Linkage mapping of the Na-K-2C1 cotransporter gene (Slcl2a1) to rat chromosome 3

Linkage mapping of the Na-K-2C1 cotransporter gene (Slcl2a1) to rat chromosome 3 Mammalian Genome 8, Brief Data Reports 379 tional Institutes of Health (Hypertension Program Project PO1 HL-35018) Linkage mapping of the Na-K-2CI and by funds provided by the Cigarette and Tobacco Surtax Fund of the cotransporter gene (Slcl2al) to rat State of Califomia through the Tobacco Related Diseases Research Pro- gram of the University of California, Grant Number 4RT-0362. Chromosome 3 References J.-M. Wang, ~ M. Pravenec, 2~ V. Kren, 2'3 T.W. Kurtz 1 1. Pravenec M, Guaguier D, Schott J-J, Buard J, Kren V, Bila V, Szpirer C, Szpirer J, Wang J-M, Huang H, St. Lezin E, Spence MA, Flodman IDepartment of Laboratory Medicine, University of Califomia, P, Printz M, Lathrop GM, Vergnaud G, Kurtz TW (1996) Mamm Ge- San Francisco, Cahfomia 94143, USA home 7, 117-127 2Institute of Physiology, Czech Academy of Sciences, Vfdenskfi 1083, 2. Manly KF (1993) Mamm Genome 4, 303-313 142 20 Prague 4, Czech Republic 3. Silver J (1985) J Hered 76, 436--440 3institute of Biology, 1st Medical Faculty, Charles University, 128 00 4. Gamba G, Miyanoshita A, Lombardi M, Lytton J, Lee WS, Hediger Prague 2, Czech Republic MA, Hebert SC (1994) J Biol Chem 269, 17713-17722 5. Quaggin SE, Payne JA, Forbush B III, Igarashi P (1995) Mamm Ge- Received: 18 October 1996 / Accepted: 28 December 1996 nome 6, 557-558 6. Simon DB, Karet FE, Hamdan JM, DiPietro A, Sanjad SA, Lifton RP Species: Rat (Rattus norvegicus) (1996) Nature Genet 13, 183-188 Locus name: Solute carrier family 12, member 1 (bumetanide- 7. Pravenec M, Guaguier D, Schott J-J, Buard J, Kren V, Bila V, Szpirer sensitive sodium-[potassium]-chloride cotransporter) C, Szpirer J, Wang J-M, Huang H, St. Lezin E, Spence MA, Flodman P, Printz M, Lathrop GM, Vergnaud G, Kurtz TW (1995) J Clin Invest Locus symbol: Slc12a1 (Nkcc2) 96, 1973-1978 Map position: The Slc12a1 gene is linked to markers on rat Chro- 8. Szpirer C, Szpirer J, Klinga-Levan K, Stahl F, Levan G (1996) Folia mosome (Chr) 3: ... D3Mit6-1.5 (0.2-6.5)-D3Mit17-1.5 (0.2- Biol (Praha) 42, 175-226 6.5)-D3Ucsf3-4.9 (1.4-15.3)-Slc12al-3.7 (0.9-12.2)-D3Mit13 ... [1] (distances between loci and 95% confidence limits are in centiMorgans). Assignment of the porcine spleen tyrosine Method of mapping: Linkage analysis of the Slc12a1 gene was carried out in the HXB and BXH recombinant inbred (RI) strains kinase gene, SYK, to Chromosome 14q14 by [1]. The Map Manager program (version 2.5b3) incorporating the fluorescence in situ hybridization statistical criteria of Silver was used to estimate the map location of the Slcl2al gene and the distances between loci [2,3]. Misaki Kojima, 1 Kousuke Ohata, 1'2 Hiroshi Yasue l'z Database deposit information: U10096 Molecular reagents: The primers used to amplify the 3'- ~Animal Genome Research Group, National Institute of Animal untranslated portion of the Slc12al were designed according to the Industry, Norindanchi P. O. Box 5, Tsukuba, Ibaraki 305, Japan published sequence [4]: the upstream primer was 5'-AAC CTC 2Faculty of Agriculture, University of Tsukuba, Tsukuba, TGG AGA CTA TCC CAC, the downstream primer was 5'-TTG Ibaraki 305, Japan ATT TGC CCC CCA CAA GC. Method of allele detection: RFLP of the PCR product digested Received: 13 October 1996 / Accepted: 13 December 199~ with the NlalII enzyme distinguished the alleles of the Slcl2al Species: Pig (Sus scrofa domestica) gene between the BN-Lx/Cub and SHR/OIa strains. The size of the Locus name: Spleen tyrosine kinase uncut PCR product was about 978 bp. After digestion with the Locus symbol: SYK NlalII restriction endonuclease, the BN-Lx/Cub DNA contained Map position: 14q14 three restriction sites, whereas the SHR/Ola DNA contained four Method of mapping: Flourescence in situ hybridization (FISH) restriction sites. Other markers used in the current study are the Molecular reagents: A porcine cosmid library was screened with same as described earlier [1]. porcine SYK cDNA, which was donated by H. Yamamura, Fukui Previously identified homologs: The Slc12a1 gene has been Medical School, Japan [1], as a probe. A cosmid clone thus ob- mapped to mouse Chr 2 [5], which is largely homologous to rat tained was subjected to sequence analysis, revealing that the se- Chr 3 and to human Chr 15q15-q21 [6]. quence corresponding to that from nucleotide number 266 to 859 Discussion: Recently, Simon and colleagues demonstrated linkage of porcine SYK cDNA (accession number: M73237) [1] was lo- of Bartter's syndrome (an autosomal recessive hypokalemic meta- cated in the clone, and that a 3-kb intron was inserted between bolic alkalosis often associated with reduced blood pressure) to the nucleotide number 698 and 699 of the cDNA sequence. These renal Na-K-2C1 cotransporter gene SLC12A1 (NKCC2) and iden- results demonstrated that the cosmid clone contains at least a part tified mutations that disrupt the amino acid sequence of the protein of SYK gene. Therefore, the DNA of cosmid clone was biotinyl- [6]. In the current study, we have mapped the Slc12a1 gene to a ated by nick translation and used as a probe for FISH to localize region of rat Chr 3. In previous studies, markers from this region the SYK gene on chromosomes, as described previously [2]. The showed no statistically significant associations with blood pressure chromosome spreads were prepared from a culture of peripheral variation in the HXB/BXH RI strains [7]. With a Pearson product- blood cells of boars (Large White). The probe DNA hybridized moment correlation, no significant relationship between the geno- with chromosomal DNA was detected by FITC-labeled streptavi- types at the S1c12al locus and blood pressure was detected in RI din/biotinylated anti-streptavidin antibody system, and the R-band strains. The linkage of the Slc12a1 gene to markers on rat Chr 3 is of the chromosomes was induced simultaneously by staining with in agreement with the previously observed homologies between rat propidium iodide (PI). FITC and PI signals were collected sepa- Chr 3 and mouse Chr 2 and also extends the homologies between rately with a cooled CCD camera and electronically overlaid. rat Chr 3 and human Chr 15q [8]. Previously identified homologs: Human SYK gene has been as- signed to human Chromosome (Chr) 9q22 [3]. Acknowledgments: The research of M. Pravenec was supported in part by an International Research Scholar's Award from the Howard Hughes Discussion: The 72-kDa tyrosine kinase, SYK, which was origi- Medical Institute. This work was also supported by a grant from the Na- nally identified in porcine spleen as a non-receptor type tyrosine Correspondence to: M. Pravenec Correspondence to: M. Kojima Mammalian Genome Springer Journals

Linkage mapping of the Na-K-2C1 cotransporter gene (Slcl2a1) to rat chromosome 3

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