Complex satellite DNA reshuffling in the polymorphic t(1;29) Robertsonian translocation and evolutionarily derived chromosomes in cattle R. Chaves1, F. Adega1, J. S. Heslop-Harrison 2, H. Guedes-Pinto1 & J. Wienberg3 1
Department of Genetics and Biotechnology, Centre of Genetics and Biotechnology ICETA-UTAD, University of Tras-os-Montes and Alto Douro, P-5000-911 Vila Real, Portugal; Tel: þ 351 259 350 543; Fax: þ 351 259 350 572; E-mail: [email protected]
; 2Department of Biology, University of Leicester, Leicester LE1 7RH, UK; 3Institute of Human Genetics, GSF Forschungsyentrum fuer Umwelt und Gesundheit and Department of Biology II, University of Munich, D-80333, Munich, Germany
Key words: Bos taurus, centromere, in-situ hybridization, satellite DNA, translocation
Abstract We have analysed and mapped physically the satellite I, III (subunits pvu and sau) and IV DNA sequences in cattle using in-situ hybridization. Four breeds were analysed including individuals with a chromosome number of 2n ¼ 60 and individuals with the widespread t(1;29) in the homozygous (2n ¼ 58) and heterozygous state (2n ¼ 59). All three satellite DNA families were present at the centromeres of the many but not all of the autosomal acrocentric chromosomes, and essentially absent from the sex chromosomes. In the translocated t(1;29) chromosome, the satellite DNA families showed a different pattern from that simply derived by fusion of the acrocentric autosomes and loss of satellite sequences, with no variation between breeds. A model of centromeric evolution is presented involving two independent events. Knowledge of mechanisms of translocation formation within cattle is important for a functional understanding of centromere and satellites, investigation of chromosomal abnormalities, and for understanding chromosomal fusion during evolution of other bovids and genome evolution in general.
breakage and reunion occurring on the short arms of both acrocentric chromosomes (Gravholt et al.1992). Here, both centromeres are retained and one is subsequently inactivated. Interest in Robertsonian translocations comes not only from their high frequency in mammals and from their in£uence on fertility (in mice, e.g. Garagna et al. 2001, and human as well as cattle) but also from their great potential as mechanistic models to study evolutionary chromosome events. The Robertsonian translocation t(1;29) is frequent in Iberian (among other) cattle breeds. Iannuzzi et al. (1987, 1992) have surmised that the t(1;29) is still evolving in cattle by progressive losses of centromeric heterochromatin. They reported the t(1;29) as being of a monocentric nature that involved loss of chromosome 29 centromere and retention of chromosome 1 centromere. Along with these data, the reduction in fertility due primarily to increased embryonic mortality, and the widespread polymorphisms in di¡erent breeds carrying t(1;29), lead us to suggest that the chromosome arises other than by a simple Robertsonian translocation only (Chaves et al.
The cattle chromosome complement includes 29 pairs of acrocentric autosomal chromosomes, plus the submetacentric X and Y sex chromosomes. Most mammalian chromosomes have satellite DNA located at or near the centromeres, and these are widely thought to play a role in centromere activity (Lee et al. 1997). Many structural chromosome rearrangements have been reported, some being widespread and having little or no e¡ect on either phenotype or fertility (Gustavsson 1974, Iannuzzi et al. 1990). Robertsonian translocations most often derive from two non-homologous acrocentric chromo- somes that fuse at or close to their centromeres to generate a biarmed, metacentric or submetacentric chromosome. They may be of monocentric or dicentric origin (Niebuhr 1972, Garagna et al. 2001). Monocentric translocations are expected to arise from fusion of two acrocentric chromosomes where the breakpoint is on the short arm in one of the chromosomes and on the long arm in the other. Dicentric translocations, however, are the result of 1
2000). At least eight families of satellite DNAs have been identi¢ed in the bovine genome, constituting about 23% of the entire genome (Schildkraut et al. 1962, Polli et al. 1966, Kurnit et al. 1973, Cortadas et al. 1977, Macaya et al. 1978, Choo 1997). Some share motifs such as 31-bp or 23-bp subrepeats, and it has been suggested that the 23-mer is evolutionarily older (Taparowsky & Gerbi 1982, Jobse et al. 1995). One of the repeats, satellite I, is widely considered as primitive in Bovidae, being present at the centromere of acrocentric chromosomes. Biarmed chromosomes that are derived from the primitive condition partially (or completely) lose satellite I from the centromeric region. Chaves et al. (2000) demonstrated that there was no detectable satellite I DNA visualized by £uorescent in-situ hybridization (FISH) on the t(1;29) chromosomes and, consequently, that there was a rearrangement of the centromere region (and complete retention of the chromosome 1 centromere structure was not likely). Chaves et al. (2000) suggested possible mechanisms of origin of the t(1;29) chromosome. Here we aimed to investigate this region with additional molecular probes. We analysed the bovine satellite IV DNA and newly isolated members of satellite III DNA family in di¡erent individuals carrying 1, 29 and t(1;29) chromosomes in homozygous and heterozygous states (2n ¼ 60, 58 and 59) by in-situ hybridization.
satellite IIIpvu and satellite IIIsau were designed following Nijman & Lenstra (2001) and are presented in Table 1. Genomic DNA was extracted from peripheral blood of a Barrosa˜ individual and ampli¢ed with the primers by a PCR carried out in a volume of 50 ml with standard methods. Between 50 and 250 ng of genomic DNA was used as the template with an initial denaturing step at 93 C for 2 min followed by 30 cycles of 92 C for 45 s, 60 C for 45 s and 72 C for 45 s. A ¢nal extension at 72 C for 10 min completed the programme. The PCR products were cloned into a plasmid pCR 4-TOPO (Invitrogen) and partially sequenced. DNA sequences were analysed using BLASTN searches of the Genbank and EMBL databases. Sequence data from the satellite IV clone (cBtGBIV-1) was already deposited in the Genbank database under Accession number AF446392. The satellite III clones were named cBtGBIIIpvu-2 and cBtGBIIIsau-3. GTG-banding and GTD-banding Air-dried slides were aged at 65 C for 6 h or overnight and were then submitted to standard G-banding with trypsin and staining with Giemsa (GTG-banding). Prior to FISH, chromosome preparations were submitted to G-banding procedures with trypsin, but, instead of staining, the slides were re¢xed in paraformaldehyde (Chaves et al. 2002) and stained with DAPI (40 ; 60 -diamidino-2-phenylindole) (GTD-banding; Figure 1). Karyotyping followed the standardization of the domestic bovid karyotypes (ISCNDB 2000).
Materials and methods Chromosome preparations
DNA probes and £uorescent in-situ hybridization (FISH)
Three Portuguese cattle (Bos taurus) breeds, Alentejana, Barrosa and Mirandesa and the Spanish breed Alistana-Sanabresa were analysed. Chromosome preparations were made from shortterm lymphocyte cultures of whole blood samples from a total of 40 individuals, 10 from each different breed (Chaves et al. 2002). All four breeds carried t(1;29).
Since no bovid chromosome paints were available, we painted cattle chromosomes with the probes for sheep chromosome 21 (Burkin et al. 1997) which is homologous to cattle chromosome 29. As described by Wienberg et al. (1997), 15 ml of the hybridization mixture (50% formamide, 2 SSC, 10% dextran sulphate, using 500 ng of each paint probe, 5 mg of sonicated sheep genomic DNA, 10 mg of salmon sperm DNA) were denatured at 70 C for 10 min, allowed to preanneal for 90 min at 37 C, dropped on denatured chromosome preparations and mounted with 22 22-mm cover slips. Slides were denatured in 70% formamide, 2 SSC at 65 C for 30 s. In-situ hybridization was performed for 48 h at 37 C. Detection of the hybridization signal was as published (Chaves et al. 2002) with the most stringent wash being in
Satellite probe isolation A satellite I cattle clone (pBtKB5) reported by Chaves et al. (2000) and with the Accession number AJ293510 (EMBL Nucleotide Sequence Database Accession) was used as probe, as it is representative of the satellite I family from Bos taurus. PCR speci¢c primers for the other three satellite DNA sequences from cattle, satellite IV, 2
50% formamide, 2 SSC at 42 C. After hybridization and washing of the slides, digoxigeninlabelled chromosome paints were detected with 5-carboxy-tetramethylrhodamine (5-TAMRA) conjugated to anti-digoxigenin (Roche) and biotin-labelled probes were detected with florescein isothiocyanate (FITC) conjugated to avidin (Vector Laboratories). Chromosomes were counterstained with DAPI and mounted in Vectashield (Vector Laboratories). After image acquisition, the slides were washed in 4 SSC, 0.05% Tween 20 at room temperature for 5 hours with agitation, dehydrated and denatured as described before, following a second round of FISH with the cattle satellite clones. Metaphases were hybridized in-situ with the sat IV, sat IIIpvu and sat IIIsau clones labelled with biotin-16-dUTP (Sigma) or digoxigenin-11-dUTP (Roche) using standard methods (Schwarzacher & Heslop-Harrison 2000). The most stringent hybridization washes were at 42 C in 2 SSC (2 SSC: 0.3 mol/L NaCl, 0.03 mol/L sodium citrate) and 50% (v/v) formamide followed by washes in 0.1 SSC. Biotin label was detected by FITC-conjugated avidin (Vector) and digoxigenin with anti-digoxigenin-rhodamine (Roche). Chromosomes were counterstained with DAPI. Preparations were analysed using a Zeiss Axioplan 2 Imaging microscope with an Axiocam digital camera and AxioVision software. Digitized photos were prepared for printing in Adobe Photoshop (version 5.0). DAPI staining was presented in red to optimise di¡erentiation in printing; contrast, overlaying and colour optimization functions were used and all a¡ected the whole of the image equally.
Chromosomal organization of centromeric satellite DNA sequences Satellite sequences were hybridized to metaphases from di¡erent animals of the breeds Alentejana, Barrosa, Mirandesa and Alistana-Sanabresa, including individuals without t(1;29) (2n ¼ 60), homozygous (2n ¼ 58) and heterozygous (2n ¼ 59) for the t(1;29). No notable di¡erences were seen in either presence or intensity of hybridization signals between individuals or the diferent breeds. Satellite I (Chaves et al. 2000) hybridized strongly to the centromeric region of all 29 pairs of autosomal centromeres (Figure 1e-j). Both families of satellite III were present on most autosomal centromeric regions, and Satellite IV was present on less than half the chromosomes, but not detectable on larger autosomes including chromosome 1 which can be identi¢ed without painting by its characteristic size and banding pattern (Figure 1a^f). None of the satellite probes was detected on the X or Y chromosomes (Figure 1). Where all three probes were present on an autosome, the order was p-terMsat IVMsat IMsat IIIMq (Figure 2); the exact position of the centromere could not be determined. Close observation indicated that the pvu and sau families were not entirely collocalized and the pvu family tended to lie closer to the sat I hybridization signal. In chromosomes such as chromosome 29 where sat III was not detected, the probes were in the order p-terMsat IVMsat IMq (Figure 2). The larger chromosomes such as chromosome 1 without sat IV had the probes in the order p-terMsat IMsat IIIMq (Figure 2), with the sat IIIpvu family tending clearly to lie closer to sat I than the sat IIIsau family. In the t(1;29) chromosome, no sat I hybridization was detected (Figure 1e^j; Chaves et al. 2000). In heterozygous 2n ¼ 59 individuals for the t(1;29) chromosome, both families of satellite III sequences were present at the centromeres of most autosomal chromosomes but not chromosome 29 (identi¢ed by painting) and some other smaller chromosomes (Figure 1a^d, g^j). The pvu family was more abundant than the sau family. On the t(1;29) chromosome, the order of probes was pMsat IVMsat IIIpvuMsat IIIsauMq (Figure 2) and this probe order was found across the four breeds.
Results Satellite sequences The satellite I clone (pBtKB5) from cattle was previously described in Chaves et al. (2000) and showed more than 95% homology with previously isolated bovine satellite I sequences (Chaves et al. 2000). We isolated and cloned three other satellite DNA sequences from cattle: satellites IIIpvu (cBtGBIIIpvu-2), sat IIIsau (cBtGBIIIsau-3) and sat IV (cBtGBIV-1) clones. The sequence of the satellite IV (cBtGBIV-1) clone showed more than 95% homology with previously isolated Bos taurus satellite sequences of the corresponding DNA family (e.g. EMBO mammalian database accession AF162507).
The submetacentric sex chromosomes in cattle, like the t(1;29), lack sat I. Sat I is associated with the primitive acrocentric chromosomes in various Bovinae (Modi et al. 1996, Gallagher et al. 1999). If sat I were unstable in a metacentric chromosome, then this would explain its evolutionary loss in the submetacentric X as well as the repeated loss in the t(1;29) (second steps of the models proposed here). Discovery of evidence for de novo translocations, representing the ¢rst step in the origin of t(1;29), would be extremely important to discriminate between the ¢rst steps of the models presented here, and would contribute to the understanding of mechanisms of Robertsonian translocations and genome evolution within and outside the Bovidae family in general.
All four cattle breeds were found to include individuals carrying the t(1;29) chromosome and there were clear polymorphisms between the translocation chromosomes (Chaves et al. 2000). Some authors have suggested that the translocation is recurrent, with a de novo origin of 1;29 (e.g. Wilson 1990), which could be supported by the level of polymorphism and by its widespread occurrence in more than 50 breeds which have presumably been through genetic bottle-necks. However, no new occurrences have been identi¢ed in cases where parents have been investigated, and both rearrangements of the centromeric heterochromatin (Rangel-Figueiredo & Iannuzzi 1993) and pericentric inversions (Eggen et al. 1994) can give polymorphisms. Sensitive studies of heterozygous individuals usually ¢nd them to be subfertile with increased embryonic mortality (for review, see Popescu & Pech 1991), although some authors ¢nd minimal fertility di¡erence within statistical and observational limitations of the studies, and no defects in eggs or gametogenesis are found (Gustavsson 1969, Logue & Harvey 1978, Bouvet & Cribiu 1990). Gustavsson, as early as 1969 considered t(1;29) a selectively neutral rearrangement. However, in domesticated mammals of economic importance, Robertsonian translocations in general could, eventually, lead to reproductive problems and hence loss of breed productivity. Figure 3 summarizes the hybridization results of the centromeric satellites in cattle (Figures 1 & 2). If the Robertsonian t(1;29) translocation is recurrent, the data presented here allows us to suggest a model for a two-step model of the dynamic evolution of the t(1;29) chromosome (Figure 3). The ¢rst event is a reciprocal translocation involving the regions of the chromosomes with satellite III in chromosome 1 and sat IV in chromosome 29. Sat I of chromosome 1 origin, probably with some sat III repeats and sat IV repeats from chromosome 29, are lost in this ¢rst step as a chromosomal fragment. A second step involves elimination of the sat I block from the intermediate chromosome. An alternative model would suggest that a pericentric inversion in chromosome 29 precedes the fusion step, where sat I in chromosome 29 would recombine with the satI from chromosome 1 and the sat I would be lost during the fusion step. In either case, two independent events occur but the fact we ¢nd no evidence for an intermediate in the four breeds, if independent, suggests that the second step occurs soon after the ¢rst.
Acknowledgements This work was supported by two grants ^ PRAXIS XXI/BD/9046/96 and SFRH/BD/3280/2000 ^ and one project ^ POCTI/P/BIA/11285/98. We would like to thank M. A. Ferguson-Smith and D. Burkin for the chromosome sheep paint probe. JSHH thanks CREST, Japan Science and Technology for support of the centromere work.
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Figure 1. (a, c, e, g, i) Metaphase chromosome preparations (chromosomal DNA stained with DAPI, presented in red pseudocolour) showing chromosome paints from sheep chromosome 21 (homologous to cattle chromosome 29, blue^purple in parts a, c. yellow^green in e, g, i) and sequential in-situ hybridization of the different combinations of satellite sequences: (b) sat IV (green hybridization signal) and IIIpvu (blue signal); (d) sat IV (green) and sat IIIsau (blue); (f) sat I (green) and sat IV (blue); (h) sat I (green) and sat IIIpvu (blue); (j) sat I (green) and sat IIIsau (blue). Chromosomes 1, 29, t(1;29) and X are indicated in one of each pair of micrographs. Bar in (c) represents 12 mm.
Figure 2. Image showing enlarged comparison of the in-situ hybridization with satellite probe sequences in Figure 1b, d, f, h, j for autosomal chromosomes 29, 1, t(1;29) and typical acrocentric chromosomes. Chromosomal DNA shown stained red and satellite probe hybridization in blue or green. Bar represents 8 mm
Figure 3. A summary showing the locations of the sat I, sat III and sat IV satellite DNA sequences in the autosomal chromosomes including 1, 29, t(1;29) and the remaining acrocentric chromosomes. A possible intermediate stage for the dynamic evolution of the centromeric sequences involved in the origin of the t(1;29) chromosome in cattle is also shown.