivation by immunofluorescence is an excellent quantitative procedure, but it is only applicable when there is a structural difference between the two chromosomes that allows them to be distinguished.     

Characterization of the adaptive response to ionizing radiation induced by low doses of X rays to human lymphocytes

In previous studies we have shown that low doses of radiation from incorporated tritiated thymidine can make human lymphocytes less susceptible to the genetic damage manifested as chromatid breakage induced by a subsequent high dose of X rays. We have also shown that this adaptive response to ionizing radiation can be induced by very low doses of X rays (0.01 Gy; i.e., 1 rad) delivered during S phase of the cell cycle. To see if a low dose of X rays could induce this response in cells at other phases of the cell cycle, human lymphocytes were irradiated with 0.01 or 0.05 Gy before stimulation by phytohemagglutinin (G0) or with 0.01 Gy at various times after stimulation (G1), followed by 1.5 Gy (150 rad) at G2 phase. Although G0 lymphocytes failed to exhibit an adaptive response, G1 cells irradiated as early as 4 h after stimulation did show the response. Experiments were also carried out to determine how long the adaptive response induced by 0.01 Gy could persist. A 0.01-Gy dose was delivered to lymphocytes in the first S phase, followed by 1.5 Gy in the same or subsequent cell cycles. Lymphocytes receiving a 1.5-Gy dose at 40, 48, or 66 h after stimulation exhibited an adaptive response, whereas those receiving a 1.5-Gy dose at 90 or 114 h did not. Duplicate cultures containing bromodeoxyuridine showed that at 40 h all the lymphocytes were in their first cell cycle after stimulation, at 48 h half of the lymphocytes were in their first cell cycle and half in their second, and at 66 h 80% of the lymphocytes were in their third cell cycle. Thus the adaptive response persists for at least three cell cycles after it is induced by 0.01 Gy of X rays. In other experiments, the time necessary for maximal expression of the adaptive response was determined by delivering 0.01 Gy at hourly intervals 1-6 h before the 1.5-Gy dose. While a 4-h interval was enough for expression of the adaptive response, shorter intervals were not.     

HIV-1 Vpr induces G2 cell cycle arrest in fission yeast associated with Rad24/14-3-3-dependent, Chk1/Cds1-independent Wee1 upregulation

Viral protein R (Vpr), an accessory protein of human immunodeficiency virus type 1 (HIV-1), induces the G2 cell cycle arrest in fission yeast for which host factors, such as Wee1 and Rad24, are required. Catalyzing the inhibitory phosphorylation of Cdc2, Wee1 is known to serve as a major regulator of G2/M transition in the eukaryotic cell cycle. It has been reported that the G2 checkpoint induced by DNA damage or incomplete DNA replication is associated with phosphorylation and upregulation of Wee1 for which Chk1 and Cds1 kinase is required. In this study, we demonstrate that the G2 arrest induced by HIV-1 Vpr in fission yeast is also associated with increase in the phosphorylation and amount of Wee1, but in a Chk1/Cds1-independent manner. Rad24 and human 14-3-3 appear to contribute to Vpr-induced G2 arrest by elevating the level of Wee1 expression. It appears that Vpr could cause the G2 arrest through a mechanism similar to, but distinct from, the physiological G2 checkpoint controls. The results may provide useful insights into the mechanism by which HIV-1 Vpr causes the G2 arrest in eukaryotic cells. Vpr may also serve as a useful molecular tool for exploring novel cell cycle control mechanisms.     

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.     

Cell-stage dependence of mutagen-induced sister-chromatid exchanges in human lymphocyte cultures

The induction of sister-chromatid exchanges (SCEs) was studied in phytohemagglutinin (PHA)-stimulated human lymphocytes exposed for 1 h to mitomycin C (MMC, 3 X 10(-6) M), ethyl methanesulphonate (EMS, 2 X 10(-2) M), or 4-nitroquinoline-1-oxide (4NQO, 3 X 10(-5) M) at various cell-cycle stages of 72-h cultures. The doses of the chemical were chosen to give about 20 SCEs per cell when treated at Go. The SCE frequency increased almost linearly with MMC or EMS treatments at later times after PHA stimulation, peaking with those at 36 h (at around the first G1/S boundary in the 2 consecutive cell cycles, which was revealed by concomitant experiments), and then decreased with subsequent treatment times. Cell-cycle kinetics and the cell stages at which the cells were treated were measured by autoradiography and sister-chromatid differential staining. The data show that MMC and EMS produce larger numbers of SCEs when treated at stages closer to the beginning of S, and that the most efficient time of treatment is the G1/S boundary in the first cell cycle of the two consecutive cycles before sampling. Pulse treatment with EMS caused about 3 times larger inductions of SCEs when done at late G1/early S(G1/S boundary) in the first cell cycle compared to that at G0/early G1, whereas identical exposure to MMC at the first G1/S boundary produced only 1.5 times larger numbers of SCEs than that at G0/early G1. EMS and MMC both, however, induced 30-40% larger numbers of SCEs when treated at the G1/S boundary in the first cell cycle than when treated at the second cell cycle before sampling. On the contrary, treatment with 4NQO led to the induction of about the same numbers of SCEs even when treated at different cell-cycle stages before the second G1/S boundary. The SCE frequency in 4NQO-treated cells then decreased with subsequent treatment times.