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.     

Cloning, characterization and physical mapping of three cervid satellite DNA families in the genome of the Formosan muntjac (Muntiacus reevesi micrurus)

Due to their high sequence diversity even among closely related species, satellite DNA sequences can be a useful molecular marker for phylogenetic and taxonomic analyses. To characterize the satellite DNA in the genome of a native muntjac species of Taiwan, the Formosan muntjac, satellite DNA clones representing three different cervid satellite DNA families from this species were isolated and analyzed. Genomic organization study of these satellite DNAs was also undertaken. Three Formosan muntjac satellite DNA clones were obtained and designated as FM-satI (1,391 bp), FM-satII (1,143 bp) and FM-satIV (1,103 bp), and found to share approximately 82, 81 and 98% sequence homology with the Chinese muntjac satellite I clone (C5), Indian muntjac satellite II clone (Mmv-0.7) and Chinese muntjac satellite IV clone (MR-1.0), respectively. These three satellite DNA families are organized in a pter<--FM-satII-FM-satIV-FM-satI-->qter orientation in the centromeric region with satII closely associated with the telomeric sequences. Satellite DNA sequence comparison, in combination with chromosome data concludes that the Formosan muntjac is likely a subspecies of M. reevesi, closely related to the Chinese muntjac. With the kinetochore satellite II DNA co-localizing with the telomeric sequences, the Formosan muntjac chromosomes could be truly telocentric.     

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.     

Localization and characterization of recombinant DNA clones derived from the highly repetitive DNA sequences in the Indian muntjac cells: their presence in the Chinese muntjac

A total of seven, highly repeated, DNA recombinant M13 mp8 clones derived from a Hpa II digest of cultured cells of the Indian muntjac (Muntiacus muntjac vaginalis) were analyzed by restriction enzymes, in situ hybridization, and DNA sequencing. Two of the clones, B1 and B8, contain satellite DNA inserts which are 80% homologous in their DNA sequences. B1 contains 781 nucleotides and consist of tandem repetition of a 31 bp consensus sequence. This consensus sequence, TCCCTGACGCAACTCGAGAGGAATCCTGAGT, has only 3 bp changes, at positions 7, 24, and 27, from the consensus sequence of the 31 bp subrepeats of the bovine 1.715 satellite DNA. The satellite DNA inserts in B1 and B8 hybridize primarily but not specifically to chromosome X, and secondarily to other sites such as the centromeric regions of chromosomes 1 and 2. Under less stringent hybridization conditions, both of them hybridize to the interior of the neck region and all other chromosomes (including chromosomes 3 and Y). The other five DNA clones contain highly repetitive, interdispersed DNA inserts and are distributed throughout the genome except for the neck region of the compound chromosome X+3. Blot hybridization results demonstrate that the satellite DNA component is also present in Chinese muntjac DNA (Muntiacus reevesi) in spite of the very different karyotypes of the Chinese and Indian muntjacs.     

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.     

Analysis and location of a rice BAC clone containing telomeric DNA sequences

BAC2, a rice BAC clone containing (TTTAGGG)_n homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG)_n-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescencein situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.     

Analysis and location of a rice BAG clone containing telomeric DNA sequences

BAC2, a rice BAC clone containing (TTTAGGG)_n homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG)_n-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescencein situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.     

Chromosomal distribution of the telomere sequence (TTAGGG)(n) in the Equidae

Telomeres are a class of repetitive DNA sequences that are located at chromosome termini and that act to stabilize the chromosome ends. The rapid karyotypic evolution of the genus Equus has given rise to ten taxa, all with different diploid chromosome numbers. Using fluorescence in situ hybridization (FISH) we localized the mammalian telomere sequence, (TTAGGG)(n), to the chromosomes of nine equid taxa. TTAGGG signal was located at chromosome termini in all species, however additional signal was seen at interstitial sites on some chromosomes in the Burchell's zebra, Equus quagga burchelli, the Hartmann's zebra, Equus zebra hartmannae, and at large heterochromatin-associated regions on the chromosomes of the donkey, Equus asinus. The interstitial signal in the zebras may be a relic of an ancient telomere-telomere fusion and mark the point at which two ancestral chromosomes may have fused. For the donkey, the heterochromatin-associated signal may represent degenerate telomere-like satellite sequences and identify a second type of satellite DNA for this taxon.     

Isolation and identification of a novel satellite DNA family highly conserved in several Cervidae species

In an attempt to amplify cervid satellite II DNA from the genomes of Indian muntjac and Chinese muntjac, a pair of primers derived from the white tailed deer satellite II DNA clone (OvDII) yielded a prominent approximately 1 kb polymerase chain reaction (PCR) product (in addition to the expected 0.7 kb satellite II DNA fragments) in both species. The approximately 1 kb products were cloned, sequenced, and analyzed by Southern blotting and fluorescence in situ hybridization (FISH). This revealed that the approximately 1 kb cloned sequences indeed represent a previously unknown cervid satellite DNA family, which is now designated as cervid satellite IV DNA. Approximately 1 kb PCR clones were also obtained from the genomes of the black tailed deer and Canadian woodland caribou with similar primer pairs. Extremely high sequence conservation (over 90% homology) was observed among the clones generated from all four deer species and PCR-Southern hybridization experiments further verified the co-amplification of two kinds of satellite DNA sequences with the same pair of primers. This satellite DNA was found to co-localize with centromeric proteins at the kinetochore by a simultaneous FISH and immunofluorescence study. Due to its high sequence conservation and close association with kinetochores, the newly identified satellite DNA may have a functional centromeric role.     

黑麂和费氏麂四种卫星DNA的克隆特征和染色体定位

近年来,分子细胞遗传学研究已基本证实了染色体的串联融合(端粒-着丝粒融合)是麂属动物核型演化的主要重排方式。尽管染色体串联融合的分子机制还不清楚,但通过染色体的非同源重组,着丝粒区域的卫星DNA被认为可能介导了染色体的融合。以前的研究发现在赤麂和小麂染色体的大部分假定的串联融合位点处存在着非随机分布的卫星DNA。然而在麂属的其他物种中,这些卫星DNA的组成以及在基因组中的分布情况尚未被研究。本研究从黑麂和费氏麂基因组中成功地克隆了4种卫星DNA(BMC5、BM700、BM1.1k和FM700),并分析了这些卫星克隆的特征以及在小麂、黑麂、贡山麂和费氏麂染色体上的定位情况。结果表明,卫星I和IIDNA(BMC5,BM700和FM700)的信号除了分布在这些麂属动物染色体的着丝粒区域外,也间隔地分布在这些物种的染色体臂上。其研究结果为黑麂、费氏麂和贡山麂的染色体核型也是从一个2n=70的共同祖先核型通过一系列的串联融合进化而来的假说提供了直接的证据。     

Cytotypes of Kirk's dik-dik (Madoqua kirkii,Bovidae) show multiple tandem fusions

Madoqua kirkii, a miniature African antelope, is noted for extensive chromosomal variation that has been categorized in four distinct cytotypes (A-D). In this investigation, we analyzed the A cytotype (2n = 46, FN = 48) using a suite of molecular cytogenetic approaches that entailed (i) whole chromosome and subchromosomal painting by fluorescence in situ hybridization (FISH), (ii) the study of Madoqua centromeric-specific DNA derived from pooled DNA obtained from the centromeric regions of the acrocentric chromosomes, and (iii) DNA from the telomere:centromere junctions of tandemly fused chromosomes. DNA from these sources was used to probe for the persistence of interstitial satellite DNA and residual centromeric sequences in the tandem and centric fusion junctions by PCR and FISH. The analyses show centromeric sequences at two of the six tandem fusion junctions. These data, and those of hybrid specimens (A × B cytotypes) in conjunction with published information permitted an interpretation of the probable sequence of chromosomal rearrangements among the M. kirkii cytotypes. We discuss the findings in the context of chromosomal evolution in these antelopes, and the implications that these hold for ex-situ breeding programs of the species.     

AT-rich sequences containing Arabidopsis-type telomere sequence and their chromosomal distribution in Pinus densiflora

Japanese red pine Pinus densiflora has 2 n=24 chromosomes and after FISH-detection of Arabidopsis-type (A-type) telomere sequences, many telomere signals were observed on these chromosomes at interstitial and proximal regions in addition to the chromosome ends. These interstitial and proximal signal sites were observed as DAPI-positive bands, suggesting that the interstitial and proximal telomere signal sites are composed of AT-rich highly repetitive sequences. Four DNA clones (PAL810, PAL1114, PAL1539, PAL1742) localized at the interstitial telomere signals were selected from AluI-digested genomic DNA library using colony blot hybridization probed with A-type telomere sequences and characterized using FISH and Southern blot hybridization. The AT-contents of these selected four clones were 60.8–76.3%, and repeat units of the telomere sequence and degenerated telomere sequences were found in their nucleotide sequences. Except for two sites of PAL1114, FISH signals of the four clones co-localized with interstitial and proximal A-type telomere sequence signals. FISH signals a showed similar distribution pattern, but the patterns of signal intensity were different among the four clones. PAL810, PAL1539 and PAL 1742 showed similar FISH signal patterns, and the differences were only with respect to the signal intensity of some signal sites. PAL1114 had unique signals that appeared on chromosomes 7 and 10. Based on results of the Southern blot hybridization these four sequences are not arranged tandemly. Our results suggest that the interstitial A-type telomere sequence signal sites were composed of a mixture of several AT-rich repetitive sequences and that these repetitive sequences contained A-type telomere sequences or degenerated A-type telomere sequence repeats.     

Karyotypic evolution of a novel cervid satellite DNA family isolated by microdissection from the Indian muntjac Y-chromosome

A minilibrary was constructed from DOP-PCR products using microdissected Y-chromosomes of Indian muntjac as DNA templates. Two microclones designated as IM-Y4-52 and IM-Y5-7 were obtained from negative screening of all three cervid satellite DNAs (satellites I, II, and IV). These two microclones were 295 and 382 bp in size, respectively, and shared 70% sequence homology. Southern blot analysis showed that the IM-Y4-52 clone was repetitive in nature with an 0.32-kb register in HaeIII digest. Sequence comparison revealed no similarities to DNA sequences deposited in the GenBank database, suggesting that the microclone sequences were from a novel satellite DNA family designated as cervid satellite V. A subclone of an Indian muntjac BAC clone which screened positive for IM-Y4-52 had a 3,325-bp insert containing six intact monomers, four deleted monomers, and two partial monomers. The consensus sequence of the monomer was 328 bp in length and shared more than 80% sequence homology with every intact monomer. A zoo blot study using IM-Y4-52 as a probe showed that the strong hybridization with EcoRI digested male genomic DNA of Indian muntjac, Formosan muntjac, Chinese muntjac, sambar deer, and Chinese water deer. Female genomic DNA of Indian muntjac, Chinese water deer, and Formosan muntjac also showed positive hybridization patterns. Satellite V was found to specifically localize to the Y heterochromatin region of the muntjacs, sambar deer, and Chinese water deer and to chromosome 3 of Indian muntjac and the X-chromosome of Chinese water deer.Y.-C. Li and Y.-M. Cheng contributed equally to this work.     

CENP-A associated complex satellite DNA in the kinetochore of the Indian muntjac

The centromere/kinetochore complex is a chromosomal assembly that mediates chromosome motility and mitotic regulation by interacting with microtubules of the mitotic spindle apparatus. Centromere protein A (CENP-A) is a histone H3 homolog that is concentrated in the chromatin of the inner kinetochore plate of human chromosomes. To identify DNA sequences associated with the inner kinetochore plate, we used anticentromere autoantibodies to immunoprecipitate CENP-A associated chromatin selectively from Indian muntjac fibroblasts. DNA was cloned from immunoprecipitated CENP-A- associated chromatin and characterized by DNA sequence and hybridization analyses. A novel centromeric satellite DNA sequence was identified and shown by fluorescence in situ hybridization analysis to be present at all centromeres of the Indian muntjac. This satellite DNA constitutes a 972 bp monomer repeat and shows partial homology with satellite II DNA of the white-tailed deer. Southern blot analysis of muntjac genomic DNA suggests that this satellite DNA is present in repetitive tandem arrays and contains complex internal arrangements. In conjunction with previous work showing the association of CENP-A with human α-satellite DNA, we conclude that the mammalian inner kinetochore plate contains a unique form of chromatin that contains CENP-A in association with complex satellite DNA. Received: 18 May 1999; in revised form: 5 July 1999 / Accepted: 20 July 1999     

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.     

Visualization of the terminal structure of rice chromosomes 6 and 12 with multicolor FISH to chromosomes and extended DNA fibers

High-resolution fluorescence in situ hybridization (FISH) on interphase and pachytene nuclei, and extended DNA fibers enabled microscopic distinction of DNA sequences less than a few thousands of base pairs apart. We applied this technique to reveal the molecular organization of telomere ends in japonica rice (Oryza sativa ssp. japonica), which consist of the Arabidopsis type TTTAGGG heptameric repeats and the rice specific subtelomeric tandem repeat sequence A (TrsA). Southern hybridizations of DNA digested with Bal31 and EcoRI, and FISH on chromosomes and extended DNA fibers demonstrated that (1) all chromosome ends possess the telomere tandem repeat measuring 3–4 kb; (2) the subtelomeric TrsA occurs only at the ends of the long arms of chromosomes 6 and 12, and measure 6 and 10 kb, which corresponds to 231 and 682 copies for these sites, respectively; (3) the telomere and TrsA repeats are separated by at most a few thousands of intervening nucleotide sequences. The molecular organization for a general telomere organization in plant chromosomes is discussed.     

Isolation and characterization of a mouse subtelomeric sequence

A mouse subtelomeric sequence, ST1, was generated from genomic DNA of the mouse HR9 (129/Sv origin) cell line by the polymerase chain reaction (PCR) using a single telomeric primer. ST1 was cloned and characterized: it is composed of 670 bp of novel DNA sequence flanked on each end by inverted telomeric hexanucleotide repeats (TTAGGG)_n. PCR amplification from BALB/c mouse DNA using this single primer gave the same major product. Southern analysis and PCR using internal ST1 primers confirmed that the ST1 sequence is present in mouse genomic DNA. In situ hybridization to metaphase chromosomes of SJL origin mapped ST1 to many, if not every, mouse telomere. PCR experiments using different combinations of the telomeric, minor satellite, and ST1 primers indicated that some ST1 copies are adjacent to minor satellite sequences, that telomeric and ST1 sequences are not generally interspersed with minor satellite sequences,and that ST1 and the minor satellite have a consistent and specific orientation relative to each other and to the telomere.by H.F. Willard     

End joining at Caenorhabditis elegans telomeres

Critically shortened telomeres can be subjected to DNA repair events that generate end-to-end chromosome fusions. The resulting dicentric chromosomes can enter breakage–fusion–bridge cycles, thereby impeding elucidation of the structures of the initial fusion events and a mechanistic understanding of their genesis. Current models for the molecular basis of fusion of critically shortened, uncapped telomeres rely on PCR assays that typically capture fusion breakpoints created by direct ligation of chromosome ends. Here we use independent approaches that rely on distinctive features of Caenorhabditis elegans to study the frequency of direct end-to-end chromosome fusion in telomerase mutants: (1) holocentric chromosomes that allow for genetic isolation of stable end-to-end fusions and (2) unique subtelomeric sequences that allow for thorough PCR analysis of samples of genomic DNA harboring multiple end-to-end fusions. Surprisingly, only a minority of end-to-end fusion events resulted from direct end joining with no additional genome rearrangements. We also demonstrate that deficiency for the C. elegans Ku DNA repair heterodimer does not affect telomere length or cause synthetic effects in the absence of telomerase.