Comparative sequence analyses reveal sites of ancestral chromosomal fusions in the Indian muntjac genome

Background

Indian muntjac (Muntiacus muntjak vaginalis) has an extreme mammalian karyotype, with only six and seven chromosomes in the female and male, respectively. Chinese muntjac (Muntiacus reevesi) has a more typical mammalian karyotype, with 46 chromosomes in both sexes. Despite this disparity, the two muntjac species are morphologically similar and can even interbreed to produce viable (albeit sterile) offspring. Previous studies have suggested that a series of telocentric chromosome fusion events involving telomeric and/or satellite repeats led to the extant Indian muntjac karyotype.

Results

We used a comparative mapping and sequencing approach to characterize the sites of ancestral chromosomal fusions in the Indian muntjac genome. Specifically, we screened an Indian muntjac bacterial artificial-chromosome library with a telomere repeat-specific probe. Isolated clones found by fluorescence in situ hybridization to map to interstitial regions on Indian muntjac chromosomes were further characterized, with a subset then subjected to shotgun sequencing. Subsequently, we isolated and sequenced overlapping clones extending from the ends of some of these initial clones; we also generated orthologous sequence from isolated Chinese muntjac clones. The generated Indian muntjac sequence has been analyzed for the juxtaposition of telomeric and satellite repeats and for synteny relationships relative to other mammalian genomes, including the Chinese muntjac.

Conclusions

The generated sequence data and comparative analyses provide a detailed genomic context for seven ancestral chromosome fusion sites in the Indian muntjac genome, which further supports the telocentric fusion model for the events leading to the unusual karyotypic differences among muntjac species.     

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     

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.     

Karyological characteristics of cell sublines of the kidney of the kangaroo rat and of skin fibroblasts of the Indian muntjac

A study was made of the karyotypic structure of sublines derived from the kangaroo rat's kidney (NBL-3) and skin fibroblasts of the Indian muntjac, available in the cell culture bank of the Institute of Cytology Acad. Sci. USSR. A comparative karyologic analysis was made of subline NBL-3 both contaminated with mycoplasma (NBLK) and decontaminated with antibiotics (NBLD). Authentic differences in cell distribution according to chromosome number in NBLK and NBLD variants were shown. Modal numbers of chromosomes are 11 and 17, respectively. The modal number for the Indian muntjac cell subline (MT) is 9. 60-80% of the cells had an identical karyotype (the main structural variant of the karyotype is MSVK). Using the G-banding technique, all the MSVK variants were shown to display constant karyotypes. In NBLK there are 5 pairs of homologous chromosomes and one metacentric. In NBLD, the number of homologous chromosomes increased in all the groups (hypotriploid karyotype). In subline MT 3 homologous chromosomes were found in groups I and IV, 2 in group III in addition to one X-chromosome. A comparison with the Indian muntjac karyotype showed the absence of marker chromosomes in MT. The analysis of additional SVK shows that the deviations from MSVK are caused mostly by changes in the number of homologous chromosomes within the groups. A study of the frequency of deviations in chromosome numbers observed in the groups from MSVK showed that different chromosomes were involved in karyotypic changes in the same way in the "low-chromosome" variants of NBLK and MT, and in different ways in NBLD. A comparison of the "premycoplasmic" variants of line NBL-3 with NBLK shows no differences in the parameters studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of the repetitive telomeric sequence (TTAGGG)n in two muntjac species and implications for their karyotypic evolution

It has been suggested that the chromosome set of the Indian muntjac, Muntiacus muntjak vaginalis (female, 2n = 6; male, 2n = 7), evolved from small acrocentric chromosomes, such as those found in the complement of the Chinese muntjac, M. reevesi (2n = 46), by a series of tandem fusions and other rearrangements. The location of the highly conserved human telomeric sequence (TTAGGG)n in the metaphase chromosomes of M.m. vaginalis and its close relative, M. reevesi, was investigated by non-radioactive in situ hybridization. The (TTAGGG)n repeat was found adjacent to the centromeres in the short arm and at the telomeres in the long arm of M. reevesi acrocentric metaphase chromosomes. Tandem fusions present in the karyotype of M.m. vaginalis chromosomes were not reflected by interstitial signals of the telomere repeat, as these chromosomes displayed hybridization signals only at the ends of the chromatids. Mechanisms that might have played a role in the evolution of the reduced karyotype of the Indian muntjac are discussed.     

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.     

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.     

Defining the orientation of the tandem fusions that occurred during the evolution of Indian muntjac chromosomes by BAC mapping

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n=6 in the female and 7 in the male, the karyotypic evolution of which through extensive tandem fusions and several centric fusions has been well-documented by recent molecular cytogenetic studies. In an attempt to define the fusion orientations of conserved chromosomal segments and the molecular mechanisms underlying the tandem fusions, we have constructed a highly redundant (more than six times of whole genome coverage) bacterial artificial chromosome (BAC) library of Indian muntjac. The BAC library contains 124,800 clones with no chromosome bias and has an average insert DNA size of 120 kb. A total of 223 clones have been mapped by fluorescent in situ hybridization onto the chromosomes of both Indian muntjac and Chinese muntjac and a high-resolution comparative map has been established. Our mapping results demonstrate that all tandem fusions that occurred during the evolution of Indian muntjac karyotype from the acrocentric 2n=70 hypothetical ancestral karyotype are centromere–telomere (head–tail) fusions.     

The G-banded chromosomes of Roosevelt's muntjac, Muntiacus rooseveltorum

The chromosomes of a female Roosevelt's muntjac (Muntiacus rooseveltorum) captured in Laos have been studied with G-banding. The diploid number is six and the karyotype is indistinguishable from that of the Indian muntjac (Muntiacus muntjak vaginalis).     

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     

Compound kinetochores of the Indian muntjac

The chromosomes of the Indian muntjac (Muntiacus muntjak vaginalis) are unique among mammals due to their low diploid number (2N=6, 7) and large size. It has been proposed that the karyotype of this small Asiatic deer evolved from a related deer the Chinese muntjac (Muntiacus reevesi) with a diploid chromosome number of 2n= 46 consisting of small telocentric chromosomes. In this study we utilized a kinetochore-specific antiserum derived from human patients with the autoimmune disease scleroderma CREST as an immunofluorescent probe to examine kinetochores of the two muntjac species. Since CREST antiserum binds to kinetochores of mitotic chromosomes as well as prekinetochores in interphase nuclei, it was possible to identify and compare kinetochore morphology throughout the cell cycle. Our observations indicated that the kinetochores of the Indian muntjac are composed of a linear beadlike array of smaller subunits that become revealed during interphase. The kinetochores of the Chinese muntjac consisted of minute fluorescent dots located at the tips of the 46 telocentric chromosomes. During interphase, however, the kinetochores of the Chinese muntjac clustered into small aggregates reminiscent of the beadlike arrays seen in the Indian muntjac. Morphometric measurements of fluorescence indicated an equivalent amount of stained material in the two species. Our observations indicate that the kinetochores of the Indian muntjac are compound structures composed of linear arrays of smaller units the size of the individual kinetochores seen on metaphase chromosomes of the Chinese muntjac. Our study supports the notion that the kinetochores of the Indian muntjac evolved by linear fusion of unit kinetochores of the Chinese muntjac. Moreover, it is concluded that the evolution of compound kinetochores may have been facilitated by the nonrandom aggregation of interphase kinetochores in the nuclei of the ancestral species.     

MNNG转化赤麂细胞株（KIZ—8401）的生长特性和核型观察

本文用MNNG(N-Methyl-N'-nitro-N-nitrosoguanidine)(甲基硝基亚硝基胍)转化赤麂肺成纤维细胞株(KIZ-7901)。转化的赤麂细胞株(KIZ-8401)具有新的核型,2n=8♂,细胞形态,细胞周期以及生物学特性都与未转化的细胞株(2n=7♂)不一样。转化细胞中还常见多倍体出现,细胞具有克隆生长能力。     

Comparative gene mapping in cattle,Indian muntjac,and Chinese muntjac by fluorescence in situ hybridization

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n = 6 in the female and 2n = 7 in the male. The karyotypic evolution of Indian muntjac via extensive tandem fusions and several centric fusions are well documented by molecular cytogenetic studies mainly utilizing chromosome paints. To achieve higher resolution mapping, a set of 42 different genomic clones coding for 37 genes and the nucleolar organizer region were used to examine homologies between the cattle (2n = 60), human (2n = 46), Indian muntjac (2n = 6/7) and Chinese muntjac (2n = 46) karyotypes. These genomic clones were mapped by fluorescence in situ hybridization (FISH). Localization of genes on all three pairs of M. m. vaginalis chromosomes and on the acrocentric chromosomes of M. reevesi allowed not only the analysis of the evolution of syntenic regions within the muntjac genus but also allowed a broader comparison of synteny with more distantly related species, such as cattle and human, to shed more light onto the evolving genome organization. For convenience and to avoid confusion we added for each species a three letter abbreviation prior to the chromosomal band location discussed in this paper: BTA, Cattle chromosome; HSA, Human chromosome; MMV, M. m. vaginalis chromosome; MRE, M. reevesi chromosome.     

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.     

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.     

Absence of true interchromosomal connectives in microsurgically isolated chromosomes

Mitotic chromosomes of the Indian muntjac were isolated from cultured fibroblast-like cells by microsurgery. The entire complement of seven chromosomes could be obtained with the radial array of chromosomes on the mitotic spindle intact. The center of the radial array was occupied by a fibrous network which stained with tubulin antiserum. This network was absent when cells were treated with colchicine or vinblastine sulfate prior to chromosome isolation, and probably represents a remnant of the mitotic spindle. Most isolated chromosomes were connected to the spindle by fibers attached to the centromeres. Such fibers did not stain for DNA and were resistant to DNases but sensitive to proteases. No interconnections were found to run from chromosome to chromosome except for occasional artifactual adhesions resulting from collisions between chromosomes which occurred during micromanipulation. We therefore found no evidence that chromosomes of the Indian muntjac are interconnected at mitosis.     

Human telomerase can immortalize Indian muntjac cells

The mechanisms of replicative senescence by telomere shortening are not fully understood. The Indian muntjac has the fewest chromosomes of all mammals, greatly simplifying the analysis of each telomere over time. In this study, telomere shortening was observed throughout the life span of cultured normal muntjac cells by quantitative fluorescence in situ hybridization and terminal restriction fragment analysis. Ectopic expression of the human telomerase catalytic subunit in these cells reconstituted telomerase activity, extended telomere lengths, and immortalized the cells, demonstrating that the Indian muntjac cells can serve as a telomere-based replicative senescence model for human cells. In one strain, two chromosome ends had significantly shorter telomeres than the other ends, which led to a variety of chromosome abnormalities. Near senescence, additional ends became telomere signal free, and chromosome aberrancies increased dramatically. Interstitial telomere sequences coincided with fragile sites, suggesting that these remnants of chromosome fusion events might contribute to genome instability. One SV40-immortalized cell line lacked telomerase, and its genetic instability was corrected by the ectopic expression of telomerase, confirming that too-short telomeres were the source of abnormalities. Indian muntjac cells provide an excellent system for understanding the mechanism of replicative senescence and the role of telomerase in the elongation of individual telomeres.     

New insights into the karyotypic relationships of Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis)

To investigate the karyotypic relationships between Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis), a complete set of Chinese muntjac chromosome-specific painting probes has been assigned to G-banded chromosomes of these three species. Sixteen autosomal probes (i.e. 6-10, 12-22) of the Chinese muntjac each delineated one pair of conserved segments in the forest musk deer and gayal, respectively. The remaining six autosomal probes (1-5, and 11) each delineated two to five pairs of conserved segments. In total, the 22 autosomal painting probes of Chinese muntjac delineated 33 and 34 conserved chromosomal segments in the genomes of forest musk deer and gayal, respectively. The combined analysis of comparative chromosome painting and G-band comparison reveals that most interspecific homologous segments show a high degree of conservation in G-banding patterns. Eleven chromosome fissions and five chromosome fusions differentiate the karyotypes of Chinese muntjac and forest musk deer; twelve chromosome fissions and six fusions are required to convert the Chinese muntjac karyotype to that of gayal; one chromosome fission and one fusion separate the forest musk deer and gayal. The musk deer has retained a highly conserved karyotype that closely resembles the proposed ancestral pecoran karyotype but shares none of the rearrangements characteristic for the Cervidae and Bovidae. Our results substantiate that chromosomes 1-5 and 11 of Chinese muntjac originated through exclusive centromere-to-telomere fusions of ancestral acrocentric chromosomes.     

A comparative study of karyotypes of muntjacs by chromosome painting

We have used a combination of chromosome sorting, degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), chromosome painting and digital image capturing and processing techniques for comparative chromosome analysis of members of the genus Muntiacus. Chromosome-specific paints from a female Indian muntjac were hybridised to the metaphase chromosomes of the Gongshan, Black, and Chinese muntjac by both single and three colour chromosome painting. Karyotypes and idiograms for the Indian, Gongshan, Black and Chinese muntjac were constructed, based on enhanced 4, 6-diamidino-2-phenylindole (DAPI) banding patterns. The hybridisation signal for each paint was assigned to specific bands or chromosomes for all of the above muntjac species. The interspecific chromosomal homology was demonstrated by the use of both enhanced DAPI banding and comparative chromosome painting. These results provide direct molecular cytogenetic evidence for the tandem fusion theory of the chromosome evolution of muntjac species.Deceased     

Characteristics of quantitative variability of karyotype in cell line of fibroblasts from indian muntjac

The numerical regularities of karyotypic variability in cell line of the Indian muntjac skin fibroblasts (Muntiacus muntjak) has been studied. It was found that the karyotypic structure of cell population is mainly determined by some number of specific variant deviations from the main structural variant of karyotype (MSVK) to be depended on internal connections between chromosomes. Specific regulations determining the karyotypic structure of cell population are: 1) nonrandom character of cell distribution according to the number of chromosomal deviations from MSVK; 2) specific character of deviations in each chromosome from MSVK; 3) presence of significant connections between individual chromosomes by simultaneous mainly single directed numeral deviations. Results presented in this investigation were thought owner by analysing the number of individual chromosomes. These results extend considerably the known ideas of regulations of karyotypic variability in cell populations in vitro.