Induction of Duplication Reversion in Human Fibroblasts, by Wild-Type and Mutated Sv40 T Antigen, Covaries with the Ability to Induce Host DNA Synthesis

Intrachromosomal homologous recombination, manifest as reversion of a 14-kbp duplication in the hypoxanthine phosphoribosyl transferase (HPRT) gene, is elevated in human cells either stably transformed or transiently transfected by the SV40 (simian virus 40) large T antigen gene. Following introduction of wild-type SV40, or any of several T-antigen point mutations in a constant SV40 background, we observed a strong correlation between the stimulation of chromosomal recombination and induction of host-cell DNA synthesis. Moreover, inhibitors of DNA replication (aphidicolin and hydroxyurea) suppress SV40-induced homologous recombination to the extent that they suppress DNA synthesis. Stable integration of plasmids encoding T antigen also augments homologous recombination, which is suppressed by aphidicolin. We infer that the mechanism by which T antigen stimulates homologous recombination in human fibroblasts involves DNA replicative synthesis.     

Expression of injected HPRT minigene DNA in mouse embryos and its inhibition by antisense DNA

We have used a highly sensitive biochemical microassay to monitor the expression of a cloned minigene for hypoxanthine phosphoribosyl transferase (HPRT, EC.2.4.2.8) in preimplantation mouse embryos. The mouse HPRT promoter and the mouse metallothionein promoter (MT-I) function equally well in embryos at the 2-cell stage whereas the viral SV40 promoter does not allow HPRT expression. Induced HPRT activity from the MT-I HPRT minigene construct occurs in cleavage embryos cultured in the presence of cadmium. In contrast, negation of enzyme expression from the injected minigene DNA is mediated by simultaneous injection of HPRT antisense DNA.     

两种含SV40T不同区段的基因构建及其转基因小鼠的建立

目的　为解决SV4 0T转基因小鼠高发瘤难保种的问题 ,构建了两种含有SV4 0T不同区段的外源基因 :Rb结合域点突变的SV4 0T基因 (SV4 0T DRb)和无p5 3结合域的SV4 0T基因 (SV4 0T Dp5 3)。方法　利用分子生物学的基因克隆手段 ,将改造的SV4 0T基因片段克隆测序 ,最终将这两个改造后的基因克隆进乳腺特异性的真核表达载体p2 0 5C3中 ,运用雄原核显微注射法制备转基因小鼠。结果　利用PCR方法检测出 6只双阳性转基因 (同时检测到SV4 0T DRb和SV4 0T Dp5 3两种基因 )小鼠 ,为避免检测结果中假阳性的发生 ,应用Southern blot方法检测出 1只双阳性转基因小鼠。结论　本试验的结果证明 ,构建的两种含SV4 0T不同区段的策略是成功的 ,其建立的阳性转基因小鼠确实是弱化了SV4 0T转基因小鼠高发瘤的特性。     

Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.     

A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus.

In order to identify the determinants responsible for the nuclear migration of simian virus 40 (SV40) polypeptide VP1, the 5'-terminal portion of the SV40 VP1 gene was fused with the complete cDNA sequence of poliovirus capsid polypeptide VP1 and the hybrid gene was inserted into an SV40 vector in place of the normal SV40 VP1 gene. Deletions of various length were generated in the SV40 VP1 portion of the hybrid gene, resulting in a set of truncated genes encoding 2-40 NH2-terminal amino acids from SV40 VP1, followed by poliovirus VP1. Monkey kidney cells were infected by the deleted hybrid viruses in the presence of an early SV40 amber mutant as helper, and the subcellular localization of the fusion proteins was determined by indirect immunofluorescence using an anti-poliovirus VP1 immune serum. The presence of the first 11 NH2-terminal amino acids from SV40 VP1 was found to be sufficient to target the fusion protein to the cell nucleus. Deletions extending from the NH2- towards the COOH-terminal end of the protein were next generated. Transport of the SV40 VP1-poliovirus VP1 fusion polypeptide to the nucleus was abolished when the first eight amino acids from SV40 VP1 were deleted. Thus the sequence of the first eight NH2-terminal amino acids of SV40 VP1 appears to contain a nuclear migration signal which is sufficient to target the protein to the cell nucleus.     

An early event in murine cytomegalovirus replication inhibits presentation of cellular antigens to cytotoxic T lymphocytes.

Cytomegalovirus (CMV) infection of simian virus 40 (SV40)-immune mice inhibits priming of SV40-specific helper and cytotoxic T lymphocytes (CTL) in vivo (A. E. Campbell, J. S. Slater, and W. S. Futch, Virology 173:268-275, 1989; J. S. Slater, W. S. Futch, V. J. Cavanaugh and A. E. Campbell, Virology 185:132-139, 1991). We now demonstrate that murine CMV (MCMV) infection of SV40-transformed macrophages and fibroblasts prevents presentation of SV40 T antigen to SV40-specific CTL. MCMV-infected macrophages failed to stimulate SV40-immune CTL precursors in vitro. In addition, MCMV-infected, SV40-transformed macrophage and fibroblast target cells lost their susceptibility to lysis by major histocompatibility complex class I-restricted, SV40-specific CTL clones. MCMV infection did not alter the synthesis of SV40 T antigen in the target cells. MCMV early gene expression was required for inhibition of SV40 T-antigen presentation; immediate-early gene expression was insufficient for this effect. Early viral gene expression also resulted in significant reduction of H-2K and H-2D molecules on the surface of MCMV-infected fibroblasts. However, this reduction occurred independently from suppression of antigen presentation to CTL. The same target cells which were resistant to lysis by SV40 CTL were susceptible to lysis by MCMV-specific CTL. MCMV early gene products therefore interfere with the processing and/or presentation of SV40 T-antigen determinants to CTL independent of alterations in the major histocompatibility complex.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.     

Carcinogen-Mediated Amplification of Specific DNA Sequences

A model experimental system based on SV40-transformed Chinese hamster embryo cells and a highly sensitive in situ hybridization procedure was designed. Exposure of the cells to different categories of chemical and physical carcinogens resulted in the induction of SV40 DNA synthesis in the treated cells. Although the carcinogen-mediated amplification of SV40 DNA sequences is regulated by the viral “A” gene, neither infectious virus nor complete viral DNA molecules were rescued from the treated cells. A heterogenous collection of DNA molecules containing SV40 sequences was generated following treatment with DMBA. Restriction enzyme analysis of the amplified DNA molecules in the Hirt supernatant revealed that not all sequences in the integrated SV40 inserts are present. The possibility that the amplification of SV40 sequences is a reflection of a general gene amplification phenomenon mediated by carcinogens is discussed.     

Enhanced expression of apoptin by the Myc-Max binding motif and SV40 enhancer for SCLC gene therapy

Apoptin is derived from chicken anemia virus (CAV) and known to induce tumor specific apoptosis but not normal cells. The aim of this study was to use increased expression of apoptin by the Myc-Max response element (MMRE) and SV40 enhancer in small-cell lung cancer (SCLC) gene therapy. To investigate the possibility of the utilization of the MMRE, apoptin, and SV40 promoter/enhancer in targeted cancer gene therapy, adenovirus vector expressing apoptin controlled by the MMRE, and SV40 promoter/enhancer was constructed. Ad-MMRE-apoptin-enh infected SCLC cells were significantly suppressed and induced apoptosis more than those of Ad-apoptin or Ad-apoptin-enh. Infection with Ad-MMRE-apoptin-enh of normal cells did not increase apoptosis. About 85% of SCLC tumors show overexpression of the myc family, so the increased expression of apoptin by MMRE and SV40 enhancer can be used in targeted SCLC gene therapy. These results indicate that apoptin expression was increased by the MMRE and SV40 promoter/enhancer, and that this strategy can be used in SCLC targeted cancer gene therapy.     

The role of plasmid constructs containing the SV40 DNA nuclear-targeting sequence in cationic lipid-mediated DNA delivery

One of the steps that limit transfection efficiency in non-viral gene delivery is inefficient nuclear import of plasmid DNA, once it has been delivered into the cytoplasm. Recently, via microinjection into the cytoplasm and in situ hybridizations into a few cell types, it was shown that a region of Simian virus 40(SV40), specifically a c. 372-bp fragment of SV40 genomic DNA encompassing the SV40 promoter-enhancer-origin of replication (SV40 DTS), could enable the nuclear import of a plasmid carrying these sequences (Dean D.A. Exp. Cell Res. 230 (1997) 293). In this report, we address the issue of the suitability of the SV40 DTS for cationic lipid-mediated gene delivery, and its capacity to improve the efficiency of the transfection process. For this study, we used transient reporter gene expression assays on various cell types. The gene expression from the plasmid constructs carrying the SV40 DTS varied with cell type and plasmid construct used. Such cell-type and plasmid-construct dependency on gene expression from plasmids containing the SV40 DTS suggests that the gene expression from plasmids is not entirely dependent on its ability to enhance the nuclear import of said plasmids.     

Stimulation of host centriolar antigen in TC7 cells by simian virus 40: requirement for RNA and protein syntheses and an intact simian virus 40 small-t gene function

Simian virus 40 (SV 40) stimulated a host cell antigen in the centriolar region after infection of African green monkey kidney (AGMK) cells. The addition of puromycin and actinomycin D to cells infected with SV40 within 5 h after infection inhibited the stimulation of the host cell antigen, indicating that de novo protein and RNA syntheses that occurred within the first 5 h after infection were essential for the stimulation. Early viable deletion mutants of SV40 with deletions mapping between 0.54 and 0.59 map units on the SV40 genome, dl2000, dl2001, dl2003, dl2004, dl2005, dl2006, and dl2007, did not stimulate the centriolar antigen above the level of uninfected cells. This indicated that an intact, functional small-t protein was essential for the SV40-mediated stimulation of the host cell antigen. Our studies, using cells infected with nondefective adenovirus-SV40 hybrid viruses that lack the small-t gene region of SV40 (Ad2+ND1, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5), revealed that the lack of small-t gene function of SV40 could be complemented by a gene function of the adenovirus-SV40 hybrid viruses for the centriolar antigen stimulation. Thus, adenovirus 2 has a gene(s) that is analogous to the small-t gene of SV40 for the stimulation of the host cell antigen in AGMK cells.     

A novel selection system for recombinational and mutational events within an intron of a eucaryotic gene.

In order to identify a poison sequence that might be useful in studying illegitimate recombination of mammalian cell chromosomes, several DNA segments were tested for their ability to interfere with gene expression when placed in an intron. A tRNA gene and its flanking sequences (267 bp) were shown to inhibit SV40 plaque formation 100-fold, when inserted into the intron in the T-antigen gene. Similarly, when the same DNA segment was placed in the second intron of the adenosine phosphoribosyl transferase (APRT) gene from CHO cells, it inhibited transformation of APRT-CHO cells 500-fold. These two tests indicated that the 267-bp DNA segment contained a poison sequence. The poison sequence did not affect replication since the replication of poisoned SV40 genomes was complemented by viable SV40 genomes and poisoned APRT genes were stably integrated into cell chromosomes. Cleavage of the poison sequence in the SV40 T-antigen intron by restriction enzymes indicated that the tRNA structural sequences and the 5' flanking sequences were not required for inhibition of SV40 plaque formation. Sequence analysis of viable mutant SV40, which arose after transfection of poisoned genomes, localized the poison sequence to a 35 bp segment immediately 3' of the tRNA structural sequences.     

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).     

Gene therapy using SV40-derived vectors: what does the future hold?

Effective genetic therapy requires both a fragment of genetic material to be used therapeutically and a means to deliver it. We began to study simian virus-40 (SV40) as a vector for gene transfer because available gene delivery vehicles did not provide for the full range of therapeutic uses. Other vectors are variably limited by immunogenicity, difficulties in production, restricted specificity, low titers, poor transduction efficiency, etc. In theory recombinant viral vectors based on SV40 (rSV40) should not, on the other hand, be similarly constrained. rSV40 vectors are easily manipulated and produced at very high titer, stable, lacking in immunogenicity, and capable of providing sustained high levels of transgene expression in both resting and dividing cells. The principle limitation of SV40-derived vectors is the size of the packageable insert (相似文献   

Amplification of hypoxanthine-guanine phosphoribosyltransferase genes in chromosome-mediated gene transferents.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme activities may be elevated in genetically unstable chromosome-mediated gene transferents selected for transfer of the HPRT gene. Increased levels of HPRT polypeptides in unstable mouse L cell gene transferents were demonstrated by two-dimensional gel electrophoresis and immunoprecipitation. No additional polypeptides were found to be overexpressed. HPRT mRNA levels were elevated 10- to 15-fold in the unstable gene transferent GT427C. Southern blot hybridization experiments showed that overexpression of HPRT correlated with a 5- to 15-fold amplification of HPRT gene sequences in two unstable cell lines. Stabilized gene transferents displayed reduced HPRT copy numbers. The amplification of HPRT gene sequences in the unstable transferent GT427C was associated with the presence of multiple minute chromosome fragments. An average of 9.6 fragments was found per metaphase, but the variation was considerable, ranging from 0 to 53. We conclude that genomic DNA sequences may be amplified in unstable chromosome-mediated gene transferents and that such amplification may be associated with the occurrence of multiple chromosomal fragments.     

利用Red同源重组系统构建兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶基因打靶载体

利用EL350基因工程菌进行同源重组,成功进行基因敲除已有报道,但利用该系统进行兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶(Hypoxanthine guanine phosphoribosyl transferase,HPRT)基因突变和基因打靶方面的研究还没有报道。本实验首先在已经筛选到含有兔全长HPRT基因BAC克隆（LBNL1-304M19）的基础上,利用Red重组系统,通过Gap-Repair方式从此克隆上将一段47Kb无启动子的HPRT基因组片段（不含有第1个外显子）克隆到pBACLinkSp质粒上,产生pBACLinkSp-rHPRT质粒。然后基于pBACLinkSp-rHPRT质粒,设计不同的同源臂,从而删除了HPRT基因的不同编码区,成功构建了三个不同的HPRT基因打靶载体。同时对利用同源重组技术敲除不同大小的DNA片段的效率进行了研究。基于本实验所构建的三个不同的兔HPRT基因打靶载体,为探索兔成纤维细胞和胚胎干细胞基因打靶的适宜条件,及进一步获得兔HPRT基因敲除动物疾病模型奠定了基础。