FISH mapping of three bovine cosmids to cattle, goat, sheep and. buffalo X chromosomes. Hereditas 126: (1997)

Transcription

1 Hereditas 126: (1997) FISH mapping of three bovine cosmids to cattle, goat, sheep and buffalo X chromosomes B. PRAKASH', I. OLSAKER', I. GUSTAVSSON' and B. P. CHOWDHARY' ' Department of Animul Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden ' Department of Morphology, Genetics and Aquatic Biology, Norwegian College of Veterinary Medicine, Oslo, Norway Prakash, B., Olsaker, I., Gustavsson, 1. and Chowdhary, B. P FISH mapping of three bovine cosmids to cattle, goat, sheep and buffalo X chromosomes.- Hereditas 126: Lund, Sweden. ISSN Received November 26, Accepted February 3, 1997 Contrary to extensive banding similarities of the autosomes. there are variations in the morphology as well as banding patterns of the X chromosomes of various species of bovidae family. We used three bovine X chromosome specific cosmid clones for fluorescence in situ hybridization mapping on cattle, goat, sheep, and river buffalo chromosomes to identify homologous regions in the genomes of the four species. The three cosmids (ciobt 314, 945, and 1489) mapped to well spaced locations on buffalo X, and both arms of cattle X chromosome. However, of the three probes, cosmids clobt 314 and 1489 showed no hybridization signal on any of the goat-sheep chromosomes. Incidentally, the two cosmids show correspondence to those regions of the buffalo X chromosome which are proposed to be lost during the structural reshuffle of the latter, leading to sheep/goat X chromosomes. The lack of hybridization signal of the two cosmids in sheep and goat, therefore, indicates that these DNA sequences are most likely absent in the two genomes and probably correspond to the segments lost during evolution of their X chromosomes. BIi(inu P. Cliowdliary, Depariinmt of' Aiiiinnl Breeding and Genetics, Sivrdish Unioersity of Agricultural Sciences, Box 7023, S Uppsalcr, Sitah. Bhanu.Chowdhary@hgen.slu.se The chromosomes of cattle, sheep, goat, and buffaloes have been studied for over two decades. A high degree of similarity in banding patterns of all the autosomes demonstrates extensive conservation of the karyotypes of the four species during evolution. However, in striking contrast to the extensive autosoma1 banding conservation among bovids, the X chromosome shows considerable variation both in morphology as well as banding patterns (BUCKLAND and EVANS 1978; HAYES et al. 1991; KAF- TANOVSKAYA and SEROV 1994; IANNUZZI and DI MEO 1995). Understanding the mechanism of this variation in the bovid X chromosome has been of special evolutionary interest to animal geneticists. Using high resolution banding comparisons of the cattle, goat, sheep, and buffalo sex chromosomes, possible structural rearrangements differentiating the X chromosome have been proposed (HAYES et al. 1991; KAFTANOVSKAYA and SEROV 1994; IANNUZZI and Dr MEO 1995). Though portions of the X chromosomes of the four species are shown to have similar banding patterns, yet the proposed schemes explaining this variation (comprising disruption of the euchromatic regions, peri- and para-centric inversions, centric placement and acquisition and/or loss of centromeric heterochromatin) lack unanimity. We have attempted to examine the validity of these schemes by using three X chromosome specific bovine cosmid clones, two of which map in the regions supposed to be involved in rearrangements. The re- sults provide molecular basis to the observed cytogenetic differences between the X chromosomes of the four species. MATERIALS AND METHODS Metaphase chromosome preparations. - Peripheral blood lymphocytes from the domestic ox (Bos taurus, BTA), domestic goat (Cupru hircus, CHI), domestic sheep (Ovis uries, OAR), and the domestic river buffalo (Bubulus bubalis, BBU) were individually cultured in RPMI 1640 medium supplemented with fetal calf serum (10 %), pokeweed mitogen (I YO), and L- glutamine, at 38 C for 72 h. Mitoses were arrested at metaphase by treating cultures with colcemid for 1 h before harvesting. Air dried chromosome slides were aged for 4 days at room temperature and then stored in deep freezer until used for in situ hybridization. Probes. - A bovine cosmid library constructed from a female of Norwegian Red cattle (NRF) breed was screened with a (GT),, oligonucleotide. Clones positive to this probe were further screened with bovine satellites 1709 and 1715, and bovine Cot1 DNA. Only clones giving a negative signal to the latter three were selected. DNAs from these clones were isolated and used for in situ hybridizations. Probe libelling, hybridization, und signal detection. - The cosmid DNAs along with the insert were biotin labelled by nick translation using biotin-1 4-dATP

2 116 B. Prakash et ul. Hereditas 126 (1997) (BioNick labelling system, Cat. No , Gibco, BRL) following manufacturer s protocol. Unincorporated nucleotides were separated from the labelled probes by spinning through sephadex G-50 columns. Fluorescent in situ hybridization (FISH) was performed essentially as described earlier (CHOWDHARY et al. 1995) with minor modifications. For example, the post hybridization washings in 50 % formamide, 2 x SSC, ph 7.0 were carried out for 3 x 10 min each, instead of the usual 3 x 15 min. Suppression hybridization of repetitive sequences in the probes was achieved by adding sheared bovine genomic DNA to the hybridization mixture. Probe signals and chromosome images were screened and processed as described earlier (CHOWDHARY et al. 1995) At least 25 metaphase spreads were analysed for each probe to determine the specific hybridization location in each species. RESULTS AND DISCUSSION In situ hybridization of the three cosmid clones (ciobt 314, 945, and 1489) to metaphase chromosomes produced fairly strong hybridization signals at three different locations on the X chromosomes of cattle and buffaloes. However, of the three cosmids, only ciobt 945 hybridized to the goat and sheep X chromosomes. No hybridization signals were obtained for cosmids ciobt 314 and 1489 on any of the goat or sheep chromosomes despite repeating the hybridization experiment 8 times and even by using higher probe concentration (double of the routinely used) and longer hybridization times (up to 72 h). The observed hybridization signals were localized only on the X chromosome of the four species. No secondary signals on any other chromosome were detected. Identification of the X chromosomes of all the four species is straightforward due to their distinct morphology in the respective karyotypes. The cattle X chromosome is the only large submetacentric chromosome in the karyotype. The goat and sheep X chromosomes have distinct very short small arms which are absent in all the acrocentric autosomes. The buffalo X chromosome is the largest acrocentric, about 25 YO longer than the largest acrocentric autosome, which makes its identification easy (YADAV 1981). Representative partial metaphase spreads with typical FISH signals (arrows) of the three probes in the four species are presented in Fig. IA. Ideograms showing the relative band locations of each mapped cosmid on the X chromosomes are given in Fig. 1B. The ideograms of cattle and buffalo X chromosomes have been taken from the respective standard karyotypes (ISCNDA 1990 and IANNUZZI 1994, respectively). Since no standard ideogram is available for the goat X chromosome, we have taken the one proposed by IANNUZZI et al. (1994). As the goat and sheep X chromosomes are identical in their morphology and banding patterns (HAYES et al. 1991; IAN- NUZZI and Dr MEO 1995), we do not provide separate ideogram for the sheep X chromosome. Buffalo us. Cuttle. -The three cosmid clones (ciobt 314, 945 and 1489), in this study, hybridized to bands q13, q33-34, and q47, respectively, of the buffalo acrocentric X chromosome. The corresponding hybridization locations of the three probes on the submetacentric cattle X chromosome are at bands p12-13, q26-31, and q42-43, respectively (Fig. IB). The cattle X chromosome is hypothesized to have originated from the buffalo X following a break in the proximal (about one third) part of the buffalo X with pericentric inversion and tandem fusion of the inverted and remaining part, and loss of the CH block of the buffalo X (IANNUZZI and Dr MEO 1995). The linear order and locations of cosmids ciobt 945 and 1489 on the distal two-third part of buffalo X chromosome correspond to their locations on the long arm of the cattle X. The cattle Xq arm has also been shown to have similar G- and R-banding patterns to the distal two thirds of the buffalo X (IAN- NUZZI and DI MEO 1995). Cosmid ciobt 314, in this study, which mapped close to the centromere on the buffalo X, hybridized also close to the centromere but on the short arm of cattle X. Thus the hybridization sites of the three cosmids in the two species are in close agreemet with the proposed rearrangement in the cattle X-chromosome, following the breakage-inversion-fusion event in its buffalo counterpart, and provide evidence of this homology at the DNA level. However, neither the FISH mapping results of this study nor the cytogenetic observations exactly pinpoint the precise locations of breakage point on the buffalo X chromosome. Buffalo us. Goat/Sheep. - Cosmid clone ciobt 945 produced distinct hybridization signal on corresponding regions of sheep and goat X chromosomes (Fig. 1A and B). However, ciobt 314 and 1489 did not show any signal despite modifying hybridization conditions as well as post hybridization washing. The observations are in contrast to the FISH mapping data of 28 other cosmid clones from the same bovine cosmid library, which produced strong hybridization signals, at identical band locations of 15 autosomal arms of cattle, goat, sheep, and buffalo (PRAKASH et al. 1996a). The accumulated comparative gene mapping data in the four species also do not show disconcordance between the banding and genetic homology.

3 Hereditas 126 (1997) FISH mapping of three bovine cosmids 11 7 A B BTA 24 I I I X BBU n!? I-314 j I X CHI C. OAR 12 j X I- 945 Fig. 1. (A) (a-h) Partial metaphase cells showing (arrows) fluorescence in situ hybridization signals of cosmid clones ciobt 314 in cattle (a) and buffalo (d), of cosmid clobt 945 in cattle (b), buffalo (e), sheep (g) and goat (h), and of cosmid ciobt 1489 in cattle (c) and buffalo (9. (B) Ideograms of G-banded cattle (top), buffalo (middle), and goatssheep (bottom) X chromosomes showing the locations of the three cosmid clones.

4 118 B. Prakash et al. Hereditas 126 (1 997) Recently, the three bovine cosmids showed distinct hybridization signals on reindeer X chromosome (PRAKASH et al. 1996b). Considering that the bovine DNA probe successfully detects homologous sequences in a relatively distantly related species (reindeer), the probability of its detecting similar sequences in closely related species (sheep and goat) is much higher. Expectations for the latter are further increased because close proximity between cytogenetic and gene mapping data is well established in these species. Hence, the most likely explanation for the absence of signal from the two bovine cosmids on sheep and goat chromosomes could be that the sequences corresponding to these probes do not exist in the goat-sheep genomes. Alternatively, the probability that the corresponding sequences are too diverged to show cross hybridization, though less likely, cannot be completely ruled out. Comparison of G- and R-banding patterns of buffalo and goat-sheep X chromosomes has shown that almost the whole goat-sheep X chromosome finds full banding homology with the buffalo X chromosome when the former is turned upside down (IANNUZZI and DI MEO 1995). Relative locations of cosmid ciobt 945 in the two species fully correspond with the cytogenetic observations. The proposed possible structural reshuffling of the buffalo X chromosome leading to the origin of goat-sheep X is described by IANNUZZI and DI MEO (1995). The authors hypothesised two breaks in the buffalo X chromosome, one proximal and the other at the telomere, followed by pericentric inversion of the centromere with very short small arms and paracentric inversion of the remaining part. The two inverted parts then fuse by centromere-telomere tandem fusion after losses of the CH block and of two smaller regions, one close to the centromere (corresponding to the localization of cosmid ciobt 314 on buffalo X) and the other at the telomere (corresponding to the map location of cosmid ciobt 1489 on buffalo X). It is probable that the DNA sequences from the two negative clones correspond to part of the segments lost during the evolution of the sheep-goat X chromosomes, thus explaining the absence of hybridization signals. The two small deleted/lost segments were recently found to be composed mainly of heterochromatic material (IANNUZZI and DI MEO 1995). Comparative FISH mapping results of this study, together with the available cytogenetic observations, suggest that contrary to the extensive autosomal conservation, intrachromosomal arm rearrangements (comprising peri- and para-centric inversions, centric placement, loss and/or acquisition of CH) have taken place in the bovid X chromosome during the evolutionary divergence of the four species. However, these comparative data are not sufficient to precisely locate the points of breaks/inversions/deletions or to determine the primitive condition of the bovid X chromosome. A detailed map of this chromosome may, therefore, be particularly useful in understanding the precise structural shuffling of the bovid X and the phylogenetic relationship both within and across bovid sub-families which represent divergent forms of the X chromosomes. Alternatively, the recently developed Zoo-FISH painting technique (SCHERTHAN et al. 1994) using band/region specific libraries of the bovine X may provide the much needed answer to the precise regional homologies and structural rearrangements of this chromosome and possibly the evolutionary divergence of bovidae sub-families. ACKNOWLEDGEMENTS This work was supported by grants from the Swedish Council of Agricultural Research. Financial assistance from the Norwegian Council of Agricultural Research (1.0.) is gratefully acknowledged. REFERENCES Buckland RA and Evans HJ, (1978). Cytogenetic aspects of phylogeny in Bovidae. I. G-banding. Cytogenet. Cell Genet. 21: Chowdhary BP, de la Sena C, Harbitz I, Eriksson L and Gustavsson I, (1995). FISH on metaphase and interphase chromosomes demonstrates the physical order of the genes GPI, CRC, and LIPE in pigs. Cytogenet. Cell Genet. 71: Hayes H, Petit E and Dutrilaux B, (1991). Comparison of RBG banded karyotypes of cattle, sheep and goats. Cytogenet. Cell Genet. 57: Iannuzzi L (Co-ordinator), (1994). Standard karyotype of the river buffalo (Bubalus bubalis L. 2n = 50). Report of the committee for the standardization of banded karyotypes of the river buffalo. Cytogenet. Cell Genet. 67: Iannuzzi L and Di Meo GP, (1995). Chromosomal evolution in bovids: a comparison of cattle, sheep and goat G- and R-banded chromosomes and cytogenetic divergences among cattle, goat and river buffalo sex chromosomes. Chromosome Res. 3: Iannuzzi L, Di Meo GP and Perucatti A, (1994). An improved characterisation of goat chromosomes by means of G- and R-band comparison. Hereditas 120: ISCNDA, (1990). International system for cytogenetic nomenclature of domestic animals. Report of the committee for the standardization of banded karyotypes of domestic animals. Cytogenet. Cell Genet. 53: Kaftanovskaya HM and Serov OL, (1994). High resolution GTG- banded chromosomes of cattle, sheep and goat: a comparative study. J. Hered. 85: Prakash B, Olsaker I, Gustavsson I and Chowdhary BP, ( Comparative chromosomal FISH mapping of 28 bovine cosmids in cattle, goat, sheep and river buffalo. 5th Workshop of Nordic Genome Initiative, Laugarvatn, Island (abstract).

5 Hereditas I26 (1997) FISH mapping of three bovine cosmids 119 Prakash B, Kuosku V, Olsaker I, Gustavsson I and Chowdhary BP, (1996b). Comparative FISH mapping of bovine cosmids to reindeer chromosomes demonstrates conservation of the X chromosome. Chromosome Res. 4: Scherthan H, Cremer T, Arnason U, Weier HU and Lima- de-faria A, (1994). Comparative chromosome painting discloses homologous segments in distantly related mammals. Nature Genet. 6: Yadav BR, (1981). Studies of chromosomes and their abnormalities in cattle and buffaloes. Ph. D. thesis, Kurukshetra University, Kurukshetra, India.