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 464-8601, Japan, or e-mail:
[email protected].
q 1999 The American Genetic Association 90:418–422
418
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.
Materials and Methods
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 1 C content was 66.1% and 67.4%
in the buffalo satellite I and II DNA, respectively.
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 pZErO2 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 mg/
ml concanavalin A (type IV-S, Sigma), 10
mg/ml lipopolysaccharide (Sigma), 2%
HA15 (Murex), and 50 mM mercaptoethanol, and were incubated at 378C in a humidified atmosphere of 5% CO2 in air. Mitogen-stimulated buffalo lymphocyte cultures were synchronized by thymidine
block, and BrdU (25 mg/ml) was incorporated during the late replication stage to
obtain differential replication staining after release from excessive thymidine. Rbanding was obtained by exposure of
chromosome slides to UV light after staining with Höechst 33258.
Fluorescence in situ hybridization
Probe DNAs were labeled with biotin-16dUTP using a nick translation kit ( Boehringer Mannheim) and ethanol precipitated
with salmon sperm DNA and E. coli tRNA.
Hybridization and detection of fluores-
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.
cence 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 470–550 nm), U-MSWG (480–550 nm),
and U-MWU (330–385 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 AB012926–AB012931. 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
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
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 1 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)
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 1 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 Cband-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 Cband-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 humana-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.
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Received April 22, 1998
Accepted December 7, 1998
Corresponding Editor: Leif Andersson