Cell-specific variation of X chromosome replication in the Syrian hamster (Mesocricetus auratus)

The sub-division of S-phase in Syrian hamsters, on the basis of BrdU/Hoechst 33258/Giemsa banding, has allowed a quantitative comparison of the replication of individual chromosome bands within defined subphases of S. This analysis has shown that in hamsters, as has been reported in humans, there are distinct patterns of early replication in vitro in the early X, the late X in fibroblasts, and the late X in lymphocytes. In addition, it has been possible to show that, although the pattern of replication of the late X in fibroblasts differs from that in lymphocytes, the time in S at which bands first appear on this chromosome is the same in the two cell types. — No significant heterogeneity can be ascribed to differences between individuals, adult or embryonic sources, culture media, or time of exposure to BrdU. — The absence of any detectable heterogeneity in the replication band frequencies in autosomal heterochromatic arms suggests that the cell-specific variability of the late-replicating X is a feature of facultative rather than constitutive heterochromatin.     

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbillus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially reinactivated, suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.Deceased on 2 Jan. 1987     

Heterochromatin organization in the nucleus of Indian muntjac (Muntiacus muntjak)

The heterochromatin in Indian muntjac (Muntiacus muntjak) is located at the periphery of primary constrictions of all the chromosomes. The X chromosome contains significantly larger amounts of heterochromatin than the rest of the complement by C-banding technique. However, the small portion of C-band region was found to be resistant by restriction endonuclease HaeIII (5'...GG decreases CC...3') and was clearly visible on the nucleus. Therefore, the position of this large heterochromatic segment is examined at somatic metaphases. The distribution of the heterochromatin of the X chromosome observed in Indian muntjac is contrary to the general pattern observed in other species, i.e., the chromosomes consisting greater amount of heterochromatin are located more peripherally than those with lesser amount. However, the smaller Y chromosome (Y1) is frequently found at the periphery. The present findings suggest that the role of heterochromatin organization in the nucleus vary between different heterochromatic segments of the same species and vary from species to species.     

Intercalary heterochromatin in Drosophila

Sites of intercalary heterochromatin (IH) in the complete set of Drosophila melanogaster polytene chromosomes were localized and studied according to the following criteria: tendency to break (weak points), ectopic pairing and late replication, the existence of repeats (in X and 2R) including those enriched with A-T bases. Correlation between these features investigated, the highest correlation coefficients found between weak point behavior, late replication, and ectopic pairing. The frequency of breaks in weak points in some IH bands was shown to be different in different tissues, strains and closely related Drosophila species. Sexual differences in morphology and manifestation of IH features were found in bands of the X chromosome: weak point behavior and participation in ectopic pairing of IH bands are an order of magnitude less frequent in male X chromosomes than in female X chromosomes. In autosomes such differences have not been observed. IH bands in male X chromosomes look more massive than the homologous ones in female X chromosomes: the DNA content of the 11A6-9 region is four times less in females than in males. The hypothesis is proposed that the specific features of intercalary heterochromatin bands are determined by tandem repetitiveness and late replication. The latter, if it occurs in a cluster of repetitions, could cause incomplete polytenization of the region and, as a consequence, breaks (or weak points) and the appearance of adhesive ends which may take part either in realization of ectopic contacts or in fixation of those occurring previously. Breaks caused by chromosome aberrations in regions with repeats may not result in a sharp decline of viability, so that break points of chromosome rearrangements in intercalary heterochromatin may be more frequent than in other regions.     

Late DNA replicaion of X chromosomes in female and pseudofemale cells of Microtus agrestis.

The late replication pattern of the short arms of the X chromosomes of Microtus agrestis was studied in female cells and in cells with 2 X chromosomes of male origin by means of the BUdR-Giemsa technique and of 3H-thymidine labelling. The light absorption of Giemsa stained chromosome sections which were unifilarly substituted with BUdR (labelled), was found to be 59.2% of that of unlabelled chromosomes. In female cells, asynchrony of DNA replication of both X chromosomes indicated the presence of facultative heterochromatin in the X2 and euchromatin in the X1. In the male cells only euchromatic X chromosomes were observed in diploid XX and XO cells as well as in triploid XXY, XX and XO cells. The results show that inactivation of an X chromosone in vitro, in cells with more than one originally active X chromosome does not occur even after a culture duration of several years.     

Obervations on the Induction of Position Effect Variegation of Euchromatic Genes in Drosophila Melanogaster

In the T(1;2)dor(var7) translocation, the 1A-2B7-8 segment of the X chromosome is brought to the vicinity of 2R-chromosome heterochromatin resulting in position effect variegation of dor, BR-C and more distal genes, as well as compaction of chromatin in this segment. By irradiation of T(1;2)dor(var7), nine reversions (rev) to a normal phenotype were recovered. In two cases (rev27, rev226), the 1A-2B7-8 section is relocated to the 19A region of the X chromosome, forming free duplications (1A-2B7-8/19A-20F-X-het). Modifiers of position effect do not change the normal expression of the BR-C and dor genes in these duplications. In five reversions (rev3, rev40, rev60, rev167, rev175), free duplications have formed from the 1A-2B7-8 fragment and X chromosome heterochromatin. In these rearrangements, modifiers of position effect (low temperature, removal of Y and 2R-chromosome heterochromatin and a genetic enhancer (E-var(3)201) induce position-effect again. Two reversions (rev45 and rev110) are associated with additional inversions in the original dor(var7) chromosomes. The inversions relocate part of the heterochromatin adjacent to the 1A-2B7-8 section into new positions. In T(1;2)dor(rev45), position-effect is seen in the 2B7-8-7A element as compaction spreading from 2B7-8 proximally in some cases as far as the 5D region. Thus, in rev45 the pattern of euchromatin compaction is reciprocal to that of the initial dor(var7) strain. Apparently, it is due to the same variegation-evoking center near the 2R centromere in both cases. In all nine revertants, weakening or complete disappearance of the position-effect is observed despite retention of the 20- kb heterochromatic segment adjacent to the 1A-2B7-8 region. Thus, a 20-kb heterochromatic sequence does not inactivate euchromatin joined to it.     

Chromosome banding in Amphibia

High-resolution replication banding patterns were induced in prometaphase and prophase chromosomes of Xenopus laevis by treating kidney cell lines with 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT) in succession. Up to 650 early and late replicating bands per haploid karyotype were demonstrated in the very long prophase chromosomes. This permits an exact identification of all chromosome pairs of X. laevis. Late replicating heterochromatin was located by analysing the time sequence of replication throughout the second half of S-phase. Neither heteromorphic sex chromosomes nor sex chromosome-specific replication bands were demonstrated in the heterogametic ZW females of X. laevis. A detailed examination of the BrdU/dT-labelled prometaphases and prophases revealed that the X. laevis chromosomes can be arranged in groups of four (quartets), most of which show conspicuous similarities in length, centromere position, and replication pattern. This is interpreted as further evidence for an ancient allotetraploid origin of X. laevis.by H.C. MacgregorThis paper is dedicated to Prof. Wolfgang Engel on the occasion of his 50th birthday     

Dynamic organization of the β-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25°?C and β-heterochromatic in X0 males at 14°?C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(1LR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin.     

Analysis of spreading of inactivation in eight X autosome translocations utilizing the high resolution RBG technique

Summary Eight X autosome translocations were studied with replication banding to localize spreading of late replication into the autosomal segments. Partial spreading into the autosomal segment was seen in four translocations and no spreading of late replication was seen in four translocations. In those translocations with partial spreading of late replication into the autosomal segment, late replication did not always spread continuously from the X chromosome breakpoint throughout the autosome. Instead, it appeared to skip some bands and affect others. The data on the pattern of replication, taken to indicate also a spread of inactivation into these autosomal segments, correlated well with the clinical data in most cases and suggest that spreading of late replication is often incomplete and may be discontinuous.     

Random/nonrandom X-chromosome inactivation in Nesokia indica: possible influence of heterochromatin

Five types of X chromosomes with different amounts of heterochromatin have been observed in Nesokia indica, the Indian mole rat. They have been found in both mosaic and nonmosaic individuals. The influence, if any, of heterochromatin on the kinetics of X-chromosome DNA replication was evaluated in bone marrow cells and peripheral blood lymphocytes of Nesokia females with variant X chromosomes. In bone marrow cells of nonmosaic females a random X-chromosome inactivation (XCI) pattern was observed, except when there was a total loss of heterochromatin from the variant X chromosome, resulting in predominantly early replication. A nonrandom pattern was observed, however, in blood and bone marrow cells of all individuals with mosaic genotypes. In these females the X chromosome with the lesser amount of heterochromatin was predominantly the active one. The amount of heterochromatin per se or, more likely, specific sequences contained in the heterochromatic region seem to influence the XCI pattern in a cis-acting manner. The observations also seem to support a process of cell selection in individuals with variant X chromosomes.     

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.     

Interline differences in morphology of the precentromeric region of polytene X-chromosome in Drosophila melanogaster salivary glands]

Morphology of the Drosophila melanogaster polytene X chromosome section 20 in normal flies, in strains carrying inversions that break pericentric heterochromatin at different points, and at the background of the Su(UR)ES mutation has been examined. In all of the strains carrying the Su(UR)ES mutation section 20 displayed a distinct banding pattern till to the section 20F, while in the wild-type strains this region was represented by beta-heterochromatin. The strains carrying different inversions substantially differed in the number and morphology of bands forming section 20. In the Su(UR)ES mutants the most proximal X chromosome euchromatin gene, su(f), is mapped to the boundary between sections 20E and F, while rDNA forming the middle part of the X chromosome mitotic heterochromatin is located in the proximal part of section 20F. All large bands observed in section 20 of the w; Su(UR)ES strain were also present in In(1)sc4; Su(UR)ES, which breaks heterochromatin in the distal part. Hence, the bands of polytene chromosome section 20 are virtually devoid of mitotic heterochromatin.     

Chronology of the replication of sex chromosome bands in lymphocytes of normal subjects and patients]

The replication sequence of the bands carried by chromosomes X and Y has been studied in normal individuals and in patients with structural abnormalities of the X. By comparing the segment with that of the autosomal bands (which had been previously studied), it was shown that the normal early X replicates in early X-phase for its R-bands and in late S-phase for its Q bands. The late X replicates entirely in late S-phase, and the sequence of band replication is not as stringent as for the early X and the autosomes. The study of fourteen cases of anomalies of chromosome X in females showed the following: in balanced reciprocal X-autosome translocations the rearranged X most often replicates early and the normal X late. Both show a normal replication sequence of their bands. In non-balanced X-autosome translocations, inactivation of the autosome fragment attached to the AUTOSOME FRAGMENT ATTACHED TO THE X may take place. In Xq- or in ter rea (X;X) (pter;pter), band p22 has a delayed replication. In iso-Xor Xp-, the long-arm-band sequence of replication shows a variation comparable to that of the late X in fibroblasts. These replication modifications are likely to induce partial inactivations or changes in activity which correspond to the so-called position effect in Drosophila.     

Late replicating bands of human chromosomes demonstrated by fluorochrome and Giemsa staining.

The addition of thymidine (TdR) to cells growing in a medium containing 5-bromodeoxyuridine (BUdR) at the end of the first replication cycle results in the incorporation of TdR into the late replicating DNA regions. These sites can be visualized by staining the metaphase chromosomes with the fluorescent dye "33258 Hoechst" or a "33258 Hoechst" Giemsa procedure. A sequence of late replication patterns has been established in metaphase chromosomes of cultured human peripheral lymphocytes. The patterns are in agreement with those obtained by the standard autoradiographic procedures, but are more accurate. As is known from autoradiography, late replicating bands are in the position of G or Q bands. The "33258 Hoechst" Giemsa staining procedure of chromosomes which have replicated in the presence of BUdR first and in TdR for the last 2 hrs of the S phase is preferable to the currently used Giemsa banding techniques: the method yields very well banded metaphases in all preparations examined, as the chromosome structure is not disrupted by the pretreatment. The bands are very distinct, even in the "difficult" chromosomes (e.g. No. 4, 5, 8 and X). In female cells the late replicating X chromosome can be identified by its size and staining pattern. In addition to the replication asynchrony, the sequence of replication within both X chromosomes in female cells is not absolutely identical. The phenomenon of a phase difference in replication between the homologues is not a peculiarity of the X chromosome, but can be found in all autosomes as well as in homologous positions on the chromatids of individual chromosomes.     

Sister chromatid exchanges in the human active and inactive X chromosomes

Four human female fibroblast strains with an i(Xq) or derivative X chromosome as a cytological marker for the inactive X chromosome were used to determine the frequency of sister chromatid exchanges (SCEs) in the active and inactive X chromosomes. No significant difference in SCE frequency between the active and inactive X chromosomes was observed. Therefore, the state of chromatin condensation and the late DNA replication in the facultative heterochromatin of the inactive X chromosome do not appear to influence the SCE frequency.     

The effect of heterochromatin on synapsis of the sex chromosomes of Peromyscus (Rodentia, Cricetidae)

The pairing behavior of the sex chromosomes in male and female individuals representing seven species of Peromyscus was analyzed by electron microscopy of silver-stained zygotene and pachytene configurations. Six species possess submetacentric or metacentric X chromosomes with heterochromatic short arms. Sex-chromosome pairing in these species is initiated during early pachynema at an interstitial position on the X and Y axes. Homologous synapsis then progresses in a unidirectional fashion towards the telomeres of the X short arm and the corresponding arm of the heterochromatic Y chromosome. The distinctive pattern of synaptic initiation allowed a late-synapsing bivalent in fetal oocytes to be tentatively identified as that of the X chromosomes. In contrast to the other species, Peromyscus megalops possesses an acrocentric X chromosome and a very small Y chromosome. Sex-chromosome pairing in this species is initiated at the proximal telomeric region during late zygonema, and then proceeds interstitially towards the distal end of the Y chromosome. These observations suggest that the presence of X short-arm heterochromatin and corresponding Y heterochromatin interferes with late-zygotene alignment of the pairing initiation sites, thereby delaying XY synaptic initiation until early pachynema. The pairing initiation sites are conserved in the vicinity of the X and Y centromeres in Peromyscus, and consequently the addition of heterochromatin during sex-chromosome evolution essentially displaces these sites to an interstitial position.     

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.     

Distribution of heterochromatin in a reconstructed karyotype of Vicia faba as identified by banding- and DNA-late replication patterns

1) The distribution pattern of heterochromatin characterized by Giemsa-banding, Quinacrine-banding and DNA-late replication has been studied in a reconstructed karyotype of Vicia faba with all chromosome pairs interdistinguishable. 2) By means of two Giemsa-banding methods both an interstitial and a centromeric Giemsa-banding pattern are described. The former one comprehends 14 marker and 18 additional bands of lower but characteristic visualization frequencies. The centromeric Giemsa-banding pattern consists of 7 bands, located in the centromeric and in the secondary constrictions of the metaphase chromosomes. Chromosomes with banding patterns intermediate between the interstitial and the centromeric Giemsa-banding have also been observed. 3) Quinacrine-banding revealed 10–12 brightly fluorescent bands and 1–2 regions of dim fluorescence. Most Q-bands occupy chromosomal positions also characterized by interstitial Giemsa bands. 4) The DNA-late replication pattern, analyzed both by autoradiography and by FPG-technique, revealed 9 late replicating chromosome regions; all of these correspond positionally to the sites of interstitial Giemsa bands. 5) The results are discussed with respect to (a) the relationships between the banding- and the DNA-late replication pattern; (b) banding and heterochromatin characteristics; (c) the correlations between the distribution of chromatid aberrations and special types of heterochromatin. — The patterns of heterochromatin distribution found are in basic conformity with the corresponding patterns reported for the standard karyotype of Vicia faba. The heterochromatin type characterized by both Giemsabanding and late replication is characteristic of all those chromosome regions which after mutagen treatments show up as aberration hot spots. Positional correlations between interstitial Giemsa marker bands and chemically induced isochromatid breaks are indicative of preferential aberration clustering in heterochromatin/euchromatin junctions.     

Early replication banding in <Emphasis Type="Italic">Leporinus</Emphasis> species (Osteichthyes,Characiformes) bearing differentiated sex chromosomes (ZW)

There are few examples of differentiated sex chromosomes in fishes. In the genus Leporinus, seven species present a highly differentiated ZW system, derived from heterochromatinization process. Cytogenetic analyses carried out in three of these fish species, Leporinus obtusidens, L. elongatus and L. reinhardti, through RBG-banding, showed late replication bands, coincident with heterochromatic regions in both Z and W chromosomes. A similar interstitial early replication segment was observed in the complex heterochromatic region along the Wq arms in the three species, which might correspond to a pseudoautosomal segment (SD, sex determining locus). Asynchrony related to the replication pattern among different Z chromosomes was not observed. When the identification of nuclear organizer regions by silver nitrate was performed over chromosomal preparations previously exposed to 5-bromo-2′-deoxyuridine (BrdU), remarkable positive signals at interstitial and telomeric position were observed on the q arms of W chromosomes in the species L. elongatus and L. reinhardti. The absence of 18S ribosomal RNA gene loci in this region, formerly demonstrated by FISH, indicates that this argentophilic behavior is putatively due to heterochromatin decondensation caused by BrdU incorporation, favoring such Ag+ reaction. Early and late replication bands were also observed in the heterochromatic portions of Z and W chromosomes, indicating that euchromatic and heterochromatic regions are interspersed. The present data suggest a significant level of heterochromatic complexity in the sex chromosomes of each species. On the other hand, the replication pattern shared by them supports a monophyletic origin.     

Sex chromosome-autosome translocations in the leaf-nosed bats, family Phyllostomidae. I. Mitotic analyses of the subfamilies Stenodermatinae and Phyllostominae

Mitotic analyses using RBA- and C-banding were performed on Stenodermatine bats with X-autosome (XY1Y2) and X- and Y- autosome (neo-XY) translocations. RBA-banded metaphases of females revealed differential replication of the inactive X chromosome. An early replicating band comprises the short arm of the X, and an intermediate replicating band is located interstitially on the long arm. The early replicating short arm has a homologous counterpart either in the form of a free autosome (the Y2) or as part of the Y. Both the "autosomal" short arm of the X and its homologue fused to the Y are C-band negative and behave autonomously from the remainder of the sex chromosomes. They are separated from X and Y chromatin by centromeric heterochromatin which presumably acts as a barrier. The intermediate replicating region of the long arm of the X is also present in the subfamily Phyllostominae. In both subfamilies this region lacks a homologous counterpart. However, it may also represent a translocated autosome which, unlike the short arm of the X, is not separated from the inactive X by centromeric heterochromatin. Its intermediate replication time may represent a retarded replication due to its juxtaposition to late replicating X chromatin. These data are discussed in light of the theory of the evolution of sex chromosome heteromorphism, specifically as it applies to mammals.