Isolation and characterization of genomic sequences involved in the regulation of the human reduced folate carrier gene (RFC1)

Decreased reduced folate carrier (RFC) activity has been associated with MT X resistance in experimental models of transport-mediated MTX resistance, a nd has been attributed to changes in the expression of RFC1, the gene that encodes a protein with this activity. RNA transcripts of RFC1 may contain a ny one of four distinct 5' untranslated regions (UTRs). We cloned a human g enomic DNA fragment upstream from the RFC1 translation start site and deter mined the nucleotide sequence of a 4.8 kb region that contained the exons c orresponding to each of the reported UTRs. To identify regulatory elements that may be involved in RFC1 transcription, we first developed a semi-quant itative RT-PCR assay using primers specific for each of the 5' UTRs to ampl ify RNA transcripts containing each of the RFC1 5' exons, and found evidenc e for differential transcription of RFC1 noncoding exons in tissues, during development, and in MTX-resistant, transport-deficient cells. We also foun d that RFC1 RNA levels are cell cycle regulated and peak at the G1 to S tra nsition. Then, using a series of RFC1 promoter-reporter fusion constructs, we identified genomic sequences that may be involved in the regulation of e xpression of exons Ib and Ic of the RFC1 gene. These studies therefore iden tify regulatory regions of RFC1 promoters and potential models of regulatio n in which these regions may exert control. (C) 1999 Elsevier Science B.V. All rights reserved.