Mutation spectrum in gamma-irradiated shuttle vector replicated in ataxia-telangiectasia lymphoblasts.

Cells from ataxia-telangiectasia (AT) patients are hypersensitive to the lethal effects of ionizing radiation. To assess radiation mutagenesis in these cells, the SV40-based shuttle vector, pZ189, was used to analyze gamma-ray-induced mutations following the plasmid's replication in AT lymphoblasts. Progenies from the AT line GM2783 exposed to 50 Gy showed a mutation frequency of 7.6 x 10(-3), 63-fold over background; surviving plasmids were 3.4% of control. Both values were essentially the same as those of irradiated plasmids replicated in a normal lymphoblast line, GM606. In addition, pZ189 exposed to 25 Gy of gamma radiation and replicated in another normal lymphoblast line and in cells of two additional AT lymphoblast lines showed similar mutation frequencies and percentages of surviving plasmids. Qualitative comparison of plasmid mutations from AT and normal cells showed no significant differences, indicating that the damaged DNA was repaired with similar fidelity in AT and normal cells. These studies suggest that there is no correlation between the enhanced sensitivity of AT cells to killing by ionizing radiation and gamma-radiation-induced mutagenesis of plasmid DNA processed in these cells.     

Analysis of mutations occurring during replication of a SV40 shuttle vector in mammalian cells

To analyse mutations that arise in mammalian cells we have used a SV40::plasmid shuttle vector containing a portion of the E. coli lacZ gene. We have found that following transfection into monkey Cos-7 cell mutations are not detected in the recovered plasmids at 24 h post transfection, but are found at 48 h post transfection, after the onset of DNA replication. Analysis of the mutant plasmids shows that in almost all cases the mutant phenotype is caused by a deletion or rearrangement of the lacZ gene in the shuttle vector.     

Comparison of spontaneous and ultraviolet-induced mutagenesis on naked SV40 DNA and SV40 virus

Molecular aspects of mutagenesis in mammalian cells have been essentially analyzed using biological probes such as viruses and shuttle vector. Although the main data concerning the specificity of carcinogen-induced mutations are similar, the observed spontaneous mutation frequencies are significantly different when using one or the other model. This frequency is considerably higher with shuttle vectors than with viruses. We have performed an analysis of mutagenesis in order to determine if the obligatory transfection step associated with shuttle vector technology was responsible for the high mutation frequency found with these molecules. For this purpose simian virus 40 (SV40) genome used as virus or as naked DNA was introduced into permissive cells by viral infection or DNA transfection respectively. Our results show that transfection alone does not induce a higher mutation frequency on SV40 DNA the virus infection. Moreover, we have shown that the ultraviolet-light induced mutation spectrum was similar on the SV40 VP1 gene after viral infection or DNA transfection.     

Abnormal processing of transfected plasmid DNA in cells from patients with ataxia telangiectasia.

In order to assess spontaneous mutability and accuracy of DNA joining in ataxia telangiectasia, a disorder with spontaneous chromosome breakage, the replicating shuttle vector plasmid, pZ189, was transfected into SV40 virus-transformed fibroblasts from ataxia telangiectasia patients. The ataxia telangiectasia fibroblasts showed elevated frequency of micronuclei, a measure of chromosome breakage. The spontaneous mutation frequency was normal with circular plasmids passed through the ataxia telangiectasia line. These results were compared to those with transformed fibroblasts from a patient with xeroderma pigmentosum, and from a normal donor. Mutation analysis revealed spontaneous point mutations and deletions in the plasmids with all 3 cell lines, however, insertions or complex mutations were only detectable with the ataxia telangiectasia line. To assess DNA-joining ability, linear plasmids which require joining of the DNA ends by host cell enzymes for survival, were transfected into the cells. We found a 2.4-fold less efficient DNA joining in ataxia telangiectasia fibroblasts (p = 0.04) and a 2.0-fold higher mutation frequency (p less than 0.01) in the recircularized plasmids than with the normal line. Plasmid DNA joining and mutation frequency were normal with the xeroderma pigmentosum fibroblasts. These findings with the ataxia telangiectasia fibroblasts of abnormal types of spontaneous mutations in the transfected plasmid and inefficient, error-prone DNA joining may be related to the increased chromosome breakage in these cells. In contrast, an EB virus-transformed ataxia telangiectasia lymphoblast line with normal frequency of micronuclei showed normal types of spontaneous mutations in the transfected plasmid and normal frequency of DNA joining which was error-prone. These data indicate that mechanisms that produce chromosome breakage in ataxia telangiectasia cells can be reflected in processing of plasmid vectors.     

Joining of linear plasmid DNA is reduced and error-prone in Bloom''s syndrome cells.

A linearized, replicating, shuttle vector plasmid, pZ189, was used to measure in vivo DNA joining ability of cells from patients with the cancer-prone, immunodeficient, chromosome breakage disorder, Bloom's syndrome (BS). The BS cell lines we studied were reported to contain reduced in vitro activity of DNA ligase I. We assessed in vivo joining ability by transfecting linear plasmids with overlapping or blunt ends (produced by EcoRI or StuI) into BS and normal fibroblast or lymphoblast host cells and measuring the amount of re-joined, replicated plasmids by their ability to transform bacteria. With plasmids having either overlapping or blunt ends we found a 1.3- to 3-fold lower (P less than 0.05) joining efficiency in BS cells than in the normal cells. The mutation frequency of the recovered plasmids was measured by screening for function of the suppressor tRNA contained in pZ189, for plasmid size, for presence of restriction sites, or by DNA sequencing. The spontaneous mutation frequency with the circular plasmid was 0.05-0.08% with both BS cell lines, values 2- to 21-fold higher (P less than 0.03) than with the normal cell lines. The mutation frequency with the linear plasmid passaged through both BS cell lines was 21-52%, values 1.4- to 5.4-fold higher (P less than 0.001) than with the normal lines. Detailed analysis of 210 recovered plasmids revealed an increase (P less than or equal to 0.001) in deletions, insertions or complex mutations at the joining sites, and in point mutations with the EcoRI cut plasmid with the BS cells in comparison to the normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

SV40-based shuttle viruses

We summarize in this paper the advantages of the shuttle virus system. These SV40-based vectors exhibit the unique properties of being packaged as SV40 pseudo-virions and of being able to infect host cells. Using these transient vectors, we show that their replication can be regulated in some monkey cell lines, in such a way that either low or very high amounts of plasmid DNA can be obtained. The stability of these infectious shuttle vectors in different conditions is analyzed by rescuing them in E. coli, using various gene mutation targets. Moreover, we describe a new series of vectors which can be produced as single-stranded DNA in bacteria. They allow the transfection of a plasmid genome into mammalian cells, as either single-stranded or double-stranded DNA.     

Repair and mutagenic potency of 8-oxoG:A and 8-oxoG:C base pairs in mammalian cells.

Replication of the oxidative lesion 8-oxo-7,8-dihydroguanine (GO) leads to the formation of both 8-oxo-7,8-dihydroguanine:adenine (GO:A) and 8-oxo-7,8-di-hydroguanine:cytosine (GO:C) pairs. The repair and mutagenic potency of these two kinds of base pairs were studied in simian COS7 and human MRC5V1 cells using the shuttle vector technology. Shuttle vectors carrying a unique GO residue opposite either a C or an A were constructed, then transfected into recipient mammalian cells. DNA repair resulting in G:C pairs and mutation frequency, were determined using resistance to digestion by the Ngo MI restriction enzyme for screening and DNA sequencing of suspect mutants. Results showed that the GO:C mismatch was well repaired since almost no mutations were detected in the plasmid progeny obtained 72 h after cell transfection. The GO:A pair was poorly repaired since only 32-34% of the plasmid progeny contained G:C whereas two thirds contained A:T at the original site. Repair kinetics measured with a non-replicating vector deleted by 13 bp at the SV40 replication origin, showed that GO:A was slowly repaired. Only 30% of the mispairs were corrected in 12 h. During this time 100% of the plasmids containing GO:A pairs were replicated as seen by the replication kinetics in a vector with an intact SV40 replication origin. These results show that, under our experimental conditions, replication is occurring before completion of DNA repair which explains the high mutagenic potency of the GO:A mispair.     

A shuttle vector plasmid for studying carcinogen-induced point mutations in mammalian cells

We have constructed a shuttle vector plasmid for studying mutagenesis in mammalian cells. The plasmid replicates in cell lines permissive for SV40 virus as well as in the bacterium Escherichia coli and carries a bacterial suppressor tRNA gene (supF) that can serve as a mutagenesis marker. The plasmid replicates as efficiently as SV40 virus in African Green Monkey kidney CV1 cells, indicating that all traces of the inhibitory sequences normally found in pBR322 and its derivatives have been removed. The design of the plasmid and the small size of the mutagenesis target gene decrease the probability of recovering spontaneous deletion mutations that have been shown to occur at high frequency during passage in mammalian cells. The frequency of spontaneous-mutant plasmids recovered after passage in CV1 cells is substantially lower than with other vectors described previously. When the plasmid DNA is treated with UV radiation before passage in CV1 cells, mutants are observed at a frequency about 20-fold above the spontaneous background.     

SV40-based shuttle viruses

We summarize in this paper the advantages of the shuttle virus system. These SV40-based vectors exhibit the unique properties of being packaged as SV40 pseudo-virions and of being able to infect host cells. Using these transient vectors, we show that their replication can be regulated in some monkey cell lines, in such a way that either low or very high amounts of plasmid DNA can be obtained. The stability of these infectious shuttle vectors in different conditions is analyzed by rescuing them in E. coli, using various gene mutation targets. Moreover, we describe a new series of vectors which can be produced as single-stranded DNA in bacteria. They allow the transfection of a plasmid genome into mammalian cells, as either single-stranded or double-stranded DNA.     

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.     

Trans replication and high copy numbers of wheat dwarf virus vectors in maize cells.

The replication of shuttle vectors derived from Wheat Dwarf Virus, a monopartite geminivirus, was studied in cultured maize endosperm cells, and in the Black Mexican Sweet (BMS) maize cell line. Using in vivo labeling and DNA methylation analysis, we showed that replication was initiated within 24 hrs after transfection, and did not require cell division in both cell lines. Copy numbers of 30,000 ds DNA molecules per cell were observed in endosperm cells after three days. The replication protein was shown to act in trans, since the wild type gene of the shuttle vector enabled replication-deficient vectors carrying mutated genes to replicate. These properties suggest that WDV may have similar applications in plants as SV40 in mammalian cells.     

SV40 T基因转化的山羊乳腺上皮细胞系及其生物学特性

目的建立能用于乳腺特异表达基因构件质量检验的山羊乳腺上皮细胞系.方法根据已发表的SV40病毒T基因序列设计引物,以整合有SV40 DNA早期基因区的COS-1细胞基因组DNA为模板,用高保真PCR扩增SV40 T基因.将获得的SV40 T基因克隆入真核表达载体,并用获得的重组表达质粒转染山羊原代乳腺上皮细胞.经有限稀释和反复传代后获得转化细胞克隆,对其生物学特性进行研究.结果扩增出序列正确的SV40T基因,重组质粒转染获得的转化细胞的对数生长期为接种后第4天,细胞群体倍增时间为23.5*!h,克隆形成率为26.7%.DNA斑点杂交试验证明转化细胞的基因组中整合有SV40 T基因,染色体核型分析试验表明转化细胞的核型无明显异常,裸鼠接种试验证明转化细胞不能形成肿瘤,软琼脂集落形成试验表明转化细胞在软琼脂中不能生长.部分细胞克隆已在体外传30代以上,保持正常乳腺上皮细胞的形态特征,在胶原基质上能形成腺泡样结构.结论本研究获得的SV40 T基因转化的山羊乳腺上皮细胞具有转化细胞系的生物学特性.     

pZ402, an improved SV40-based shuttle vector containing a T-antigen mutant unable to interact with wild-type p53

Shuttle vectors are useful tools for studying DNA replication and mutagenesis. SV40-based shuttle vectors are popular because of their ease of use and quick results. However, one complication with the use of SV40-based shuttle vectors is the interaction of cellular p53 protein with the T-antigen of SV40. Wild-type, but not mutant, p53 has been shown to be involved in DNA replication and DNA repair. To address this concern, we have modified an SV40-based shuttle vector, pZ189, by exchanging the wt T-antigen for a mutant SV40 T-antigen, which is unable to bind with p53. This shuttle vector, pZ402, provides us with a tool to study DNA replication and genomic instability in cells with varying genetic backgrounds without interference from the interaction of T-antigen with p53.     

Molecular mechanism of mutagenesis induced by olaquindox using a shuttle vector pSP189/mammalian cell system

Olaquindox, a quinoxaline 1,4-dioxide derivative from quindoxin, is widely used as an animal growth promoter in China. We tested olaquindox as a mutagen in a SV40-based shuttle vector pSP189 and African green kidney cell (Vero E6 cell line) system to define the safety of olaquindox as a food-additive for animals. When applied at 6.6 microg/ml, olaquindox caused 12 times higher mutation frequency in comparison to untreated controls. More than 70% of base substitutions happened at G:C base pairs featuring G:C to T:A or G:C to A:T conversions. Frequency of point mutations for in vitro modified plasmids was also dramatically increased from the spontaneous background level. Olaquindox-induced mutations did not occur randomly along the supF shuttle vector, but instead, had a hot spot at base pair #155 which accounts for 37% of total mutations. Olaquindox-induced mutations also showed sequence-specificity in which most point mutations occurred at site N in a 5'-NNTTNN-3' sequence while most tandem bases deletion and rearrangement were seen at the 5'-ANGGCCNAAA-3' sequence. We conclude that olaquindox induces DNA mutation, therefore, should not be used as an additive to promote animal growth.     

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.     

Cellular mutation mediates T-antigen-positive revertant cells resistant to simian virus 40 transformation but not to retransformation by polyomavirus and adenovirus type 2.

T-antigen-positive transformation revertant cell lines were isolated from fully simian virus 40 (SV40)-transformed Fisher rat embryo fibroblast cells (REF 52 cells) by methionine starvation. Reversion of the transformed cells (SV-52 cells) was caused by a mutation within the cellular genome. To demonstrate this, we isolated SV40 DNA from the host genome, inserted it into plasmid pSPT18 DNA, cloned it in Escherichia coli, and microinjected it into the nuclei of the REF 52 cells. Fully transformed cells were obtained with the same efficiency (20 to 25%) as after microinjection of wild-type SV40 DNA I. Furthermore, the revertant cells were resistant to retransformation by SV40. Following microinjection of wild-type SV40 DNA I, 42 independent cell lines were isolated. Cells of all analyzed lines acquired additional SV40 DNA copies, but changes in the cell morphology or growth characteristic were not demonstrable. However, the revertants were retransformable with a high efficiency after polyomavirus and adenovirus type 2 infections or microinjection. Also, fusion of the revertant cells with the grandparental REF 52 cells led to restoration of the transformed state.     

Persistence of phage lambda DNA in genomes of mouse cells transformed by lambda-carrying SV40 vectors.

To test the suitability of simian virus 40 (SV40) DNA as a vector for inserting DNA segments into the chromosomes of mammalian cells, an EcoRI-A fragment of bacteriophage lambda DNA was covalently joined to a fragment of SV40 DNA and used to transform mouse cells in culture. Three independent, morphologically transformed clones were obtained that were positive for SV40 T-antigen by immunofluorescence staining. DNA from each transformant was examined by restriction enzyme analysis and found to contain both lambda and SV40 sequences. Co-migration of some fragments containing lambda and SV40 sequences following digestion of transformed cell DNA by each of four different restriction enzymes indicated that part of the retained lambda and SV40 DNA was linked in two of the three lines. In the third line, however, none of the restriction fragments had both lambda and SV40 sequences. Although the presence of non-integrated lambda DNA was not excluded, at least some of the lambda DNA appeared to be linked to host cell DNA. Results of digestion by EcoRI suggested that in some cases the transforming linear molecule had probably circularized prior to integration.     

Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants.

To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants.