These data suggest that regulated polyadenylation site choice of male germ cell-specific polyadenylation sites requires one or more factors that are absent from somatic cells. Finally, mutation of the male germ cell-specific poly(A) signal to a somatic poly(A) signal resulted in more efficient polyadenylation in somatic cells. Using this assay, we demonstrated that male germ cell-specific polyadenylation cassettes were not polyadenylated at the expected site in somatic cells, but rather at aberrant sites upstream of the sites used in male germ cells. We also developed a sensitive, 3' RACE-based assay to analyze polyadenylation site choice. We showed that in somatic cells, somatic polyadenylation cassettes were efficiently polyadenylated, while male germ cell-specific polyadenylation cassettes were not. To do this, we developed a luciferase reporter system in which luciferase activity correlated with polyadenylation efficiency. We tested whether pre-mRNA sequences surrounding male germ cell-specific polyadenylation sites (polyadenylation cassettes) could be used to direct polyadenylation efficiently in somatic cells. We therefore hypothesized that male germ cell-specific polyadenylation sites would be inefficiently used in somatic cells. These differences suggested that male germ cell-specific polyadenylation sites may be poor substrates for polyadenylation in somatic cells. Finally, a number of mRNAs used a different polyadenylation site in male germ cells than in somatic cells.
Second, the polyadenylation sites chosen in male germ cells tended to be nearer the 5' end of the mRNA than those chosen in somatic cells. First, male germ cells showed a lower incidence of the sequence AAUAAA (an important element for somatic polyadenylation site choice) near the polyadenylation site choice.
This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications).We have previously noted that there were differences in somatic and male germ cell polyadenylation site choices. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS).
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