DNA base sequence changes induced by ethyl methanesulfonate in a chromosomally integrated shuttle vector gene in mouse cells

We have analyzed the specificity of mutations induced by ethyl methanesulfonate (EtMes) in mouse cells carrying a selectable bacterial gene. The target gene was the Escherichia coli gptgene contained within a retroviral shuttle vector integrated into mouse chromosomal DNA. Following mutagenesis by EtMes, cells with mutations in the gptgene were selected as resistant to 6-thioguanine. Shuttle vector sequences were recovered from the mutant cell lines following fusion with monkey COS cells and introduced into bacteria as part of a bacterial plasmid. The DNA base sequences of the mutant genes were directly determined from plasmid DNA. All of the EtMes-induced mutations involving single base changes were found to be G:C to A:T transitions.     

Mutational specificity in a shuttle vector replicating in chromium(VI)-treated mammalian cells.

An SV40-based shuttle vector, pZ189, was used to characterize the mutation specificity and to explore the mechanism of chromium mutagenesis in mammalian cells. We showed previously that mutagenic DNA damage is induced by the treatment of plasmid with chromium(VI) plus glutathione in vitro. The induced mutation pattern suggested that chromium mutagenesis can be induced by the generation of reactive oxygen intermediates. To further investigate the mechanism of chromium mutagenesis, we treated cultured mammalian cells containing normal pZ189 vector with chromium(VI). Mutations were induced by Cr(VI) in a dose-dependent manner. The majority of base substitution mutations were widely distributed across the supF mutation target gene and occurred mainly at GC basepairs. Overall, the mutation spectra were not significantly different from each other except for a mutation hot spot at position 43, observed only in plasmids from Cr(VI)-treated cells. The characteristics of Cr(VI)-induced mutations were similar to those observed in the mutation spectra induced by H2O2 treatment of the pZ189 plasmid or plasmid-containing cells. These results are consistent with the hypothesis that induction of mutations by chromium in cultured cells occurs through the generation of oxidative DNA damage.     

Ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum lymphoblastoid cells and fibroblasts

In order to examine possible cell-type specificity in mutagenic events, a shuttle-vector plasmid, pZ18, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in Epstein-Barr virus transformed lymphoblastoid cell lines from a patient, XP12BE, with xeroderma pigmentosum (XP), group A, and a normal control. XP is a skin-cancer-prone disorder with UV hypersensitivity and defective DNA repair. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of E. coli. An earlier report on this data [Seetharam et al., (1990) J. Mol. Biol., 212, 433] indicated lower survival and higher mutation frequency with the UV-treated plasmid passed through the XP12Be(EBV) line. In the present report, sequence analysis of 198 mutant plasmids revealed a predominance of G:C → A:T transitions with both lymphoblastoid cell lines. This finding is consistent with the bias of polymerases toward insertion of an adenine opposite non-coding photoproducts (dinucleotides or other lesions). Transversion mutagenesis, non-adjacent double mutations, and triple-base mutations may involve other mechanisms. These results were compared to similar data from a fibroblast line from the same patient [Bredberg et al., (1986) Proc. Natl. Acad. Sci. (U.S.A.), 83, 8273]. The frequency of G:C → A:T transitions was higher, and there were fewer plasmids with multiple-base substitutions and with transversion mutations with both XP lymphoblasts and fibroblasts than with the normal lymphoblasts and fibroblasts. There were no significant differences in classes or types of mutations in the UV-treated plasmid replicated in the XP lymphoblasts and the XP fibroblasts. This suggests that the major features of UV mutagenesis in different cell types from the same individual are similar.     

Genetic evidence for a chromosomally integrated multiresistance plasmid in Salmonella dublin

Of 1099 isolates of Salmonella dublin during 1985-86, 11 (1%) were resistant to three or more antibiotics. Strain S4659/85, a multiresistant isolate, lacked autonomous R plasmids but showed incompatibility with incH2 plasmids and donated resistance determinants in matings. Transconjugants acquired incomplete R plasmids which integrated stably into a specific chromosomal site. These data provide an insight into the behaviour of R plasmids in S. dublin.     

Investigation of the mutagenic specificity of X-rays using a retroviral shuttle vector in CHO cells.

In some biological systems ionizing radiation appears to induce large deletions and rearrangements, while in others point mutations predominate the mutational spectrum. Moreover, while the point mutations are often randomly distributed, some systems exhibit "hot spots." Retroviral shuttle vectors are particularly useful for investigating the basis of these differences since the genetic target can be conveniently analyzed in a variety of host backgrounds and genomic locations. We have studied the mutational specificity of X-rays in a Chinese hamster ovary cell line (CHO) containing a stably integrated retroviral shuttle vector, carrying the CHO aprt cDNA as the genetic target. Cells were irradiated with 7 Gy using a 180 kVp X-ray source. The predominant mutation (87% of all APRT mutants), as determined by Southern analysis, was the complete deletion of the shuttle vector construct. In addition, 23 APRT mutants, carrying an apparently intact shuttle vector, were characterized at the sequence level: 5 were transitions, 9 were transversions, 3 were small deletions or insertions, 4 were frameshifts, and 2 were small rearrangements. Although the type and the location of the point mutations characterized appeared largely random, small deletions, insertions, and frameshifts were frequently associated with direct sequence repeats.     

Analysis of mammalian diamine oxidase genes

Inflammation Research -     

Frequent spontaneous deletions at a shuttle vector locus in transgenic mice

Transgenic mice carrying multiple copies of a recoverable lambdaphage shuttle vector (     

Use of a bicistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells

 Transient transfection of ion channels into mammalian cells is a useful method with which to study ion channel properties. However, a general problem in transient transfection procedures is how to select cells that express the transfected cDNA. We have constructed a bicistronic vector, pCINeo/IRES-GFP, which utilises a red-shifted variant of Green Fluorescent Protein as an in vivo cell marker. Incorporation of an ion channel cDNA into the bicistronic unit allows coupled expression of the ion channel and Green Fluorescent Protein. After transient transfection of COS cells with pCINeo/IRES-GFP containing a rat delayed rectifier K⁺ channel cDNA (RCK1, Kv1.1), all green cells (n = 32) expressed the RCK1 channel as identified by the well known kinetics, K⁺ selectivity and pharmacology of Kv1.1. In contrast, non-fluorescent cells (n = 24) were negative with respect to RCK1 expression. It is concluded that the bicistronic pCINeo/IRES-GFP vector provides an efficient and non-invasive way of identifying cells which express ion channels after transfection. This novel method should greatly facilitate functional studies of ion channels transfected into mammalian cells. Received: 17 March 1997 / Received after revision: 14 May 1997 / Accepted: 15 May 1997     

Use of a human minichromosome as a cloning and expression vector for mammalian cells.

A natural human minichromosome (MC1) derived from human chromosome 1 was shown to be linear and to have a size of 5.5 Mb. Human IL-2 cDNA and the neo gene were co-transfected into a MC1-containing human-CHO hybrid cell line. Integration of the foreign genes was directed to the pericentromeric region of MC1 by co-transfection of chromosome 1-specific satellite 2 DNA. A number of G418-resistant transfectants were obtained and expression of IL-2 was determined. FISH analysis demonstrated co-localization in the minichromosome of the IL-2 gene and of the satellite 2 DNA. An IL-2-producing clone was used in cell fusion experiments with IL-2-dependent murine CTLL cells to generate CTLL-human hybrids containing the modified minichromosome (MC1- IL2 ). The hybrids were able to grow in medium lacking IL-2 for 17 mean population doublings (MPD), indicating that expression of the cytokine was sufficient to relieve the IL-2 dependence of CTLL proliferation. Endogenous IL-2 production delayed the onset of apoptosis in the IL-2-dependent CTLL cells. Mitotic stability was shown to be 100% in the human-CHO hybrids and 97% per MPD in CTLL cells. These results demonstrate that a natural human minichromosome can be utilized as a cloning and expression vector for mammalian cells and that the MC1 minichromosome can be engineered to deliver IL-2 to two types of cells, fibroblasts and lymphocytes.     

腺病毒载体介导HBsAg基因在哺乳动物细胞中的表达

目的　探讨腺病毒载体介导乙型肝炎病毒 (HBV)前S2 S(preS2/S)基因在几种哺乳动物细胞中的表达。方法　制备携带HBVpreS2/S基因的非复制型重组腺病毒Ad-HBs ,体外转染人胚肾细胞 (2 93)、绿猴肾细胞 (Vero)、肝癌细胞系 (HepG2 )和间质干细胞 (MSCs) ,荧光显微镜观察EGFP的表达 ,同时采用RT-PCR检测目的基因的转录 ,ELISA法检测目的基因的表达。结果　MOI为 2 0的重组腺病毒Ad-HBs转染 2 93、Vero、HepG2 细胞和MSCs ,4 8h后 90 %以上的细胞表达EGFP ,同时细胞表达高滴度的HBsAg(A值 >3 2 2 9)。结论　腺病毒载体不仅能够介导HBVpreS2 S基因于连续细胞系细胞中高效表达 ,也能介导HBVpreS2 S基因于干细胞中高效表达     

Analysis of spontaneous and induced mutations in transgenic mice using a lambda ZAP/lacI shuttle vector.

A short term, in vivo mutagenesis assay has been developed utilizing a lacl target gene contained within a lambda ZAP shuttle vector which has been incorporated into transgenic mice. Following chemical exposure, the target gene was recovered from mouse genomic DNA by mixing the DNA with in vitro lambda phage packaging extract. Mutations within the lacl target were identified by infecting host E. coli with the packaged phage and plating on indicator plates containing Xgal. Phage plaques with mutations in the lacl appeared blue, while intact phage were colorless. The ratio of blue plaques to colorless plaques is a measure of the mutagenicity of the compound. This system was used to obtain significant induction (up to 74-fold) over background levels for a variety of compounds, including N-ethyl-N-nitrosourea, benzo(a)pyrene (BaP), cyclophosphamide, and methylnitrosourea. Sequence analysis of selected mutant clones derived from this system was accomplished through the use of partial filamentous phage origins which allow rapid transfer of the target gene from phage to plasmid. Sequence analysis of spontaneous mutants derived from the mice primarily found of base substitutions, differing markedly from the previous data for spontaneous mutations in lacl derived from E. coli, where the preponderance of mutations were found at a single site, a repeated tetramer sequence. Upon sequence analysis of BaP derived base substitutions, only transversions were obtained, consistent with the known mechanism of BaP mutagenesis. Use of the well-characterized lacl gene in transgenic mice should allow for extrapolation of the extensive pool of in vitro data to whole animals, as well as provide insight into the tissue specific effects of mutagenic compounds.     

Adeno-associated viruses having nonsense mutations in the capsid genes: growth in mammalian cells containing an inducible amber suppressor.

When an adeno-associated virus (AAV) genome contained in a recombinant plasmid is transfected into adenovirus-infected cells, infectious AAV particles are efficiently generated. We previously described the construction of a conditional lethal mutant of AAV having an amber termination codon inserted in the rep gene. This mutant was propagated on a monkey kidney cell line (SupD12) having an inducible amber suppressor tRNAser. We now describe the construction and propagation of two additional conditional lethal mutants of AAV having amber codons affecting all three capsid proteins (AAV Capam) or only the VP1 capsid protein (AAV VP1am). Suppression of the amber mutations in the capsid proteins was demonstrated directly by immunoblot analysis. The efficiency of amber suppression on the SupD12 cell was about 6 to 10% for AAV VP1am and 4 to 5% for AAV Capam. The reversion frequency of either mutant was apparently less than 10(-5). On nonsuppressing cells AAV VP1am exhibited an Lip (Inf) phenotype, whereas AAV Capam exhibited a Cap phenotype.     

Introduction of YACs containing a putative mammalian replication origin into mammalian cells can generate structures that replicate autonomously

Yeast artificial chromosomes (YACs) containing or lacking a biochemically defined DNA replication origin were transferred from yeast to mammalian cells in order to determine whether origin-dependent autonomous replication would occur. A specialized YAC vector was designed to enable selection for YACs in mammalian cells and for monitoring YAC abundance in individual mammalian cells. All of eight clones made with linear and circularized YACs lacking the origin and seven of nine clones made with linear and circularized YACs containing the origin region contained single copies of the transfected YAC, along with various amounts of yeast DNA, integrated into single but different chromosomal sites. By contrast, two transformants derived from circularized YACs containing the putative replication origin showed very heterogeneous YAC copy number and numerous integration sites when analyzed after many generations of in vitro propagation. Analysis of both clones at an early time after fusion revealed variously sized extrachromosomal YAC/yeast structures reminiscent of the extrachromosomal elements found in some cells harboring amplified genes. The data are consistent with the interpretation that YACs containing a biochemically defined origin of replication can initially replicate autonomously, followed by integration into multiple chromosomal locations, as has been reported to occur in many examples of gene amplification in mammalian cells.     

Mutations induced in a shuttle vector plasmid exposed to monofunctionally activated mitomycin C

Reductive activation of mitomycin C leads to its covalent binding to DNA, forming monoadducts and cross-links. The cytotoxicity of mitomycin C has been attributed to cross-link formation, whereas monoadducts are assumed to cause mutagenicity. We have developed a ³²P-postlabeling technique to measure mitomycin C DNA adducts. Using this technique, we have measured monoadduct formation in the shuttle vector plasmid pSP189 and have determined mutations induced by monoadduct formation. The shuttle vector plasmid was incubated with mitomycin C under conditions favoring monofunctional activation of mitomycin C. The plasmid was then replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence of pSP189. One major mitomycin C/DNA adduct was observed by ³²P-postla-beling and was characterized as a monoadduct ofguanine. When pSP189 was exposed to monofunctionally activated mitomycin C, increases in adduct levels and mutation frequency were found to be related to mitomycin C concentration. The majority of the mutations involved single bases, with base substitutions making up 59.1% of the total mutations observed. Of the base substitutions, 67.2% were transversions and 32.8% were transitions, with nearly 80% of all base substitutions involving G:C base pairs. Deletions, either as single bases or large deletions, also involved G:C base pairs the majority of the time. The observed bias of mutations atG:C and the formation of a mitomycin C/DNA monoadduct involving guanine suggests that monoadduct formation may be responsible for the mutations. Environ. Mol. Mutagen. 29:143–151, 1997. © 1997 Wiley-Liss, Inc.