Indian Muntjac Research Articles

Study of chromosomal replication of red muntjac (Muntiacus muntjac).

The technique of cell fusion, the method of sister chromatid differentiation (SCD), and the silver staining method modified by us that can show nucleolus organizing region (NOR) and sister chromatid differentiation at the same time have been used in the study of red muntjac chromosomes. Differential staining of sister chromatid has been observed in metaphase chromosomes and in premature condensed chromosomes (PCC) at various stages of the interphase. Our silver staining method shows that the NOR in the dark staining chromatid is Ag-positive, while that in the light staining chromatid is Ag-negative. The chromatid differentiation staining and the NOR sister staining suggest that the structure of metaphase chromosomes is similar to that of the premature condensed chromosomes of the interphase cell. The chromosomal replication of red muntjac conforms with the semiconservative replication mechanism and the replication of rDNA is also semiconservative.

Immunological visualization of 5-methylcytosine-rich regions in Indian muntjac metaphase chromosomes

Regions rich in 5-methylcytosine were localized in male metaphase chromosomes of the Indian muntjac deer ( Muntiakus muntjak). Chromosomes were ultraviolet irradiated and subsequently photooxidized in the presence of methylene blue to induce maximum DNA denaturation. Following treatment with anti 5-methylcytosine antibody (anti 5-MeC), regions of antibody binding were visualized by an immunofluorescence or immunopreoxidase staining procedure. All chromosomes showed some level of antibody binding along their length and at centromeric regions, with intense binding evident in the centromere of chromosome 3 and the elongated centromeric “neck” of chromosome 3-X. The Y chromosome displayed low levels of antibody binding. The banding pattern observed with anti 5-MeC is the reverse of that obtained by quinacrine staining.

Electrical fusion of cells and nucleiin vitro: A preliminary note

The electrical fusion of male skin fibroblasts of the Indian muntjac (Muntiacus muntjak)in vitro is described. Fusion of cell nuclei in the resulting multinucleated giant cells was observed after application of up to ten additional DC pulses. Electrical fusion of bovine erythrocytes and alveolar macrophages is also described. Cell fusion can be obtained without the use of protease pretreatment, but the yield of fused cells is about 20 times lower than with prior use of proteases.

Cytogenetic studies in mammalian cells after treatment with insect pathogenic viruses [Baculoviridae]. II.In vitro studies with mammalian cell lines

Mammalian cell-lines from Chinese hamster, Indian muntjac and mouse were inoculated with infectious supernatant ofAutographa californica (Speyer) nuclear polyhedrosis virus (Ac NPV) replicated inMamestra brassicae (L.) cell cultures (IZD-Mb-0503). There was no adverse effect on cell proliferation, nor was a cytopathogenic effect (CPE) induced in such cultures. Cytogenetic data indicate that uptake ofAc-NPVs into the cytoplasm of mammalian cells, as shown by an electron microscopic study, induced neither numerical or structural chromosome aberrations nor sister chromatid exchange (SCE) events.

Autographa californica nuclear polyhedrosis virus (AcNPV) DNA does not persist in mass cultures of mammalian cells

Autographa californica nuclear polyhedrosis virus (AcNPV) is one of the most extensively studied baculoviruses. We have investigated whether AcNPV or its DNA can replicate and/or persist in cultures of mammalian cells. Human HeLa cells or primary human embryonic kidney cells, simian CV1 cells, hamster BHK21(B3) cells or Muntiacus muntjak cells growing in monolayer cultures were used in these studies. Cells were inoculated with AcNPV at multiplicities ranging from 0.1 to 100 PFU/cell. Subsequently, the inoculated cells were investigated for virus production and for the replication and the persistence of viral DNA. Extracts of inoculated cells were also screened for the occurrence of AcNPV-specific RNA. AcNPV does not multiply in any of the cell lines studied. Viral DNA replication or transcription could not be detected by blotting and nucleic acid hybridization experiments using nick-translated, cloned viral probes. Furthermore, there was no evidence for the persistence of viral DNA or of fragments of viral DNA in mass cultures of mammalian cells. A puzzling homology between pBR322 plasmid DNA and human, simian, and hamster DNAs was detected. Since mammalian cells can take up and integrate any foreign DNA at very low frequency, it cannot be ruled out by the approach chosen that a very small number of cells might have incorporated and fixed viral DNA in their genomes. As this caveat is always pertinent for any population of cells exposed to foreign DNA, this reservation does not appear to be of particular significance in safety considerations when working with baculoviruses or any virus for that matter.

The taxonomic status of feral muntjac deer (Muntiacussp.) in England

Summary Two species of muntjac deer, the Indian and the Reeves', were introduced into England in the nineteenth and the present centuries. Feral muntjac deer now occur widely over the southern half of England. Their identity, however, is uncertain with claims being made for both species and for hybrids. Skulls and jaws of the two species of muntjac deer from their native countries and from feral animals from eight counties in England have been examined. From these measurements and from chromosome studies it is concluded that the feral muntjac deer in England are Reeves' muntjac. No evidence has been found to substantiate claims for the presence of either the Indian muntjac or hybrids.

Chromosome rearrangement between the Indian muntjac and Chinese muntjac is accompanied by a delection of middle repetitive DNA.

The organizations of the genomes of two related species of Asian deer, the Indian (2n = 6 female, 7 male) and Chinese muntjac (2n = 46), were compared at the cytogenetic and molecular levels. These dramatically different karyotypes preserve little apparent G-banding homology. The difference in chromosome number is coincident with a 22% reduction in haploid DNA content from 2.7 to 2.1 pg in the Chinese and Indian muntjac, respectively. The kinetics of reassociation of the Indian muntjac (equivalent Cot = 4285 M-1. s-1) and Chinese muntjac DNA (equivalent Cot - 4362 M-1.s-1) in 2.4 M tetraethylammonium chloride suggests conservation in amount of "single-copy" DNA. Two middle repetitive DNA sequence classes differ in both amount and in degree of repetition between the two species. A middle repetitive frequency component (935-fold repeated) represents 13% of the Indian muntjac DNA. A similar component (644-fold repeated) represents 17% of the Chinese muntjac DNA. Low repetition DNA sequence components (repeated 5- and 50-fold) represent 30 and 40% of the Indian and Chinese muntjac DNAs, respectively. These differences quantitatively account for the 0.6 pg haploid DNA content variation between species. The deletion of middle repetitive DNA has not substantively altered the distribution of restriction endonuclease DNA base composition classes as defined by buoyant density in cesium chloride. These results represent the first time that middle repetitive DNA has been directly implicated in a chromosome rearrangement within the vertebrates.

Simian virus 40 causes persistent infection of muntjac cells in the absence of virus transformation.

SV40 infection of Muntiacus muntjak cells (ATCC, CCL: 157) resulted in abortive transformation with formation of T antigen and induction of cellular DNA replication in the absence of virus production. These cells were resistant to stable transformation by SV40 regardless of the route of infection, including microinjection of virus into cell nuclei. The present studies show that T antigen-containing cells persist and that the number of T antigen-positive cells remains constant in infected cultures, which is reflected in the nearly constant fraction of T antigen-positive cells in stationary cultures and a decrease of the fraction of T antigen-positive cells in proliferating cultures. These data suggest that following cell division only one daughter cell on the average can maintain a detectable level of T antigen. The cell cycle kinetics of proliferating muntjac cultures were not altered by the abortively transforming infection, and infectious progeny was made following cell fusion with permissive CV-1 cells. It is suggested that the few copies of viral DNA that do persist are present in a "plasmid-like' state as non-integrated DNA. In this way SV40 can appear to be lost from the majority of the cells in a culture but actually be conserved in a small population of cells for a long time. In such a state a papovavirus might go undetected in vitro as well as in vitro by even the most sensitive immunological and molecular techniques.

Arrangement of prematurely condensed chromosomes in cultured cells and lymphocytes of the Indian muntjac.

Premature chromosome condensation (PCC) was induced in order to study the arrangement of muntjac chromosomes in the interphase nuclei of proliferating and resting cells with respect to their polarity and the spatial relationship between them. The data were compared with the situation in in situ fixed and colcemid blocked metaphases. It appears that in rapidly dividing cells almost all G1- and G2 interphase chromosomes exhibit the Rabl type polarized orientation. This pattern still predominates in G0 lymphocytes which may have been arrested at this stage for some months or even years. - The location of the small chromosome Y2 was found to be "central" in normal metaphases but "peripheral" in colcemid blocked mitoses. The behavior in the premature condensed chromosome preparations was intermediate. Measurements of centromere distances between all possible pairs of chromosomes as well as on the relative position of chromosomes in circular spreads revealed no evidence for homologous somatic association during interphase and metaphase or any other suprachromosomal ordering principle. Interphase chromosome orientation seems to be solely the result of chromosome arrangement of the foregoing anaphase. Association between heterochromatic regions or the nucleolus organizers did not substantially influence this pattern. There is no support for speculations that in mammalian cells close proximity of homologous sites is instrumental in functional cooperation.

Synaptonemal complex of the sex-autosome trivalent in a male indian muntjac.

Bright-field microscopy of silver-stained pachytene spermatocytes of a male Indian muntjac, Muntiacus muntjak revealed that (a) the synapsis between the autosomal homologs, including the long arm of the X and Y2, was normal, (b) the nucleolus organizer regions were present in both the No. 1 bivalent and the long arm of the X and Y2, (c) the accessory structures of the X chromosome short arm in the forms of light and dark thickenings and the hairpin-like bend were present despite the X-autosome translocation, (d) a short synaptonemal complex was present between the Y1 (real Y) and the short arm of the X chromosome, and (e) the centromeric orientation of the Y1 and Y2 chromosomes was in Cis configuration as opposed to the X chromosome.

Sister chromatid differentiation and isolabeling of chromosomes.

Isolabeling observed during sister chromatid differentiation (SCD) was studied from human skin fibroblasts by the fluorescence-plus-Giemsa (FPG) technique. Bromodeoxyuridine (BrdU) was fed to exponentially dividing cells for 52 h to enable completion of two consecutive cycles of DNA replication. During this period, the late-replicating regions of some chromosomes were able to go through three replication cycles. These chromosome regions had evidently incorporated BrdU bifiliarly in both chromatids and hence, on staining with FPG, appeared isostained (isolabeled). Thus, incubation of exponentially dividing cells with BrdU for a period longer than that required for two cell cycles appears to be a suitable method for revealing the late-replicating regions of the genome, such as the X chromosome in a human female, as isolabeled. In another experiment with Indian muntjac chromosomes, isolabeled segments were darkly stained, which suggested unifilar incorporation of BrdU. In this case, unequal crossing-over or an unequal distribution of thymine residues probably is responsible for the isolabel.

Gametogenesis in a male Indian muntjac x Chinese muntjac hybrid.

Histological and cytogenetic examinations of testicular preparations from a male hybrid between an Indian muntjac x Chinese muntjac showed a certain degree of chromosomal homology, as evidenced by the synaptic behavior. The male hybrid is considered sterile because spermatogenetic stages were arrested at early prophase. Silver staining demonstrated that the nucleolus organizer regions of both parents were expressed, but no synaptonemal complex and sex vesicle were observed.

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.

An ecological survey of the Royal Karnali-Bardia Wildlife Reserve, Nepal: Part III: Ungulate populations

Population density and biomass estimates for wild and domestic ungulates in the Royal Karnali-Bardia Wildlife Reserve, Nepal, are presented and compared with values obtained from other reserves in South Asia. Wild ungulate population densities varied in relation to habitat diversity; large herbivores were most numerous where early and intermediate successional stages intergraded with one another and only sparsely distributed in continuous climax sal Shorea robusta forest. Chital Axis axis was the most abundant wild ruminant in the reserve. It and the nilgai Boselaphus trago-camelus constituted 88% of the total wild herbivore biomass in the southwestern corner of Karnali-Bardia, where an intensive study was carried out. The biomass of domestic stock utilising the same study area in 1975 was computed to be 15–17 times higher than the levels attained by wild ungulates in April of 1977, nearly two years after the reserve borders had been closed to domestic grazing. Observations during the hot-dry season indicated that both wild and domestic ruminants competed for the same forage species. Among the wild ungulates, chital were decidedly the most gregarious, commonly associating in feeding aggregations of over 20 individuals on the open savannahs. Nilgai and wild boar Sus scrofa formed smaller groups while barking deer Muntiacus muntjak and hog deer Axis porcinus were primarily solitary. Chital exhibited a peak in rutting activity during the late hot-dry season and a subsequent birth peak in February. Nilgai exhibited a different pattern, breeding in January and calving during the monsoon, when optimum foraging conditions prevailed.

Sister-chromatid exchanges and cytotoxicity in cultured indian muntjac cells treated with alkylating agents

6 monofunctional alkylating mutagens/carcinogens — 4 N-nitroso and 2 methanesulfonate compounds — differed in their efficiency for inducing sister-chromatid exchanges (SCEs) when normalized on cytotoxicity. SCE induction occurred only at highly cytotoxic doses. Ethylating agents were, on a molar basis, generally less potent inducers of SCEs, and they were also less cytotoxic than the corresponding methylating agents. The observed differential action spectrum of the 6 alkylating agents is discussed in the light of recent ideas on the interaction between alkylating mutagens with DNA and the role of specific DNA lesions in the formation of SCEs.

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.

Separation of large quantities of chromosomes by velocity sedimentation at unit gravity

A chromosome sorting technique is described based on velocity sedimentation at unit gravity which combines simplicity with good resolution. Large quantities of chromosomes (up to 10 10) can be sorted for further biological and biochemical research. Male Indian muntjac cells were first synchronized and subsequently accumulated in mitosis with vinblastine. After hypotonic treatment, mitotic cells were disrupted and the crude chromosomal preparation was layered as a thin film onto a two-step linear sucrose gradient in a specially designed sedimentation chamber (area 127.6 cm 2). After 18 h of sedimentation at unit gravity, fractions were collected and the chromosomal composition of the fractions was analyzed by flow cytometry and microscopy.

Persistent infection of Muntiacus muntjak cells with adenovirus type 2 and abortive infection with adenovirus type 12

The Indian deer Muntiacus muntjak has the lowest chromosome number reported for mammals, six for the female and seven for the male animal. Adenovirus type 2 (Ad2) can replicate efficiently in these cells. When inoculated at low multiplicity, however, Ad2 establishes a persistent infection. The viral DNA persists in these cells apparently in a free form and replicates continuously. Tests for viral antigens and infectious center assays have revealed that about 1 to 2% of the cells produce Ad2. The persistent infection can be abolished by treating the infected cells with rabbit anti-Ad2 serum. As shown by the Southern blotting technique, the viral DNA is lost from the cells which were cured by antiserum treatment. Judging from the unabated replication of Sindbis virus or vesicular stomatitis virus in persistently Ad2-infected Muntiacus muntjak (M.m.) cells, the persistence of Ad2 is not accompanied by the production of interferon. Nor is there evidence for an Ad2-specific inhibitory factor, since persistently infected cells can be superinfected with high multiplicities of Ad2. Adenovirus type 12 (Ad 12) cannot multiply in M.m. cells. A very low level of replication of unit length Ad12 DNA can be detected. M.m. cells abortively infected with Ad12 continue to grow, and the viral DNA is eventually lost from the bulk of the cells. Ad12 infection leads to chromosomal abnormalities in M.m. cells. At 24 hr postinfection, 30–40% of the cells could be shown to be T antigen positive by immune fluorescence.

DNA methylation and viral gene expression in adenovirus-transformed and -infected cells.

The level of DNA methylation in adenovirus type 2 (Ad2) and type 12 (Ad12) DNA was determined by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes HpaII and MspI. As previously reported virion DNA of Ad2 and Ad12 is not methylated. Parental or newly synthesized Ad2 DNA in productively infected human KB or HEK cells is not methylated either, nor is the integrated form of Ad2 DNA in productively infected cells. Hamster cells and Muntiacus muntjak cells are abortively infected by Ad12. We have not detected methylation of Ad12 DNA in hamster or Muntiacus muntjak cells. An inverse correlation between the level of methylation and the extent of expression of viral DNA in Ad12-transformed hamster cells has been described earlier. A similar relation has been found for the EcoRI fragment B of Ad2 DNA which is not methylated but is expressed as the Ad2 DNA-binding (72K) protein in the Ad2-transformed hamster line HE1. Conversely, the same segment is completely methylated in lines HE2 and HE3, and there is apparently no evidence for the expression of the 72K protein in these cell lines.

An ecological survey of the Royal Karnali-Bardia Wildlife Reserve, Nepal. Part II: Habitat/animal interactions

The habitat utilisation by ungulates in a newly created tiger sanctuary of the southwestern Nepalese Terai is analysed. While most of the Royal Karnali-Bardia Wildlife Reserve is covered by a rather homogeneous belt of moist subtropical deciduous forest, one section of the reserve supported a wide variety of habitat types. In this area, flood plain, savannah, and several riverine forest associations intergraded with stands of the dominant Shorea robusta forest. Free-living mammals responded to this ecological heterogeneity, permitting an analysis of habitat preferences by the following species: chital Axis axis, nilgai Boselaphus tragocamelus, hog deer Axis porcinus, barking deer Muntiacus muntjak, swamp deer Cervus duvauceli, sambar Cervus unicolor, and two primate species, common langur Presbytis entellus, and rhesus monkeys Macaca mulatta. Differences in feeding and anti-predator strategies offered a degree of ecological separation between most of the ungulates studied. Changes in plant distribution and phenology affected ungulate food habits, energy budgets, movements, and seasonal distribution. A special feeding relationship between certain ungulates and langur and rhesus monkeys was observed. The sambar, an ungulate capable of exploiting a feeding niche in continuous climax forest, appears to be best adapted to the habitat types which dominate most of Karnali-Bardia, while only 30% of the reserve could be considered prime habitat for the chital, the most abundant grazing ungulate in the reserve. Proposals to improve habitat conditions for grazing ungulates through an experimental programme integrating controlled burns, water hole development, and the creation of openings in continuous climax forest are examined.