Identification of an Indian muntjac DNA fragment preferentially hybridizing to the X-chromosome

Short notes

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SHORT NOTE Identification

of an Indian

HELEN Department

Muntjac DNA Fragment to the X-chromosome

VASILIKAKI-BAKER of Biology

Preferentially

and YUTAKA

and Centre for Human Montreal, Quebec, Canada

Genetics, H3A IBI

Hybridizing

NISHIOKA* McGill

University,

Upon digestion of DNA from male and female Indian muntjac (Muntiacus muntjak) tibroblasts with the restriction enzyme Hae III or Alu I, a prominent fragment of DNA (>20 kb in length) was observed. This excluded DNA (ex-DNA) appeared not to contain sequences recognized by a variety of restriction enzymes and constituted about 0.6% of the total DNA in the female genome. For equal amounts of DNA digested, female DNA contained more of this material. In situ hybridization indeed revealed strong hybridization of the ex-DNA to the entire X chromosome with a few less intense sites of hybridization on other chromosomes. Hybridization studies against total muntjac DNA indicated the presence of repetitive sequences in the ex-DNA. These repetitive sequences did not crosshybridize with human or mouse DNA.

The Indian muntjac deer (Muntiucus munrjak) has been used extensively as a subject in cytogenetic studies due to its low diploid chromosome number combined with large easily identifiable homologue pairs [l]. We have applied restriction enzyme analysis in an attempt to further characterize the muntjac genome. Upon digestion of muntjac DNA with the restriction enzyme Hae III, a prominent band of high molecular weight (~20 kb) was generated. Comparison of male and female digests showed that for equal amounts of DNA digested, the high molecular weight or excluded material (ex-DNA) was more abundantly present in female DNA. This observation suggested an X chromosomal location and prompted further investigation. Here, we report the localization of the ex-DNA to the muntjac X chromosome by in situ hybridization and the presence of a subset of repetitive sequences within the ex-DNA band. The muntjac X chromosome is joined to one of the autosomes by a narrow elongated “neck”, an event reported to have occurred by centric fusion during evolution [ 1, 21. The ex-DNA described in this communication may prove useful in the study of muntjac X chromosome evolution. Materials and Methods CeNs. Male muntjac fibroblasts were purchased from American Type Culture Collection. Female muntjac flbroblasts were obtained from Dr W. Peterson of the Child Research Center of Michigan. DNA preparation. Cells were lysed with 0.5% SDS and DNA was extracted with phenol and chloroform. DNA was digested with various restriction enzymes under the conditions recommended by the suppliers (Bethesda Research Laboratories, New England BioLab, and Amersham) and analyzed by electrophoresis on 1% agarose slab gel run horizontally in a buffer containing 40 mM Bis (pH 7.2), 80 mM sodium acetate and 1 mM EDTA. To isolate the ex-DNA, 300-900 g DNA was * To whom offprint requests should be sent. Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/84 $03.00

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digested with Hae III and electrophoresed on a 0.8 % low melting point agarose gel (type VII, Sigma). The portion of the gel containing the ex-DNA was cut out and melted at 70°C. The melted agarose was cooled at 45°C NaCl was added to 0.5 M and extracted twice with phenol. The ex-DNA was precipitated with ethanol, resuspended into water and stored at -20°C. About 1 pg of ex-DNA could be routinely obtained from 300 pg of female DNA. In situ hybridization. The ex-DNA isolated from female muntjac Iibroblasts was nick-translated with [‘H]dATP (17 Ci/mmole) and [3H]dCTP (30 Wmmole) using a nick-translation kit (Bethesda Research Laboratories). The probe’s specific activity was 4.2~ 10’ cpm/g DNA. Slides of fixed metaphase chromosomes were prepared as described previously [3]. Slides were then treated with RNase (100 pg/ml) in 2xSSC for 1 h at 37”C, rinsed thoroughly in 2xSSC and dehydrated in ethanol. Chromosomal DNA was denatured by immersing slides in a solution containing 70% formamide in 2xSSC at 70°C for 2 min. The 3H-labelled probe was denatured by heat (70°C) for 5 min in a solution containing 50% formamide, 2xSSC, 0.04 M NaHPO.,. 10% dextran sulfate and 50 &ml of sonicated E. coli DNA. A portion of the solution was applied onto each slide (0.25 ml/slide), and incubated at 37°C for 12-18 h. Slides were then rinsed three times in 50% formamide, 2xSSC at 40°C washed thoroughly in 2XSSC and dehydrated in ethanol. Autoradiography was performed according to a procedure described by Pardue & Gall [4]. Briefly, slides were covered with Kodak NTB2 nuclear track emulsion and incubated in a light-tight container with dessicant at 4°C for 7-12 days. Slides were developed in Kodak D-19 developer for 2-4 min, fixed in Kodak fixer and rinsed with distilled water. Chromosomes were stained with Giemsa (0.75 in 50% methanol), examined with a Zeiss photomicroscope and photographed on Kodak Tri X-Pan film. DNA blot hybridization.Hae III and Alu I digests of male and female muntjac total DNA were fractionated on a 1% agarose gel and transferred to nitrocellulose filters as described by Southern [5]. Filters were pretreated with 50 @ml of denatured herring sperm DNA in a solution containing 3xSSC, 10X Denhardt’s [6], 0.1 % SDS and 10 @ml poly(A), and hybridized to the 32P-labelled probe in the same solution overnight at 65°C. Filters were washed three times in 50 ml of 3xSSC, IOX Denhardt’s, and 0.1% SDS at 65°C followed by a stringent wash in 100 ml of 0.1 xSSC and 0.1% SDS at 65°C. Filters were then air-dried and exposed to Kodak X-Omat film with Cronex intensifying screens at -70°C.

Results Restriction endonuclease digestions. DNA from both male and female fibroblasts

of Indian muntjac was cleaved with a variety of restriction enzymes and specific patterns of DNA fragments were observed. For example, Eco RI and Hind III produced a gradient of DNA fragments mainly larger than 4.8 kb, whereas Barn HI formed a similar gradient of DNA fragments with lengths mainly above 7.6 kb. Sau 3A, on the other hand, generated mainly smaller fragments below 3.3 kb in length (not shown). The most distinct pattern was obtained with Hae III or Alu I (fig. 1). These enzymes generated a prominent band of large MW (ex-DNA) which co-migrated with the largest Eco Rl band of phage lambda marker DNA (21.8 kb) in a 1% agarose gel. Visual comparison of male and female DNA digested with Hae III or Alu I showed that for equal amounts of DNA digested, female DNA contained more of this material (fig. 1). This initial observation suggested its location on the X chromosome and prompted further investigation of this ex-DNA. In an attempt to detect the presence of any restriction enzyme sites within the ex-DNA, DNA from both sexes were digested with Hae III and Barn HI, Hind III, Alu I, Hpa II or Msp I. None of these double digestions were observed to have an appreciable effect on the intensity or position of the ex-DNA fragment (not shown). Using Exp Cell Res 152 (1984)

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Fig. 1. Hae III and Alu I digestion of muntjac DNA. I, Lambda DNA digested with EcoRl ; 2, female DNA digested with Hae III; 3, male DNA digested with Hae III; 4, female DNA digested with Alu I; 5, male DNA digested with Alu I. The arrow points to the ex-DNA. Fig. 3. DNA blot hybridization. 1, Female Hae III digested DNA; 2, female DNA digested with Alu I. The arrow points to the ex-DNA.

microdensitometry, we estimated that the ex-DNA comprised about 0.6%, of total DNA in the female muntjac genome. In situ hybridization. The chromosomal localization of the ex-DNA was examined by in situ hybridization. Fig. 2 shows chromosomes of the muntjac, probed with the ‘H-labelled ex-DNA after a 7-day exposure. As expected, grains were preferentially localized over the X chromosome. This pattern was observed consistently in almost 100% of cells examined. In addition, the presence of minor sites of hybridization at other locations was recognized. In particular, grains were often observed near the centromere or over the mid-arm of chromosome 1. DNA blot hybridization. In order to investigate the organization of the ex-DNA in relation to the muntjac genome, the female ex-DNA was labelled with 32P and hybridized to Hae III or Alu I digests of female muntjac DNA. Fig. 3 shows the pattern of hybridization with female Hae III digests. There was an intense band near the top corresponding to the ex-DNA itself. The smear of hybridization suggests the presence of repetitive sequences within the ex-DNA. It was of interest to note that the hybridization pattern did not correspond to the distribution of DNA fragments. Instead, intense hybridization occurred in the upper part of the blot, where the presence of DNA was barely visible by ethidium bromide staining. Results of hybridization to Alu I digests are also shown in fig. 3. The pattern resembled that obtained with Hae III. No cross-hybridization was observed between the muntjac ex-DNA and mouse or human DNA (not shown). &p

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Fig. 2. In situ hybridization. 3H-labelled ex-DNA hybridized to metaphase chromosomes of male muntjac ceils. Arrows point to the dark grains localized over the X chromosome.

Discussion

The action of some restriction enzymes, including Hae III, on muntjac DNA has previously been described [7]. However, the ex-DNA generated by Hae III has not been characterized. An interesting property of the muntjac ex-DNA is its apparent resistance to further digestion by a number of restriction enzymes. The lack of recognition sites within such a large molecule is unusual and indicates the presence of highly repetitive sequences, as demonstrated by DNA-blot hybridization. Since the pattern of hybridization does not correspond to that obtained by ethidium bromide staining, these sequences represent a subset(s) of muntjac repetitive sequences. Based on the observations that (a) in both Hae III and Alu I digests, the female DNA contained more of the ex-DNA; (b) double digestion with Hae III and Alu I had no observable effect on the ex-DNA and (c) the Hae III generated ex-DNA cross-hybridized to the Alu I-generated ex-DNA, it can be concluded that the Hae III and Alu I ex-DNAs contain the same repeating sequences. A band corresponding in size to the muntjac ex-DNA was also observed in mouse [8] and human [9] DNA but a quantitative difference between sexes was not apparent in these species. Results of hybridization in situ confirmed our inference that the ex-DNA was mainly derived from the X chromosome. Bogenberger et al. [IO] isolated a repetitive sequence from the muntjac genome which was a Ban Hl fragment of 800 bp and hybridized exclusively to the “neck” or region of fusion between the muntjac X and one of the autosome. Unlike the sequence described by Bogenberger et al. [lo], the ex-DNA (1) does not contain Barn Hl sites; and (2) hybridizes to the X chromosome but not to the centromeric “neck”. The exDNA exhibited less intense hybridization to other chromosomes, especially Erp Cell Res 152 (1984)

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chromosome 1. This observation is analogous to that reported by Schmeckpeper et al. [l II of human DNA fragments carrying repetitive sequences common to both autosomes and the human X chromosome. Alternatively, the ex-DNA may contain at least two classes of repetitive sequences: the X chromosome-specific and autosome-specific sequences. Cloning is necessary to further classify sequences in the ex-DNA. The gene content and linkage of the X chromosome has been remarkably well conserved during evolution [12]. However, most of human X chromosomal DNA sequences did not hybridize to rodent DNA under the conditions generally used [13-171. It seems that the conservation of gene linkage has been achieved without a strong conservation of DNA sequence. Furthermore, in light of the current view that some repetitive sequences are nomadic and not under functional constrain [18, 191, it is not surprising that the muntjac ex-DNA failed to crosshybridize with mouse or human DNA. We thank Dr W. Peterson of the Child Research Center of Michigan and Dr N. Sonenberg of McGill University for female muntjac fibroblasts and cloned chicken actin c-DNA, respectively. We are most grateful to MS Fran Langton for her assistance in the preparation of this manuscript. This work was supported by a grant from the Medical Research Council of Canada. H. V.-B. was the recipient of a Fellowship from the Natural Sciences and Engineering Research Council of Canada.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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Received October 25, 1983 Revised version received January 11, 1984

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Res 152 (1984)