Analysis of 5' flanking sequences and intron-exon boundaries of the rat prolactin gene.

Abstract

A rat genomic DNA clone containing the 5’ flanking region, three exons, two introns, and a portion of a third intron of the rat prolactin gene was isolated and characterized. Sequence determinations were used to identify the exon-intron boundaries and analyze the DNA region upstream from the initiator methionine codon. Another genomic DNA clone that we previously characterized (Gubbins, E. J., Maurer, R. A., Lagrimini, M., Erwin, C. R., and Donelson, J. E. (1980) J. Biol. Chem. 255, 8655-8662) contains two additional exons and the 3’ flanking region of the prolactin gene. These five exons account for the complete coding sequence of rat preprolactin. Their relative locations in the two genomic DNA clones indicate that the five exons and four intervening sequences (introns) of the rat prolactin gene comprise approximately 10 kilobases of DNA. The sequence at the Intron A-Exon I1 boundary was found to contain two separate splicing sites offset by three nucleotides. These two splicing sites can explain a discrepancy of one codon in two published preprolactin cDNA sequences. The 5’ terminus of prolactin mRNA was mapped using the S1 nuclease protection procedure. The results suggest that transcription is initiated 54 nucleotides upstream from the initiator methionine codon. Analysis of the sequence preceding this site reveals (i) the sequence TATAAA at positions -21 to -22 which, by analogy with other systems, is likely involved in initiation of prolactin gene transcription and (ii) the palindrome, TGATTATATATATATTCA, at positions -62 to -45 which may be a site involved in the regulation of prolactin gene transcription.