Structure of the murine mb-1 gene encoding a putative sIgM-associated molecule

Genomic DNA clones containing the B cell-specific murine mb-1 gene were isolated and a 5.6-kb BamH I fragment was characterized. It is 5629 bp long and contains five exons: an exon containing the 5' untranslated and the coding sequence of the signal peptide, an exon of 294 bp, which contains most of the extracellular sequence of the MB-1 protein, a 119-bp long exon coding mainly for the transmembrane portion, and two exons of 69 bp and 427 bp encoding the cytoplasmic domain and the 3'-untranslated region, respectively. The mb-1 gene does not contain a "TATA box" found in many eukaryotic promoters. The 5'-flanking region has sequence stretches homologous to IgVH 5'-promoter regions and a bcl 2 intron sequence. It contains the decanucleotide sequence (ATGGCAAATA) almost identical to the octamer motif of IgVH promoters. A B cell-specific DNase I-hypersensitive site was found in the 3'-flanking region indicating that this region might be involved in B cell-specific expression of mb-1. Southern blot analysis of genomic liver DNA with the cloned mb-1 cDNA suggests the existence of another mb-1-related gene segment.     

Studies on the promoter region of the c-Ha-ras gene in FRTL5 rat thyroid cells

The functional activity of the promoter region of the rat c-Ha-ras gene was examined in FRTL5 rat thyroid cells, the cell type from which this promoter was cloned. A plasmid (p035-ras-CAT) was constructed containing the untranslated-1 exon as well as 172 base pairs (bp)5' to this exon inserted upstream of the chloramphenicol acetyl transferase (CAT) reporter gene. These 172 bp of 5'-flanking region contain two 10 bp GC box consensus sites and two CAAT boxes. Very weak promoter activity was observed in experiments involving transient transfection of FRTL5 cells with this plasmid, as well as with another plasmid (p5kb-ras-CAT) containing a much more extensive (3.5 kb) 5'-flanking region of the gene. In contrast, strong promoter activity was observed when the same plasmids were transfected into mouse 3T3 fibroblasts. When other promoters (pfos, RSV, and MMTV) were used to drive CAT activity, CAT activity in FRTL5 cells was about 10-fold less than in NIH-3T3 cells and rat embryo fibroblasts. However c-Ha-ras promoter activity was reduced out of proportion in FRTL5 thyroid cells relative to the other cell types (approximately 50-fold less). DNA gel-shift assays performed using crude extracts of FRTL5 and 3T3 nuclear proteins revealed quantitatively similar binding to the same promoter region in the c-Ha-ras 5'-flanking sequence. These data demonstrate that promoter activity of the rat c-Ha-ras gene is contained within the 172 bp 5'-flanking region of the gene. This promoter activity is expressed at a much lower level in slow-growing FRTL5 cells relative to other more rapidly growing cell types.     

The human IGF-I gene contains two cell type-specifically regulated promoters.

The human insulin-like growth factor-I (IGF-I) gene contains two alternative leader exons: exons 1 and 2. We have identified, by transient transfection experiments, the putative promoters P1 and P2 upstream of these leader exons. The promoter regions were cloned in front of the luciferase reporter gene and their promoter activities were measured in transfected SK-N-MC (human neuroepithelioma) and OVCAR-3 (human ovarian carcinoma) cells. Both of these cell lines express the IGF-I gene endogenously, resulting in normally sized IGF-I mRNAs of 7.6, 1.3 and 1.1 kb. In SK-N-MC cells, in which P1 is the most active IGF-I promoter, P2 displayed a three times lower promoter activity than P1. However, in OVCAR-3 cells, P2 is four times more active than P1, resulting in an overall 12-fold difference in the relative promoter activities of the two IGF-I gene promoters in these two cell types. This indicates that the IGF-I promoters show a cell type-specific expression pattern.     

Conserved regulatory elements in the promoter region of the N-CAM gene.

Genomic clones containing 5'-flanking sequences, the first exon, and the entire first intron from the chicken N-CAM gene were characterized by restriction mapping and DNA sequencing. A > 600-bp segment that includes the first exon is very G + C-rich and contains a large proportion of CpG dinucleotides, suggesting that it represents a CpG island. SP-1 and AP-1 consensus elements are present, but no TATA- or CCAAT-like elements were found within 300 bp upstream of the first exon. Comparison of the chicken promoter region sequence with similar regions of the human, rat, and mouse N-CAM genes revealed that some potential regulatory elements including a "purine box" seen in mouse and rat N-CAM genes, one of two homeodomain binding regions seen in mammalian N-CAM genes, and several potential SP-1 sites are not conserved within this region. In contrast, high CpG content, a homeodomain binding sequence, an SP-1 element, an octomer element, and an AP-1 element are conserved in all four genes. The first intron of the chicken gene is 38 kb, substantially smaller than the corresponding intron from mammalian N-CAM genes. Together with previous studies, this work completes the cloning of the chicken N-CAM gene, which contains at least 26 exons distributed over 85 kb.     

Structural characterization of exon 6 of the rat IGF-I gene.

In rat liver, insulin-like growth factor I (IGF-I) mRNAs exist as two major size classes of 7.5-7.0 kb and 1.2-0.9 kb. The 7.5- to 7.0-kb IGF-I mRNAs predominate in some nonhepatic tissues of the rat. Because the previously reported sequences of rat IGF-I cDNAs and genomic clones account for only 2.1 kb of sequence, the majority of the sequence of 7.5- to 7.0-kb rat IGF-I mRNAs was unknown. Using a combination of nucleotide sequencing of genomic DNA and cDNA clones and Northern hybridization and RNase protection, we have characterized a 6,354-base-long 3' exon (exon 6) of the rat IGF-I gene. The sequence of exon 6 establishes the previously unknown sequence of the 3' end of the 7.5- to 7.0-kb rat IGF-I mRNAs, comprised predominantly of an unusually long 3' untranslated sequence (3'UT). The long 3'UT contains multiple ATTTA, A(T)nA, and (T)nA sequences, as well as inverted repeats. These sequences may contribute to the shorter half-life of the 7.5- to 7.0-kb rat IGF-I mRNAs relative to the 1.2- to 0.9-kb forms that have been demonstrated previously in vitro and in vivo. We also demonstrate that the 7.5- to 7.0-kb rat IGF-I mRNAs are localized to the cytoplasm of rat liver, providing indirect evidence that they are mature and functional mRNAs.     

Human cystathionine β-synthase: gene organization and expression of different 5′ alternative splicing

The human cystathionine β-synthase (CBS) gene spans in excess of 30 kb and consists of 19 exons, with three different 5′ untranslated regions including three different exons 1 (exons 1 a, b, and c). Exon la and 1b are 390 bp apart from each other and are linked to exon 2 in cDNA « a » and cDNA « b ». Exon 1c, which linked to exon 5 in cDNA « c », is 7 kb apart from exon 1b. All splice sites conform to the GT/AG rule, including those from exon la or 1b to exon 2 and from exon 1c to exon 5. Upstream of exons la and 1b, we found two putative promoter sequences with high C + G nucleotide content, one CAAT box at —70 nucleotides (for exon lb), no TATA box, several Sp1 binding regulatory consensus sequences, and some other regulatory sequences. Human adult and fetal Northern blots hybridized with total cDNA containing exon 1b, or specific probes from exons 1 (b and c) showed mRNAs of 2.5 kb, 2.7 kb, and 3.7 kb. These results suggest that the mRNAs containing the different exons 1 are under the control of different promoters.