Determination of Transcription Start Site and Analysis of Promoter Sequence, Splice Junction Sites, Intron Sequence and Codon Usage Bias of Rat Liver-specific Organic Anion Transporter-1 (rlst-1/Oatp-4/Slc21a10) Gene

Abstract

The full-length coding sequence of rat liver specific organic anion transporter-1 (rlst-1/Oatp4/Slc21a10) was cloned by our group (Choudhuri et al., 2000) and by Cattori et al. (2000). We also cloned a splice variant of rlst-1 (Choudhuri et al., 2000). Another splice variant was cloned by Kakyo et al. (1999). We had also obtained a BAC clone from screening a BAC-Rat library, and determined the exon breakpoints to be able to determine the origin of the splice variants. Since our original publication, we have further characterized the rlst-1 gene. In this communication, we report the sequence of about 2.3 kb of the 50-flanking sequence of the rlst-1 gene that contains the promoter. We have mapped the transcription start site to define the precise location of the promoter. We also report here the splice junction sequence, and the partial sequences and lengths of introns of the rlst-1 gene. Finally, we have analyzed the codon usage bias in the rlst-1 coding sequence. Transcription start site was determined by capsite cloning as described by Choudhuri et al. (2001) with two major modifications. First, 20mg total RNA (instead of polyA mRNA) and secondly, Gene Racer RLM RACE system (In Vitrogen, CA), were used for this experiment. Sequence of the gene specific reverse primer (GSP), and nested gene specific reverse primer (nGSP) used, was as follows: