The stability of telomereless chromosome fragments in adult androgenetic rainbow trout
1 Department of Evolutionary Genetics, University of Warmia and Mazury in
Olsztyn, 10-957 Olsztyn, Poland
2 Department of Animal Biochemistry and Biotechnology, University of Warmia
and Mazury in Olsztyn, 10-957 Olsztyn, Poland
3 Department of Fisheries and Natural Sciences, Bodø Regional
University, 8049 Bodø, Norway
4 Department of Salmonid Research, Inland Fisheries Institute in Olsztyn,
Rutki, 83-330 Zukowo, Poland
5 Department of Fish Biology and Farming, University of Warmia and Mazury in
Olsztyn, 10-957 Olsztyn, Poland
* Author for correspondence at present address: Laboratoire de Genetique des Poissons, Institut National de la Recherche Agronomique, Centre de Recherche de Jouy-en-Josas, Domaine de Vilvert, F-78352 Jouy-en-Josas Cedex, France (e-mail: kocale{at}diamant.jouy.inra.fr)
Accepted 1 April 2004
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Summary |
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Key words: androgenesis, centromere, chromosome, fish, gamma radiation, telomere
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Introduction |
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Ends of eukaryotic chromosomes are capped with nucleo-protein complexes
named telomeres, which serve many functions, such as protecting chromosome
ends from degradation during the cell cycle and preventing chromosome
end-to-end fusions. The DNA component of a telomere is built of short,
tandemly repeated G-rich sequences, such as TTAGGG in vertebrates
(Blackburn, 2001). Telomeric
probes are widely used to investigate chromosomal rearrangements because in
many vertebrates they appear frequently at interstitial sites of chromosomes
(Meyne et al., 1990
). These
interstitial telomeric sites (ITS) can be remnants of chromosome
rearrangements (fusions or inversions) occurring in genome evolution
(Go et al., 2000
;
Castiglia et al., 2002
;
Slijepcevic, 1998
) or
aberrations (Natarajan et al.,
1996
; Slijepcevic et al.,
1997
). Telomeres allow the repairing system to distinguish between
natural chromosome ends and chromosomal breaks such as double strand breaks
(DSB). These can occur spontaneously during DNA replication, formation of
dicentric chromosomes followed by breakage during chromosome segregation or by
recombination. Also, DSB can be induced experimentally by subjecting DNA to
damaging agents, such as radiation, alkylating agents, certain antibiotics or
endonucleases (Obe et al.,
2002
). The naked ends of chromosomes arrest the cell cycle, and
the repairing process starts before chromosome aberrations become irreparable
and kill the cell (Crompton,
1997
).
Cellular repairing mechanisms can fix the breaks de novo by
healing the chromosome end with a new telomere built by a telomerase, addition
of telomere-associated DNA sequences or through specific retrotransposons, or
recombination (Biessmann and Mason,
1997). Fusion of chromosomes that have lost their telomeres or
fusion during subsequent cell divisions, termed the breakagefusion
bridge (B/F/B) cycle, is another repair mechanism
(McClintock, 1941
).
Most of the data concerning chromosome fragmentation and cell response to
this event were obtained through irradiating cell lines with mild radiation
(Natarajan et al., 1996;
Slijepcevic et al., 1996
).
Much higher irradiation doses are applied to inactivate nuclear DNA in fish
gametes during induced artificial gynogenesis or androgenesis, i.e. in order
to produce individuals possessing exclusively maternal or paternal nuclear
DNA, respectively (Thorgaard,
1986
). These manipulations include oocyte (androgenesis) or
spermatozoa (gynogenesis) irradiation with UV, gamma rays or X rays, followed
by fertilization and diploidization of a haploid zygote with thermal or
hydrostatic pressure shock, which in turn results in retention of the second
polar body (heterozygous gynogenesis) or blockage of the first mitotic
division (homozygous gynogenesis and androgenesis) of the zygote
(Thorgaard, 1986
). Although
the radiation used for induced gynogenesis or androgenesis is believed to
eliminate paternal or maternal nuclear DNA from the subsequent zygote
entirely, chromosome fragments have been found in some cells of androgenetic
or gynogenetic embryos and larvae. They are supposed to be residues of the
irradiated genome (Parsons and Thorgaard,
1985
; Chourrout,
1986
; Lin and Dabrowski,
1998
); however, to our knowledge no thorough investigation on
their characteristics has been reported so far. As gyno- and androgenetic
individuals are viable, they seem to be a good in vivo model for
investigating fate of chromosome fragments during cell division as well as the
cell response to chromosome breaks and mechanism of replication of chromosome
fragments. In the present study, we performed a cytogenetic analysis of
chromosomal fragments, putative maternal remnants in the genome of
androgenetic rainbow trout. Androgenetic individuals were adult and normally
developed. This ensured that the replication process of chromosome fragments
was active through multiple cell divisions. Using conventional and molecular
cytogenetic techniques, we have identified telomereless chromosome fragments
and investigated chromosomal rearrangements that occurred as a consequence of
fusions of putatively maternal chromosome fragments and intact paternal
chromosomes. Mechanisms of chromosome fragment stability and repair of the
chromosome breaks are discussed.
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Materials and methods |
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Cytogenetic analysis
Metaphase chromosomes were obtained according to the standard air-dry
procedure (Rab and Roth, 1988)
or short-term culture of blood cells
(Martinez et al., 1991
). To
estimate the chromosome number, metaphase plates were stained with 10% Giemsa
solution (Sigma, Poznan, Poland) for 10 min. Chromosomes were stained with
4',6-diamidino-2-phenylindole (DAPI) to identify AT-rich regions
(Ocalewicz et al.,
2003
). Three drops of antifade solution (Vectashield; Vector
Laboratories, Burlingame, USA) containing DAPI (1.5 µg
ml1) were dropped onto a slide and covered with a coverslip.
Above 30 metaphases per individual were examined.
Primed in situ labelling (PRINS) was performed using two systems:
rhodamine PRINS reaction set kit (Roche, Mannheim, Germany;
Ocalewicz and Babiak, 2003) or
PRINS reaction with FITC-labelled dUTP. In both cases, (CCCTAA)7
primers were used. The first procedure followed manufacturer's protocol. In
the latter procedure, chromosomes were denatured by incubation in 70%
formamide in 2xSSC at 70°C for 3 min and quenched in ice-cold
2xSSC and 70% ethanol. Then, the slides were dehydrated using an
ice-cold ethanol series (70%, 80%, 96%), for 2 min each, and air dried. The
PRINS reaction mixture consisted of dATP, dGTP, dCTP and FITC-dUTP (Roche; 0.5
µl each), 2.5 µl of glycerol (Sigma), 5 µl of Taq polymerase buffer
(Promega, Madison, USA), 3 µl of (CCCTAA)7 telomere primer (100
pmol µl1), 0.5 µl of Taq polymerase (Promega; 5 U
µl1) and 37 µl of dH2O. The mixture was
dropped on a slide and covered with a coverslip. Slides were placed in a humid
chamber at 65°C for 35 min in order to anneal primers and extend the new,
labelled DNA strand. After reaction, coverslips were gently removed and the
slides were transferred to stop buffer (50 mmol l1 EDTA, pH
8) heated to 60°C. After 5 min of incubation in stop buffer, slides were
washed three times for 5 min each in a washing buffer [4xSSC/0.05% Tween
20 (ICN Biomedicals, Aurora, USA), pH=7] and once in phosphate-buffered saline
at room temperature. Chromosomes were air dried in the dark at room
temperature and counterstained with 10 µl of antifade solution
(Vectashield) containing propidium iodide (PI; Vector Laboratories).
Metaphase plates were analyzed under a Nikon Optiphot microscope equipped with a fluorescent lamp and digital camera. Chromosomes were scored under fluorescent light and filters: UV-1A (DAPI), B-2A (FITC/PI), FITC/rhodamine/DAPI filter (rhodamin).
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Results |
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Chromosome fragments were found in cells of five individuals (31%), and their number ranged from 1 to 3 per specimen. These fragments were smaller than the smallest rainbow trout acrocentric chromosomes (Table 1; Fig. 2), but in individuals 14 and 15, chromosome fragments were bigger than chromosome fragments in other fish (Fig. 2). DAPI banding showed distinct bright, fluorescent patterns of AT-rich chromatin on intact chromosomes as well as fluorescent spots on some of the chromosome fragments (Table 1; Fig. 2). Specifically, all chromosome fragments in individuals 2, 10 and 12 were stained bright (Fig. 2A,C), whereas DAPI-negative chromosome fragments were found in individuals 14 and 15 (Fig. 2B,D). PRINS signals were observed on both ends in all intact chromosomes but only in some chromosome fragments, namely in two of three fragments in individual 15 (Fig. 3). In specimen 12, ITS were found in a paracentromeric position on four metacentric chromosomes (Fig. 4A,C). No ITS were found in chromosomes of control individuals (Fig. 4B).
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Discussion |
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Some of the observed fragments showed bright DAPI-positive signals
(Fig. 2) similar to centromeres
in normal rainbow trout metacentric or X chromosomes
(Ocalewicz, 2002). Function of
kinetochores could be retained in centromeres of the chromosome fragments. The
DAPI-negative fragments in fishes 14 and 15 could be centromeric remnants of
chromosomes with no AT-rich chromatin in centromeric regions, similar to those
observed in acrocentric chromosomes
(Ocalewicz et al., 2003
).
Centromeres are heterochromatic structures and are less sensitive to damage
caused by irradiation (Blackburn and
Szostak, 1984
), therefore higher resistance to the radiation in
observed fragments could be attributed to their centromeric origin.
Different diploid chromosome numbers in androgenetic individuals result
from Robertsonian rearrangements, which are common in rainbow trout. Fish from
the Rutki strain show Robertsonian polymorphism in chromosome number, which
varies from 59 to 62 (Ocalewicz,
2002). The father of examined individuals probably possessed 59
chromosomes. His haploid spermatozoa contained either 29 or 30 chromosomes.
After diploidization in the course of androgenesis, resulting progeny should
have 58 or 60 chromosomes. Specimens having 62 chromosomes differ from this
simple segregation model, however. It might be because of abnormal meiotic
disjunctions in testes of the father. Such anomalies in males having odd
numbers of chromosomes (2n=59, 61) were reported by Nakayama and Chourrout
(1993
).
It is hypothesized that centromeres, telomeres and replication origins are
necessary for retaining chromosomal functionality during the cell cycle
(Blackburn and Szostak, 1984).
Our results indicate that telomereless chromosome fragments having centromeric
regions are as stable as regular chromosomes. These fragments apparently do
not disturb the cell cycle and function. Also, the fragments do not trigger
repairing mechanisms to rebuild the telomeric structures on their naked ends.
In yeast, ends of artificially broken chromosome arrest the cell cycle in the
absence of functioning repair mechanisms, although some cells eventually
re-enter the cell cycle (Sandel and
Zakian, 1993
). Our study demonstrates for the first time the
stability of telomereless fragments in a vertebrate.
The fusion of chromosome fragments as revealed by ITS appearance is
frequently observed in irradiated mammalian somatic cells
(Slijepcevic et al., 1997). In
the present study, the chromosome fragments missing centromeres might have to
undergo a process of DNA incorporation into intact chromosomes. ITS found on
metacentric chromosomes (fish No. 12) could be the remnants of such a process
(Fig. 4).
In conclusion, the lack of telomere sequences on chromosome fragments in androgenetic rainbow trout did not disturb the replication process and segregation of the fragments into daughter cells. We hypothesize that the stability of such fragments is possible because of the presence of active centromeres. Fragments lacking functional centromeres could incorporate into intact chromosomes, forming ITS.
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Acknowledgments |
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References |
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