Enhancer of RNA polymerase III gene transcription.

A protein responsible for enhanced transcription by RNA polymerase III was identified in extracts from Xenopus oocytes. This protein, called EP3, interacts with a specific DNA sequence adjacent to the 3'-end of a Xenopus somatic 5S RNA gene and forms a distinct band shift complex with a unique DNase I footprint. Enhanced transcription was observed from both 5S RNA and tRNA reporter genes when EP3 binding sites were inserted at different locations and orientations. Removal of the EP3 protein from an oocyte extract abolished this enhanced transcription. In addition, EP3 was shown to stimulate transcription by increasing the rate of transcription complex assembly. EP3 directly discriminates between the somatic and oocyte 5S RNA gene families and may play a significant role in their differential expression during early Xenopus development.

Interaction of Xenopus TFIIIC with a 5S RNA gene.

Using fractionated Xenopus transcription factors we have identified and characterized a unique protein-DNA complex formed between TFIIIA, TFIIIC and a 5S RNA gene. The formation of this complex was blocked by specific competitor DNAs and by the inactivation of TFIIIC using two different methods. In addition, TFIIIC activity was retained when the complexes were affinity purified using a reversibly immobilized DNA template. The TFIII(A+C)-5S RNA gene complex has a distinct electrophoretic mobility on band-shift gels and a unique DNase I footprint. The characteristic feature of the DNase I footprint is a TFIIIC-dependent extension of the TFIIIA footprint an additional 25 bp toward the 5' end of the gene. This indicates a direct interaction between Xenopus TFIIIC and the template DNA.

Calcium-dependent inactivation of RNA polymerase III transcription.

Extracts from whole oocytes of Xenopus laevis are widely used as an efficient in vitro system for the transcription of cloned genes by RNA polymerase III. We have found that these extracts no longer support RNA polymerase III transcription in response to a brief incubation in the presence of Ca2+. However, when transcription complexes were first formed on the genes, a subsequent incubation in the presence of Ca2+ had little effect. Fractionation of extracts was used to show that transcription factors (TF) IIIC and, to a lesser extent, TFIIIB, but not RNA polymerase III, were targets of the Ca(2+)-dependent inactivation process. An additional component (not present in fractionated TFIIIC or TFIIIB) was required for the Ca(2+)-dependent destruction of transcription factor activity. The Ca(2+)-dependent inactivation process was blocked by protease inhibitors that inhibit known Ca(2+)-dependent proteases called calpains. These results suggest that TFIIIC and TFIIIB are inactivated by an endogenous calpain. The common use of Ca2+ as a second messenger and the widespread distribution of calpains suggest that the proteolytic degradation of transcription factors may be a general mechanism for the regulation of gene expression.

Alcoholism and treatment in airline aviators: one company's results.

Airline transport pilots are at risk for alcoholism, although prevalence statistics are not known. (Alcoholism is used to mean alcohol dependence as defined in DSM-III-R.) Whether their prevalence of alcoholism is consistent with that of the general population's, less, or possibly enhanced by psychological vulnerabilities, is considered. However, the development and implementation of this job-based, peer-oriented alcohol treatment can be an asset to pilot career progression and airline pilot retention. According to a retrospective analysis of identified aviators, 87% of alcoholic pilots of this major airline returned to flight duties after substance abuse treatment. Relapse occurred in 13% of those accepting treatment. Early identification and treatment of the substance-abusing aviator can be enhanced by encouraging teamwork between pilots, union, airline management, flight surgeons, and employee assistance program professionals.