Chromosome Research 8: 593^601, 2000. # 2000 Kluwer Academic Publishers. Printed in the Netherlands
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Spontaneous occurrence of a Robertsonian fusion involving chromosome 19 by single whole-arm reciprocal translocation (WART) in wild-derived house mice J. Catalan1 , J.-C. Auffray1 , F. Pellestor2 & J. Britton-Davidian1 Institut des Sciences de l'Evolution, UMR 5554, Laboratoire Ge¨ne¨tique et Environnement, CC65, Universite¨ Montpellier II, 34095 Montpellier cedex 5, France; Tel: ( 33) 4 67 14 39 10; Fax: ( 33) 4 67 14 36 22; E-mail:
[email protected]; 2 Institut de Ge¨ne¨tique Humaine, CNRS UPR 1142, 141 rue de la Cardonille, 34396 Montpellier cedex 5, France 1
Received 12 May 2000; received in revised form and accepted for publication by A. Sumner 11 July 2000
Key words: chromosome 19, house mouse, mosaicism, WART
Abstract Chromosomal races of the house mouse (Mus musculus domesticus) bear Robertsonian (Rb) fusions, which consist of centric translocations between two non-homologous acrocentric chromosomes. The high level of diversity of these fusions in house mice is generated by de-novo formation of Rb fusions and subsequent whole-arm reciprocal exchanges (WARTs). This paper describes the spontaneous occurrence of a new Rb fusion, Rb(4.19), in progeny of wild-derived house mice segregating for Rb(4.12). The chromosomal mutation was traced to a female which exhibited germline and somatic mosaicism indicating an early embryonic origin of the mutation. FISH analysis of centromerically-located ribosomal genes suggested that no modi¢cation was observed on chromosomes 12 and 19 prior to or following the occurrence of Rb(4.19). Distribution of telomeric sequences showed that both Rb fusions lacked telomeres in their centromeric regions. It is argued that this spontaneous mutation most likely originated by single whole-arm reciprocal translocation (WART) between Rb(4.12) and an acrocentric chromosome 19, resulting in Rb(4.19) and a neo-acrocentric chromosome 12. Sequences required for centromeric function and proximal telomeres would have been transferred to the neo-chromosome 12 from chromosome 19 during the translocation. The existence of such WARTs which generate derived acrocentric chromosomes has several implications for chromosomal evolution in house mice.
Introduction House mice belonging to the western European subspecies Mus musculus domesticus are characterized by a 40 all-acrocentric karyotype except where Robertsonian fusions (Rb) occur leading to chromosomal races distributed throughout Europe and North Africa (Nachman & Searle
1995). Rb fusions consist of the joining of two non-homologous chromosomes by the centromere thereby producing a metacentric chromosome and reducing the diploid number (Capanna 1982). Under wild conditions, all autosomes are involved in Rb fusions although with varying frequency (Nachman & Searle 1995), the extreme case being that of chromosome 19 which has only recently
594 been found in Rb fusions in mice from Madeira (Britton-Davidian et al. 2000). The large diversity of Rb fusions, more than 90 (Nachman & Searle 1995), and the age of the races, less than 5000 years (Auffray 1993), suggest that chromosomal mutation and/or ¢xation rates are high in this subspecies. This has led several authors to investigate chromosomal mutation mechanisms and patterns in this taxon (Redi et al. 1990, Nachman & Searle 1995, Hauffe & Pia¨lek 1997, Riginos & Nachman 1999, Serakinci et al. 1999). The most common process is the fusion of two acrocentric chromosomes (John & Freeman 1975, Holmquist & Dancis 1980, Schubert et al. 1992, Nanda et al. 1995); when several Rb fusions are present in a population, additional ones may be generated by whole-arm reciprocal translocation (WART) where two Rb fusions exchange one arm producing two newly-derived ones (Winking 1986, Capanna & Redi 1995, Garagna et al. 1995, Castiglia & Capanna 1999). In crosses between wild-derived house mice carrying the Rb(4.12) fusion, a different Rb fusion was observed in some of the progeny. This observation motivated the present study with the aim to: (1) characterize this new Rb fusion, (2) analyse its distribution in the progeny, (3) investigate the stage and mechanism of the mutation event, and (4) discuss its relevance to the dynamics of chromosomal evolution in house mice.
Materials and Methods Crosses Crosses involved wild mice trapped in Belgium in 1992. Eighty-¢ve standard mice (2n 40) were captured in six localities south of Namur (Space, Natoye, Assesse, Ohey, Gesves, Maillen) and 67 Rb mice (2n 38) homozygous for Rb(4.12) in one locality north of Namur (Corroy-Le-Grand; see Bauchau et al. 1990 and Dallas et al. 1998 for additional information on the distribution of Rb races in Belgium). Reciprocal interracial crosses were performed to produce an F1 generation of mice with 2n 39 chromosomes, as well as intraracial ones to generate F1 standard mice. Fifty-nine pairs between these two types of F1 mice were set up to produce a backcross gener-
J. Catalan et al. ation in which transmission of Rb(4.12) was analysed. A total of 1865 progeny were karyotyped. Chromosomal analyses Mitotic metaphases were obtained by the air-drying technique from bone marrow cells after yeast stimulation (Lee & Elder 1980). The presence/absence of Rb(4.12) was scored on conventionally stained preparations. Identi¢cation of chromosomal arms in the Rb fusions was performed by G-banding according to the technique of Seabright (1971) and the nomenclature of Cowell (1984). All observations were made with a Zeiss Axiophot £uorescent microscope. Karyotying was performed by image analysis using the Genevision software (Applied Imaging). Fluorescent in-situ hybridization (FISH) Prior to FISH, identi¢cation of chromosomes was performed by G-banding. Forty or more banded metaphase plates were scored for each individual and ¢led by image analysis. Slides were then destained in several changes of 3 : 1 methanol : acetic acid and absolute alcohol, air-dried, post¢xed with 3.7% formaldehyde for 15 min, washed twice in PBS, and air-dried (Klever et al. 1991). Cloned gene fragments of the mouse 28S (BE2-pSP64, 1.5 kb; Hassouna et al. 1984) and 18S (SalC-pSP64, 2 kb; Raynal et al. 1984) were labelled separately with biotin-14-dATP by nick translation according to the Gibco BRL protocol and then added to the same hybridization solution. The hybridization and staining procedures were performed according to Eyche©ne et al. (1992) and Muleris et al. (1996) except for denaturation of the G-banded slides which was reduced to 1 min. Following hybridization, slides were incubated successively with antibiotin antibody and £uorescein-conjugated IgG, counterstained with propidium iodide and mounted in Vectashield antifade solution (Vector Laboratories, Burlingame, CA). Fluorescent signals were localized on G-banded metaphases (Zeiss ¢lter: 487909) and photographed at 1000 magni¢cation on Kodak Ektachrome 400 colour slide ¢lm. Images were obtained from scanned colour
Single arm translocation in house mice
595 (0.02 mg/ml) in Vectashield antifade solution (Vector Laboratories, Burlingame, CA). Visualization of signals and photomicrographs followed the same procedure as in the FISH analysis.
slides and printed with Corel Photo Paint on an Hewlett Packard 2000C printer. Primed in-situ (PRINS) labelling Slides of freshly-prepared metaphase spreads were denatured by immersion in 70% formamide, 2 SSC, pH 7.2 at 72 C for 2 min, dehydrated in a series of ice-cold ethanol washes (70%, 90%, 100%) and then air-dried. Telomere repetitive sequences were labelled by PRINS using the telomeric consensus primer (CCCTAA)7 . For each slide, a reaction mixture was prepared in a ¢nal volume of 50 ml containing the oligonucleotide (200 pmol), 0.1 mmol/L each of dATP, dCTP, dGTP, 0.002 mmol/L dTTP, 0.025 mmol/L of £uorescein-12-dUTP (Boehringer Mannheim, Meylan, France), 50 mmol/L KCl, 10 mmol/L Tric^HCl, pH 8.3, 1.5 mmol/L MgCl2 , 0.01% bovine serum albumin and 2 units of Taq DNA polymerase (Perkin-Elmer, Cetus, Norwalk, CT). The reaction was performed on a programmable temperature cycler Hybaid Omnigene, ¢tted with a £at plate block. Denatured slides were put on the plate block, the reaction mixes placed on the slides and overlaid with coverslides (22 30 mm). The reaction programme consisted of two steps: 10 min at the speci¢c annealing temperature of the primer (60 C) and 30 min at 72 C in order to allow nucleotide chain elongation. The reaction was arrested by immersing the slides in a stop solution (500 mmol/L NaCl/50 mmol/L EDTA, pH 8) at 70 C for 2 min. The slides were then washed in 4 SSC ^ 0.5% Tween 20 at room temperature and directly counterstained with propidium iodide
Results Identi¢cation and distribution of the spontaneous mutation The chromosomal analysis by conventional staining of the backcross progeny revealed that 974 mice carried the standard karyotype and 891 an Rb fusion. Among the latter, four individuals presented a new Rb fusion which was identi¢ed by G-banding as Rb(4.19). These mice were siblings belonging to several litters of a male (2n 40) female (2n 39) pair (Table 1). The distribution of karyotypes within the 49 progeny of this pair showed that 8% carried Rb(4.19), 31% inherited the Rb(4.12) fusion and 61% presented the standard karyotype. These results suggest that the spontaneous Rb fusion had occurred in one of the parents. A reanalysis of the parental karyotypes showed that the 2n 39 mother was a mosaic with two types of cells (Figure 1a, b): among 106 bone marrow cells, 24% carried Rb(4.19) and 76% Rb(4.12). No cells carrying both Rb fusions were observed. Chromosome preparations from the father, six siblings of the mother and the wild grandparents were reanalysed and revealed no discrepancy from the expected karyotypes. These results suggest that the chromosomal mutation occurred in the female parent at an early stage of development since both
Table 1. Diagram of the mating scheme showing segregation of Rb fusions. Wild grandparents Parents
2n 40 2n 40 2n 40 r
2n 40 2n 38 u 2n 39 mosaic u
Karyotype
Sex
1
2
Litters 3
4
5
6
2n 40
u r
1 1
3 2
4 4
4 3
2 2
4 0
2n 39 Rb (4.12)
u r
2 1
2 0
1 1
2 1
0 2
2 1
2n 39 Rb (4.19)
u r
0 0
0 1
0 0
0 0
1 0
2 0
596
J. Catalan et al.
Figure 1. G-banded metaphases from bone marrow cells of the mosaic female showing a metaphase with (a) Rb(4.19) and (b) Rb(4.12) indicated by arrowheads. Bar indicates 10 mm.
bone marrow and germline cells were chromosome mosaics (Drost & Lee 1998). Characterization of the centromeric regions of the Rb fusions and of chromosomes 12 and 19 As Rb fusions involve centromeric regions of acrocentric chromosomes, the occurrence of Rb(4.19) may be related to or have resulted in structural modi¢cations of these regions in chromosomes 12 and 19. Two types of centromerically located sequences were analysed to characterize the centromeric regions of these chromosomes. The ¢rst one involved in-situ hybridization of rDNA probes of ribosomal genes which are organized in chromosomal clusters of tandemly repeated units. This was performed on the mosaic female and a daughter carrying Rb(4.19). Results showed that the ¢ve major centromeric rDNA-bearing chromosomes common to M. m. domesticus (i.e. 12, 15, 16, 18 and 19; Dev et al. 1977, Kurihara et al. 1994) as well as an additional site with a very low copy number on one chromosome 5, were present in
the two mice (Figure 2a^d). In both females, rDNA genes were always detected on both chromosomes 19 whether in a metacentric or an acrocentric state. However, in the mosaic female, differences in labelling were observed between the chromosome 12 homologues. In Rb(4.12) cells, a £uorescent signal was always observed on the metacentric chromosome, whereas labelling of the acrocentric chromosome 12 was less frequent (65% of cells analysed). These relative differences in signal detection suggest that the two chromosomes 12 could be distinguished by the number of rDNA sequence copies, the metacentric chromosome carrying a larger number than the acrocentric one (Maluszynska & Heslop-Harrison 1991, Leitch & Heslop-Harrison 1992). Such rDNA polymorphisms have previously been described in house mice (Suzuki et al. 1990), Similar differences in labelling were found between the two acrocentric chromosomes 12 in Rb(4.19) cells of the mosaic female (100% labelling of one chromosome 12, 60% of the other) (Figure 2a, c). FISH analysis of chromosome preparations from the Rb(4.19) heterozygous daughter revealed
Figure 2. Opposite. (a^d) Sequential G-banding and FISH of rDNA probes of a mosaic female Rb(4.19) cell (a, c) and her Rb(4.19) daughter (b, d); arrowheads indicate rDNA labelling and corresponding chromosomes. Notice that labelling is absent in one of the chromosomes 12 of the mother (c). (e) G-banding of chromosomes 12 in the father, mother and daughter. Brackets indicate slightly extended centromeric regions. (f) PRINS of telomeric sequences of the Rb(4.19) son; no telomeric signal is detected in the centromeric region of the Rb fusion (arrowhead). Bars indicate 10 mm.
Single arm translocation in house mice
597
598 that both chromosomes 12 were labelled in all cells, suggesting probable inheritance of the high rDNA copy number chromosome 12 from the mosaic mother (Figure 2b, d). The results of the FISH analyses thus suggested that no modi¢cation of the centromerically located ribosomal genes was observed either prior to or following the occurrence of Rb(4.19) and allowed us to trace the new acrocentric chromosome 12 to the original metacentric form. Furthermore, banding analysis of the daughter revealed a difference in structure of the two acrocentric chromosomes 12, one with a standard pattern and the other showing a slightly extended centromere. However, G-banding of the parental karyotypes indicated that the latter chromosome was inherited from the 2n 40 father and not related to the Rb-carrying lineage (Figure 2e). As Rb fusions typically lack centromeric telomeres in house mice (Schubert et al. 1992, Nanda et al. 1995, Garagna et al. 1995), a second analysis was performed using the PRINS technique to determine the distribution of telomeric sequences in the mosaic female and the son carrying Rb(4.19). In both mice, no telomeric sequences were detected in the centromeric regions of either Rb(4.12) or Rb(4.19), whereas they were present at the distal ends of these chromosomes as well as at both ends of the acrocentric ones (Figure 2f). Discussion In house mice, chromosomal differentiation is known to occur by Rb fusion of two acrocentric chromosomes (John & Freeman, 1975), and further diversi¢cation by whole-arm reciprocal exchanges between existing Rb fusions (WARTs; Winking 1986, Capanna & Redi 1995, Garagna et al. 1995, Castiglia & Capanna 1999). In laboratory crosses between Belgian Rb mice segregating for Rb(4.12), a new fusion Rb(4.19) occurred in a 2n 39 mosaic female and was transmitted to its progeny. This new fusion may have originated by one of the two mechanisms mentioned above. Following the ¢rst one, de-novo formation of Rb(4.19) would have occurred in a Rb(4.12)bearing cell by fusion of the remaining acrocentric chromosome 4 and one of the chromosomes 19.
J. Catalan et al. However, such a process is highly unlikely as Rb(4.19) never occurred simultaneously with Rb(4.12) in the same cells, suggesting that these two Rb fusions were not independent events. The second mechanism would imply an exchange of chromosomal arms between Rb(4.12) and chromosome 19 resulting in Rb(4.19) and an acrocentric chromosome 12. This is supported by the FISH analysis which identi¢ed a higher rDNA copy number on the acrocentric chromosomes 12 in Rb(4.19) cells, similar to the metacentric one in Rb(4.12) cells. The only other case relating a similar event is the identi¢cation of several intercentric translocations in the progeny of Rb-carrying laboratory house mice following exposure to X-rays (Crocker & Cattanach 1981). Recent studies on house mice have demonstrated that the centromeric regions of Rb fusions lack telomeres and a varying amount of the centromeric minor satellite DNA sequences mice (Schubert et al. 1992, Nanda et al. 1995, Garagna et al. 1995). These results suggest that different Rb fusions involve breakage and rejoining at various sites within the minor satellite DNA regions of acrocentric chromosomes. Both Rb(4.12) and Rb(4.19) conform with these results since the PRINS analysis showed that no telomeric sequences were detected in the centromeric regions of either of these metacentrics. However, the same analysis showed that the proximal ends of acrocentric chromosomes bore telomeric repeats which are required for the maintenance of chromosomal stability and integrity (Zakian 1995). This suggests that these sequences must have been reacquired by the neoacrocentric chromosome 12 (neo-12) during the process. Two sources of telomeres are possible: (1) a translocation of repeats from chromosome 19 to chromosome 12 during the exchange, or (2) de-novo formation at the end of the neo-12. The latter has been shown to occur through healing of chromosomal broken ends by seeding of telomeric sequences, and requires that telomerase be expressed in the cell tissue (Cooke 1995). Several studies have indicated that, in most mammalian adult cells, telomerase is not expressed and telomeres are progressively shortened during each cell cycle due to conventional DNA replication mechanisms. In speci¢c
Single arm translocation in house mice tissues such as the gonads, however, telomerase is active and replaces lost telomeric sequences (Prowse & Greider 1995). The existence of a somatic and germinal mosaicism in the female carrier indicates that the mutation occurred at a very early stage of embryonic development during which telomerase activity is also known to be present at least up to the blastocyst cell stage in human embryos (Wright et al. 1996). The ability of cells to seed telomeric repeats onto chromosomal fragments varies, but mouse embryonic stem cells are known to be ef¢cient healers (Cooke 1995). These data suggest that the proximal telomeres present on the neo-12, may have originated by either of the two processes. However, Rb fusions in wild mice lack a large fraction of minor satellite DNA (Garagna et al. 1995), sequences of which may be associated with centromeric function (Mitchell 1996, Karpen & Allshire 1997). This suggests that, in addition to one functional centromere (Haaf et al. 1989), Rb fusions probably have no latent or inactive centromeric material available. In this case, transfer of these sequences from chromosome 19 to the neo-12 chromosome would be required to restore its centromeric function. Such arguments provide support for a reciprocal translocation event between Rb(4.12) and chromosome 19. Such an exchange has been termed a WART (Searle 1990) and in this case involves not two Rb fusions but one Rb fusion and an acrocentric chromosome (see type-b WART in Hauffe & Pia¨lek 1997). The existence of a somatic and germ cell mosaicism following a spontaneous chromosomal mutation is the ¢rst described in wild-derived house mice and indicates that the rearrangement appeared before germline determination (Drost & Lee 1998). This spontaneous chromosomal rearrangement has three important implications for chromosomal evolution. The ¢rst one is that the occurrence of the mutation early during development may allow for a higher transmission frequency than one appearing during the meiotic stages of gametogenesis, as a much larger number of mutant cells may be involved (Capanna & Redi 1994, Drost & Lee 1998, Selby 1998). Such early cleavage mutations may thus contribute to a high rate of ¢xation of Rb rearrangements in house mice (Woodruff & Thompson 1992, Woodruff
599 et al. 1996). Additionally, Castiglia & Capanna (1999) recently suggested that hybridization events between all-acrocentric and Rb races or between the latter may trigger the occurrence of WARTs in house mice. Our results support this view as the mosaic female is a ¢rst-generation hybrid between wild homozygous chromosomal races. The third evolutionary contribution lies in the production of a new functional acrocentric chromosome. Whereas Rb ¢ssions are considered as highly unlikely in house mice due to the loss of proximal telomere and minor satellite DNA sequences (Garagna et al. 1995), our results suggest that such single WARTs may be the most likely mechanism allowing reversion to an ancestral acrocentric form. However, as the number of Rb fusions remains constant, this type of WART must be considered as an additional mechanism for chromosomal diversi¢cation rather than a means of reversal to an all-acrocentric karyotype. An additional outstanding feature of this spontaneous mutation is the presence of chromosome 19 which is the only autosome to be very rarely observed in Rb fusions in wild mice (Nachman & Searle 1995, Britton-Davidian et al. 2000). Several authors have ascribed the extremely infrequent occurrence of Rb fusions involving chromosome 19 in wild mice, to their high selective disadvantage (Nachman & Searle 1995). Recent morphological studies using £uctuating asymmetry as a measure of developmental stability, were not able to uncover differences between heterozygotes carrying Rb(4.19) or Rb(4.12) and all-acrocentric mice, suggesting a similar level of developmental ¢tness (Auffray et al. in press). Fertility assays remain to be performed to assess the nature and extent of counterselection associated with Rb fusions involving chromosome 19. Acknowledgements We are very grateful to H. Croset and J. Grobert for their enthusiastic and continuous contributions to this project. Many thanks are extended to S. Garagna and P. Bachellerie for providing DNA probes. This work was supported by the Programme du Ministe©re de l'Education Nationale (Approche interdisciplinaire et de¨veloppement
600 me¨thodologique: ``Etude des perturbations de l'environnement cellulaire sur la transmission d'une fusion centrique''), as well as grants from the De¨partement Biologie Evolution Environnement of the University Montpellier II, and from the CNRS and the University Montpellier II to UMR5554. We gratefully acknowledge the CNRS-SDV and la Re¨gion Languedoc-Roussillon for ¢nancing the Zeiss photomicroscope and image analysis systems. This is publication ISEM No. XX-XXX. References Auffray J-C (1993) Chromosomal divergence in house mice in the light of palaeontology: a colonization-related event? Quarternary Int 19: 21^25. Auffray J-C, Fontanillas P, Catalan J, Britton-Davidian J (2000) Developmental stability in house mice heterozygous for single Robertsonian fusions. J Hered (in press). Bauchau V, Smets S, Viroux M-C, Nootens D, de Caritat A-K (1990) Robertsonian translocation in free-living populations of the house mouse in Belgium. Biol J Linnean Soc 41: 193^201. Britton-Davidian J, Catalan J, Ramalhinho M-G et al. (2000) Rapid chromosomal evolution in island mice. Nature 403: 158. Capanna E (1982) Robertsonian numerical variation in animal speciation: Mus musculus, an emblematic model. In: Barigozzi C, ed. Mechanisms of Speciation. New York: Alan R. Liss Inc., pp 155^177. Capanna E, Redi CA (1994) Chromosomes and the microevolutionary processes. Boll Zool 61: 285^294. Capanna E, Redi CA (1995) Whole-arm reciprocal translocation (WART) between Robertsonian chromosomes: ¢nding of a Robertsonian heterozygous mouse with karyotype derived through WARTs. Chromosome Res 3: 135^137. Castiglia R, Capanna E (1999) Whole-arm reciprocal translocation (WART) in a feral population of mice Chromosome Res 7: 493^495. Cooke H (1995) Non-programmed and engineered chromosome breakage. In: Blackburn EH, Greider CW, eds. Telomeres. Cold Spring Harbor Laboratory Press, pp 219^245. Cowell JK (1984) A Photographic representation of the variability of G-banded structure of the chromosomes of the mouse karyotype. Chromosoma 89: 294^320. Crocker M, Cattanach BM (1981) X-ray induction of translocations in mice carrying metacentrics (Robertsonian fusions); detection of whole arm chromosome exchanges. Mutation Res 91: 353^357. Dallas JF, Bonhomme F, Boursot P, Britton-Davidian J, Bauchau V (1998) Population genetic structure in a Robertsonian race of house mice: evidence from microsatellite polymorphism. Heredity 80: 70^77.
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