Characterization and Chromosomal Distribution of Satellite DNA Sequences of the Water Buffalo (Bubalus bubalis)

Transcription

1 Characterization and Chromosomal Distribution of Satellite DNA Sequences of the Water Buffalo (Bubalus bubalis) K. Tanaka, Y. Matsuda, J. S. Masangkay, C. D. Solis, R. V. P. Anunciado, and T. Namikawa Satellite DNA sequences were isolated from the water buffalo (Bubalus bubalis) after digestion with two restriction endonucleases, BamHI and StuI. These satellite DNAs of the water buffalo were classified into two types by sequence analysis: one had an approximately 1,400 bp tandem repeat unit with 79% similarity to the bovine satellite I DNA; the other had an approximately 700 bp tandem repeat unit with 81% similarity to the bovine satellite II DNA. The chromosomal distribution of the satellite DNAs were examined in the river-type and the swamp-type buffaloes with direct R-banding fluorescence in situ hybridization. Both the buffalo satellite DNAs were localized to the centromeric regions of all chromosomes in the two types of buffaloes. The hybridization signals with the buffalo satellite I DNA on the acrocentric autosomes and X chromosome were much stronger than that on the biarmed autosomes and Y chromosome, which corresponded to the distribution of C-bandpositive centromeric heterochromatin. This centromere-specific satellite DNA also existed in the interstitial region of the long arm of chromosome 1 of the swamptype buffalo, which was the junction of the telomere-centromere tandem fusion that divided the karyotype in the two types of buffaloes. The intensity of the hybridization signals with buffalo satellite II DNA was almost the same over all the chromosomes, including the Y chromosome, and no additional hybridization signal was found in noncentromeric sites. From the Laboratory of Animal Genetics, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan (Tanaka, Matsuda, Solis, Anunciado, and Namikawa), and the College of Veterinary Medicine, University of the Philippines at Los Baños, College, Laguna, The Philippines (Masangkay, Solis, and Anunciado). The authors wish to thank A. Kuroiwa and K. Amano of Nagoya University and J. Matsuda of Fujita Health University for their helpful cooperation to complete the present work. Address correspondence to Dr. Takao Namikawa, Laboratory of Animal Genetics, School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya , Japan, or tnami@agr.nagoya-u.ac.jp The American Genetic Association 90: Highly repetitive DNA sequences are characteristic of the eukaryotic genomes. Two types of highly repetitive DNA sequences in mammalian genomes are known: one is interspersed DNA, in which the repeated DNA sequences are dispersed throughout the genome; the other is satellite DNA, which is characterized by a long tandem array and consistent association with constitutive heterochromatin (see Singer 1982). The satellite DNA sequences are preferentially localized to the centromeric region of chromosomes (Willard and Waye 1987). Its biological function remains obscure, although it has been proposed that it is implicated in centromere condensation, sister chromatid pairing, chromosome association with the mitotic spindle, karyotype evolution, and chromosome rearrangement (Miklos and John 1979; Singer 1982). The domestic buffalo (Bubalus bubalis) has been classified into two general types according to geographical distribution: one is the river-type buffalo, raised in most areas from India to Egypt and some south and east European countries; the other is the swamp-type buffalo of Southeast Asia (Mason 1974). The karyotypes differ in the two types of buffalo, and their diploid chromosome numbers are 48 and 50 in the swamp-type buffalo and the river-type buffalo, respectively ( Fischer and Ulbrich 1968). The karyotypes of the two types of buffaloes differ due to a tandem fusion translocation: the swamp-type chromosome 1 resulted from a telomerecentromere tandem fusion between the river-type chromosomes 4p and 9, with a loss of the centromere of river-type chromosome 9 (Bongso and Hilmi 1982; Di Berardino and Iannuzzi 1981). In this study we have molecularly cloned and characterized two types of satellite DNA sequences of the swamp-type buffalo. We have also investigated the chromosomal distribution of the satellite DNA sequences in the swamp-type and the river-type buffaloes by fluorescence in situ hybridization ( FISH), and discuss the correlation between chromosomal location of these satellite DNA sequences and karyotype evolution in the buffaloes. 418

2 Materials and Methods Molecular Cloning of Satellite DNA Tissue samples of the swamp-type buffalo were collected in the Philippines and genomic DNA was prepared from the tissue samples using standard technique (Sambrook et al. 1989). Satellite DNAs are often observed as satellite bands when mammalian genomic DNA is digested with appropriate restriction endonucleases and electrophoresed through agarose gel. Genomic DNA from a male swamp-type buffalo was digested with 20 restriction endonucleases, size fractionated by 1.5% agarose gel electrophoresis, and stained with ethidium bromide. The satellite DNA bands were eluted from the gel and cloned into plasmid pzero-2 ( Invitrogen). The satellite DNA fragments inserted in pzero- 2 were sequenced with an ABI PRISM Dye Primer Cycle Sequencing Kit with M13 forward (-21) and reverse primers using an ABI 373S DNA Sequencer (Perkin Elmer). Cell Culture and Chromosome Preparation for Replication R-Banding Blood samples were collected in heparinized vacutainers (10 ml) from a male rivertype buffalo (Murrah breed) and a male swamp-type buffalo (Carabao) at the Philippine Carabao Center of the University of the Philippines at Los Baños. Buffalo lymphocyte cultures for replication R-banding were established following methods for human lymphocytes ( Takahashi et al. 1990) with slight modification. Mononuclear cells were separated by using Lymphoprep ( Nycomed), transferred into culture flasks containing RPMI 1640 medium supplemented with 20% fetal calf serum, 3 g/ ml concanavalin A (type IV-S, Sigma), 10 g/ml lipopolysaccharide (Sigma), 2% HA15 (Murex), and 50 M mercaptoethanol, and were incubated at 37 C in a humidified atmosphere of 5% CO 2 in air. Mitogen-stimulated buffalo lymphocyte cultures were synchronized by thymidine block, and BrdU (25 g/ml) was incorporated during the late replication stage to obtain differential replication staining after release from excessive thymidine. R- banding was obtained by exposure of chromosome slides to UV light after staining with Höechst Figure 1. A photograph of electrophoresis of total buffalo DNA digested with BamHI or StuI in 1.5% agarose slab gel. StyI digest of lambda DNA was used as a molecular size marker. Fluorescence in situ hybridization Probe DNAs were labeled with biotin-16- dutp using a nick translation kit (Boehringer Mannheim) and ethanol precipitated with salmon sperm DNA and E. coli trna. Hybridization and detection of fluorescence signals was performed according to Matsuda and Chapman (1995). Fluorescence images were observed by an Olympus BX-60 epifluorescence microscope with Olympus filter set U-MWIB (excitation at nm), U-MSWG ( nm), and U-MWU ( nm), and photographed with Kodak Ektachrome ISO 100 films. Results Sequence Analysis and Characterization of Satellite DNAs It was found that BamHI and StuI produced two and four satellite bands, respectively (Figure 1). The 700 bp and 1,400 bp BamHI satellite DNA bands, and the 300 bp, 400 bp, 600 bp, and 800 bp StuI satellite DNA bands (Figure 1) were isolated from the gel, cloned into plasmid pzero-2, and then sequenced. The nucleotide sequence data of each one clone from the six satellite DNA bands have been incorporated into the DDBJ, EMBL, and Gene Bank Nucleotide Sequence databases with the accession numbers AB AB These DNA sequences were compared with the DDBJ database and extensive homologies to bovine satellite DNA sequences were found. These six satellite-type DNA fragments were classified into two different types of tandem repeats: one (1,400 bp BamHI, 600 bp and 800 bp StuI satellite DNA bands) had a 1,400 bp tandem repeat unit with 79% similarity to the bovine satellite I (1.715 satellite) DNA reported by Taparowsky and Gerbi (1982), and the other (700 bp BamHI, 300 bp and 400 bp StuI satellite DNA bands) had a 700 bp tandem repeat unit with 81% similarity to the bovine satellite II (1.723 satellite) DNA (Buckland 1985). The two types of tandem repeats isolated in this study were named as buffalo satellite I and II DNA, respectively. The G C content was 66.1% and 67.4% in the buffalo satellite I and II DNA, respectively. Chromosomal Distribution of Buffalo Satellite I and II DNA Sequences in the River-Type and Swamp-Type Buffaloes FISH analysis was applied to localize these satellite-type DNA sequences to the rivertype and swamp-type buffalo chromosomes. We used the 1,400 bp BamHI and the 700 bp BamHI DNA clones, which included the full length of tandem repeat units of buffalo satellite I and II sequences, respectively, as DNA probes. The hybridization patterns of the satellite DNA sequences on metaphase chromosomes of the swamp-type and rivertype buffaloes are shown in Figure 2. The buffalo satellite I DNA was distributed in centromeric heterochromatin blocks of all the chromosomes in both types of buffaloes, while the intensity of the hybridization signals quantitatively differed in the chromosomes ( Figure 2a,b). Strong hybridization signals were found on all the acrocentric autosomes and the X chromosome, whereas the signals were much weaker in the biarmed autosomes. A very weak signal was detected on the Y chromosome with this probe (Figure 2g,h). An additional signal of the buffalo satellite I DNA existed in the interstitial region of the long arm of chromosome 1 of the swamptype buffalo. This region was a junction of telomere-centromere tandem fusion between chromosomes 4p and 9 of the rivertype buffalo (Figures 2a,e,f and 3). The intensity of this additional signal was relatively weak compared with the signals of its ancestral acrocentric chromosome 9 of the river-type buffalo. The centromeric region was also labeled with the buffalo satellite II DNA in all the chromosomes of the two types of buffaloes and the intensity of the hybridization signals was weaker than that of buffalo satellite I DNA except on the Y chromosome (Figure 2c,d). The intensity of the hybridization signals was almost the same Tanaka et al Characterization of Satellite DNA of Water Buffalo 419

3 over all the chromosomes including the Y chromosome and no additional signal was focal in the interstitial region of any chromosomes ( Figure 2c,d). Discussion Sequence Evolution of Buffalo Satellite I and II DNA The bovine satellite I DNA was shown to be a 1.4 kb tandem repeat that contained imperfect variants of G C-rich 31 bp motif sequences (Plucienniczak et al. 1982). Southern blot hybridization with this 31 bp motif sequences of the bovine satellite I DNA indicated that various species in Bovidae and Cervidae have a homologous sequence in their genome (Modi et al. 1996). The repeat unit of buffalo satellite I DNA (1,400 bp BamHI clone) had approximately 79% similarity with bovine satellite I DNA and contained this type of 31 bp motif sequences. The satellite I DNAs of goat and sheep also contained this type of 31 bp motif sequence, however, the length of their tandem repeat units are much shorter (about 820 bp) than the bovine and the buffalo satellite I DNAs (about 1,400 bp) ( Buckland 1983; Jobse et al. 1995). Deepika and Sher (1996) reported a 1,378 bp satellite DNA sequence of rivertype buffalo that had 95% similarity (maximum matching) with our buffalo satellite I DNA (1,400 bp BamHI clone) isolated from swamp-type buffalo. The sequence similarity between the different DNA clones of the buffalo satellite I DNA (1,400 bp BamHI clone and 800 bp StuI clone or 600 bp StuI clone) was more than 97%, thus the DNA sequence of the satellite I DNA of river-type buffalo seemed to be differentiated from that of the swamp-type buffalo though the number of the analyzed sequence was very limited. Figure 2. Partial metaphase cells after direct R-banding FISH with following probes. (a) Swamp-type buffalo satellite I. (b) River-type buffalo satellite I. (c) Swamptype buffalo satellite II. (d) River-type buffalo satellite II. Chromosome 1 of swamp-type buffalo with the buffalo satellite I probe (e) and its G-banding pattern (f): #, points to centromeric region; *, points to interstitial signal. Y chromosome of river-type buffalo with feeble signal of buffalo satellite I (g) and its G-banding pattern (h). Figure 3. Comparisons of the informative chromosomes between the swamp-type and the river-type buffalo, which involved in the telomere-centromere tandem fusion. (a) Direct R-banding FISH with buffalo satellite I probe. (b) G-banding pattern of the same chromosomes set. (c) Diagrammatic representation of G-bands (redrawn from Iannuzzi 1994). 420 The Journal of Heredity 1999:90(3)

4 Unlike satellite I DNA, any internal repetition was not found in the published satellite II DNA sequences of Bovidae (cattle, buffalo, sheep, and goat) and Cervidae (white-tailed deer), and the length of their repeat units was very conservative ( Buckland 1985; Qureshi and Blake 1995). The length of the repeat units (about 700 bp) and the G C content in the buffalo satellite II DNA were very similar to other artiodactyl satellite II DNA (about 67%). These results suggest that concerted evolution of the satellite II DNA family in Bovidae and Cervidae occurred mainly by base substitution from an ancestral 700 bp tandem repeat. Chromosomal Distribution of the Satellite DNAs The centromeric regions in the two types of domestic buffalo portrayed considerable difference between acrocentric and biarmed chromosomes in the amount of C- band-positive heterochromatin; acrocentric chromosomes including the X chromosome exhibited a large amount of heterochromatin, whereas biarmed chromosomes showed a relatively small amount of heterochromatin, and Y chromosome was C negative ( Di Berardino and Iannuzzi 1981). This extensive variation in the size of C-bands was quite coincident with the chromosomal distribution and intensity variation of the hybridization signals of buffalo satellite I DNA, suggesting that the buffalo satellite I DNA sequences occupy considerable parts of the centromeric heterochromatin blocks ( Figure 2a,b). Modi et al. (1996) reported the cross-hybridization of the bovine satellite I (1.715 satellite) DNA sequence to the chromosomes of the river-type buffalo. Excluding Y chromosomes, the distribution pattern of the hybridization signals of the probe was almost identical to the present result with the buffalo satellite I DNA (Figure 2b). It was reported that buffalo Y chromosome was not labeled with bovine satellite I DNA, while very feeble signals of the buffalo satellite I were detected in this study (Figure 2g,h). This difference might simply be due to the difference of hybridization efficiency to quite a small number of copies of buffalo satellite I DNA sequences on the Y chromosome. Comparing the intensity of the signals in the river-type buffalo with the swamp-type buffalo, the rivertype X chromosome was much more intensely labeled with the buffalo satellite I DNA, indicating that the copy number of this satellite sequence was much larger on the river-type X chromosome. Karyotype analysis with chromosome banding in swamp-type and river-type buffalo suggested that chromosome 1 of the swamp-type buffalo arose out of a telomere-centromere tandem fusion of chromosomes 4 and 9 of the river-type buffalo (Di Berardino and Iannuzzi 1981). The present data with high-resolution R-banding confirmed the fusion of the two chromosomes. In situ hybridization with the buffalo satellite I DNA demonstrated the additional interstitial hybridization signals in the junction region of the fusion in the chromosome 1 of the swamp-type buffalo (Figure 3), where the presence of a pale C- band-positive region was reported ( Di Berardino and Iannuzzi 1981). The satellite DNA sequences retained in the long arm of the swamp-type buffalo chromosome 1, derived from chromosome 9 of the river-type buffalo, represented a relic of the telomerecentromere fusion of two ancestral chromosomes in the evolution of swamp-type buffalo chromosome 1 (Figure 3). On the other hand, any hybridization signals of the buffalo satellite II DNA was not detected in this region ( Figure 2c). The disappearance of the buffalo satellite II sequence may be closely related with inactivation of this inert centromere. The copy numbers of the buffalo satellite II DNA were relatively low in the chromosome of the two types of buffalo, and there was little variation in the intensity of hybridization signals ( Figure 2c,d). This result suggested that the buffalo satellite II DNA distributed equally regardless of the considerable variation in the amount of the centromeric heterochromatin on the chromosomes. These differences in the chromosomal distribution pattern between the buffalo satellite I DNA and buffalo satellite II DNA resembled that of mouse major and minor satellite DNA sequences (Matsuda and Chapman 1995; Wong and Rattner 1988). The difference in the chromosomal distribution of the satellite DNAs might result from functional differences, though their biological roles are not well understood. The mouse major satellite was known as the major component of centromeric heterochromatin block and the mouse minor satellite had a close relationship with centromere activity (Broccoli et al. 1990; Singer 1982). It has been reported that some centromere-specific satellite DNA sequences (e.g., mouse minor satellite and human -satellite) contain 17 bp motif called CENP-B box which bind with centromere-associated protein CENP-B ( Kipling et al. 1994, 1995; Masumoto et al. 1989). We tried to find the consensus sequence of CENP-B box ( Kipling et al. 1995) in the buffalo satellite I and II DNA sequences, but any related sequences were not found in the clones isolated in this study. References Bongso TA and Hilmi M, Chromosome banding homologies of a tandem fusion in river, swamp and crossbred buffalo (Bubalus bubalis). 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