Independent regulation of thymidine kinase mRNA and enzyme levels in serum-stimulated cells.

We have studied the regulation of thymidine kinase mRNA and protein/enzyme expression in quiescent and serum-stimulated rat cells transfected with a human TK cDNA clone expressed from a number of promoters. Our results indicate that while the pattern of mRNA expression is a function of the promoter used, the pattern of protein/enzyme expression is not. When the gene is expressed from the homologous human TK promoter both mRNA and enzyme levels remain low throughout G1 and increase as the cells enter S phase. When it is expressed from the heterologous SV40 early promoter, mRNA levels are high throughout G1, but enzyme and protein levels remain low until 8-10 h following serum stimulation. Thus, protein levels appear to be uncoupled from mRNA levels in this system, suggesting the presence of translational and/or posttranslational regulation. An analysis of mutant cDNA clones indicates that this regulation is not dependent upon sequences at the 5' or 3' end of the cDNA, including the entire 5'-untranslated region, the authentic AUG and the first 48 nucleotides of the coding region.     

Role of the promoter in the regulation of the thymidine kinase gene.

To identify the regulatory elements of the human thymidine kinase (TK) gene, we have established stable cell lines carrying different chimeric constructs of the TK gene. Our results can be summarized as follows. (i) When the TK coding sequence is under the control of the calcyclin promoter (a promoter that is activated when G0 cells are stimulated by growth factors), TK mRNA levels are higher in G1-arrested cells than in proliferating cells; (ii) when the TK coding sequence is under the control of the promoter of heat shock protein HSP70, steady-state levels of TK mRNA are highest after heat shock, regardless of the position of the cells in the cell cycle; (iii) the bacterial CAT gene under the control of the human TK promoter is maximally expressed in the S phase; (iv) the TK cDNA driven by the simian virus 40 promoter is also maximally expressed in the S phase; and (v) TK enzyme activity is always at a maximum in the S phase, even when the levels of TK mRNA are highest in nonproliferating cells. We conclude that although the TK coding sequence may also play some role, the TK promoter has an important role in the cell cycle regulation of TK mRNA levels.     

A requirement for Zn2+ for the induction of thymidine kinase but not ornithine decarboxylase in 3T3 cells stimulated from quiescence.

In 3T3 cells stimulated from quiescence by serum, impaired thymidine incorporation caused by inadequate supply of Zn2+ was associated with both decreased thymidine kinase activity and a comparable decrease in its mRNA concentration. In contrast, the amount of mRNA for ribosomal protein S6 was not affected, nor was the earlier increase in the activity of ornithine decarboxylase.     

Role of small t antigen in the acute transforming activity of SV40

A plasmid, pHR402, containing SV40 sequences that include a truncated early region bearing an intact t-coding sequence and a functionally intact late region, was introduced into thymidine kinase deficient (tk-) mouse L cells by cotransformation with a cloned tk gene. tk+ cotransformants synthesized SV40 t but not T antigen, and no truncated T-coding sequence products were detected. The viral sequences of pHR402 were reconstituted as a virus in COS1 cells, and acute infection of untransformed mouse cells with this viral stock (SV402) also led to the appearance of t but not T or a truncated T. Abortive transformation assays of such infected cells were negative, as were those performed on the same cells infected with either of two viral mutants (dl883 and dl884), each of which leads to T but not t synthesis. However, mixed infection with SV402 and either dl883 or dl884 led to a clear abortive and permanent transformation response. Thus, at least in part, t and T appear to function in a complementary fashion in eliciting transformation expression by SV40-infected cells.     

High recombination rate of an Epstein-Barr virus-simian virus 40 hybrid shuttle vector in human cells

The stability of an Epstein-Barr virus (EBV)-simian virus 40 (SV40) hybrid shuttle vector, the p205-GTI plasmid, was analyzed in human cells during EBV- or SV40-type replication mode. When the p205-GTI plasmid was maintained as an episomal EBV vector in the human 293 cell line, no rearrangement was detected. To induce the SV40 replication mode, cells containing the episomal p205-GTI plasmid were either transfected with vectors carrying the T antigen gene or infected with SV40. Surprisingly, we observed both production and amplification of different classes of recombinant molecules. Particular types of modifications were found in most of the recombinants. The most striking rearrangement was a duplication of the promoter and enhancer regions of SV40 which was inserted in the thymidine kinase (TK) promoter. This recombination process involved a few bases of homology, and one of the recombination junctions implicated the GC boxes which constitute the essential components of the TK and SV40 early promoters. Our results suggest that a combination of a low level of base homology and a specific DNA sequence function (promoter and enhancer sites) leads to a very high level of recombinational activity during T-antigen-dependent plasmid replication.     

The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). 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. 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 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). 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. 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. 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).     

Effect of butyrate on the expression of microinjected or transfected genes

We have studied the effect of sodium n-butyrate on the expression of specific genes. For this purpose, tk-ts13 cells (a thymidine kinase-deficient mutant originating from Syrian hamster cells) were microinjected or transfected with pC2, a plasmid containing the entire SV40 genome and the herpes simplex virus thymidine kinase gene (HSV-TK), cloned in pBR322. As a measure of the expression of these two genes, one of which is spliced (SV40) and the other one (HSV-TK) which is not, we have taken the protein levels (amount of T antigen for SV40 and incorporation of [3H]thymidine for HSV-TK) and the levels of RNA (by dot blot hybridization). The expression of the microinjected genes was inhibited when tk-ts13 cells were exposed to butyrate, actinomycin D, cycloheximide, and mitomycin C, but not when the cells were treated with insulin or dexamethasone. Further studies showed that a decrease in the percentage of T-positive cells occurs at lower concentrations of butyrate than a decrease in the levels of specific mRNA. In tk-ts13 cells transfected with pC2 and treated with butyrate at a concentration of 3 mM, SV40 mRNA levels are not decreased but the percentage of T-positive cells is decreased 50%. At 5 mM, the amount of T antigen/cell is decreased a further 40%. These results indicate that butyrate may have at least two sites of action, one at the level of mRNA amount and a second at the level of protein amount. In addition, our studies show that the use of microinjected or transfected genes offers certain unique possibilities for studies on the effects of environmental manipulations on gene expression.     

Activation of an enhancerless gene by chromosomal integration.

Expression of enhancerless (E-) and enhancer-containing (E+) genes that are chromosomally integrated was examined. An E- plasmid (pE-cat) containing a chloramphenicol acetyltransferase (cat) gene linked to the simian virus 40 (SV40) early promoter or its E+ counterpart plasmid (pE+-cat) containing the SV40 enhancer was cotransfected into thymidine kinase (TK)-deficient L cells with a cloned tk gene. A number of TK+ transformants were isolated, and expression of the cointegrated cat gene in these cell lines was quantitatively determined by the assay of CAT activity. The results indicated unexpectedly that the E- cat gene was as actively expressed as the E+ cat gene. Analysis of CAT mRNA by primer extension indicated that the E- cat gene, as well as the E+ cat gene, was transcribed from the "native" initiation site contained in the SV40 early promoter region. The active expression of the E- cat gene was maintained in secondary TK+ transformants that arose by transfection with genomic DNA from the primary transformant. These results suggest that expression of the integrated E- cat gene is activated by endogenous enhancer elements.     

Demethylation and expression of methylated plasmid DNA stably transfected into HeLa cells.

In vitro methylation at CG dinucleotides (CpGs) in a transfecting plasmid usually greatly inhibits gene expression in mammalian cells. However, we found that in vitro methylation of all CpGs in episomal or non-episomal plasmids containing the SV40 early promoter/enhancer (SV40 Pr/E) driving expression of an antibiotic-resistance gene decreased the formation of antibiotic-resistant colonies by only approximately 30-45% upon stable transfection of HeLa cells. In contrast, when expression of the antibiotic-resistance gene was driven by the Rous sarcoma virus long terminal repeat or the herpes simplex virus thymidine kinase promoter, this methylation decreased the yield of antibiotic-resistant HeLa transfectant colonies approximately 100-fold. The low sensitivity of the SV40 Pr/E to silencing by in vitro methylation was probably due to demethylation upon stable transfection. This demethylation may be targeted to the promoter and extend into the gene. By genomic sequencing, we showed that four out of six of the transfected SV40 Pr/E's adjacent Sp1 sites were hotspots for demethylation in the HeLa transfectants. High frequency demethylation at Sp1 sites was unexpected for a non-embryonal cell line and suggests that DNA demethylation targeted to certain aberrantly methylated regions may function as a repair system for epigenetic mistakes.     

Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat.

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.