Isolation, sequencing, and the genomic organization of the reduced folate carrier gene in the murine system

Abstract

Defective closure of the neural tube during embryogenesis results in serious and common birth defects such as spina bifida and anencephaly. Neural tube defects (NTDS) occur at an average rate in the U.S. of 1 per 1000 live-births with selected ethnic groups having higher or lower prevalence rates. Several investigators have shown a marked decrease in the occurrence and recurrence rates of NTDs with the administration of periconceptional supplemental folic acid. Intracellular folates are believed to be responsible for the reduction in NTDs due to their role in facilitating single-carbon transfers in critical biochemical pathways leading to DNA synthesis. Alterations in the folate pathway could compromise DNA synthesis and cellular proliferation, potentially disrupting normal developmental events. To this end, the genetic regulation of folate internalization is being investigated. The reduced folate carrier (RFC) delivers 5-methyltetrahydrofolate from the folate receptors to the cytoplasm of the cell. As a first step in defining the relationship between NTDs and aberrant folate internalization, we isolated and partially sequenced the RFC gene in the mouse. Screening of a murine ES cell genomic library produced eight positive clones; three were selected for subcloning. An approximately 1OKb positive clone was subcloned into pbluescript and has been divided into smaller subclones. Utilizing each subc[one independently, and deriving sequencing primers from the known CDNA sequence of the gene, followed by chromosome walking, 5.6Kb of the genomic sequence was derived. The gene contains eight exons and seven introns. The gene forms at least four splicing variants with alternate exons located 5'of exons 1 and 5, and as an extension of exon 3a. Future studies will be initiated to determine the tissue specific expression of the RFC gene in relation to the folate binding proteins. The RFC variant expression will be localized and a knock-out mouse constructed. Utilizing this model system, correlations between altered RFC function, folate supplementation, and the occurrence of NTDs may be elucidated.