| First Author | Patek CE | Year | 1994 |
| Journal | Mouse Genome | Volume | 92 |
| Issue | 4 | Pages | 696-698 |
| Mgi Jnum | J:22097 | Mgi Id | MGI:69988 |
| Citation | Patek CE (1994) DYEpal: a novel mouse Y chromosome-specific repetitive DNA element. Mouse Genome 92(4):696-698 |
| abstractText | Full text of Mouse Genome contribution: DYEpal: A NOVEL MOUSE Y CHROMOSOME-SPECIFIC REPETITIVE DNA ELEMENT. C.E. Patekl, E. Olszewska2, D. Kipling3, J.F. Armstrongl, P.G. Sealey3, M. Lee3, R.M. Speed3, M.L. Hooperl and K.W. Jones2. 1 CRC Laboratories, Department of Pathology, The University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, Scotland; 2Division of Biological Sciences, Institute of Cell, Animal and Population Biology, Ashworth Laboratories, The University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JT, Scotland; 3 MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Scotland. DYEpal is a mouse Y chromosome-specific 940 bp repetitive DNA fragment which, after cloning into the Hind site of pBR322 as a linear array of three copies (plasmid previously designated pM34- 2/0.6t), has been used to identify male cells in XX-XY chimaeric tissues (1, 2). The present report concerns the isolation, localization and nucleotide sequence composition of DYEpal. DYEpal is derived from M34-2, a 2.7 kbp EcoRI fragment derived from M34 (3, 4). M34 was originally isolated by screening a male mouse genomic DNA library (laboratory strain CBA/Ca) firstly with uncloned Banded krait minor satellite DNA (Bkm, derived from the heterogametic W chromosome of the Elapid snake, Bungarus fasiatus) and subsequently with the Bkm-related Drosophila melanogaster probe 2 (8), which consists largely of repeats of the tetranucleotide GATA. M34-2 hybridises extensively to the mouse Y chromosome but shows weak hybridisation to other chromosomes besides the Y chromosome (3). Digestion of M34-2 with HindIII resulted in three fragments of 530 bp , 940 bp and 1230 bp. All three hybridised to the Y chromosome, but whereas the 530 bp and 1230 bp fragments also hybridised to other chromosomes (data not shown), the 940 bp fragment, designated DYEpal, hybridised exclusively to the Y chromosome (Fig lA), and specifically to Yq (Fig 1B). However no signal was detected at the distal end of Yq which includes the telomere and pseudoautosomal region (5). The nucleotide sequence of both strands of DYEpal was determined using the 'Sequenase Version 2.0' DNA sequencing kit (United States Biochemical). The sequence (Fig 2) was used to search the EMBL DNA database using the BLASTN programme (6). The sequence was also conceptually translated in all six possible reading frames and compared to the NBRF protein database using the BLASTX programme (7). In neither case was any significant nucleotide or amino acid similarity detected. DYEpal therefore identifies a novel mouse repetitive sequence family. Approximately the first half of the sequence (nucleotides 1-479) has an A+T content of 55% while the remainder (nucleotides 480-940) is characterised by a particularly high A+T content of 74%. Overall, the 940 bp sequence has an A+T content of 64%. The sequence is devoid of any striking structural features and there are no perfect direct or indirect repeats greater than 15 nucleotides in length. However, a number of shorter repetitive motifs were identified, including short stretches of A and T and a total of nine GATA and six GACA repeats on both strands (Fig 2). While these repeat numbers do not differ significantly from the mean value of 7.34 expected in a random 940 bp sequence, there is a statistically significant cluster of eight GATA repeats between nucleotides 15 and 238 (P = 0.002). Figure 1. (Legend). Fluorescence in situ hybridisation, performed according to the method described in detail by Fantes et a1 (8), showing (A) metaphase chromosome spread from the male mouse embryonic stem cell line, CGR8 (laboratory strain 129/Ola) hybridised with DYEpal: hybridisation signal is exclusively on the Y chromosome, and (B) high power view of part of a metaphase spread showing the distribution of DYEpal sequences on the Y chromosome: hybridisation signal is localised on the proximal region of Yq. The centromere and Yq telomere, identified by DAPI staining, are indicated by small and large arrows respectively. DYEpal was nick-translated using digoxygenin-11-dUTP (Boehringer Mannheim) and hybridisation signal detected using FITC anti-digoxygenin raised in sheep (Boehringer Mannheim) followed by FITC anti-sheep (Vector). Figure 2. (Legend). DNA nucleotide sequence composition of DYEpal. Bkm-related GATA (single underline) and GACA (double underline) repeats are indicated. The sequence has been submitted to the EMBL database and assigned the accession number:Z 32745 Mus musculus M34-2/940. As for Drosophila and snakes, the major component of mouse Bkm DNA is the tetranucleotide GATA with a minor GACA contribution (9-11). However the GATA repeats in DYEpal are unlikely to account for hybridisation to Yq for several reasons: (A) the GATA-rich Drosophila-derived Bkm-2 (8) probe hybridises to the mouse Y pericentric region but not to Yq (4, 12), (B) the oligonucleotide (GATA)4 probe hybridises to mouse Yp and the centromere but not Yq (13-15), and (C) Bkm-derived probes hybridise extensively to the overall mouse chromosome complement, and particularly to autosomes 4 and 17 (9, 12, 16), but the DYEpal sequence is Y chromosome-specific. Thus it would appear that hybridisation of DYEpal to Yq is driven by sequences other than its GATA repeats. At first sight the results appear contradictory in that DYEpal, in spite of its GATA cluster, fails to hybridise to Yp (which is rich in Bkm-related sequences), while Bkm-related probes (including 2 (8) and the (GATA)4 oligonucleotide) fail to hybridise to Yq. This apparent paradox can be explained by differences in sequence composition. For example, in the 420 bp mouse Bkm-positive clone, M3.1, which hybridises to Yp, the GATA repeats are arranged as long contiguous tandem stretches with 20 of the 22 GATA repeats clustered in a 123 bp fragment (10), whereas in DYEpal the GATA repeats are dispersed and less numerous (Fig 2). These differences in organisation and number of GATA repeats would affect hybridization specificity. Thus DYEpal and Bkm-related probes, including M3.1, may share only limited sequence homology and any weak hybridisation of DYEpal to Yp and of Bkm-rich probes to Yq might normally go undetected. Alternatively, the failure of DYEpal to hybridise to Yp may arise because hybridisation (to Yq) is driven by a repetitive sequence which either lacks or contains very few GATA repeats. Thus while DYEpal contains a GATA cluster it remains to be determined whether GATA clusters are enriched or even present onYq. If they were not, this would account for the failure of Bkm-related probes to hybridise to Yq. These issues can only be resolved once the nature of the consensus sequence identified by DYEpal and repeated on Yq is known. Acknowledgements. We thank Judith Fantes for advice regarding FISH analysis and Austin Smith for CGR8 cells. The work was supported by the CRC and the MRC. References. (1) Ansell, J. D., Samuel, K., Whittingham, D.G., Patek, C.E., Hardy, K., Handyside, A.H., Jones, K.W., Muggleton-Harris, A.L., Taylor, A.H., Hooper, M.L. (1991) Development. 112:489-498. 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