ORGANIZATION OF THE MURINE REDUCED FOLATE CARRIER GENE AND IDENTIFICATION OF VARIANT SPLICE FORMS

RT-PCR analysis of the reduced folate carrier (RFC) from L1210 and mur ine erythroleukemia cells led to the identification of three clones wh ich appeared to result from the use of alternative splice sites. The n ucleotide sequence of each splice form predicts a protein that contain s at least the first 7 transmembrane domains of the parental RFC prote in followed by a novel hydrophilic carboxyl terminus of 33, 72, or 105 amino acid residues. Sequence analysis of cDNA clones isolated from m urine liver and the results of 5'-RACE from L1210 cells indicated that RFC also utilizes alternate 5'-terminal exons. To understand how the alternatively spliced RFC transcripts and multiple 5'-termini were gen erated, the genomic organization of RFC was determined. The gene is co mprised of at least 8 exons,the first two of which encode the alternat ive 5' termini. Based on sequence identity with cDNAs encoding RFC fro m hamster and rat, however, it appears that additional 5' exons may be present. Two of the RFC splice variants result from the use of a cryp tic splice donor site within exon 4 and the third results from the use of a cryptic splice acceptor site within exon 5. In addition, the spl ice variant form that encodes the largest protein also utilizes an alt ernative exon located between exons 5 and 6. The apparent use of alter native transcriptional start sites and the identification of several R FC splice forms raises the possibility that unique RFC molecules may b e generated that exhibit tissue-or cell line-specific distribution. (C ) 1997 Elsevier Science B.V.