Increased transcriptional activity of the c-Ha-ras gene product is correlated with induction of several important human tumor types. For this reason, we have investigated the nature of the c-Ha-ras promoter and the factors that regulate its expression. Using Si and primer extension analysis of c-Ha-ras RNA from EJ cells, we have identified 18 initiation sites within an upstream exon (exon -1) whose 3' end (the donor splice site [DI) is located 1,105 base pairs (bp) upstream of the ATG codon. The furthest-upstream initiation site is located -191 bp relative to D, and the furthest downstream is located -16 bp relative to D. Transient expression assays, in which a series of mutants spanning this region were ligated to a promoterless chloramphenicol acetyltransferase vector, functionally confirmed the position and extent of this promoter. Mutational analysis further located a 47-bp element located between -243 and -196 relative to D that up-regulated transcriptional activity of the promoter region by 20-to 40-fold. This region contained both a GC box known to bind SP1 and a CCAAT box. Insertion of a simian virus 40 enhancer 5' to the promoter up-regulated transcription from each initiation site by approximately 10-to 20-fold. We have also localized, both by chloramphenicol acetyltransferase assay and by SI analysis, a strong promoter operating in the direction opposite that of the gene and originating immediately 5' to the 47-bp regulatory region. The reverse promoter was found to have nine initiation sites between -248 and -278 relative to D.The members of the ras gene family were first identified as cellular homologs of the Harvey and Kirsten sarcoma virus oncogenes (21). Genes bearing a high degree of homology to the mammalian ras family have been identified in organisms from virtually every evolutionary group, including Drosophila (51, 59), yeasts (18), and slime mold (56). In mammals, the ras genes appear to be expressed in all cell types and at all developmental stages (48, 49). The high degree of evolutionary conservation and constitutive expression of these genes in different cell types has led to the suggestion that they fulfill some essential cellular functions (9).The human ras gene family (Ha-ras, K-ras, and N-ras) is highly conserved in the human genome and encodes structurally related proteins of approximately 21,000 daltons (60). The p21 proteins are localized at the plasma membrane (67), bind guanine nucleotides (58), and have weak GTPase activity (43). Furthermore, there is some structural homology between ras proteins and the G protein known to regulate hormone-sensitive adenylate cyclase activity (28). Again, this suggests a crucial role for these genes in cellular control. Direct support for this suggestion has come from several experimental systems. It has been shown that the ras proteins can induce cellular proliferation (5,22,50) and can also influence cellular differentiation (3, 31).Members of the ras oncogene family have been implicated in the etiology of a variety of naturally occurring...
We have identified a short, highly repetitive element within intron -1 of the human c-Ha-ras gene. This element was found to be transcribed in both orientations and to be homologous to heterogeneous nonpolyadenylated transcripts. The repetitive element blocked transcriptional readthrough from a strong upstream viral promoter but allowed unimpaired readthrough from the c-Has-ras promoter. We suggest that it may serve to prevent excessive transcription into the coding region of the gene under such circumstances as viral insertion.Elevated expression of the c-Ha-ras gene is associated with many important tumor types, such as breast (28), colon (13), and gastric (4). In addition, elevated expression of the normal gene under the control of a retrovirus long terminal repeat has been shown to induce tumorigenic transformation of 3T3 cells (7), and activation of the c-Ha-ras oncogene in an avian nephroblastoma has been attributed to proviral insertion (30).The potential importance of c-Ha-ras transcriptional deregulation in tumor progression led us to examine the nature of the c-Ha-ras promoter and the factors which regulate its expression (20), and we have recently identified a bidirectional c-Ha-ras promoter whose 3' end (the donor splice site) is located 1,105 base pairs (bp) upstream of the ATG codon. The direct promoter contains 18 initiation sites between -196 and -16 bp relative to the donor splice site (D). The reverse promoter contains nine initiation sites between -248 and -278 bp relative to D. A 43-bp element between -243 and -196 up regulates activity of the direct promoter by 20-to 40-fold (20; summarized in Fig. lc).A highly repetitive, transcribed element in c-Ha-ras intron -1. To confirm that the far upstream exon -1 promoter gives rise to mature 1,400-bp c-Ha-ras mRNA, we performed Northern (RNA) blot analysis, using probes derived from exon -1 and the coding exon 1. Total RNA was prepared from EJ bladder carcinoma cells (23) and the human erythroleukemia cell line K562 (26). Probes derived both from exon -1 and exon 1 detected a major 1,400-bp c-Ha-ras mRNA species and a minor species of approximately 5.5 kilobase pairs (kb) (Fig. la). To determine whether the larger RNA species represented a splicing intermediary, a 1.16-kb Sau3A fragment containing intron -1 was used as a hybridization probe. Surprisingly, this probe hybridized to highly repetitive heterogeneous RNA species ranging in size from approximately 7 kb to 800 bp. We proceeded to localize the DNA sequence coding for repetitive RNA by using a series of probes derived from intron -1. The 837-bp XmaIII-Sau3A probe (containing the 3' end of intron -1 and coding exon 1) hybridizes to c-Ha-ras 1,400-bp mRNA, whereas the 305-bp XmaIII probe * Corresponding author. detects the repetitive species. A 130-bp XmaIII-SstII fragment from the extreme 5' end of intron -1 also hybridizes to repetitive RNA, whereas the 170-bp SstII-XmaIII probe does not. The repetitive RNA species are mainly represented in the nonpolyadenylated RNA fraction. Using multiple ...
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