The genomic structure of the human SPEC1 gene reveals complex splicing and close promoter proximity to the AF1q translocation gene.

SPECs are small Cdc42 signaling molecules. In mammals, two genes, SPEC1 and SPEC2, encode proteins of 79 and 84 amino acid residues, respectively. Here we report the expression and genomic organization of the human SPEC1 gene. Using Northern blot analysis, three major SPEC1 mRNA transcripts of 1.6, 3.3, and 6.3 kb were detected. Identification and sequencing of different sized SPEC1 cDNA clones revealed that the transcript size heterogeneity was due to alternative splicing in the 3'-untranslated region. In addition, a distinct SPEC1 splice variant from within the coding sequence, SPEC1-beta, was identified and detected in a variety of human tissues. Analysis of the genomic organization of SPEC1 revealed that the coding sequence of the SPEC1 isoform was derived from exons 2, 3 and 4, while the SPEC1-beta isoform was derived from exon 2 and a read-through event of intron 2. Examination of the 5'-end of the SPEC1 genomic sequence revealed that AF1q, a previously identified gene involved in translocations with the MLL (mixed-lineage leukemia) gene, was 631 bp away in a head-to-head orientation. This intergenic sequence containing the putative promoter region for both SPEC1 and AF1q genes did not contain a TATA box or CAAT box. Transfection experiments using an AF1q promoter luciferase reporter construct in a variety of cells including Cos1 cells, Jurkat T-cells, MCF-7 breast cancer cells, and NIH-3T3 fibroblasts showed no promoter activity. In contrast, a SPEC1 promoter luciferase reporter construct showed high levels of reporter activity in Cos1 and MCF-7 cells, low activity in NIH-3T3 fibroblasts and no activity in Jurkat T-cells. These promoter analyses suggest that although SPEC1 and AF1q genes share the same promoter region, they are not coordinately regulated.

Dissection of RAS downstream pathways in melanomagenesis: a role for Ral in transformation.

Cutaneous malignant melanoma is considered one of the most deadly human cancers, based on both its penchant for metastatic spread and its typical resistance to currently available therapy. Long known to harbor oncogenic NRAS mutations, melanomas were more recently reported to be frequent bearers of activating mutations in BRAF, one of the effectors situated downstream of wild-type NRAS. NRAS and BRAF mutations are rarely found in the same melanoma, suggesting that they may possess important overlapping oncogenic activities. Here, we compare and contrast the oncogenic roles of the three major NRas downstream effectors, Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine exchange factor (RalGEF), using genetically engineered Arf-deficient immortalized mouse melanocytes as a model system. Although no single downstream pathway could recapitulate all of the consequences of oncogenic NRas expression, our data indicate a prominent role for BRaf and PI3K in melanocyte senescence and invasiveness, respectively. More surprisingly, we discovered that constitutive RalGEF activation had a major impact on several malignant phenotypes, particularly anchorage-independent growth, indicating that this often overlooked pathway should be more carefully evaluated as a possible therapeutic target.

The encephalomyocarditis virus 3C protease is rapidly degraded by an ATP-dependent proteolytic system in reticulocyte lysate.

The encephalomyocarditis virus 3C protease has been observed to undergo rapid degradation, both in vivo in mouse cells and in vitro in reticulocyte lysate. Experiments were carried out to characterize the turnover of the 3C protease in reticulocyte lysate. 3C protease prepared in reticulocyte lysate by in vitro translation and processing of a precursor polyprotein could be separated from the proteolytic activity responsible for its degradation. This implies the 3C protease is not directly involved in its own proteolysis. Active 3C protease flanked by only a few amino acids was degraded at a rate identical to that of a similar protein containing an inactivated catalytic site. This indicates that 3C protease activity is not indirectly required for the proteolytic process. Other viral proteins, including the 3D polymerase and capsid proteins, were relatively stable in the lysate. In addition, polyprotein precursors containing 3C protease with an inactive catalytic site and various flanking proteins displayed distinctly different stabilities. These results suggest that the reticulocyte proteolytic system functions in a selective manner toward the viral proteins. The effects of several proteolytic inhibitors on the lysate proteolytic system were evaluated. The results of these experiments indicate that the rapid degradation of the EMC virus 3C protease requires the hydrolysis of ATP.