scholarly journals Determination of the spectrum of mutations induced by defined-wavelength solar UVB (313-nm) radiation in mammalian cells by use of a shuttle vector.

1988 ◽  
Vol 8 (12) ◽  
pp. 5425-5431 ◽  
Author(s):  
S M Keyse ◽  
F Amaudruz ◽  
R M Tyrrell
Keyword(s):  
Hot Spots ◽  
Mammalian Cells ◽  
Important Determinant ◽  
Shuttle Vector ◽  
Common Mechanism ◽  
Site Specificity ◽  
Base Pairs ◽  
Dna Photoproducts ◽  
Supf Gene

Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189). We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation. Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced. In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm. These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength. Lastly, we observed that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously. Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts.

Determination of the spectrum of mutations induced by defined-wavelength solar UVB (313-nm) radiation in mammalian cells by use of a shuttle vector

1988 ◽  
Vol 8 (12) ◽  
pp. 5425-5431
Author(s):  
S M Keyse ◽  
F Amaudruz ◽  
R M Tyrrell
Keyword(s):  
Hot Spots ◽  
Mammalian Cells ◽  
Important Determinant ◽  
Shuttle Vector ◽  
Common Mechanism ◽  
Site Specificity ◽  
Base Pairs ◽  
Dna Photoproducts ◽  
Supf Gene

Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189). We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation. Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced. In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm. These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength. Lastly, we observed that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously. Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts.

scholarly journals Kinds and spectrum of mutations induced by 1-nitrosopyrene adducts during plasmid replication in human cells.

1988 ◽  
Vol 8 (8) ◽  
pp. 3364-3372 ◽  
Author(s):  
J L Yang ◽  
V M Maher ◽  
J J McCormick
Keyword(s):  
Hot Spots ◽  
Shuttle Vector ◽  
Human Fibroblasts ◽  
Parent Compound ◽  
Human Cells ◽  
Dna Adduct ◽  
Base Substitution ◽  
Base Pairs ◽  
Cold Spots ◽  
Supf Gene

1-Nitropyrene has been shown in bacterial assays to be the principal mutagenic agent in diesel emission particulates. It has also been shown to be mutagenic in human fibroblasts and carcinogenic in animals. To investigate the kinds of mutations induced by this carcinogen and compare them with those induced by a structurally related carcinogen, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetra-hydrobenzo [a]pyrene (BPDE) (J.-L. Yang, V. M. Maher, and J. J. McCormick, Proc. Natl. Acad. Sci. USA 84:3787-3791, 1987), we treated a shuttle vector with tritiated 1-nitrosopyrene (1-NOP), a carcinogenic mutagenic intermediate metabolite of 1-nitropyrene which forms the same DNA adduct as the parent compound, and introduced the plasmids into a human embryonic kidney cell line, 293, for DNA replication to take place. The treated plasmid, pZ189, carrying a bacterial suppressor tRNA target gene, supF, was allowed 48 h to replicate in the human cells. Progeny plasmids were then rescued, purified, and introduced into bacteria carrying an amber mutation in the beta-galactosidase gene in order to detect those carrying mutations in the supF gene. The frequency of mutants increased in direct proportion to the number of DNA-1-NOP adducts formed per plasmid. At the highest level of adduct formation tested, the frequency of supF mutants was 26 times higher than the background frequency of 1.4 X 10(-4). DNA sequencing of 60 unequivocally independent mutant derived from 1-NOP-treated plasmids indicated that 80% contained a single base substitution, 5% had two base substitutions, 4% had small insertions or deletions (1 or 2 base pairs), and 11% showed a deletion or insertion of 4 or more base pairs. Sequence data from 25 supF mutants derived from untreated plasmids showed that 64% contained deletions of 4 or more base pairs. The majority (83%) of the base substitution in mutants from 1-NOP-treated plasmids were transversions, with 73% of these being G . C --> T . A. This is very similar to what we found previously in this system, using BPDE, but each carcinogen produced its own spectrum of mutations. Of the five hot spots for base substitution mutations produced in the supF gene with 1-NOP, two were the same as seen with BPDE-treated plasmids. However, the three other hot spots were cold spots for BPDE-treated plasmids. Conversely, four of the other five hot spots seen with BPDE-treated plasmids were cold spots for 1-NOP-treated plasmids. Comparison of the two carcinogens for the frequency of supF mutants induced per DNA adduct showed that 1-NOP-induced adducts were 3.8 times less than BPDE adducts. However, the 293 cell excised 1-NOP-induced adducts faster than BPDE adducts.

Kinds and spectrum of mutations induced by 1-nitrosopyrene adducts during plasmid replication in human cells

1988 ◽  
Vol 8 (8) ◽  
pp. 3364-3372
Author(s):  
J L Yang ◽  
V M Maher ◽  
J J McCormick
Keyword(s):  
Hot Spots ◽  
Shuttle Vector ◽  
Human Fibroblasts ◽  
Parent Compound ◽  
Human Cells ◽  
Dna Adduct ◽  
Base Substitution ◽  
Base Pairs ◽  
Cold Spots ◽  
Supf Gene

1-Nitropyrene has been shown in bacterial assays to be the principal mutagenic agent in diesel emission particulates. It has also been shown to be mutagenic in human fibroblasts and carcinogenic in animals. To investigate the kinds of mutations induced by this carcinogen and compare them with those induced by a structurally related carcinogen, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetra-hydrobenzo [a]pyrene (BPDE) (J.-L. Yang, V. M. Maher, and J. J. McCormick, Proc. Natl. Acad. Sci. USA 84:3787-3791, 1987), we treated a shuttle vector with tritiated 1-nitrosopyrene (1-NOP), a carcinogenic mutagenic intermediate metabolite of 1-nitropyrene which forms the same DNA adduct as the parent compound, and introduced the plasmids into a human embryonic kidney cell line, 293, for DNA replication to take place. The treated plasmid, pZ189, carrying a bacterial suppressor tRNA target gene, supF, was allowed 48 h to replicate in the human cells. Progeny plasmids were then rescued, purified, and introduced into bacteria carrying an amber mutation in the beta-galactosidase gene in order to detect those carrying mutations in the supF gene. The frequency of mutants increased in direct proportion to the number of DNA-1-NOP adducts formed per plasmid. At the highest level of adduct formation tested, the frequency of supF mutants was 26 times higher than the background frequency of 1.4 X 10(-4). DNA sequencing of 60 unequivocally independent mutant derived from 1-NOP-treated plasmids indicated that 80% contained a single base substitution, 5% had two base substitutions, 4% had small insertions or deletions (1 or 2 base pairs), and 11% showed a deletion or insertion of 4 or more base pairs. Sequence data from 25 supF mutants derived from untreated plasmids showed that 64% contained deletions of 4 or more base pairs. The majority (83%) of the base substitution in mutants from 1-NOP-treated plasmids were transversions, with 73% of these being G . C --> T . A. This is very similar to what we found previously in this system, using BPDE, but each carcinogen produced its own spectrum of mutations. Of the five hot spots for base substitution mutations produced in the supF gene with 1-NOP, two were the same as seen with BPDE-treated plasmids. However, the three other hot spots were cold spots for BPDE-treated plasmids. Conversely, four of the other five hot spots seen with BPDE-treated plasmids were cold spots for 1-NOP-treated plasmids. Comparison of the two carcinogens for the frequency of supF mutants induced per DNA adduct showed that 1-NOP-induced adducts were 3.8 times less than BPDE adducts. However, the 293 cell excised 1-NOP-induced adducts faster than BPDE adducts.

scholarly journals Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative.

1987 ◽  
Vol 7 (10) ◽  
pp. 3561-3565 ◽  
Author(s):  
M M Seidman
Keyword(s):  
Resistance Gene ◽  
Mammalian Cells ◽  
Restriction Analysis ◽  
Shuttle Vector ◽  
Recombination Reaction ◽  
Neomycin Resistance Gene ◽  
African Green Monkey Cells ◽  
The Individual ◽  
Neomycin Resistance

Intermolecular recombination in mammalian cells was studied by coinfecting African green monkey cells in culture with two shuttle vector plasmids, each carrying an incomplete but overlapping portion of the gene for neomycin resistance. The region of homology between the two plasmids was about 0.6 kilobases. Recombination between the homology regions could reconstruct the neomycin resistance gene, which was monitored by analysis of progeny plasmids in bacteria. The individual plasmids carried additional markers which, in combination with restriction analysis, allowed the determination of the frequency of formation of the heterodimeric plasmid which would be formed in a conservative recombination reaction between the homologous sequences. Reconstruction of the neomycin resistance gene was readily observed, but only 1 to 2% of the neomycin resistance plasmids had the structure of the conservative heterodimer. Treatment of the plasmids which enhanced the frequency of the neomycin resistance gene reconstruction reaction did not significantly increase the relative frequency of conservative product plasmids. The results support nonconservative models for recombination of these sequences.

scholarly journals Binding of transcription factors creates hot spots for UV photoproducts in vivo

1992 ◽  
Vol 12 (4) ◽  
pp. 1798-1804
Author(s):  
G P Pfeifer ◽  
R Drouin ◽  
A D Riggs ◽  
G P Holmquist
Keyword(s):  
Transcription Factors ◽  
Hot Spots ◽  
Mammalian Cells ◽  
Phosphoglycerate Kinase ◽  
Promoter Sequence ◽  
Mean Value ◽  
Naked Dna ◽  
Dna Photoproducts ◽  
Uv Photoproducts

Cyclobutane dipyrimidines and less than mean value of 6-4 dipyrimidines are the two major classes of mutagenic DNA photoproducts produced by UV irradiation of cells. We developed a method to map cyclobutane dipyrimidines at the DNA sequence level in mammalian cells. The frequency of this class of photoproducts was determined at every dipyrimidine along the human phosphoglycerate kinase-1 (PGK1) promoter sequence and was compared to the UV-induced frequency distribution of mean value of 6-4 dipyrimidines. After irradiation of living cells containing active or inactive PGK1 genes, enzymatic or chemical cleavage at UV photoproducts, and amplification by ligation-mediated polymerase chain reaction, photofootprints were seen in all regions which bind transcription factors and appear as DNase I footprints. Photoproduct frequency within transcription factor binding sites was suppressed or enhanced relative to inactive genes or naked DNA with enhancements of up to 30-fold. Since photoproducts are mutagenic, this indicates that photoproduct (mutation?) hot spots may be tissue specific in mammals.

Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells

1986 ◽  
Vol 6 (1) ◽  
pp. 277-285 ◽  
Author(s):  
J Hauser ◽  
M M Seidman ◽  
K Sidur ◽  
K Dixon
Keyword(s):  
Dna Synthesis ◽  
Hot Spots ◽  
Mammalian Cells ◽  
Shuttle Vector ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Mutation Induction ◽  
Sequence Specificity ◽  
Induced Mutations ◽  
Double Base

A simian virus 40-based shuttle vector was used to characterize UV-induced mutations generated in mammalian cells. The small size and placement of the mutagenesis marker (the supF suppressor tRNA gene from Escherichia coli) within the vector substantially reduced the frequency of spontaneous mutations normally observed after transfection of mammalian cells with plasmid DNA; hence, UV-induced mutations were easily identified above the spontaneous background. UV-induced mutations characterized by DNA sequencing were found primarily to be base substitutions; about 56% of these were single-base changes, and 17% were tandem double-base changes. About 24% of the UV-induced mutants carried multiple mutations clustered within the 160-base-pair region sequenced. The majority (61%) of base changes were the G . C----A . T transitions; the other transition (A . T----G . C) and all four transversions occurred at about equal frequencies. Hot spots for UV mutagenesis did not correspond to hot spots for UV-induced photoproduct formation (determined by a DNA synthesis arrest assay); in particular, sites of TT dimers were underrepresented among the UV-induced mutations. These observations suggest to us that the DNA polymerase(s) responsible for mutation induction exhibits a localized loss of fidelity in DNA synthesis on UV-damaged templates such that it synthesizes past UV photoproducts, preferentially inserting adenine, and sometimes misincorporates bases at undamaged sites nearby.

Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative

1987 ◽  
Vol 7 (10) ◽  
pp. 3561-3565
Author(s):  
M M Seidman
Keyword(s):  
Resistance Gene ◽  
Mammalian Cells ◽  
Restriction Analysis ◽  
Shuttle Vector ◽  
Recombination Reaction ◽  
Neomycin Resistance Gene ◽  
African Green Monkey Cells ◽  
The Individual ◽  
Neomycin Resistance

Intermolecular recombination in mammalian cells was studied by coinfecting African green monkey cells in culture with two shuttle vector plasmids, each carrying an incomplete but overlapping portion of the gene for neomycin resistance. The region of homology between the two plasmids was about 0.6 kilobases. Recombination between the homology regions could reconstruct the neomycin resistance gene, which was monitored by analysis of progeny plasmids in bacteria. The individual plasmids carried additional markers which, in combination with restriction analysis, allowed the determination of the frequency of formation of the heterodimeric plasmid which would be formed in a conservative recombination reaction between the homologous sequences. Reconstruction of the neomycin resistance gene was readily observed, but only 1 to 2% of the neomycin resistance plasmids had the structure of the conservative heterodimer. Treatment of the plasmids which enhanced the frequency of the neomycin resistance gene reconstruction reaction did not significantly increase the relative frequency of conservative product plasmids. The results support nonconservative models for recombination of these sequences.

scholarly journals Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells.

1988 ◽  
Vol 8 (9) ◽  
pp. 3943-3946 ◽  
Author(s):  
E Roilides ◽  
P J Munson ◽  
A S Levine ◽  
K Dixon
Keyword(s):  
Sequence Analysis ◽  
Mitomycin C ◽  
Dna Sequence ◽  
Mammalian Cells ◽  
Shuttle Vector ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Twofold Increase ◽  
Mechanisms Of Mutagenesis ◽  
Supf Gene

When monkey cells were treated with mitomycin C 24 h before transfection with UV-irradiated pZ189 (a simian virus 40-based shuttle vector), there was a twofold increase in the frequency of mutations in the supF gene of the vector. These results suggest the existence of an enhancible mutagenesis pathway in mammalian cells. However, DNA sequence analysis of the SupF- mutants suggested no dramatic changes in the mechanisms of mutagenesis due to mitomycin C treatment of the cells.

Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells

1988 ◽  
Vol 8 (9) ◽  
pp. 3943-3946
Author(s):  
E Roilides ◽  
P J Munson ◽  
A S Levine ◽  
K Dixon
Keyword(s):  
Sequence Analysis ◽  
Mitomycin C ◽  
Dna Sequence ◽  
Mammalian Cells ◽  
Shuttle Vector ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Twofold Increase ◽  
Mechanisms Of Mutagenesis ◽  
Supf Gene

When monkey cells were treated with mitomycin C 24 h before transfection with UV-irradiated pZ189 (a simian virus 40-based shuttle vector), there was a twofold increase in the frequency of mutations in the supF gene of the vector. These results suggest the existence of an enhancible mutagenesis pathway in mammalian cells. However, DNA sequence analysis of the SupF- mutants suggested no dramatic changes in the mechanisms of mutagenesis due to mitomycin C treatment of the cells.

Sign in / Sign up

Export Citation Format

Share Document