Paradoxical Effect of Eukaryotic Expression Vectors on Reporters

A critical issue in transfection or cotransfection experiments is to define the appropriate controls. In most cases, a corresponding empty vector is used as one control. We report a paradoxical effect of empty mammalian expression vectors on different reporters. We have found that different empty vectors can inhibit or stimulate the same reporter. In addition, the same vector can have different effects on different reporters. This situation is further complicated by the observation that the effects of a vector on a reporter can vary depending on the cells used

RNA polymerase I transcription in confluent cells: Rb downregulates rDNA transcription during confluence-induced cell cycle arrest

When 3T6 cells are confluent, they withdraw from the cell cycle. Concomitant with cell cycle arrest a significant reduction in RNA polymerase I transcription (80% decrease at 100% confluence) is observed. In the present study, we examined mechanism(s) through which transcription of the ribosomal genes is coupled to cell cycle arrest induced by cell density. Interestingly with an increase in cell density (from 3–43% confluence), a significant accumulation in the cellular content of hyperphosphorylated Rb was observed. As cell density increased further, the hypophosphorylated form of Rb became predominant and accumulated in the nucleoli. Co-immunoprecipitation experiments demonstrated there was also a significant rise in the amount of hypophosphorylated Rb associated with the rDNA transcription factor UBF. This increased interaction between Rb and UBF correlated with the reduced rate of rDNA transcription. Furthermore, overexpression of recombinant Rb inhibited UBF-dependent activation of transcription from a cotransfected rDNA reporter in either confluent or exponential cells. The amounts or activities of the rDNA transcription components we examined did not significantly change with cell cycle arrest. Although the content of PAF53, a polymerase associated factor, was altered marginally (decreased 38%), the time course and magnitude of the decrease did not correlate with the reduced rate of rDNA transcription. The results presented support a model wherein regulation of the binding of UBF to Rb and, perhaps the cellular content of PAF53, are components of the mechanism through which cell cycle and rDNA transcription are linked.This study was supported in part by National Institute of Health grants GM48991 (LI Rothblum), DK15658 (LS Jefferson) and DK13499 (LS Jefferson), the Juvenile Diabetes Foundation JDFI195051 (LS Jefferson) and an award from the Geisinger Foundation (LI Rothblum). All imaging experiments were performed at the Whitehead Institute Microscopy Facility. BK Kennedy is supported by a Leukemia Society of America Postdoctoral Fellowship. Dr Muramatsu of the Saitama Medical School of Japan, Department of Biochemistry graciously provided the original sample of PAF53 antisera used in these studies