Vol. 56, no. 2: 213-216, 2003
CARYOLOGIA
B chromosomes of the Chinese raccoon dog
(Nyctereutes procyonoides procyonoides Gray) contain
inactive NOR-like sequences
SZCZERBAL I. and SWITONSKI M.*
Department of Genetics and Animal Breeding, August Cieszkowski Agricultural University of Poznan, Wolynska 33, 60-637
Poznan, Poland.
Abstract - Application of the FISH technique with the human 28S rDNA probe
on metaphase spreads of the Chinese raccoon dog confirmed the presence of the
NOR sequences on three A autosome pairs and the Y chromosome. Moreover,
NOR-like sequences were found on the B chromosomes in the telomeric region
of the long arm and in the proximal half of these chromosomes.
Key words: Chinese raccoon dog, B chromosome, NOR.
INTRODUCTION
In the raccoon dog, belonging to the family
Canidae, two subspecies are recognized: the Chinese raccoon dog (Nyctereutes procyonoides procyonoides Gray) 2n=54 + B, and the Japanese
raccoon dog (Nyctereutes procyonoides viverrinus
Temminck) 2n=38 + B. In spite of differences in
the chromosome number and their morphology,
extensive chromosome arm homology was
revealed by G-banding (WARD et al. 1987).
Study on the molecular nature of the B chromosomes, performed by the FISH technique with
the use of the telomeric DNA probe, revealed
the presence of these sequences throughout the
length of the B chromosomes in both subspecies
of the raccoon dog (WURSTER-HILL et al. 1988).
Recently, B chromosomes of the Japanese raccoon dog were studied by the chromosome painting approach. It was shown that the painting
probes derived from the microdissected proximal
* Corresponding author: fax +48 61 8487148; e-mail:
[email protected]
and distal fragments hybridized to the same fragments of the B chromosomes and not to other
chromosomes (TRIFONOV et al. 2002). In the present study we show that B chromosomes of the
Chinese raccoon dog contain unevenly distributed inactive NOR-like sequences.
MATERIAL AND METHODS
Blood samples were collected from four unrelated males originating from a local fur animal farm.
Metaphase spreads were obtained from mitogenic
stimulated blood lymphocytes cultures. Chromosome slides were made according to standard airdrying procedure for in vitro cultured lymphocytes.
Q-banding technique was applied to identify chromosomes prior to FISH. The staining was performed in 0.005% quinacrine mustard for 90 s.
Active nucleous organizer regions (NORs) were
detected by the Ag-I technique. Slides were incubated in distilled water (37°C) for one hour, than
50% silver nitrate was added on a slide and covered with coverslip. Slides were incubated in a moist
chamber for 20 hours at 60°C.
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and SWITONSKI
Fig. 1 – Metaphase spreads of the Chinese raccoon dog. Two partial male Q-banded metaphase spreads
(a and c) prior to FISH (b and d), and a silver stained female metaphase spread (e). Large white arrows
(b, d) indicate B chromosomes and small ones show hybridization signals on other autosomes and the
Y chromosome. Black arrows (e) show silver stained NORs.
B-CHROMOSOMES OF THE CHINESE RACCOON DOG
Standard protocol of the fluorescence in situ
hybridisation (FISH) was applied for the human 28S
rRNA gene probe (SYLVESTER et al. 1986) labelled
with biotin-16-dUTP by the random priming method.
Slides were analysed under a fluorescence microscope
(Nikon E 600 Eclipse) equipped with a CCD camera,
driven by computer aided software Lucia.
Chromosome nomenclature for the Chinese raccoon dog proposed by PIENKOWSKA et al. ( 2002) was
followed.
RESULTS AND DISCUSSION
The hybridisation signals appeared on: three A
autosome pairs, the Yq chromosome and B chromosomes. Among seventy analysed metaphase
spreads two types of B chromosomes (BI and BII),
distinguished due to their size and Q-banding patterns, were identified. Hybridisation patterns on
both types of the B chromosomes were similar. A
pronounced hybridisation signal was present in
the telomeric part of the q arm and the proximal
halves of the q arms were evenly covered with
weaker signals (Fig. 1a-d). Almost in all metaphase
spreads (69 out of 70) two B chromosomes were
found. However, different combinations of the B
chromosomes occurred in the studied animals:
animal 1 – two BI chromosomes; animal 2 and 3 one BI and one BII chromosome, and animal 4 –
a mosaic karyotype with three cell lines: two BI
chromosomes, BI + BII chromosomes and one BI
chromosome (one spread only). The distribution
of the signals on the A autosomes and on the Y
chromosome confirmed earlier data of PIENKOWSKA and ZAGALSKA (2001) on the distribution of
active NORs, visualised by silver staining (Fig. 1e).
Our observations indicate that B chromosomes
of the Chinese raccoon dog contain not only
telomeric-like sequences, as it was shown by
WURSTER-HILL et al. (1988), but also NOR-like
sequences which are inactive, since their presence
was not detected by the silver staining. However,
it should be emphasised that we can not unambiguously state what sequences were recognised
by the probe applied in this study, since sequence
of the probe was not described. In our former
studies this probe hybridised to NORs in karyotypes of the pig and coypu (SOLINAS-TOLDO et
al. 1992), and the dog (PIENKOWSKA and SWITONSKI 1997). It is well known that human spacer
sequences, which occur between rRNA genes,
demonstrate extensive homology with primates
215
and little with other mammals (SYLVESTER et al.
1986). Thus, we can assume that the probe contains at least part of the 28S rDNA sequence. The
presence of the inactive NOR-like sequences in B
chromosomes is not a unique phenomenon, since
it was also found in other mammalian species,
including some rodents (STITOU et al. 2000). We
suggest that during the evolution of the Chinese
raccoon dog karyotype accumulation and then
inactivation of the NOR-like sequences took
place.
In the family Canidae there is also another
species – red fox (Vulpes vulpes) – carrying from
one to eight small B chromosomes (2n=34 + B).
Recent studies, performed with the use of chromosome painting approach, revealed that these
structures contain centromer-like DNA sequences
(YANG et al. 1999). The authors showed that a
paint probe derived from the B chromosomes
hybridised to themselves and centromeric regions
of other chromosomes. These observation supports earlier hypothesis of GUSTAVSSON and SUNDT
(1967) that B chromosomes of the red fox are centromeric remains of the centric fusion events which
took place during karyotype evolution of this
species.
Interestingly, in both species – the raccoon
dog and red fox, in spite of difference in size and
DNA content of the B chromosomes, similar
pairing behaviour during pachytene of the meiotic prophase I was observed. Visualisation of
the synaptonemal complexes in an electron
microscopy revealed multivalent configuration
of the Bs if more than two B chromosomes were
found or selfpairing if only one B chromosome
was present (SWITONSKI et al. 1987; SHI et al.
1988).
Taking into consideration all the data mentioned above we assume that B chromosomes of
the raccoon dog contain similar DNA sequences,
rich in telomeric-like and NOR-like sequences, in
spite of the observed size differences between
the both types of the Bs (type B-I and B-II).
Moreover, it can be concluded that in karyotype
evolution of the family Canidae different mechanisms were responsible for the occurrence of B
chromosomes in the raccoon dog and red fox.
Acknowledgements – This study was supported by the Foundation for Polish Science (contract
13/2000). We wish to thank Prof. Dr.G. Stranzinger
and Dr. S. Solinas-Toldo for providing the rDNA
probe.
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SZCZERBAL
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Received October 7, 2002; accepted January 28, 2003