Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells.

Development of transcriptional pulsing approaches using the c-fos and Tet-off promoter systems greatly facilitated studies of mRNA turnover in mammalian cells. However, optimal protocols for these approaches vary for different cell types and/or physiological conditions, limiting their widespread application. In this study, we have further optimized transcriptional pulsing systems for different cell lines and developed new protocols to facilitate investigation of various aspects of mRNA turnover. We apply the Tet-off transcriptional pulsing strategy to investigate ARE-mediated mRNA decay in human erythroleukemic K562 cells arrested at various phases of the cell cycle by pharmacological inhibitors. This application facilitates studies of the role of mRNA stability in control of cell-cycle dependent gene expression. To advance the investigation of factors involved in mRNA turnover and its regulation, we have also incorporated recently developed transfection and siRNA reagents into the transcriptional pulsing approach. Using these protocols, siRNA and DNA plasmids can be effectively cotransfected into mouse NIH3T3 cells to obtain high knockdown efficiency. Moreover, we have established a tTA-harboring stable line using human bronchial epithelial BEAS-2B cells and applied the transcriptional pulsing approach to monitor mRNA deadenylation and decay kinetics in this cell system. This broadens the application of the transcriptional pulsing system to investigate the regulation of mRNA turnover related to allergic inflammation. Critical factors that need to be considered when employing these approaches are characterized and discussed.

The influence of ideological interest groups on congressional financial support of international family planning

The World Health Organization estimates there are about 585,000 maternal deaths each year, with 98% of the maternal deaths in developing countries. Access to family planning methods is one method to decrease maternal mortality and morbidity. ^ The U.S. was the leader in providing financial and technical assistance to developing countries to enable women to have this access. The election of Ronald Regan changed the course of U.S. support; abortion became a central factor in the political decision-making with regards to the financial support of international family planning. ^ One factor that may sway policy-makers' decisions is the influence of ideological interest groups, Political Action Committees funding of candidates in relation to votes on bills that impacted on financial support of international family planning and the amount spent by these groups on lobbyist was reviewed. Pro-choice funding of candidates was greater for the four of the fives votes supporting family planning. Pro-Choice lobbyist spending was $185,000 vs. $8,184,000 spent by Pro-Life ideological groups. ^