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.     

Karyotype,DNA replication and origin of sex chromosomes in Anopheles atroparvus

Anopheles atroparvus has two pairs of autosomes similar in length and morphology and two sex chromosomes with equal, heterochromatic, late replicating long arms with homologous C-, G-, and Q-bands. The short arm of the Y is shorter than that of the X and both are euchromatic. The mean number of chiasmata per cell in the male is 3.2. During mitosis there is a high grade of somatic pairing but X and Y, which form a heteropycnotic mass in the interphase nucleus, have a differential behaviour. The chronology of DNA replication was studied in spermatogonia and brain cells by autoradiography. It is hypothesized that the present sex chromosomes of A. atroparvus evolved by accumulation of sex determining factors and gene deterioration resulting in heterochromatinization of the long arms, followed by structural rearrangements.—The homology of the two sex chromosomes requires limited dosage compensation which is achieved either as in Drosophila by modifier genes or by accumulation on the short arm of the X, only of female determining factors which do not require dosage compensation.     

Constitutive heterochromatin,G-bands and nucleolus-organizer regions in four species of Didelphidae (Marsupialia)

Chromosomes of Didelphis albiventris, D. marsupialis, Philander opossum and Lutreolina crassicaudata, four species of marsupials with very similar karyotypes and 2n=22 were studied. All the chromosomes were acrocentrics except the X in L. crassicaudata, which is a metacentric.The G-band patterns of these species are similar but the distribution of constitutive heterochromatin differs among them as shown by C-banding. The hypothesis that the X in L. crassicaudata might be an isochromosome derived from the acrocentric X in the other species is discarded since G-and C-banding patterns differ in the two arms.In D. marsupialis the Ag-NORs are terminal and located in both arms of one pair and in the long arms of two pairs of medium-sized autosomes. In P. opossum the NOR-bearing chromosomes could be precisely identified through simultaneous silver staining and G-banding. The Ag-NORs are terminal and located at the short arm of pair 5 and the long arm of pair 7.     

Patterns of replication in the neo-sex chromosomes ofDrosophila nasuta albomicans

Drosophila nasuta albomicans (with 2n = 6), contains a pair of metacentric neo-sex chromosomes. Phylogenetically these are products of centric fusion between ancestral sex (X, Y) chromosomes and an autosome (chromosome 3). The polytene chromosome complement of males with a neo-X- and neo-Y-chromosomes has revealed asynchrony in replication between the two arms of the neo-sex chromosomes. The arm which represents the ancestral X-chromosome is faster replicating than the arm which represents ancestral autosome. The latter arm of the neo-sex chromosome is synchronous with other autosomes of the complement. We conclude that one arm of the neo-X/Y is still mimicking the features of an autosome while the other arm has the features of a classical X/Y-chromosome. This X-autosome translocation differs from the other evolutionary X-autosome translocations known in certain species ofDrosophila.     

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.     

The replication of human heterochromatin in serial culture

The human C group chromosomes late to start replication in asynchronous and in FUdR synchronized cell lines are X chromosomes. These same chromosomes are also heterochromatic during interphase. During metaphase these allocyclic Xs cannot be identified simply by metaphase position or morphology and show a wide range of measurements for arm ratio, centromere index and total length. Replication starts in the short arm and extends over the entire chromosome during the 2nd and 3rd hr of S until by the 4th hr distinction from other C group chromosomes cannot be made by means of the labeling pattern. When the allocyclic X chromosomes start replication the pattern of H³TdR label over interphase sex chromatin and non-specific heterochromatin shifts from unlabeled to labeled in FUdR synchronized human cell lines. The overall time required for replication of the allocyclic X is less than that for the other chromosomes in both asynchronous and FUdR treated cells. A hypothesis is presented for a direct relation between the delay of onset of replication in heterochromatin and its degree of interphase condensation.The present study was supported by research grants: No. HD-00777 from the National Institutes of Health and No. E-487 from the American Cancer Society, Inc.     

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.     

Cytological identification of two X-chromosome types in the wood lemming (Myopus schisticolor)

In the wood lemming (Myopus schisticolor) three genetic types of sex chromosome constitution in females are postulated: XX, X^*X and X^*Y (X^*=X with a mutation inactivating the male determining effect of the Y chromosome). Males are all XY. It is shown in the present paper that the two types of X chromosomes, X and X^*, exhibit differences in the G-band patterns of their short arms. In addition, it was demonstrated in unbanded chromosomes that the short arm in X^* is shorter than in X. The origin of these differences is still obscure; but they allow to identify and to distinguish the individual types of sex chromosome constitution, as of XX versus X^*X females and of X^*Y females versus XY males, on the basis of G-banded chromosome preparations from somatic cells.     

Induction of a high incidence of damage to the X chromosomes of Rattus (Mastomys) natalensis by base analogues,viruses, and carcinogens

Cultured embryonic cells from Rattus (Mastomys) natalensis were treated with the base analogues 5-bromodeoxyuridine and 2-aminopurine, the viruses herpes simplex virus and adenovirus type 12, and the carcinogens 7,12-dimethylbenz(a)anthracene and urethan. Treatment with these agents, except urethan, causes an increase in the incidence of chromosome aberrations. The induced aberrations are not randomly distributed among the chromosomes or within a chromosome. The X chromosome, especially its long arm, is more sensitive to these agents than is any other chromosomes in the complement. — A possible relationship among the high incidence of damage, the positive heteropycnosis, and the late replication in the X chromosome of R. natalensis is discussed.     

Asynchronous Duplication of Chromosomes in Cultured Cells of Chinese Hamster

Chromosome duplication (DNA synthesis) was studied in cultured cells of Chinese hamsters by means of autoradiography following thymidine-H³ incorporation. The technique used was to expose an asynchronously dividing population of rapidly growing cells for a 10 minute interval to a medium with thymidine-H³. Cells were then transferred to a medium with excess unlabeled thymidine. The population was sampled at intervals thereafter and studies made of the frequency of labeled interphases and division figures, and the patterns of labeling of specific chromosomes. The average generation time during these experiments was about 14 hours. DNA synthesis occurred during an interval of about 6 hours and stopped 2 to 3 hours before metaphase. After metaphase the chromosomes usually begin duplication again within 5 to 6 hours. Grain counting, to estimate the amount of tritium incorporated after a short contact with thymidine-H³ and at intervals after transfer to a medium with excess unlabeled thymidine, indicated that the intracellular pool of labeled precursors was diluted within less than a minute so that further labeling would not be detected. The chromosomes labeled during the contact period retained their precise pattern of labeling through another duplication cycle and no turnover of DNA or loss of tritium was detectable. Five or 6 chromosomes of the complement have segments typically late in duplication. Two of these are the X and Y chromosomes. The long arm of the X chromosome and the whole Y chromosome are duplicated in the last half of the interval of DNA synthesis. The short arm of the X chromosome in a male strain is duplicated in the first half of the interval. In another strain (female), one X chromosome had the same timing, but the other one was all duplicated in the last half of the period of DNA synthesis. The DNA in the short arms of 2 medium sized chromosomes, as well as most of the DNA in 1 or 2 of the smallest chromosomes of the complement was replicated late. The study has led to the hypothesis that various chromosomes or parts of chromosomes have a genetically controlled sequence in duplication which may have some functional significance.     

The origin of tertiary monosomics in the tomato

Six aneuploid tomato plants with 2n–1=23 chromosomes were observed in populations grown from the seedlings treated with thermal neutrons and from seeds treated with X-rays. Four of the aneuploids were tertiary monosomics in which, as a result of centromeric interchanges between two different chromosomes, two whole arms were missing from the complement and two arms connected at the centromere. In one aneuploid, as a result of centromeric breakage, the two short arms of a homologous pair were missing from the complement and the two long arms connected to the long arm and the short arm respectively of another chromosome in which breakage had occurred also at the centromere. In one aneuploid, the interchange has occurred in the arms, and not in the centromere. Here the aneuploid condition is due to the loss of an arm with a centromere and a short piece of the other arm.In most of the tertiary monosomics the missing arms were either the short arms of sub-metacentric chromosomes or any of the arms of metacentric chromosomes. However, in one case the long arms of two submetacentric chromosomes were lost from the complement. That in spite of such large chromosomal deletions the sporophyte can survive, may be due to the fact that the aberrant plants are mostly chimeras.This study was part of a project resulting from a contract between the Association Euratom-I.T.A.L., and the Agricultural University of Wageningen.     

MAMMALIAN CHROMOSOMES IN VITRO : XVIII. DNA Replication Sequence in the Chinese Hamster

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H³-thymidine labeling and the method of 5-fluorodeoxyuridine block with H³-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.     

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.     

Reversion of XO to XY sex chromosome mechanism in a phasmid

Summary The sex chromosomes of the male phasmid Isagoras schraderi Rehn comprise an X and a Y, — each with a submedian kinetochore, and one euchromatic and one heterochromatic arm. At meiosis X and Y form an unequal sex bivalent in which the euchromatic arms are terminally associated. Relatively recent reversion from the XO-XX mechanism characteristic of the Phasmidae is indicated by the presence of the euchromatic arm in both X and Y. The diploid number of the male is 34.Unequal autosomal bivalents are found at meiosis in two other species of Isagoras — Isagoras subaquiles Rehn and Isagoras sp. — and in Pseudophasma menius Westwood. The chromosome complements of these species are described.     

Deletion und Translokation heterochromatischer Chromosomenabschnitte bei Microtus agrestis

Zusammenfassung In unbehandelten Nierenepithel-und Fibroblastenkulturen von Microtus agrestis wurden Brüche, Deletionen und Translokationen an den heterochromatischen langen Armen von X-und Y-Chromosomen in 2% aller Mitosen eines weiblichen und 3% der Mitosen eines männlichen Tieres gefunden. In tetraploiden Mitosen sind Deletionen häufiger als in diploiden zu finden. Die Deletionen treten sowohl auf dem X₁ und X₂ des Weibchens als auch auf dem X und Y des Männchens auf. Längenmessungen an normalen und deletierten Chromosomen ergaben, daß die bevorzugte Bruchlokalisation beim X-Chromosom am Übergang vom proximalen zum mittleren Drittel der langen Arme liegt, beim Y in der Mitte der langen Arme. Es wurden Translokationen der azentrischen Fragmente auf die langen Arme von X-Chromosomen und Fusion deletierter X-Chromosomen zu dizentrischen Chromosomen beobachtet, jedoch keine Translokation auf euchromatische Chromosomen. Das häufigere Auftreten von ein oder zwei deletierten Chromosomen in tetraploiden Zellen wird durch Fusion zweier diploider (Schwester-) Kerne in zweikernigen Zellen erklärt die durch Mitose ohne nachfolgende Plasmateilung einer diploiden Zelle mit Chromatidbruch oder deletiertem Chromosome entstanden sind.

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Deletion and translocation of heterochromatic chromosome segments of Microtus agrestis

Summary Breaks, deletions and translocations of the heterochromatic long arms of the X and Y chromosomes were found in 2% of all female mitoses and 3% of male mitoses of untreated kidney epithelial cell and fibroblast cultures of Microtus agrestis. Deletions are more frequent in tetraploid than in diploid mitoses. Deletions were found in the X₁ and X₂ of the female as well as in the X and Y of the male. Length measurements of normal and deleted chromosomes showed that the breaks in the X are preferentially located between the proximal and middle third of the long arm, whereas in the Y chromosome they are near the middle of the long arm. Translocations of acentric fragments to the long arms of X chromosomes and fusions of deleted X chromosomes resulting in dicentric chromosomes were also observed, but no translocations to euchromatic chromosomes could be found. The relatively high frequency of one or two deleted chromosomes in tetraploid cells is explained by a fusion of two diploid (sister-) nuclei in binucleated cells resulting from mitosis without cytoplasmic division of diploid cells with a break of a chromatid or a deleted chromosome.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Chromosomes of five artiodactyl mammals

Somatic chromosomes of six specimens belonging to the following five species of artiodactyls (Artiodactyla: Mammalia) are described: A female nilgai (Boselaphus tragocamelus), 2n=46; male baresingha (Rucervus duvauceli), two specimens, 2n=56; a female Himalayan tahr (Hemitragus jemlahicus), 2n=48; a female Kirk's dik-dik (Rhynchotragus kirki), 2n=46; and a male sambar (Cervus unicolor), 2n=58. In the baresingha and the sambar, one or more acrocentric chromosomes carried satellites on their long arms. ³H-thymidine radioautographs of cultured cells of the Himalayan tahr showed a long acrocentric chromosome to be late-replicating, suggesting that it is an X chromosome.     

Selective staining of X chromosome segments in Schistocerca gregaria after denaturation and reassociation procedures

The DNA of fixed mitotic and meiotic chromosomes and of spermatides of Schistocerca gregaria males was heat denaturated and then differentially reassociated in a Giemsa buffer or in acridine orange buffer solution. After this procedure, two to three large, selectively stained regions are seen in the X chromosome of spermatocytes and spermatides. Denaturation and reassociation experiments have shown that after differential reassociation such a selective stainability of chromosome regions is characteristic for the presence of fast-reassociating, i.e., repetitive DNA (Stockert and Lisanti, 1972). The possible presence of repetitive DNA in the X chromosome regions concerned can not be the only reason for the occurrence of the heavily stained segments after reassociation because (1) these segments are obtained in positively heteropycnotic X chromosomes, but not in negatively heteropycnotic Xs and (2) they do not occur in positively heteropycnotic X chromosomes when the histones have been extracted before the denaturation and reassociation processes. Contrary to the latter statement, the heavily stained X chromosomal regions are preserved when the histones are removed after the denaturation and reassociation steps. — It is assumed that the heavily stained X chromosome segments represent DNA reassociation complexes which are only formed if histones are present. It is discussed whether the formation of the X chromosome complexes depends on a specific chromatin configuration within positively heteropycnotic X chromosomes.     

Incomplete sister chromatid separation of long chromosome arms

Chromosome segregation ensures the equal partitioning of chromosomes at mitosis. However, long chromosome arms may pose a problem for complete sister chromatid separation. In this paper we report on the analysis of cell division in primary cells from field vole Microtus agrestis, a species with 52 chromosomes including two giant sex chromosomes. Dual chromosome painting with probes specific for the X and the Y chromosomes showed that these long chromosomes are prone to mis-segregate, producing DNA bridges between daughter nuclei and micronuclei. Analysis of mitotic cells with incomplete chromatid separation showed that reassembly of the nuclear membrane, deposition of INner CENtromere Protein (INCENP)/Aurora B to the spindle midzone and furrow formation occur while the two groups of daughter chromosomes are still connected by sex chromosome arms. Late cytokinetic processes are not efficiently inhibited by the incomplete segregation as in a significant number of cell divisions cytoplasmic abscission proceeds while Aurora B is at the midbody. Live-cell imaging during late mitotic stages also revealed abnormal cell division with persistent sister chromatid connections. We conclude that late mitotic regulatory events do not monitor incomplete sister chromatid separation of the large X and Y chromosomes of Microtus agrestis, leading to defective segregation of these chromosomes. These findings suggest a limit in chromosome arm length for efficient chromosome transmission through mitosis.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A Drosophila melanogaster cell line tested for the presence of active NORs by silver staining

Silver staining was used to detect active NORs in a Drosophila melanogaster cell line (C1 82) characterized by dimorphic X chromosomes (XX_L), one of the two Xs showing a marked increase in heterochromatin where the nucleolar organizer (NO) is located. The Q-banding technique was used to determine the karyotype characteristics of the line. Ag-positive NORs appeared only on structurally changed X chromosomes (X_L), both in diploid and tetraploid cells, indicating that rRNA genes of X_L are more active or numerous than those on normal homologues. A possible relationship between NOR stainability, the presence of an increased heterochromatic portion and the selective advantage of XX_L cells, recurrent in numerous Drosophila female lines, is discussed.