Nucleotide sequence and characterization of the 5' flanking region of the rat Ha-ras protooncogene.

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Abstract
Thyrotropin and cAMP stimulate growth of FRTL5 rat thyroid cells and increase c-Ha-ras mRNA levels. To study the mechanism by which thyrotropin enhances c-Ha-ras expression in the thyroid, we constructed a genomic library of FRTL5 DNA in the bacteriophage vector EMBL3. Using a v-Ha-ras probe (0.7-kilobase Sst I-Pst I fragment), we isolated eight clones containing portions of the FRTL5 c-Ha-ras gene. Restriction mapping of one of these clones revealed a structure very similar to that previously reported for the rat c-Ha-ras gene. We determined the nucleotide sequence of exon 1 as well as 1.15 kilobases upstream from exon 1. Blot-hybridization analysis of FRTL5 thyroid cell mRNA was performed with three DNA fragments upstream of exon 1. Two of these probes were Pst I-Pst I fragments 0.4 and 0.55 kilobases long, 1.15 and 0.6 kilobases upstream of exon 1, respectively. The third probe, a 0.6-kilobase Pst I-HindIII fragment, was immediately upstream of exon 1 and included 54 base pairs of the 5'' end of exon 1. The data revealed an upstream ("-1") exon consistent with the homology between the nucleotide sequences of our clone and the human c-Ha-ras gene in this region. Primer extension of a synthetic 30-mer oligonucleotide probe complementary to exon +1 on a FRTL5 mRNA template revealed three transcription start (cap) sites, 182, 169, and 153 bases upstream of the ATG codon. "CCAAT boxes" are present 65 and 100 bases upstream from the initial cap site. Three "GC boxes" and two C + G rich-inverted repeats characteristic of the binding site for the transcription regulation factor Sp1 are present in the 177 base pairs upstream of the initial cap site. Comparison of the rat and human c-Ha-ras -1 exons showed an area of poor homology immediately upstream of the human cap sites, followed further upstream by a region of close homology (.apprxeq. 60 base pairs). The nucleotide sequence of the rat -1 exon is more similar to the v-ras sequence than is the human. The v-ras gene is truncated relative to both human and rat -1 exons.