New evidence for tandem chromosome fusions in the karyotypic evolution of Asian muntjacs

A clone of highly repetitive DNA, designated C5, was isolated from DNA of female Chinese muntjac cells. The nucleotide sequence of this clone is 80%–85% homologous to that of the satellite IA clone and other highly repetitive DNA clones previously obtained from the Indian muntjac. Using C5 as a probe for in situ hybridizations to chromosome preparations of cells of both the Chinese and Indian muntjacs, we were able to show that these repeated sequences occur in centromeric heterochromatin of the chromosomes of both Chinese and indian muntjac species. More significantly, non-random clusters of hybridization signals were detected on the arms of chromosomes of the Indian muntjac. These latter hybridization sites are postulated to be regions of interstitial heterochromatin and could be the remnants of centromeric heterochromatin from ancestral Chinese muntjac chromosomes. Our observations provide new supportive evidence for the tandem chromosome fusion theory that has been proposed for the evolution of the Indian muntjac karyotype.by P.B. Moens     

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.     

Cervid satellite DNA and karyotypic evolution of Indian muntjac

Five satellite DNA families (designated as satellite I?CV) have been identified in the Cervidae so far. Among those, satellite I, II and IV are centromere specific. Satellite I and II are shared by large number of deer species, where satellite IV is highly conserved among several deer species examined. Satellite III was initially thought to be roe deer specific but later identified in Chinese water deer as well. SatelliteV is Y-chromosome specific for several Asian deer species examined but also found in the pericentric region of Indian muntjac chromosome 3 and in X chromosome of Chinese water deer. The observation of interstitial hybridization sites on Indian muntjac chromosomes with satellite DNA I probe generated from Chinese muntjac provides the first molecular evidence supporting the tandem fusion theory that 2n=6??/7??of Indian muntjac karyotype could derive from an ancestral Chinese muntjac-like species with 2n=46. Interspecies chromosome painting study and the maximum number of interstitial hybridization detected with satellite I and satellite II DNA probes lend support to the hypothesis that the Indian muntjac karyotype could evolve directly from an ancestral Chinese water deer-like species with 2n=70. Such hypothesis is further substantiated by the finding of satellite V signals presented in specific chromosome regions between the Chinese water deer and the Indian muntjac chromosomes.     

The clinical significance of pericentric inversion of the human Y chromosome: a rare "third" type of heteromorphism

A 28-year-old normal East Indian was found to have a pericentric inversion of the Y chromosome. After reviewing the literature, it was concluded that an inverted Y chromosome does not impede the production of normal sperm and does not predispose to non-disjunction of other chromosomes in the progeny. Thus, the earlier concept of nondisjunction was rejected, and it is suggested that aberrant cases with aneuploidy and an inverted Y are fortuitous. The pericentric inverted Y is inherited from generation to generation and has no clinical significance. The prevalence of males with pericentric Y inversion in the general population is approximately 1 per 1000. It is suggested that a pericentric inversion of the Y chromosome is a rare chromosomal heteromorphism and should be called type III.     

Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with ³H-thymidine and chromosome samples were prepared for the next 32 h at 1–2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.     

Distribution of sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac

The frequency of sister chromatid exchanges (SCEs) was determined for the chromosomes (except Y2) of the Indian muntjac stained by the fluorescence plus Giemsa (FPG) or harlequin chromosome technique. The relative DNA content of each of the chromosomes was also measured by scanning cytophotometry. After growth in bromodeoxyuridine (BrdU) for two DNA replication cycles. SCEs were distributed according to the Poisson formula in each of the chromosomes. The frequency of SCE in each of the chromosomes was directly proportional to DNA content. A more detailed analysis of SCEs was performed for the three morphologically distinguishable regions of the X-autosome composite chromosome. The SCE frequency in the euchromatic long arm and short arm were proportional to the amount of DNA. In contrast, the constitutive heterochromatin in the neck of this chromosome contained far fewer SCEs than expected on the basis of the amount of DNA in this region. A high frequency of SCE, however, was observed at the point junctions between the euchromatin and heterochromatin.     

Comparative gene mapping in the species Muntiacus muntjac.

An extreme case of chromosomal evolution is presented by the two muntjac species Muntiacus muntjac (Indian muntjac, 2n = 6 [females], 7 [males]) and M. reevesi (Chinese muntjac, 2n = 46). Despite disparate karyotypes, these phenotypically similar species produce viable hybrid offspring, indicating a high degree of DNA-level conservation and genetic relatedness. As a first step toward development of a comparative gene map, several Indian muntjac homologs of known human type I anchor loci were mapped. Using flow-sorted, chromosome-specific Southern hybridization techniques, homologs of the protein kinase C beta polypeptide (PRKCB1) and the DNA repair genes ERCC2 and XRCC1 have been assigned to Indian muntjac chromosome 2. The male-specific ZFY gene was presumptively mapped to Indian muntjac chromosome Y2. Ultimate generation of a comparative physical map of both Indian and Chinese muntjac chromosomes will prove invaluable in the study of mammalian karyotype evolution.     

Anti-kinetochore staining for single laser, bivariate flow sorting of Indian muntjac chromosomes.

Flow cytometric chromosome sorting typically relies upon dual-laser, bivariate analysis after staining with two different base pair-specific dyes for resolution of chromosomes with similar DNA content. The availability of FITC-conjugated antibodies offers the possibility of single-laser bivariate analysis when combined with propidium iodide (PI) DNA staining, but requires exploitable antigenic differences between chromosomes of interest. A technique was developed for indirect immunofluorescent anti-kinetochore staining of Indian muntjac chromosomes in suspension. Primary antibody binding within permeabilized whole cells minimized centrifugation-induced loss of chromosomal integrity. Subsequent FITC-conjugated second antibody binding was not affected by concurrent PI-counterstaining. Anti-kinetochore staining facilitated resolution of chromosomes No. 2 and X, which formed a doublet peak upon univariate DNA content analysis, as well as recognition of the Y2 peak which was indistinguishable from debris by univariate analysis. The technique allowed greater than 90% purification of each Indian muntjac chromosome.     

Longitudinal differentiation of metaphase chromosomes of Indian muntjac as studied by restriction enzyme digestion,in situ hybridization with cloned DNA probes and distamycin A plus DAPI fluorescence staining

The longitudinal differentiation of metaphase chromosomes of the Indian muntjac was studied by digestion with restriction enzymes, in situ hybridization with cloned DNA probes and distamycin A plus DAPI (4-6-diamidino-2-phenylindole) fluorescence staining. The centromeric regions of chromosomes 3 and 3 + X of a male Indian muntjac cell line were distinct from each other and different from those of other chromosomes. Digestion with a combination of EcoRI^* and Sau3A revealed a pattern corresponding to that of C-banding. Digestion with AluI, EcoRII or RsaI yielded a band specific to the centromeric region only in chromosomes 3 and 3 + X. Furthermore, HinfI digestion yielded only a band at the centromeric region of chromosome 3, whereas DA-DAPI staining revealed a single band limited to the extreme end of the C-band heterochromatin of the short arm of 3 + X. These results suggest that centromeres of Indian muntjac chromosomes contain at least four different types of repetitive DNA. Such diversity in heterochromatin was also confirmed by in situ hybridization using specific DNA probes isolated and cloned from highly repetitive DNA families. Heterozygosity between chromosome homologs was revealed by restriction enzyme banding. Evidence is presented for the presence of nucleolus organizer regions (NORs) on the long arm of chromosome 1 as well as on the secondary constrictions of 3 and 3 + X.Abbreviations DA distamycin A - DAPI 4-6-diamidino-2-phenylindole - NOR(s) nucleolus organizer region(s) - PBS phosphate-buffered saline - PI propidium iodide     

Folate deficiency induces aneuploidy in human lymphocytes in vitro-evidence using cytokinesis-blocked cells and probes specific for chromosomes 17 and 21

Folate plays a critical role in the prevention of chromosome breakage and hypomethylation of DNA. Deficiency in this vitamin may lead to demethylation of heterochromatin causing structural centromere defects that could induce abnormal distribution of replicated chromosomes during nuclear division. Because aneuploidy of chromosomes 17 and 21 is often observed in breast cancer and leukaemia and increased risk for these cancers is associated with folate deficiency, we hypothesized that folate deficiency may lead to aneuploidy of chromosomes 17 and 21. To test these hypotheses we cultured lymphocytes from eight female volunteers (aged 40-48 years) in RPMI 1640 medium containing 12 or 120nM of folic acid (FA) or 5-methyltetrahydrofolate (MF) for 9 days. Chromosomes 17 and 21 aneuploidies induced by folate deficiency were measured in mononucleated (MONO) and cytokinesis-blocked binucleated (BN) lymphocytes after dual-color fluorescent in situ hybridization (FISH) with a digoxigenin-labeled probe for the alphoid satellite sequence of chromosome 17 and a biotin-labeled probe for the pericentric region of chromosome 21. The results showed that 12nm of MF or FA caused a significant 26-35% increment in frequency of aneuploidy of chromosome 17 (P = 0.0017) and aneupoidy of chromosome 21 (P = 0.0008) relative to 120nM MF or FA. The pattern of aneuploidy in binucleated cells was significantly correlated with that observed in mononucleated cells (R = 0.51-0.75, P < 0.0004) and was consistent with a model based on chromosome loss or partial aneusomy rescue as the cause rather than non-disjunction, although the latter mechanism could not be excluded. MF was not more efficient than FA in preventing aneuploidy in this in vitro system. We conclude that folate deficiency is a risk factor for chromosomes 17 and 21 aneuploidy.     

Rumex acetosa Y chromosomes: constitutive or facultative heterochromatin?

Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw.) Steam roots. It was shown that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.     

Characterization of nuclear compartments identified by ectopic markers in mammalian cells with distinctly different karyotype

The functional organization of chromatin in cell nuclei is a fundamental question in modern cell biology. Individual chromosomes occupy distinct chromosome territories in interphase nuclei. Nuclear bodies localize outside the territories and colocalize with ectopically expressed proteins in a nuclear subcompartment, the interchromosomal domain compartment. In order to investigate the structure of this compartment in mammalian cells with distinctly different karyotypes, we analyzed human HeLa cells (3n+=71 chromosomes) and cells of two closely related muntjac species, the Chinese muntjac (2n=46 chromosomes) and the Indian muntjac (2n=6/7 chromosomes). The distribution of ectopically expressed intermediate filament proteins (vimentin and cytokeratins) engineered to contain a nuclear localization sequence (NLS) and a nuclear particle forming protein (murine Mx1) fused to a yellow fluorescent protein (YFP) was compared. The proteins were predominantly localized in regions with poor DAPI staining independent of the cells karyotype. In contrast to NLS-vimentin, the NLS-modified cytokeratins were also found close to the nuclear periphery. In Indian muntjac cells, NLS-vimentin colocalized with Mx1-YFP as well as the NLS-cytokeratins. Since the distribution of the ectopically expressed protein markers is similar in cells with distinctly different chromosome numbers, the property of the delineated, limited compartment might indeed depend on chromatin organization.     

Expression of heterochromatin by restriction endonuclease treatment and distamycin A/DAPI staining of Indian muntjac (Muntiacus muntjak) chromosomes

The constitutive heterochromatin of the Indian muntjac (Muntiacus muntjak) was examined following digestion with various restriction endonucleases (AluI, HaeIII, HinfI, and MboI), as well as by selective fluorescence staining with distamycin A plus 4'-6-diamidino-2-phenylindole. Distinct areas within the C-bands were found to have characteristic staining patterns which were more conspicuous in the sex chromosomes. Two dot-like structures resistant to AluI were found in the X and Y1 chromosomes in the same position as the nucleolus organizer regions.     

Studies of mitotic and centromeric abnormalities in Roberts syndrome: Implications for a defect in the mitotic mechanism

Roberts syndrome is an inherited human condition that is of particular interest because separation of centromeres and constitutive heterochromatin is observed in metaphase chromosomes. In this study we investigated the frequency of other cytological abnormalities in three Roberts syndrome patients. Our findings when taken with previous cytological reports emphasize that there are other features that are equally characteristic of Roberts syndrome: (1) aneuploidy with random chromosome loss and (2) micronuclei and/or nuclear lobulations of 8%–24% of interphase cells. We observed abnormal chromosome movement involving one or all the chromosomes during anaphase. Evidence is presented suggesting that aneuploidy, micronuclei and abnormal nuclear morphology are a direct result of lagging chromosomes. The cytological features documented for Roberts syndrome indicate that this is a human mitotic mutant.by T.C. Hsu     

Decondensation of pericentric heterochromatin alters the sequence of centromere separation in mouse cells

The centromeres of a genome separate in a sequential, nonrandom manner that is apparently dependent upon the quantity and quality of pericentric heterochromatin. It is becoming increasingly clear that the biological properties of a centromere depend upon its physicochemical makeup, such as its tertiary structure, and not necessarily on its particular nucleotide sequence. To test this idea we altered the physical state of the AT-rich pericentric heterochromatin of mouse with Hoechst 33258 (bis-benzimidazole) and studied a biological parameter, viz., sequence of separation. We report that an alteration in the physical state of heterochromatin, i.e., decondensation, is accompanied by aberrations in the pattern of centromere separation. The most dramatic effect seems to be on chromosomes with large blocks of heterochromatin. Many chromosomes with large blocks of heterochromatin that, in untreated cells, separate late tend to separate early. Decondensation with Hoechst 33258 does not seem to alter the sequence of separation of inactive centromeres relative to that of active centromeres. These data indicate that alteration in the physical parameters of the pericentric heterochromatin might dispose the centromeres to errors. It is likely that this aberration results from early replication of the pericentric heterochromatin associated with active centromeres. Received: 24 August 1998; in revised form: 24 August 1998 / Accepted: 28 August 1998     

Purification of the chromosomes of the Indian muntjac by flow sorting.

Metaphase chromosomes were isolated from a male Indian muntjac cell line, were stained with ethidium bromide and were analyzed by flow microfluorometry to establish a deoxyribonucleic acid (DNA)-based karyotype. Five major peaks were evident on the chromosomal DNA distribution corresponding to the five chromosome types in this species. The amount of DNA in each chromosome was confirmed by cytophotometric measurements of intact metaphase spreads. The five chromosome types were separated by flow sorting at rates up to several hundred chromosomes per second. The sorted chromosomes were identified by morphology and by Giemsa banding patterns. The automsomes, Numbers 1, 2 and 3, and the X + 3 composite chromosome were separated with a high degree of purity (90%). The centromere region of the X + 3 chromosome was fragile to mechanical shearing, and during isolation a small proportion of these chromosomes broke into four segiments: the long arm, the short arm, the short arm plus centromere and the centromere region. A large fraction of the constitutive heterochromatin of this species is present in the centromere region of the X + 3 chromosome and in the Y chromosome; these two regions possess similar amounts of DNA and therefore sort together. Chromosome flow sorting is rapid, reproducible and precise; it allows the collection of microgram quantities of purified chromosomes.     

Structural organization of chromosomes of the Indian muntjac (Muntiacus muntjak)

The identification, morphology, and banding pattern of the chromosomes of the Indian muntjac (Muntiacus muntjak) are described. A diagrammatic representation of the banding pattern as revealed by various techniques is presented following the nomenclature suggested by Paris Conference (1971) for human chromosomes. The Y2 chromosome and the neck of the X chromosome are late replicating based on observations made with the use of a bromodeoxuridine plus Giemsa technique. Most of the G-bands are early replicating, contrary to earlier findings based on autoradiography.     

Comparative Chromosome Painting in Mammals: Human and the Indian Muntjac (Muntiacus muntjak vaginalis)

We have used human chromosome-specific painting probes forin situhybridization on Indian muntjac (Muntiacus muntjak vaginalis,2n= 6, 7) metaphase chromosomes to identify the homologous chromosome regions of the entire human chromosome set. Chromosome rearrangements that have been involved in the karyotype evolution of these two species belonging to different mammalian orders were reconstructed based on hybridization patterns. Although, compared to human chromosomes, the karyotype of the Indian muntjac seems to be highly rearranged, we could identify a limited number of highly conserved homologous chromosome regions for each of the human chromosome-specific probes. We identified 48 homologous autosomal chromosome segments, which is in the range of the numbers found in other artiodactyls and carnivores recently analyzed by chromosome painting. The results demonstrate that the reshuffling of the muntjac karyotype is mostly due to fusions of huge blocks of entire chromosomes. This is in accordance with previous chromosome painting analyses between various Muntjac species and contrasts the findings for some other mammals (e.g., gibbons, mice) that show exceptional chromosome reshuffling due to multiple reciprocal translocation events.     

High-density comparative BAC mapping in the black muntjac (Muntiacus crinifrons): molecular cytogenetic dissection of the origin of MCR 1p+4 in the X1X2Y1Y2Y3 sex chromosome system

The black muntjac (Muntiacus crinifrons, 2n = 8[female symbol]/9[male symbol]) is a critically endangered mammalian species that is confined to a narrow region of southeastern China. Male black muntjacs have an astonishing X1X2Y1Y2Y3 sex chromosome system, unparalleled in eutherian mammals, involving approximately half of the entire genome. A high-resolution comparative map between the black muntjac (M. crinifrons) and the Chinese muntjac (M. reevesi, 2n = 46) has been constructed based on the chromosomal localization of 304 clones from a genomic BAC (bacterial artificial chromosome) library of the Indian muntjac (M. muntjak vaginalis, 2n = 6[female symbol]/7[male symbol]). In addition to validating the chromosomal homologies between M. reevesi and M. crinifrons defined previously by chromosome painting, the comparative BAC map demonstrates that all tandem fusions that have occurred in the karyotypic evolution of M. crinifrons are centromere-telomere fusions. The map also allows for a more detailed reconstruction of the chromosomal rearrangements leading to this unique and complex sex chromosome system. Furthermore, we have identified 46 BAC clones that could be used to study the molecular evolution of the unique sex chromosomes of the male black muntjacs.     

Characterization of ancestral chromosome fusion points in the Indian muntjac deer

Tandem fusion, a rare evolutionary chromosome rearrangement, has occurred extensively in muntjac karyotypic evolution, leading to an extreme fusion karyotype of 6/7 (female/male) chromosomes in the Indian muntjac. These fusion chromosomes contain numerous ancestral chromosomal break and fusion points. Here, we designed a composite polymerase chain reaction (PCR) strategy which recovered DNA fragments that contained telomere and muntjac satellite DNA sequence repeats. Nested PCR confirmed the specificity of the products. Two-color fluorescence in situ hybridization (FISH) with the repetitive sequences obtained and T₂AG₃ telomere probes showed co-localization of satellite and telomere sequences in Indian muntjac chromosomes. Adjacent telomere and muntjac satellite sequences were also seen by fiber FISH. These data lend support to the involvement of telomere and GC-rich satellite DNA sequences during muntjac chromosome fusions.Communicated by E.A. NiggAccession numbers: AY322158, AY322159, AY322160