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C F Menck

Publications and source records attributed to C F Menck.

18 recordsLinked to original sources
PubMed1992

Singlet oxygen induced mutation spectrum in mammalian cells.

In order to characterize the molecular nature of singlet oxygen (1O2) induced mutations in mammalian cells, a SV40-based shuttle vector (pi SVPC13) was treated with singlet oxygen arising from the thermal decomposition of the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). After the passage of damaged plasmid through monkey COS7 cells, the vector was shuffled into E. coli cells, allowing the screening of supF mutants. The mutation spectrum analysis shows that single and multiple base substitutions arose in 82.5% of the mutants, the others being rearrangements. The distribution of mutations within the supF gene is not random and some hotspots are evident. Most of the point mutations (98.4%) involve G:C base pairs and G:C to T:A transversion was the most frequent mutation (50.8%), followed by G:C to C:G transversion (32.8%). These results indicate that mutagenesis in mammalian cells, mediated by 1O2-induced DNA damage, is targeted selectively at guanine residues.

Animals↗
PubMed1992

DNA synthesis blocking lesions induced by singlet oxygen are targeted to deoxyguanosines.

In vitro DNA synthesis on single stranded templates damaged by singlet oxygen was investigated in the supF tRNA gene sequence, using several DNA polymerases. Singlet oxygen was generated by the thermal decomposition of the water soluble with the endoperoxide of disodium 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). The data demonstrated that damage at deoxyguanosine residues interrupts DNA polymerization. Modified T7 phage and Thermus aquaticus DNA polymerases were found to synthesize DNA fragments which terminated opposite deoxyguanosine, while T4 phage DNA polymerase and avian myeloblast virus reverse transcriptase were blocked one nucleotide 3' to deoxyguanosine positions on the template. DNA polymerase I (Klenow fragment) from Escherichia coli was inhibited at both positions, before and at the putative damaged sites. The blocking lesions, induced by 5 mM NDPO2, were estimated to be approximately 1.5 per 260 nucleotides, corresponding to 2% of deoxyguanosines. The distribution of lesions in the supF gene did not reveal any specific sequence context which showed distinct susceptibility to the attack of singlet oxygen.

Bacterial Proteins↗
PubMed1992

Description of a new amplifiable shuttle vector for mutagenesis studies in human cells: application to N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation spectrum.

In order to analyze the mechanisms of mutagenesis in human cells, we have established a human 293 cell-derived line containing a permanent mutagenesis target, the bacterial lacZ' gene, on an episomal EBV/SV40-based shuttle vector. This plasmid was maintained at a low copy number per cell which rendered it closer to an endogenous gene as compared to the usual transient shuttle vectors. Transient amplification of vectors, inside the host cell due to expression of the SV40 T-antigen, allowed the recovery of a large number of bacterial colonies transformed by plasmids extracted from human cells. Mutations produced in human host cells on the lacZ' locus were easily and rapidly scored and identified in bacteria using the blue/white color assay. Over a 6-month period in culture, we have shown that the lacZ' gene exhibited a low background frequency of point mutations (< 4.8 x 10(-6)). The efficiency of our system for detecting genotoxic-induced mutations was investigated by treating cells with a potent mutagen, the direct alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A significant increase (< 230-fold) in the frequency of single-base substitutions was observed after MNNG treatment. In total, 63 MNNG-induced independent mutations were characterized. All substitutions but one involved G:C base pairs with 89% being G:C to A:T transitions which is consistent with the MNNG mutagenic specificity already reported in bacteria and mammalian cells. Mutations were distributed along the two strands of the lacZ' gene and there was no obvious influence of either the 5' or the 3' flanking base near the G:C to A:T transition sites. The low spontaneous point mutation frequency on the mutagenesis locus and the ability to detect induced point mutations indicate that this system could be readily used in human mutagenesis studies at the molecular level.

Base Sequence↗
PubMed1992

Singlet oxygen induced DNA damage.

Singlet oxygen generated by photoexcitation and by chemiexcitation selectively reacts with the guanine moiety in nucleosides (kq + kr about 5 x 10(6) M-1s-1) and in DNA. The oxidation products include 8-oxo-7-hydro-deoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Singlet oxygen also causes alkali-labile sites and single-strand breaks in DNA. The biological consequences include a loss of transforming activity as studied with plasmids and bacteriophage DNA, and mutagenicity and genotoxicity. Employing shuttle vectors, it was shown that double-stranded vectors carrying singlet oxygen induced lesions seem to be processed in mammalian cells by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells. Biological protection against singlet oxygen is afforded by quenchers, notably carotenoids and tocopherols. Major repair occurs by excision of the oxidized deoxyguanosine moieties by the Fpg protein, preventing mismatch of 8-oxodG with dA, which would generate G:C to T:A transversions.

Animals↗
PubMed1992

Spontaneous and ultraviolet-induced mutations on a single-stranded shuttle vector transfected into monkey cells.

The shuttle vector plasmid PCF3A, carrying the supF target gene, can be transfected into monkey COS7 cells as single-stranded or double-stranded DNA. Single strand-derived plasmid progeny exhibited a 10-fold higher spontaneous mutation frequency than double strand-derived progeny. The location of spontaneous mutations obtained after transfection of the single-stranded vector shared similarities with that for double-stranded vectors. However, the nature of base changes was very different. Single-stranded PCF3A DNA was used to study ultraviolet-induced mutagenesis. An earlier report (Madzak and Sarasin, J. Mol. Biol., 218 (1991) 667-673) showed that single-stranded DNA exhibited a lower survival and a higher mutation frequency than double-stranded DNA after ultraviolet irradiation. In the present report, sequence analysis of mutant plasmids is presented. The use of a single-stranded vector allowed us to show the targeting of mutations at putative lesion sites and to determine the exact nature of the base implicated in each mutation. Frameshift mutations were more frequent after transfection of control or irradiated plasmid as single-stranded DNA than as double-stranded DNA. Multiple mutations, observed at a high frequency in the spontaneous and ultraviolet-induced mutation spectra following single-stranded DNA transfection, could be due to an error-prone polymerisation step acting on a single-stranded template.

Animals↗
PubMed1992

Singlet oxygen induced DNA damage and mutagenicity in a single-stranded SV40-based shuttle vector.

The effects of singlet oxygen (1O2), generated by the thermal decomposition of water soluble NDPO2 (endoperoxide of the disodium 3,3'-(1,4-naphthylidene) dipropionate), on a single-stranded shuttle vector were analysed. 1O2 induces a much higher level of breaks in the phosphodiester backbone of single-stranded than double-stranded DNA. This may be due to a higher accessibility of guanine residue, primarily damaged by 1O2. The damaged vector was transfected into monkey COS7 cells where single-stranded DNA was converted to the double-stranded replicative form DNA. After 3 days, extrachromosomal DNA was extracted and the plasmids rescued in E. coli to study mutagenesis. There is a significant increase in mutation frequency of damaged single-stranded DNA in comparison to untreated DNA. It is concluded that 1O2 induces breaks in the backbone of single-stranded DNA and that the 1O2-damaged molecules are mutated after passage through mammalian cells.

Animals↗
PubMed1990

Constraints in simian virus 40 (SV40) encapsidation, as determined by SV40-based shuttle viruses.

Simian virus 40 (SV40)-based shuttle vectors, containing the SV40 late genes, can be packaged as infectious pseudovirions. In terms of their function as bacterial plasmids, modifications in the overall size of these plasmids can be tolerated within a very wide range, which has allowed us to determine the requirements for SV40 encapsidation, free of the more stringent limitations of SV40 virus. Monkey COS7 cells were transfected with over- and undersized SV40-based shuttle virus plasmids and their progeny have been analysed to follow the stability and evolution of these genomes. Two of the three plasmids analysed undergo recombination, generating molecules with sizes of between 4.0 and to 4.8 kb which were selected after multiple lytic cycles. This size range may correspond to the DNA lengths preferentially packaged in SV40 capsids. The structure of the rearranged plasmids indicates that there is a strong selective pressure for genomes that retain the functions necessary for replication and virus production. Depending on the parent DNA, two main classes of rearrangements were generated: duplications in tandem with the SV40 origin of replication and deletions. Both classes are probably a result of selective size and replicative advantages, which are then biologically amplified during plasmid transmission as virus particles.

Animals↗
PubMed1990

Singlet molecular oxygen induced mutagenicity in a mammalian SV40-based shuttle vector.

We have determined the deleterious effects of singlet oxygen (1O2), generated by thermal decomposition of the water-soluble endoperoxide 3,3'-(1,4-naphthylidene)dipropionate (NDPO2), on plasmid DNA. By following the electrophoretic mobility of DNA on agarose gels, we detected single and double strand breaks induced by treatment with NDPO2. The vector employed was a mammalian shuttle vector and the mutagenic consequences of these damages were investigated, using as mutation target the supF suppressor tRNA gene. A high increase of the mutation frequency, over the background, was observed in plasmids transfected in bacteria or after passage through mammalian cells. Trapping agents and quencher effects and other controls confirm the involvement of 1O2 in DNA damage and mutagenicity. These findings indicate that 1O2 can induce DNA lesions which are repaired by an error-prone process in prokaryotic and eukaryotic cells.

Animals↗
PubMed1989

Analysis of single-stranded DNA stability and damage-induced strand loss in mammalian cells using SV40-based shuttle vectors.

The fate and stability of fully or partially single-stranded DNA molecules transfected into mammalian cells have been analysed. For this, we constructed a simian virus 40 (SV40)-based shuttle vector containing the f1 bacteriophage replication origin in the two possible orientations (pi SVF1-A and pi SVF1-B). This vector contains the SV40 origin of replication, the late viral genes and DNA sequences for replication and selection in Escherichia coli. It also carries the lacO sequence, which permits the analysis of plasmid stability. Single-stranded DNA from pi SVF1-A and pi SVF1-B were produced in bacteria and annealed in vitro to form a heteroduplex molecule. We showed that, in monkey kidney COS7 cells, single-stranded vectors replicate to form duplex molecules. After transfection of the three forms of molecules (single-stranded, heteroduplex or double-stranded), replicated DNA was rescued in E. coli. Vector stability was analysed by checking for plasmid rearrangements and screening for lacO mutants. The single-stranded pi SVF1 has a lower rearrangement level, while the spontaneous mutation frequency (on the lacO target) is in the same range as for the double-stranded vector. In contrast, the level of spontaneous mutagenesis is higher for the heteroduplex than for the single- and double-stranded forms. In addition, we found that replication of heteroduplex with one strand containing ultraviolet light-induced lesions yields progeny molecules in which the irradiated strand is mostly lost. This result indicates for the first time the specific loss of the damaged strand in mammalian cells.

Animals↗
PubMed1988

Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands.

The molecular mechanisms of in vivo inhibition of mammalian DNA replication by exposure to UV light (at 254 nm) was studied in monkey and human cells infected with simian virus 40. Analysis of viral DNA by electron microscopy and sucrose gradients confirmed that the presence of UV-induced lesions severely blocks DNA synthesis, and thus the conversion of replicative intermediates (RIs) into fully replicated form I DNA is inhibited by UV irradiation. These blocked RI molecules present several special features when visualized by electron microscopy. (i) In excision repair-proficient monkey and human cells they are composed of a double-stranded circular DNA with a double-stranded tail whose size corresponds to the average interpyrimidine dimer distance, as determined by the dimer-specific T4 endonuclease V. (ii) In excision repair-deficient human cells from patients with xeroderma pigmentosum, UV-irradiated RIs present a Cairns-like structure similar to that observed for replicating molecules obtained from unirradiated infected cells. (iii) Single-stranded gaps are visualized in the replicated portions of UV-irradiated RI molecules; such regions are detected and clearly distinguishable from double-stranded DNA when probed by a specific single-stranded DNA-binding protein such as the bacteriophage T4 gene 32 product. Consistent with the presence of gaps in UV-irradiated RI molecules, single-strand-specific S1 nuclease digestion causes a shift in their sedimentation properties when analyzed in neutral sucrose gradients compared with undamaged molecules. These results are in agreement with and reinforce the model in which UV lesions are a barrier to the replication fork movement when present in the template for the leading strand; when lesions are in the template for the lagging strand they inhibit synthesis or completion of Okazaki fragments, leaving gaps opposite the lesion. Moreover, cellular DNA repair-linked endonucleolytic activity may induce double-stranded breaks in the blocked region of the replication forks, resulting in the tailed structures observed in viral DNA molecules obtained from excision repair-proficient cell lines.

Centrifugation, Density Gradient↗
PubMed1987

SV40-based Escherichia coli shuttle vectors infectious for monkey cells.

We describe SV40-based Escherichia coli shuttle vectors which can be packaged as pseudovirions without excision of plasmid sequences and which can be rescued in bacteria. These vectors replicate and are transmitted as virus in monkey COS cells without requiring a helper virus. Extrachromosomal vector DNA isolated from infected cells can be rescued in E. coli, so that DNA alterations can be easily screened. Indeed, some of the constructions give rise to very stable plasmids with no detectable rearrangements after multiple lytic cycles in COS cells. The spontaneous mutation frequency measured in bacteria, on the lacO target, is smaller than those usually found with shuttle vectors. We also constructed an expression vector derived from one of our infectious viruses by inserting the chloramphenicol acetyl transferase gene, expressed from the SV40 early promoter, which is efficiently transduced to cells by infection. In this system, the shuttle virus combines the convenience of plasmid rescue and analysis in bacteria, with the advantages of infectious virus.

Acetyltransferases↗
PubMed1987

Strategies to analyse mutagenesis in mammalian cells using simian virus 40 or shuttle vectors.

The use of exogeneous DNA probes, which replicate extrachromosomally, is proposed in order to study spontaneous and induced mutagenesis in mammalian cells. Simian virus 40 has already proved to be very useful, since it has provided much important information in this field. Recently, several shuttle vectors have been designed for this purpose; however, it seems that these molecules have high spontaneous mutation frequencies when replicating in mammalian cells. We have developed new alternative systems, such as Epstein-Barr virus-based shuttle vectors that can be episomally maintained in human cells. Furthermore, we have constructed packageable shuttle vectors, which appear to be stable in the host cell and thus suitable for analysis of mutagenesis.

Animals↗
PubMed1986

Escherichia coli xthA mutant is not hypersensitive to ascorbic acid/copper treatment--an H2O2 generating reaction.

Ascorbate (vitamin C) in the presence of copper yields H2O2, which seems to be responsible for its toxic effects in bacteria. However, we found that the Escherichia coli xthA mutant strain, which is hypersensitive to H2O2, has almost the same sensitivity as the wild-type strain to ascorbate and copper treatment. Our results suggest that the DNA damage induced in E. coli by H2O2 generated in oxidized ascorbate solutions is different from that induced by direct H2O2 treatment.

Ascorbic Acid↗
PubMed1986

Damages induced in lambda phage DNA by enzyme-generated triplet acetone.

Exposure of lambda phage to triplet acetone, generated via the oxidation of isobutanal by peroxidase, leads to genome lesions. The majority of these lesions are detected as DNA single-strand breaks only under alkaline conditions, and so true breaks do not occur. Also, no sites sensitive to UV-endonuclease from Micrococcus luteus were found in DNA from treated phage. The participation of triplet acetone in the generation of such DNA damage is discussed.

1-Propanol↗
PubMed1986

Ascorbate-copper induced DNA lesions and repair in Escherichia coli K12 cells.

Multiple lines of evidence show that oxidation products of ascorbic acid (vitamin C) are capable of inducing a variety of genetic alterations in microbial and mammalian cells. We have studied the inactivation kinetics in repair proficient and deficient Escherichia coli K12 cells treated with oxidized solutions of ascorbic acid, in the presence of catalytic amounts of copper. Our results suggest that the repair pathways controlled by the recA and uvrA gene products (the latter in a recA strain) contribute to cell survival. However, the lack of beta-galactosidase induction, in the SOS chromotest, implies a role for the RecA protein other than SOS induction. Catalase and thiourea suppress the toxic effects of oxidized ascorbate solutions, confirming that H2O2 and hydroxyl radicals are intermediate agents in the damaging action. Single-strand breaks were detected in DNA from treated cells.

Ascorbic Acid↗
PubMedDate not supplied

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.

Animals↗