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.     

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.     

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.     

DNase I sensitivity of Microtus agrestis active,inactive and reactivated X chromosomes in mouse-Microtus cell hybrids

We isolated Microtus agrestis-mouse somatic cell hybrid clones which had retained either the active or the inactive M. agrestis X chromosome. In both hybrid clones the X chromosomes retained their original chromatin conformation as studied by the in situ nick translation technique — the active X chromosome retained its high sensitivity to DNase I while the inactive one remained insensitive. A clone in which the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene had been spontaneously reactivated was isolated from the hybrid containing the inactive X chromosome. The in situ nick translation technique was used to study possible DNA conformation changes in the euchromatin of the inactive X chromosome with special reference to the reactivated HPRT locus. We found that the euchromatin in this X chromosome exhibited the same low sensitivity to DNase I as is characteristic of the inactive X chromosome.Professor Marcus passed away on 2 January 1987     

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     

Localization of repetitive DNA in the chromosomes of Microtus agrestis by means of in situ hybridization

Heterochromatin in the European field vole, Microtus agrestis, was studied using a special staining technique and DNA/RNA in situ hybridization. The heterochromatin composed the proximal 1/4 of the short arm and the entire long arm of the X chromosome, practically the entire Y chromosome and the centromeric areas of the autosomes. By using the DNA/RNA in situ hybridization technique, repeated nucleotide sequences are shown to be in the heterochromatin of the sex chromosomes.Supported in part by Research Grants DRG-1061 and 269 from the Damon Runyon Memorial Fund for Cancer Research, G-373 and G-267 from the Robert A. Welch Foundation.     

Microfluorometric analysis of DNA replication in human X chromosomes

BUdR-sensitive fluorescence of the dye 33258 Hoechst allows microfluorometric analysis of replication in human chromosomes. Comparison of the fluorescence patterns of male and female X chromosomes obtained with this technique reveals differences that may reflect regional alterations in DNA synthesis kinetics.     

Tissue-specific heterogeneity in DNA replication patterns of human X chromosomes

Regional DNA replication kinetics in human X chromosomes have been analysed using BrdU-33258 Hoechst-Giemsa techniques in five cell types from human females: amniotic fluid cells, fetal and adult skin fibroblasts, and fetal and adult peripheral lymphocytes. In all cell types, the late-replicating X chromosome can be distinguished from its active, earlyreplicating homologue, and both the early and late X exhibit temporally and regionally characteristic internal sequences of DNA replication. The replication pattern of the early X in amniotic fluid cells and skin fibroblasts is similar to that of the early X in lymphocytes, although certain discrete regions are later-replicating in these monolayer tissue culture cells than are the corresponding regions in lymphocytes. However, DNA replication kinetics in late X chromosomes from amniotic fluid cells and skin fibroblasts are strikingly different from those observed in lymphocytes with respect both to the initiation and termination of DNA synthesis. The predominant late X pattern observed in 80–95% of lymphocytes, in which replication terminates in the long arm in bands Xq21 and Xq23, was never seen in amniotic fluid cells or skin fibroblasts. Instead, in these cell types, bands Xq25 and Xq27 are the last to complete DNA synthesis, while bands Xq21 and Xq23 are earlier-replicating; this pattern is similar to the alternative replication sequence observed in 5–20% of lymphocyte late X chromosomes. This replication sequence heterogeneity is consistent with the existence of tissue-specific influences on the control of DNA replication in human X chromosomes.     

Isolation,fractionation and biochemical analysis of the metaphase chromosomes of Microtus agrestis

A method has been developed for isolating metaphase chromosomes from Microtus agrestis fibroblasts in relatively large quantities with recovery of about 50% of the chromosomes present in the metaphase cells. The method employs pressure homogenisation to release the chromosomes from the cells. The average chemical composition of the Microtus chromosome preparations is 24.6% DNA, 19.9% RNA and 55.5% protein. The isolated chromosomes were fractionated by sedimentation velocity in a density gradient into three size groups in one of which 75–80% of the chromosomes were the large sex-chromosomes. The relative composition of this fraction containing most of the heterochromatin of the cell was DNA: 100, RNA: 59, acid-soluble protein: 54, acid-insoluble protein: 178. — Disc electrophoresis studies revealed no significant difference in the histone patterns between the euchromatic and heterochromatic chromosomes of the three chromosome size-groups. Metaphase chromosomes appear to have a lower lysine-rich histone content than interphase nuclei.     

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.     

Patterns of variation in Microtus arvalis and Microtus agrestis populations from Middle to Late Pleistocene in southwestern Europe

Fifteen paired fossil populations of Microtus arvalis and Microtus agrestis from southwestern Europe have been analysed from a morphological and morphometric point of view. The sites under consideration are located in the northern Iberian Peninsula and southern France, from the Middle Pleistocene to the end of the Late Pleistocene. The aim of this study is to stress once again the importance of keeping these two species separated in the fossil record in order to recognize specific trends of evolution and divergence and to obtain more precise information on palaeoclimatic and palaeoenvironmental conditions. It was possible to observe remarkable intraspecific differences between Middle and Late Pleistocene populations of both species. Furthermore, in synchronic co-specific populations from the Late Pleistocene, climatic and geographic conditions seem to exert a major influence in shaping intraspecific changes in dental pattern.     

Fluorescence banding pattern of the chromosomes of Microtus agrestis with a benzimidazol derivative

Summary The fluorescent banding pattern of the chromosomes of Microtus agrestis following staining with a benzimidazol derivative (Hoechst 33258) has been studied. All chromosomes allow easy identification because of their characteristic longitudinal differentiation. The X chromosomes of both sexes show intense fluorescence of the long arm and of a small proximal segment of the short arm, while the rest of the short arm reveals banding patterns. In the Y chromosome, the very short arm is nonfluorescent and the entire long arm shows bright fluorescence. Examination of the interphase nuclei of cultured fibroblasts suggests that the facultative and the constitutive heterochromatin fluoresce intensely only when strongly condensed. In contrast to some other species, in which heterochromatic chromosomal segments show a characteristic staining behaviour, i.e. either positive or negative, with the fluorochrome benzimidazol derivative, this compound behaves rather indifferently in the case of the heterochromatic contents in Microtus agrestis. The staining effects of the benzimidazol dye have also been compared with the various Giemsa and the Quinacrine staining techniques.

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Zusammenfassung Bei Verwendung des Benzimidazol-Derivat Hoechst 33 258 läßt sich an den Chromosomen von Microtus agrestis ein Fluorescenz-Bandenmuster beobachten, das an den Autosomen eine Identifizierung der einzelnen Elemente ermöglicht. Die X-Chromosomen beider Geschlechter weisen eine starke Fluorescenz des linken Arms und eines kleinen proximalen Segments des kurzen Arms auf, während der übrige kurze Arm ein Bandenmuster zeigt. Am Y-Chromosom ist der gesamte lange Arm hell fluorescierend, sein sehr kleiner kurzer Arm bleibt ungefärbt. Die Untersuchung von Interphasekernen in vitro kultivierter Fibroblasten spricht dafür, daß die Fluorescenz des konstitutiven und fakultativen Heterochromatins überwiegend auf seiner etwas stärkeren Kondensation beruht. Während das Benzimidazol-Derivat bei andren Species das konstitutive Chromatin elektiv entweder durch positive oder durch negative Fluorescenz darstellt, verhält es sich bei Microtus agrestis indifferent.

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Supported by Alexander von Humboldt Foundation.     

Late DNA replication in rhesus monkey (Macaca mulatta) chromosomes

A study of the pattern of late DNA replication in rhesus monkey chromosomes showed evident similarities with man. This must be a consequence of the evolutionary conservation of replication patterns in primate chromosomes, as it has been demonstrated in the great apes, in Cebus, and man. However, the pattern of late replication of the allocyclic X chromosome in lymphocytes of female rhesus monkey was identical with the fibroblast pattern in man, and with the pattern found in only 5 to 20% of human lymphocytes.     

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.     

A highly divergent mitochondrial DNA lineage of Microtus agrestis in southern Europe

The Mediterranean peninsulas constitute important areas for endemism and intraspecific variation, and are likely places for cryptic biodiversity. We assessed the phylogeographic pattern of field voles (Microtus agrestis) in southern and central Europe by sequence analysis of a 385-bp fragment of the mitochondrial cytochrome b gene in 74 specimens from 44 localities. The majority of samples consisted of skulls collected from owl pellets. The data revealed a highly distinct cytochrome b lineage in an area ranging from Portugal to Hungary. This southern field vole phylogroup differed by a sequence divergence of 5.6-7.1% from the remaining haplotypes, a level of divergence comparable to that found between known Microtus sibling species. However, this ancient phylogeographic break that dates back many glacial cycles has not been recognised previously by either morphology or karyotype. The southern cytochrome b lineage was further divided into two well-defined sublineages that appear to have derived from different glacial refugia in the Iberian Peninsula.     

The repeated DNA sequences of Microtinae : I. Microtus agrestis, Microtus pennsylvanicus and Ellobius lutescens

A comparison has been made of the repeated nucleotide sequences from 3 Microtinae which possess varying amounts of constitutive heterochromatin per cell nucleus. Eight repetitive fractions of DNA, ranging in C_ot values from 10⁻³ to 10⁻³, were obtained by reassociation of sheared, denatured DNA and fractionation on hydroxyapatite. At C_ot values of less than 1, 3 fractions were isolated that amounted to 18.7, 10.0 and 7.4 % of the total DNA of Microtus agrestis, Microtus pennsylvanicus and Ellobius lutescens, respectively, in agreement with the amounts of heterochromatin in these species. At C_ot values higher than 1, the amounts of repeated sequences were more comparable and constituted about 12 to 14 % of the DNA. Thermal denaturation profiles of all the repetitive fractions showed a good deal of order in the reassociated duplexes, with an average hyperchromicity of 20 %. Upon density gradient centrifugation in neutral CsCl, the fractions from M. pennsylvanicus and E. lutescens yielded almost identical patterns and differed significantly from those of M. agrestis. In M. agrestis a fraction of fast-intermediate repetitiveness (reassociating at C_ot values between 10⁻² and 1) was isolated, amounting to about 12 % of the total DNA. This fraction has a base composition comparable to that of total DNA and represents the major component of the constitutive heterochromatin of giant sex chromosomes that have been isolated by the disruption of brain and liver nuclei and differential centrifugation.