SHORT COM M UNICATION Identification of a Testis-Expressed Creatine Transporter Gene at 16p11.2 and Confirmation of the X-Linked Locusto Xq28 GEETA S. IYER ,* R ALF KRAHE ,² L YNNE A. GOODWIN,‡ NORMAN A. DOGGETT,‡ M ICHAEL J. S ICILIANO,² VICKY L. F UNANAGE ,* AND R OY P ROUJANSKY* ,1 * Department of Clinical Science, Nemours Research Programs, Alfred I. duPont Institute, Wilmington, Delaware 19899, and Department of Pediatrics, Jefferson Medical College, Philadelphia, Pennsylvania; ‡Life Sciences Division and Center for Human Genome Studies, Los Alamos National Laboratory, Los Alamos, New Mexico; and ² Department of Molecular Genetics, M.D. Anderson Cancer Center, Houston, Texas Received December 14, 1995; accepted February 28, 1996 quenced by standard methods (2). A 2.6-kb clone Creatine and creatine phosphate act as a buffer sys- (2A1.1) was isolated from a human small intestinal tem for the regeneration of ATP in tissues with fluctu- cDNA library and was sequenced, revealing 100% ating energy demands. Following reports of the clon- agreement with the reported cDNA sequence for the ing of a creatine transporter in rat, rabbit, and human, human creatine transporter, CT1 (11). we cloned and sequenced a creatine transporter from A panel of somatic cell hybrids (BIOS Labs, New a human intestinal cDNA library. PCR amplification Haven, CT) was initially used to map the human cre- of genomic DNAs from somatic cell hybrid panels local- atine transporter utilizing PCR primers CTF7 and ized two creatine transporter (CT) genes: CT1 to Xq26 – CTR7 (Table 1). PCR was performed by standard meth- q28 and CT2 to 16p11.2. Refinement of CT1 to Xq28 was ods (3). Comparison of the PCR products with the chro- confirmed by FISH. Identification of CT2 sequences in mosome content of the hybrid panel identified the X YACs and cosmid contigs that had been ordered on chromosome as showing the lowest discordance fre- human chromosome 16 enabled its assignment to the quency (5%); the only discordant hybrid was one that proximal end of 16p11.2. Sequencing of the CT2 gene had chromosome 16 as its sole human chromosome identified sequence differences between CT1 and CT2 (CY18) (data not shown). Subsequent mapping by FISH transcripts that were utilized to determine that CT2 clearly demonstrated localization of CT1 to Xq28 (data is expressed in testis only. CT2 is the most proximally not shown), confirming previous studies (8). identified gene on chromosome 16p to date. The exis- Regional assignment of the CT sequence on chromo- tence of an autosomal, testis-specific form of the hu- some 16 (designated CT2) was made with the following man creatine transporter gene suggests that creatine somatic cell hybrids containing deletion derivatives of transporter activity is critical for normal function of chromosome 16: CY18, CY14, CY12, CY180A, CY7, spermatazoa following meiosis.  1996 Academic Press, Inc. CY6, CY3, CY2, CY153, CY199, CY152, and CY145(P) (1). CT2 was assigned to 16p11.2, between the somatic Creatine entry into tissues in which it is not synthe- cell hybrid breakpoints CY152/CY153 and CY199. sized occurs as a function of a sodium-dependent cre- Primer pair CTF7/R7 was then used to screen YACs atine transporter (CT1), the gene for which has been that were previously mapped to the interval defined cloned in rat (12), rabbit (9), and, more recently, human by the breakpoints CY180A and CY145(P) (5) by PCR. (11, 13). We identified significant creatine kinase activ- CTF7/CTR7 primers produced amplification products ity in human intestinal epithelium (manuscript in of the correct size only in CEPH YACs y895G9 and preparation). This suggested the presence of a creatine y662D12 from this region. It was previously shown that transporter in human intestine. PCR primers, designed STS s318A1, derived from cosmid c318A1 in contig utilizing the reported sequences of the rat and rabbit C177.1, was contained in the same two YACs. We then creatine transporters (9, 12), were used to amplify a tested all cosmids from contig C177.1 and found that 358-bp PCR product from oligo(dT)-primed cDNA from cosmids c318A1, c51C10, c329B6, c10D9, and c79D9 human intestine. This PCR product was then used to contained the region defined by CTF7/CTR7, but cos- recover clones from a human small intestinal cDNA mid 27F4 did not (Fig. 1). These YAC and cosmid clones library (Clontech, Palo Alto, CA), which were then se- are the most proximally placed clones on chromo- some 16p (5). 1 To whom correspondence should be addressed at Division of Gas- PCR primers (Table 1), designed to amplify overlap- troenterology & Nutrition, Alfred I. duPont Institute, 1600 Rockland ping regions of the CT1 cDNA sequence, were used to Road, Wilmington, DE 19899. Telephone: (302) 651-5928. Fax: (302) 651-5838. amplify the CT2 gene from both y895G9 and y662D12. 143 GENOMICS 34, 143–146 (1996) ARTICLE NO. 0254 0888-7543/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved. AID Genom 4077 / 6r15$$$$41 04-18-96 01:18:40 gnmxa AP: Genomics