All articles available online at http://www.idealibrary.com on BRIEF MAPPING REPORTS Chromosomal Mapping of the Tankyrase Gene in Human and Mouse Lingxiang Zhu,* Susan Smith,† Titia de Lange,† ,1 and Michael F. Seldin* FIG. 1. Chromosomal localization of Tnks. The segregation of these genes among flanking genetic loci on mouse chromosome 8 in *Rowe Program in Genetics, Department of Biological Chemistry and [(C3H/HeJ-gld 3 Mus spretus)F1 3 C3H/HeJ-gld] interspecific back- Department of Medicine, University of California at Davis, cross mice is shown. Filled boxes represent the homozygous C3H Davis, California 95616; †The Rockefeller University, pattern and open boxes the F1 pattern. The number of mice with New York, New York 10021 each haplotype is shown in each column. The mapping of the refer- Received November 16, 1998; accepted January 27, 1999 ence loci as well as the relationship to other mouse chromosomal markers is available on the Internet: use htpp://www.informatics. jax.org/crossdata.html to enter DNA Mapping Panel Data Sets from MGD, then select the Seldin cross and Chromosome. A consensus map position of 18 cM on mouse chromosome 8 is derived from Functional gene description: Tankyrase (TNKS) is a interpolation using the most recent composite map (http://www. novel gene with homology to both ankyrins and the cata- informatics.jax.org/bin/ccr/current/index). lytic domain of poly(ADP-ribose) polymerase (PARP) that was recently cloned using a yeast two-hybrid screen with the telomere-specific DNA binding protein TRF1 (1). Tankyrase protein is located at telomeres, centrosomes, Flanking markers used: The human location was de- and nuclear pore complexes (1; S. Smith and T. de Lange, fined relative to the large number of markers typed in the in preparation). In vitro studies have demonstrated that Stanford G3 radiation hybrid panel (http://www-shgc. tankyrase has PARP activity and that both tankyrase and stanford.edu/RH/index.html). The strongest linked marker TRF1 can function as acceptors for ADP ribosylation. Since was AFM193xh4. For the mouse, the mapping of the ref- ADP ribosylation of TRF1 decreases its telomeric DNA erence loci (Fig. 1) as well as the relationship to other binding activity in vitro, the TNKS gene may function by mouse chromosomal markers is available on the Internet: modulating telomere length. use htpp://www.informatics.jax.org/crossdata.html to en- Description of clone or DNA: For human TNKS map- ter DNA Mapping Panel Data Sets from MGD, then select ping, a PCR specific for the TNKS gene was utilized. The the Seldin cross and Chromosome. primers (forward, 59CGAAGTGCTAGGGGAGTCCG; re- Method of mapping: The human chromosomal loca- verse, 59GTGGGAGAGGCTGGG GTGGT) amplified a tion of TNKS was defined using the Stanford G3 radiation 169-bp fragment from the TNKS gene (Accession No. hybrid map. The mouse chromosomal location was defined AFO82556). For mouse Tnks mapping, a mouse EST (Acces- using a panel of DNA samples from a well-characterized sion No. AA415426) that had 98% sequence homology with interspecific cross. Informative restriction fragments that human TNKS was used. segregated in the interspecific cross were identified on Source: The source of cDNA, as described above, was Southern blot hybridization using a cDNA probe specific sequence from a human cDNA clone and a mouse cDNA for Tnks according to methods previously described (2): clone. PstI fragments (C3H/HeJ-gld, 1.3 kb; Mus spretus, 3.5 kb). Results: For human, TNKS was positioned at 17.6 Method used to validate gene identity: Gene identity cR on human chromosome 8 with a LOD of 8.2 on the was validated by specific amplification and, for the mouse 10000 G3 map. The 1000:1 confidence interval is 6–30 cM (Ge´ne´- EST, stringent hybridization to a single disparate-sized re- thon position) of chromosome 8. For the mouse, comparison striction fragment in the two species of mouse used in the of the haplotype distribution of the Tnks-specific polymor- mapping panel. phisms and those previously defined in this interspecific cross (Fig. 1) indicated the following relationships (6stan- Supported by HG00734 (M.F.S.) and grants to T.d.L. from the NIH dard error): on mouse chromosome 8, centromere–Adrb3– (GM49046 and CA76027), the Rita Allen Foundation, and the San- 7.9 6 2.5 cM–Tnks, D8Mit24–10.5 6 2.9 cM–Glra3. This doz Foundation. T.d.L. is a recipient of the Burroughs Wellcome corresponds at a consensus position of 18 cM on mouse Fund Scholar Award in Toxicology. S.S. is a Leukemia Society of America Special Fellow. Sequence data from this article have been chromosome 8 and helps define the homology relationship deposited with the Mouse Genome Database under Accession No. between regions of human and mouse chromosome 8 (see J:51381. http:/www.ncbi.nlm.nih.gov/Homology/http://www.ncbi. 1 To whom correspondence should be addressed. Telephone: (530) nlm.nih.gov/genemap/).Interestingly TNKS/Tnks map to 754-6017. Fax: (530) 754-6015. E-mail: [email protected]. positions close to ankyrin 1 (ANK1/Ank1) located on hu- Genomics 57, 320–321 (1999) 320 Article ID geno.1999.5771 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. BRIEF MAPPING REPORTS 321 man chromosome 8 (60.0–65.8 cM) and mouse chromo- the complete NBC3 gene (SLC4A7) and mapped it to chro- some 8 (9.5 cM). TNKS is more closely related to the ANK1 mosome 3p22. gene than to other ank repeat-containing genes; the ank Name of clone or DNA: A BAC human genomic DNA repeat is 39% identical to the repeat domain in ANK1, clone, 15, containing the entire human NBC3 gene was used suggesting a common derivation. It is notable that these for FISH analysis. results exclude Tnks as a candidate gene for the recently identified locus on distal mouse chromosome 2 that has Description of clone: Primers designed for screening 9 been implicated in telomere regulation in the mouse (3). the BAC human library were 5 -CTGCAACAGTGTCAA- TAAGTCATG-39 and 59-ACTGAAGTCATGAACACA- Homologies: Homologies are as indicated above. GAGAGGC-39. The 160-bp PCR product was derived from exon 20. References Source: The BAC clone 15 was isolated using a PCR- 1. Smith, S., Giriat, I., Schmitt, A., and de Lange, T. (1998). based screen of a human genomic BAC DNA library (Genome Tankyrase, a poly(ADP-ribose) polymerase at human telomeres. Systems). Science 282: 1484–1487. 2. Watson, M. L., and Seldin, M. F. (1994). Application of mouse Method used to validate gene identity: A series of crosses towards defining the genetics of disease phenotypes. primers was designed based on the known mNBC3 cDNA Methods Mol. Genet. 5: 369–387. sequence (5). These primers were used for cycle sequencing of 3. Zhu, L., Hathcock, K. S., Hande, P., Lansdorp, P. M., Seldin, the BAC clone 15. Sequences at the intron–exon boundaries M. F., and Hodes, R. J. (1998). Telomere length regulation in of the NBC3 gene were determined by aligning the cDNA mice is linked to a novel chromosome locus. Proc. Natl. Acad. Sci. sequence with the genomic sequence. USA 95: 8648–8653. Flanking markers: No flanking markers were used. Method of mapping: The chromosomal localization of the NBC3 gene was determined by fluorescence in situ hy- Mapping of the Human NBC3 bridization using as a probe the BAC genomic clone 15 con- taining this gene. DNA from clone 15 was identified by se- (SLC4A7) Gene to Chromosome 3p22 quencing and was then labeled with digoxigenin–dUTP by nick-translation. Labeled probe was combined with sheared Alexander Pushkin, Natalia Abuladze, Ivan Lee, human DNA and hybridized to normal metaphase chromo- 1 Debra Newman, James Hwang, and Ira Kurtz somes derived from phytohemagglutinin-stimulated periph- eral blood lymphocytes. Specific hybridization signals were Division of Nephrology, Center for Health Sciences, detected by incubating the hybridized slides in fluorescein- University of California at Los Angeles School of Medicine, Los Angeles, California 90095-1698 ated antidigoxigenin antibodies followed by counterstaining with DAPI for one-color experiments. Probe detection for Received December 2, 1998; accepted February 3, 1999 two-color experiments was accomplished by incubating the slides in fluoresceinated antidigoxigenin antibodies and Texas red avidin followed by counterstaining with DAPI. A total of 80 metaphase cells were analyzed with 74 exhibiting Functional gene description: NBC3, designated SLC4A7, specific labeling. is a new member of the sodium bicarbonate cotransporter Results: The initial experiment resulted in specific label- (NBC) family (6) cloned from a human muscle cDNA library ing of the short arm of a group A chromosome, which was (5). Muscle NBC3 (mNBC3) encodes a 1214-residue polypep- believed to be chromosome 3 on the basis of size, morphology, tide with 12 putative membrane-spanning domains. mNBC3 and banding pattern. A second experiment was conducted in is 78% homologous to NBC2 (3) and 39 and 46% homologous which a biotin-labeled probe that is specific for the centro- to pNBC1 and kNBC1, respectively (1, 2). The mNBC3 tran- mere of chromosome 3 was cohybridized with clone 15. This script is strongly expressed in skeletal muscle with less ex- experiment resulted in specific labeling of the centromere pression in heart. To begin to understand the genetic factors and the arm of chromosome 3. Measurement of 10 specifically responsible for this pattern of expression, we have isolated labeled chromosomes 3 demonstrated that NBC3 maps to a position that is 64% of the distance from the centromere to This work was supported by NIH Grant DK46976, the Iris and B. Gerald Cantor Foundation, the Max Factor Family Foundation, the the telomere of chromosome arm 3p, an area that corre- Verna Harrah Foundation, the Richard and Hinda Rosenthal Foun- sponds to band 3p22 (Fig.