Organization and regulation of the human rasGAP gene.

ras GTPase activating protein (rasGAP) is highly conserved among mammalian species and is required for normal cardiovascular system development. Expression of this protein exhibits both quantitative and qualitative variability among tissues. Using a combination of DNA sequencing and database analyses, we have determined that the human rasGAP gene spans 122 kb and is composed of 25 exons; the size of each intron and the intron/exon junctions also have been elucidated. With one exception, all intron/exon boundaries conform to the GT/AG rule; the splice donor site of intron 3 is GC/AG. Results of RNA ligase mediated rapid amplification of cDNA ends followed by sequence determination indicate that the transcription start point (TSP) is approximately 588 bp upstream from the translational start site and is uninterrupted by introns; this extremely long 5' untranslated region is continuous with the first coding exon. Analysis of 1 kb of sequence upstream of the TSP did not identify any of the typical promoter elements (TATA or CAAT boxes). Sequential deletions of this 1 kb region followed by secreted alkaline phosphatase reporter gene analysis revealed that transcription is supported by this region of the rasGAP gene. Because the highest efficiency is demonstrated by a 213 bp sequence just upstream from the TSP (-786 to -584), this region is identified as containing the rasGAP minimal promoter. Sequence analysis of this 213 bp sequence shows few candidate sites for transcription factor binding. A 406 bp fragment surrounding the TSP exhibits characteristics of a CpG island (68% C+G; observed/expected ratio of CpG=0.95). RapidScan analysis revealed that high levels of rasGAP transcript are present in placenta and testis, but transcript is not detectable in kidney and intestinal tract. These data suggest that rasGAP transcription is regulated by an atypical mechanism capable of producing quantitative variability among tissue types.

Sodium arsenite inhibits and reverses expression of adipogenic and fat cell-specific genes during in vitro adipogenesis.

Arsenic causes cancer in humans, but its mechanism of action is unique among known carcinogenic agents. As a naturally occurring component of sediments and ground water, human exposure to arsenic is inevitable, necessitating the establishment of exposure limits. Because cancer is characterized as an imbalance between cell growth and differentiation, it has been hypothesized that arsenic exerts its carcinogenic effect, in part, by perturbing the balance between these antagonistic processes. Previous work in this laboratory has demonstrated that sodium arsenite prevents adipocytic differentiation of C3H 10T1/2 cells, leading to the hypothesis that the underlying mechanism involves downregulation of genes associated with adipogenesis. In support of this hypothesis, it was found that mRNA levels of peroxisome proliferative-activated receptor gamma (PPAR gamma), CCAAT-enhancer binding protein alpha (C/EBP alpha), and adipocyte-selective, fatty acid-binding protein (aP2) are decreased in arsenic-treated cells; arsenic-induced phenotypic reversion of differentiated adipocytes correlates with reduced aP2 expression. Arsenic also blocks upregulation of p21(Cip1/Waf1), a factor whose expression is tightly regulated during adipogenesis. The differentiating effect of pioglitazone, which induces adipogenesis by activating PPAR gamma, is inhibited by arsenic, suggesting that arsenic interferes with adipogenic signaling at or below the level of PPAR gamma. Because C/EBP alpha is important in the expression of certain keratinocyte-specific genes, the negative effect of arsenic on C/EBP alpha might also contribute to the development of skin cancer. PPAR gamma, C/EBP alpha, and p21(Cip1/Waf1) are important in numerous normal and pathological processes, including carcinogenesis, leading us to postulate that perturbation of these factors by arsenic might contribute to the carcinogenic effect of this metalloid.