The Structural Organization of the Human Na+/Myo-inositol Cotransporter (SLC5A3) Gene and Characterization of the Promoter (original) (raw)

adenylation signals, and promoter of the human Na / / bers of the family, the electrochemical gradient for Na / myo-inositol cotransporter (SLC5A3) gene have been provides the driving force for active MI transport. In elucidated through cloning, sequencing, mRNA analyaddition, the SLC5A3 gene plays an important role in ses, and reporter gene assays. The gene consists of one mammalian osmoregulation, allowing cells to maintain promoter and two exons spanning approximately 26 cell volume regulation but not at the expense of perkb. Exon 1 contains 175 bp of 5 untranslated sequence turbing cellular ion concentrations (Thurston et al., and is 15 kb upstream of exon 2. The 9.5-kb exon 2 1989; Strange et al., 1991, 1994; Handler and Kwon, contains the entire 2157-bp open reading frame and 1996). a large 3 untranslated sequence with seven putative We recently cloned the human SLC5A3 gene and sepolyadenylation signals. Multiple messages with difquenced the coding region (Berry et al., 1995). Like the ferent-sized 3 untranslated regions can be detected on canine and bovine SLC5A3 genes (Rim et al., 1997; Northern blots. Hypertonic stress caused mRNA levels, Berry et al., 1995; Mallee et al., 1995), it is atypical and primarily that of the full-length 9.5-kb transcript, in that the coding region is intron-free and contained to increase in cultured melanoma cells; ribonuclease within an exceptionally large exon. The human gene protection analysis demonstrated that the transcriphas been fine-mapped to chromosome 21q22.1 (Berry tion start site was the same in stressed as in control et al., 1996). It is expressed in many tissues including cells. The SLC5A3 gene functions in cellular osmoregubrain, kidney, and placenta (Berry et al., 1995). The lation and is expressed in many human tissues includmajority of patients with trisomy 21 (Epstein, 1995) ing the brain, kidney, and placenta. It is localized to may be unable to repress the expression of the three chromosome 21q22.1. An overexpression of the SLC5A3 gene deserves consideration as a factor in the patho-SLC5A3 genes so that both energy-dependent uptake physiology of Down syndrome. ᭧ 1997 Academic Press and facilitated efflux of MI could be increased in some or all cells that express SLC5A3 genes (Fruen and Lester, 1990, 1991). To identify potential genetic factors MATERIALS AND METHODS the precursor of the signal-transducing phosphoinosi-Isolation of genomic clones. The first human SLC5A3 genomic clone that was isolated was from a bacteriophage lambda FIX II Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. AF027153. library from Stratagene Cloning Systems (La Jolla, CA) (Berry et al., 1995). Primers corresponding to a portion of the 3 untranslated J. J. Mallee and M. G. Atta contributed equally to this work. 1 To whom correspondence should be addressed at Division of Bio-region were used to screen a human bacteriophage P1 library from Genome Systems, Inc. (St. Louis, MO) using PCR (Berry et al., 1995). chemical Development and Molecular Diseases, The Children's Hospital of Philadelphia, 34th Street & Civic Center Boulevard, Philadel-Two overlapping clones (P1 3283 and P1 3284) were isolated from this library with average inserts of 90 kb. The P1 clones were di-phia, Pennsylvania 19104. Telephone: (215) 590-3372. Fax: (215) 590-3364. gested as described below, and fragments were subcloned in pBlue-459