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.     

5-Azadeoxycytidine induced undercondensation in the giant X chromosomes of Microtus agrestis

Fibroblasts of female Microtus agrestis were treated with 5-azadeoxycytidine (5-aza-dCyd) at a final concentration of 10^–5 M during the last 2 h of culture. This cytidine analogue induces distinct undercondensation of the constitutive heterochromatin in the giant X chromosomes. The undercondensed heterochromatic thread exhibits longitudinal segmentation reminiscent of a chromomere pattern. In the late-replicating X chromosome, 5-aza-dCyd also inhibits condensation of the genetically inactivated euchromatin (facultative heterochromatin). The described effects of 5-aza-dCyd on the X chromosome structure appear to be incorporation independent.     

Differential response of constitutive and facultative heterochromatin in the manifestation of mitomycin induced chromosome aberrations in Chinese hamster cells in vitro

The distribution of chromatid aberrations induced by mitomycin C among the individual chromosomes of female and male Chinese hamster cells in vitro was studied. The aberrations were found to be non-randomly distributed. Among the autosomes, the chromosomes possessing constitutive heterochromatin were more often involved in aberrations as well as in homologous exchanges. The inactivated X chromosomes in the female cells offer a situation where the short arm is facultatively heterochromatic and the long arm constitutively heterochromatic, thus enabling an analysis of their response for aberration formation. The short arm was seldom found to be involved in the aberration. The long arm of the inactivated X was more often affected (5 to 10 times) than the long arm of the functional X though both are constitutively heterochromatic. The possible role of (a) structure of heterochromatin, (b) the chromocenter formation and their association, (c) allocycly, and (d) the qualitative differences in the DNA of different types of heterochromatin are discussed in relation to the formation of chromatid aberrations.     

Asynchronous replication of constitutive heterochromatin on X chromosomes in female Mus dunni

Euchromatin DNA of one X chromosome in mammalian females, which becomes facultatively heterochromatinized, is known to replicate asynchronously late in S phase compared to its active homologue. In the females of a pygmy mouse species Mus dunni, which has prominent segment of constitutive heterochromatin as the short arm of its submetacentric X chromosome, we have observed asynchronous replication of c-heterochromatin arm as well, predominant number of cells showing the segment associated with the facultatively heterochromatic X to be terminating later. The preferential later termination of replication of the c-heterochromatic arm on the lyonized X appears to be due to the influence of facultative heterochromatin on the adjacent constitutive heterochromatin.     

Chromosomes and DNA of Microtus. II. Confirmation of deletion of constitutive heterochromatin in M. agrestis cells in vitro

The distribution of constitutive heterochromatin has been examined by C-banding in two somatic cell lines, grown in vitro, from a female Microtus agrestis. One line retains one intact X chromosome together with the short arm of the other X chromosome, while the other cell line retains only the short arm of one X chromosome. Thus, each cell line has lost substantial amounts of heterochromatin from the sex chromosomes, but this material has been deleted from the cells, and not translocated to other chromosomes. Nonetheless, both cell lines continue to propagate well in vitro.     

Polymorphic patterns of heterochromatin distribution in guinea pig chromosomes

The chromosome complement and patterns of heterochromatin distribution (as demonstrated by the DNA d-r method) were studied from three different guinea pigs. Karyotype analyses showed that one of the females had a heteromorphic sex pair formed by a submetacentric X chromosome and a subterminal X chromosome originated by a shortening of the short arm (x-chromosome). The heterochromatin was mainly found in the pericentromeric areas of the autosomes and X chromosomes and in the short arm of pair 7. The Y chromosome exhibited a degree of heterochromatinization different from that of pericentromeric areas.—The analysis of the heterochromatin distribution in the X chromosomes showed that the smaller size of the heteromorphic x-chromosoine was probably due to a lack of heterochromatin in its short arm. Moreover, two out of the three animals studied had a heteromorphic pattern of heterochromatinization in the pair 21 characterized by heterochromatinization of the pericentromeric area in one chromosome and almost complete heterochromatinization of the other homologue.—It is suggested that most of the heterochromatin disclosed by the DNA d-r method is formed by repetitious DNA; and that the Y chromosome and perhaps some autosome regions in guinea pigs are formed by a type of heterochromatin with properties different from those of the constitutive and facultative heterochromatin (intermediate heterochromatin).Supported in part by NIH Grant 5-501-RR05672-02 and by NIH contract 70-2299.     

DNase I sensitivity in facultative and constitutive heterochromatin

In situ nick translation allows the detection of DNase I sensitive and insensitive regions in fixed mammalian mitotic chromosomes. We have determined the difference in DNase I sensitivity between the active and inactive X chromosomes inMicrotus agrestis (rodent) cells, along both their euchromatic and constitutive heterochromatic regions. In addition, we analysed the DNase I sensitivity of the constitutive heterochromatic regions in mouse chromosomes. InMicrotus agrestis female cells the active X chromosome is sensitive to DNase I along its euchromatic region while the inactive X chromosome is insensitive except for an early replicating region at its distal end. The late replicating constitutive heterochromatic regions, however, in both the active and inactive X chromosome are sensitive to DNase I. In mouse cells on the other hand, the constitutive heterochromatin is insensitive to DNase I both in mitotic chromosomes and interphase nuclei.     

Tritiated uridine induced chromosome aberrations in relation to heterochromatin and nucleolar organisation in Microtus agrestis L.

Microtus agrestis is characterised by long sex chromosomes, most of which are constitutively heterochromatic, and thus supposedly, genetically inactive. A method to assess the template activity of the chromosomes is to study the distribution of chromatid aberrations produced by H³UdR, among and within the chromosomes. In such a study, in female Microtus agrestis cells in culture, it was found that, a large number of localised chromatid aberrations was induced in the constitutively heterochromatic regions of both X chromosomes. The frequency distribution and types of aberrations were found to be cell cycle dependent. With differential staining it has been possible to demonstrate that the constitutive heterochromatin of the sex chromosomes are involved in the nucleolar organisation in this species, thus containing the ribosomal RNA cistrons.     

Chromosome banding in Amphibia

The mitotic and meiotic chromosomes of the marsupial frog Gastrotheca riobambae were analysed with various banding techniques. The karyotype of this species is distinguished by considerable amounts of constitutive heterochromatin and unusual, heteromorphic XY sex chromosomes. The Y chromosome is considerably larger than the X chromosome and almost completely heterochromatic. The analysis of the banding patterns obtained with GC- and AT-base-pair-specific fluorochromes shows that the constitutive heterochromatin in the Y chromosome consists of at least three different structural categories. The only nucleolus organizer region (NOR) of the karyotype is localized in the short arm of the X chromosome. This causes a sex-specific difference in the number of NOR: female animals have two NORs in diploid cells, male animals one. No cytological indications were found for the inactivation of one of the two X chromosomes in the female cells. In male meiosis, the heteromorphic sex chromosomes form a characteristic sex-bivalent by pairing their telomeres in an end-to-end arrangement. The significance of the XY/XX sex chromosomes of G. riobambae for the study of X-linked genes in Amphibia, the evolution of sex chromosomes and their specific DNA sequences, and the significance of the meiotic process of sex chromosomes are discussed.     

Pattern of repetitive DNA of the chromosomes of Microtus agrestis

The distribution of repetitive DNA in the chromosomes of Microtus agrestis was studied with the method for demonstrating constitutive heterochromatin given by Yunis et al. (1971) and the reassociation technique described by Schnedl (1971). All autosomes can be individually recognized by means of the position of their bands. The euchromatic segment of the X1 chromosome shows the same banding pattern as the corresponding segment of X2 which consists of facultative heterochromatin. The short arms of the Y chromosome are not deeply stained with either method and therefore do not contain noticeable amounts of repetitive DNA. The relative distances between the bands remain constant during chromosome contraction in mitosis.     

Simple GA C T A repeats characterize the X chromosomal heterochromatin of Microtus agrestis,European field vole (Rodentia,Cricetidae)

The sex chromosomes of Microtus agrestis are extremely large due to the accumulation of constitutive heterochromatin. We have identified two prominent satellite bands of 2.0 and 2.8 kb in length after HaeIII and HinfI restriction enzyme digestion of genomic DNA, respectively. These satellites are located on the heterochromatic long arm of the X chromosome as shown using Microtus x mouse somatic cell hybrids. By in-gel hybridization with oligonucleotide probes, the organization of the two satellites was studied: among the many copies of the simple tandem tetranucleotide repeat GATA are interspersed rare single GACA tetramers. One of the satellites also harbours related GGAT simple tandem repeats. In situ hybridizations with plasmid-carried or oligonucleotide GA _C ^T A probes show clustered silver grains on the long and short arm of the X chromosome. Interspersion of differently organized (GATA)_n elements is also demonstrable in the autosomal complement and on the Y chromosome. These results are discussed in the context of the evolution of vertebrate sex chromosomes in relation to heterochromatin and simple repetitive DNA sequences.     

The distribution of SCEs within and between the genomes of an interspecific hybrid

The distribution of sister chromatid exchanges has been examined in the chromosomes of a hybrid male wallaby (Macropus rufogriseus x Wallabia bicolor ), and in the X chromosomes of M. parryi and M. rufus. Comparisons were made of SCE frequency between the two genomes of the hybrid, only one of which has an appreciable amount of constitutive heterochromatin, and between the euchromatic and heterochromatic regions of the M. rufogriseus genome. The frequency of SCEs is closely correlated with the DNA content of the individual chromosomes. The distribution of the SCEs between the euchromatin and heterochromatin in the M. rufogriseus genome showed a deficiency of SCEs observed in the heterochromatin compared with the euchromatin. —A substantial excess of SCEs occurred at the nucleolar organiser region of the M. rufogriseus X chromosome. This excess was absent from the nucleolar organiser region of the X chromosome of the two other macropodine species studied and is accounted for by the presence of an adjacent euchromatin-heterochromatin junction.     

Karyotypic analysis of the cotton boll weevil, Anthonomus grandis Boheman

The diploid chromosome number of the cotton boll weevil, Anthonomus grandis Boheman, is 44. Both C‐ and N‐banding techniques of mitotic cells demonstrated constitutive heterochromatin in the p arm of the eight largest chromosomes, the p arm of the X chromosome, and the centromeric region of autosomal groups A–D. Neither the y nor the group E autosomes appeared to contain constitutive heterochromatin. Supernumerary chromosomes were not found in the boll weevil. Restriction endonuclease banding of primary spermatocytes revealed a rod‐shaped Xy tetrad in which the X and y were terminally associated. The p arm of the large, submetacentric X was C‐band positive. While two of the autosomal tetrads were typically ring‐shaped in primary spermatocytes, the remaining 19 autosomal tetrads were rod‐shaped. This revised version was published online in July 2006 with corrections to the Cover Date.     

DNA replication,3H-cRNA in situ hybridization and C-band patterns in the polycentric chromosomes ofLuzula purpurea Link

DNA late-replication,³H-cRNA in situ hybridization, and C-band distribution patterns were studied inLuzula purpurea Link chromosomes (2n=6). With each technique it was possible to identify homologous chromosomes. DNA late-replicating regions were present at the ends and in the middle of one chromosome pair (pair 1), on both ends of another chromosome pair with one end having more late-replicating regions than the other end (pair 2), and all along the length of the final pair (pair 3). The distribution of label following in situ hybridization of³H-cRNA complementary to Cot 1-reassociated DNA was similar to the DNA late-replication patterns. One chromosome pair had grains concentrated at the ends and in the middle of the chromosomes; another pair had grains at both ends with a greater grain concentration at one end; the final chromosome pair had grains distributed all along the length. C-band distribution patterns were also similar to the DNA late-replication and³H-cRNA in situ-hybridized ones. The results demonstrate that the constitutive heterochromatin ofL. purpurea polycentric chromosomes is similar to the constitutive heterochromatin of monocentric animal chromosomes in that it consists of highly repeated DNA sequences which are replicated late in the S stage of interphase.     

The location of highly repetitious DNA in the somatic chromosomes of Drosophila melanogaster

In situ hybridization of Drosophila melanogaster somatic chromosomes has been used to demonstrate the near exact correspondence between the location of highly repetitious DNA and classically defined constitutive heterochromatin. The Y chromosome, in particular, is heavily labeled even by cRNA transcribed from female (XX) DNA templates (i.e., DNA from female Drosophila with 2 Xs and 2 sets of autosomes). This observation confirms earlier reports that the Y chromosome contains repeated DNA sequences that are shared by other chromosomes. In grain counting experiments the Y chromosome shows significantly heavier label than any other chromosome when hybridized with cRNA from XY DNA templates (i.e., DNA from male Drosophila with 1 X and 1 Y plus 2 sets of autosomes). However, the preferential labeling of the Y is abolished if the cRNA is derived from XX DNA. We interpret these results as indicating the presence of a class of Y chromosome specific repeated DNA in D. melanogaster. The relative inefficiency of the X chromosome in binding cRNA from XY and XYY DNA templates, coupled with its ability to bind XX derived cRNA, may also indicate the presence of an X chromosome specific repeated DNA.     

Chromosome banding in amphibia

The distribution of constitutive heterochromatin on the chromosomes of Triturus a. alpestris, T. v. vulgaris and T. h. helveticus (Amphibia, Urodela) was investigated. Sex-specific chromosomes were determined in the karyotypes of T. a. alpestris (chromosomes 4) and T. v. vulgaris (chromosomes 5). The male animals have one heteromorphic chromosome pair, of which only one homologue displays heterochromatic telomeres in the long arms; the telomeres of the other homologue are euchromatic. This chromosome pair is always homomorphic and without telomeric heterochromatin in the female animals. There is a highly reduced crossing-over frequency between the heteromorphic chromosome arms in the male meiosis of T. a. alpestris; in T. v. vulgaris no crossing-over at all occurs between the heteromorphic chromosome arms. No heteromorphisms between the homologues exist on the corresponding lampbrush chromosomes of the female meiosis. In T. h. helveticus no sex-specific heteromorphism of the constitutive heterochromatin could be determined. The male animals of this species, however, already possess a chromosome pair with a greatly reduced frequency of chiasma-formation in the long arms. The C-band patterns and the pairing configurations of the sex-specific chromosomes in the male meiosis indicate an XX/XY-type of sex-determination for the three species. A revision of the literature about experimental interspecies hybridizations, gonadic structure of haploid and polyploid animals, and sex-linked genes yielded further evidence in favor of male heterogamety. The results moreover suggest that the heterochromatinization of the Y-chromosome was the primary step in the evolution of the sex chromosomes.     

Differential sensitivity of constitutive and facultative heterochromatin in orthopteran chromosomes to digestion by DNaseI

In situ pancreatic DNaseI digestions were used as probes to study the structural organization of facultative and constitutive heterochromatin during both mitotic and meiotic divisions. Three different types of heterochromatic regions from three insect species were chosen for this study. These regions had been previously characterized by in situ treatments with restriction endonucleases (AT and GC rich DNA sequences). Progressive increase in DNaseI concentration (from 10 to 200 ng/ml) or in incubation time (from 5 to 30 min) revealed a specific pattern of sequential digestion of the constitutive heterochromatic regions, the centromeric ones (AT-rich DNA) being the most resistant to DNaseI action. The interstitial C-bands (with AT or GC-rich DNA) were more sensitive to DNaseI, and the band 4.4 from Baetica ustalata was the most resistant of the non-centromeric bands. Similar results were obtained during meiosis, but increased accessibility to DNAseI was observed compared to mitosis. DNA methylation in the non-centromeric band 4.4 of B. ustulata could be responsible for its differential digestion with respect to the remaining intercalar heterochromatin. Facultatively heterochromatic regions (X chromosomes) were found to exhibit a differential response to DNaseI attack from mitosis to meiosis. While they behaved as cuchromatin during mitosis, they were the most resistant together with centromeric heterochromatin regions, during metaphase I and II. The different responses to digestion of the X chromosome and X-derived regions between somatic and meiotic divisions are probably a consequence of the changes in the organization of this chromosome during the facultative heterochromatinization process.