Cytogenet Cell Genet 90:321–322 (2000) Assignment 1 of TRA1 encoding ppk98 to pig chromosome 5 by fluorescent in situ hybridization and confirmation by somatic cell hybrid analysis J.H. Calvo, a N.L. Lopez-Corrales, c S.I. Anderson, c A. Robic, b P. Zaragoza, a A.L. Archibald, c and R. Osta a a Laboratorio de Genética Bioquı ´mica, Facultad de Veterinaria, Zaragoza (Spain); b Laboratoire de Génétique Cellulaire, INRA, Castanet-Tolosan (France); c Roslin Institute (Edinburgh), Roslin, Midlothian, Scotland (UK) 1 To our knowledge this is the first time this gene has been mapped in pig. J.H.C. was supported by a doctoral grant from the M.E.C. (AP97). R.O. and P.Z. were supported by project UZ210-47. S.I.A., N.L.L-C., and A.L.A were supported by the Biotechnology and Biological Sciences Research Council (UK). Received 13 July 2000; manuscript accepted 30 August 2000. Request reprints from Jorge H. Calvo, Laboratorio de Genética Bioquı ´mica, Facultad de Veterinaria, c/ Miguel Servet, 50013 Zaragoza (Spain); telephone: 34 97 676 1619; fax: 34 97 676 1612; e-mail jhcalvo@posta.unizar.es Present address of N.L.L.-C: Manitoba Institute of Cell Biology, 100 Olivia St, Winnipeg, Manitoba (Canada). ABC Fax + 41 61 306 12 34 E-mail karger@karger.ch www.karger.com © 2000 S. Karger AG, Basel 0301–0171/00/0904–0321$17.50/0 Accessible online at: www.karger.com/journals/ccg Rationale and significance Porcine protein kinase 98 (ppk98) is a stress-inducible gly- coprotein that is known to be constitutively and ubiquitously expressed in the endoplasmic reticulum of mammalian cells (Dechert et al., 1994). The genomic DNA sequence of the ppk98 gene has been determined by Konig et al. (1997). Sequence analyses revealed domains that are highly conserved with the human tumor rejection antigen (TRA1) or glucose-reg- ulated protein (GRP94) genes. These findings suggest that these proteins are either identical or represent a family of close- ly related proteins (Konig et al., 1997). The human and mouse TRA1 genes have been located to human (HSA) chromosome 12 and mouse (MMU) chromosome 10, but, as yet, the porcine ppk98 gene (TRA1) has not been mapped. We report the local- ization of the pig ppk98 gene (TRA1) to porcine chromosome 5 by fluorescent in situ hybridization and somatic cell hybrid panel. This assignment supports the hypothesis that the ppk98 gene is the porcine homologue of the human TRA1 gene. Materials and methods Isolation of a large fragment genomic clone for ppk98 (TRA1) A BAC clone containing the pig ppk98 gene (TRA1) was identified by PCR screening of the PigE BAC library (Anderson et al., 2000), using specific primers designed from the described pig TRA1 sequence (Konig et al., 1997; EMBL accession: X90848): 5)-CTGTCATGGACTCAAGTTCC-3) 5)-CTGCTTCACAAAGCTTATAG-3) PCR amplification was performed using 25 ng of pig DNA. Cycling con- ditions were 94 ° CC for 30 s, 55 ° C for 30 s and 72 ° C for 30 s for 30 cycles. Partial sequence analysis of the BAC clone (43F15 from the PigEBAC library) confirmed the presence of ppk98 (TRA1) sequences (EMBL acces- sion: X90848). Fluorescence in situ hybridization (FISH) Probe isolation was carried out essentially as described previously by Lichter et al. (1990), using chromosome spreads from 2–3 unrelated adult pigs. FISH experiments were carried out twice, using duplicate slides. A total of 40 R-banded metaphase spreads were scored for FISH signals in each experiment. Pig R-banded chromosomes were obtained according to Le- mieux et al. (1992). BAC DNA from the ppk98 (TRA1) clone (43F15) was labeled with biotin by nick-translation using a BioNick kit (GIBCO) and purified on Sephadex columns (5prime-3prime). Hybridization signals were detected with FITC-avidin (Boehringer Mannheim) and chromosomes were counter-stained with propidium iodide (Sigma) and PPD (Sigma) adjusted to pH 11 as antifade solution Probe name: 43F15 from the pPigE BAC library Probe type: genomic DNA Vector: pBeloBAC11 Insert size: 150 kb Proof of authenticity: DNA sequencing Gene reference: X90848 Somatic cell hybrid panel A porcine rodent somatic cell hybrid panel (Yerle et al., 1996) was screened for the pig ppk98 (TRA1) gene by PCR. The primers and PCR conditions used were as described above. Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/10/2018 5:13:03 AM