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.     

Anthramycin-induced sister-chromatid exchange and caffeine potentiation in the chromosomes of Indian muntjac

Cell-cycle kinetics, sister-chromatid exchange (SCE) and chromosome aberrations have been studied from the skin fibroblasts of the Indian muntjac after treatment with 100 micrograms/ml of caffeine and 0.05 microgram/ml of anthramycin. The cultures were incubated for a period which was sufficient for the completion of two consecutive cell cycles and both the drugs appeared to produce a slight inhibitory effect. When anthramycin-treated cells were however post-treated with caffeine, the cells did not proceed beyond one cycle and exhibited a mitotic block. The SCE frequency in the control and the experiments with caffeine and anthramycin was 8.63, 18.32 and 34.88 per cell respectively. The SCEs were randomly distributed amongst all chromosomes unlike a non-random distribution within the X chromosomes. Caffeine and anthramycin produced only 0.5% and 3.1 cells with chromosome aberrations respectively. Potentiation of chromosome aberrations was observed when the anthramycin-treated cells were post-treated with caffeine. Caffeine potentiation presumably results from an inhibition of the cells to cycle and a failure to repair the effect of the mutagen on DNA.     

Optimization of conditions for isolating chromosomes in flow sorting

Factors for purity and efficiency of flow chromosome sorting were analysed on the base of quantitative analysis. The sorting rate and relative purity of individual chromosome fractions are determined both by the quality of initial chromosome suspension, instrument parameters and gate position on experimental histograms. The described procedure of analysing sorting efficiency and fraction purity allowed to formulate general tips for optimization of sorting conditions depending on a given strategy: maximization of quantity of obtained material, or achieving maximum purity for sorted fraction. The analysis is carried out on the bases of chromosome distribution parameters: their relative halfwidths and distances. These parameters can be obtained by the quantitative analysis programs for flow cytometry data. It is shown that the critical parameter for sorting purity is the level of contaminated objects in a zone of sorted chromosome signal registration. In addition, the fraction purity depends on the cover extent between different chromosome distributions. Created procedure allows to build up nomograms linking the sorting efficiency with fraction purity, depending on the position of sorting gates. These monograms permit to determine the position of sorting gates in relation to one or another strategy: 1) maximum rate for chromosome material obtaining, 2) maximum fraction purity, or 3) compromise between the two. The presented analysis allows to optimize the chromosome sorting process for subsequent genome investigations providing chromosome material with controlled characteristics.     

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.     

Patterns of silver staining on NORs of prematurely condensed muntjac chromosomes following RNA inhibition

Prematurely condensed chromosomes of muntjac G0 lymphocytes as well as contact-inhibited and Actinomycin D (actD)-treated fibroblasts have been stained with silver nitrate to estimate the correlation between RNA suppression and the NOR staining. The results demonstrate that actD treatment for up to 36 h does not significantly affect the staining. Only partial suppression occurs in contact-inhibited cells, whereas complete abolition is obtained in long quiescent lymphocytes. We conclude that the reduction of the staining occurs only gradually from the NORs over a number of days or even weeks. We assume that the silver staining proteins may be associated with rDNA having a regulatory or structural role to play in rDNA activity.     

Microsurgically-extracted metaphase chromosomes of the Indian muntjac examined with phase contrast and scanning electron microscopy.

Metaphase chromosomes are extracted from Indian muntjac cultured fibroblasts either through the use of microneedles or by the application of a droplet of silicone oil onto the cell surface. Interconnecting fibers among the chromosomes allow the entire diploid complement to be extracted from the cell. The seven muntjac chromosomes are brought to the surface of a glass coverslip for analysis. Each chromosome can be identified on the basis of morphology, and particular chromosomes or chromosome parts can be isolated. Many of the fibers which interconnect the chromosomes may be attributed to adhesions formed between the sticky chromosome surfaces during extraction. However, when interchromosomal contacts are avoided during extraction, the chromosomes are found to be arranged radially with the centromeres near the center and interconnected by fibers. This arrangement is similar to that seen inside muntjac cells at metaphase. Scanning electron microscopy reveals the chromosome surfaces to consist of looping fibers, except for regions near the centromeres and the secondary constrictions. Chromosome fibers at these sites are organized into parallel bundles. Chromosome interconnections are strands composed of multiple fibers which seem to be continuous with chromosome fibers.     

Radioadaptation in Indian muntjac fibroblast cells induced by low intensity laser irradiation.

Earlier reports have indicated that an adaptive, protective response to ionizing radiation is inducible by pre-treatment with low intensity laser irradiation (LILI). We have investigated the potential of LILI to induce an adaptive response against the damaging effects of ionizing radiation in Indian muntjac fibroblasts. LILI at 660, but not 820 nm, at 11.5 and 23.0 J/cm2, induced an apparent adaptive response in the form of a reduction in the frequency of radiation-induced chromosome aberrations, but not in cell survival. There was also a trend towards a reduction in the level of single-stranded and double-stranded DNA breaks induced by ionizing radiation when cells were preconditioned with LILI. However, this did not contribute to the reduced chromosome aberration frequency. Further analysis revealed that the reduced aberration frequency was caused by a laser-induced extension of G2 delay. The adaptive response was therefore the result of cell cycle modulation by LILI, at a wavelength where there is no known DNA damaging effect to induce the checkpoint mechanisms that are normally responsible for altering cell cycle progression.     

Regional differences in immunostainability of isolated metaphase chromosomes of Indian muntjac with anti-Z-DNA antibody

The distribution of Z-form DNA along the length of metaphase chromosomes of Indian muntjac was studied by indirect immunofluorescence procedures using an antibody specific to the Z-DNA conformation. Several fixation conditions were compared for reproducible detection of Z-DNA in isolated metaphase chromosomes. Fixation of chromosomes with 45% acetic acid alone gave reproducible reactivity with the antibody. When fixation was done either with Carnoy's solution (3:1 methanol:acetic acid) or with 75% alcohol alone, the antibody binding was at background level. Acetic acid-fixed chromosomes exhibited intense fluorescence both at C-band heterochromatin and at nucleolus organizer regions (NORs). The euchromatic regions had weakly, but clearly, stained bands, which were quite similar to the chromomycin A₃ R-bands. After treatment with topoisomerase I, the immunofluorescence at NORs and R-bands disappeared, but only a slight decrease in immunofluorescence intensity was observed at C-band regions. We suggest that this difference in the immunoreactivity of NORs and R-bands from C-bands reflects a difference in gene activity among these regions. Possible molecular mechanisms involved in Z-DNA immunoreactivity are discussed, based on SDS-polyacrylamide gel electrophoretic analysis of chromosomal proteins after extraction of metaphase chromosomes with different fixative solutions.Abbreviations PI propidium iodide - NOR(s) nucleolus organizer region(s) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Deceased, April 23, 1988     

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 homologoues sites is instrumental in functional cooperation.     

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.     

Visible light observations on the kinetochore of the Indian muntjac, Muntiacus muntjac, Z

We report here a silver stain technique (Kt stain) for locating the kinetochore (centromere body) without concomitant staining of C-band material. We compare our observations with those obtained from C-banding, Cd (centromeric dot) banding, and electron micrographs, and we report preliminary observations on Indian muntjac centromeres.     

System for flow sorting chromosomes on the basis of pulse shape

Sorting on the basis of the complex features resolved by chromosome slit-scan analysis requires rapid and flexible pulse shape acquisition and processing for determining sort decisions before droplet breakoff. Fluorescence scans of chromosome morphology contain centromeric index and banding information suitable for chromosome classification, but these scans are often characterized by variability in length and height and require sophisticated data processing procedures for identification. Setting sort criteria on such complex morphological data requires digitization and subsequent computation by an algorithm tolerant of variations in overall pulse shape. We demonstrate here the capability to sort individual chromosomes based on their morphological features measured by slit-scan flow cytometry. To do this we have constructed a sort controller capable of acquiring an 128 byte chromosome waveform and executing a series of numerical computations resulting in an area-based centromeric index sort decision in less than 2 ms. The system is configured in a NOVIX microprocessor, programmed in FORTH, and interfaced to a slit-scan flow cytometer data acquisition system. An advantage of this configuration is direct control over the machine state during program execution for minimal processing time. Examples of flow sorted chromosomes are shown with their corresponding fluorescence pulse shapes.     

小麂、黑麂、赤麂精母细胞联会复合体的比较研究

本工作以界面铺张——硝酸银染色技术,对小麂(Muntiacus reeuesi)、黑麂(M.crinifrons)和赤麂(M.muntjak)的精母细胞联会复合体(Syna ptonemal complex,SC)进行亚显微结构的比较研究。结果表明: 1.SC的平均相对长度和臂比指数同有丝分裂细胞相应染色体的数值有很好的一致性。根据SC的相对长度和臂比指数绘制了三种麂的SC组型图。雄性黑麂减数分裂前期形成一个复杂的易位多价体,意味着其核型的演化过程涉及两次染色体易位和一次臂间倒位。 2.在减数分裂前期,性染色体的形态和行为同常染色体的有明显差异,如性染色体嗜银性较强,配对延迟等。XY的配对起始于早粗线期,在中粗线期,Y的全长均同X配对;XY-SC开始解体于晚粗线期。 3.在粗线期,X染色体未配对区域出现自身折叠,形成“发夹”状结构。这种“发夹”结构的形成,可能是在性染色体的进化过程中,X染色体通过不对称易位得到的重复片段在减数分裂前期同源配对的一种细胞学表现。     

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).     

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.     

Purifying human Y chromosomes by flow cytometry and sorting

A method of producing an enriched sample of human Y chromosomes from peripheral blood lymphocytes is described. Metaphase chromosomes were prepared from peripheral blood lymphocytes donated by 17 normal male individuals. A suspension of chromosomes in a polyamine buffer was produced from each sample, stained with the fluorescent dye Hoechst 33258, and passed through a flow cytometer and sorter. Following analysis of the 17 fluorescence distributions, a single donor was found giving a separate peak corresponding to the Y chromosome. Seventy percent of the chromosomes sorted from this peak were identified as Y chromosomes. Batches of a million Y chromosomes were produced from each of several 40 ml donations of peripheral blood. These were assessed for the amount of Y DNA present and used to construct a DNA library.     

Locations of 18S and 28S ribosomal genes on the chromosomes of the Indian muntjac

The locations of genes coding for 18S and 28S ribosomal RNA have been mapped on metaphase chromosomes of the Indian muntjac M. muntjak by in situ hybridization with (3H)rRNA from the toad X. laevis. The results show that, in the muntjac, rDNA clusters are associated with the prominent secondary constrictions on the X and the Y1 chromos. In addition a cluster of rDNA is found near the tip of one arm on the longest pair of autosomes. The autosomal cluster of rDNAs usually does not express as a secondary constriction at metaphase.     

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     

Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S-phase fraction, and surface phenotype using single laser instrumentation.

We have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7-amino-actinomycin D. PHA-activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin-labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti-BrdUrd monoclonal antibody followed by a FITC-conjugated goat anti-mouse antibody and incubated with 7-amino-actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained-fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (greater than 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.     

Improved BrdUrd-Hoechst bivariate cell kinetic analysis by helium-cadmium single laser excitation.

In laser based flow cytometers, UV excitation of Hoechst 33258 and propidium iodide (PI) or ethidium bromide (EB) is performed with 351/364 nm high power lines of UV-capable argon ion lasers, which are expensive and short-lived. In this paper we note for the first time that helium-cadmium lasers emitting 10 to 30 mW at 325 nm are even more superior for cell kinetic bivariate bromodeoxyuridine (BrdUrd)/Hoechst PI or EB cell cycle analysis. HeCd single laser UV excitation gives comparable CVs for cell cycle distributions, and almost normal G2M/G1 ratios of 1.9 to 2.0 for all cell cycles. This is shown for synchronous and asynchronous cell populations on a FACStar+ and an Ortho Cytofluorograf. Therefore we recommend helium-cadmium lasers as low-power, cheap, and long-lived UV excitation sources for the cytochemically simple but high resolution multiparameter BrdUrd-Hoechst cell kinetic analysis.