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

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5425-5431.1988 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5425 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5425-5431 ◽

Cited By ~ 41

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.

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Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.84.11.3782 ◽

1987 ◽

Vol 84 (11) ◽

pp. 3782-3786 ◽

Cited By ~ 123

Author(s):

D. E. Brash ◽

S. Seetharam ◽

K. H. Kraemer ◽

M. M. Seidman ◽

A. Bredberg

Keyword(s):

Hot Spots ◽

Human Cells ◽

Major Determinant ◽

Base Substitution ◽

Cold Spots

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Family Practices and Temporality at Breakfast: Hot Spots, Convenience and Care

Sociology ◽

10.1177/00380385211015563 ◽

2021 ◽

pp. 003803852110155

Author(s):

Daniela Pirani ◽

Vicki Harman ◽

Benedetta Cappellini

Keyword(s):

Hot Spots ◽

Hot Spot ◽

Family Meals ◽

Personal Integrity ◽

Structured Interviews ◽

Time Saving ◽

Family Time ◽

Dominant Feature ◽

Family Practices ◽

Cold Spots

Drawing on 34 semi-structured interviews, this study investigates the temporality of family practices taking place in the hot spot. It does so by looking at how breakfast is inserted in the economy of family time in Italy. Our data show that breakfast, contrary to other meals, allows the adoption of more individualised and asynchronous practices, hinged on the consumption of convenience products. These time-saving strategies are normalised as part of doing family. Although the existing literature suggests that convenience and care are in opposition, and consumers of convenience products can experience anxiety and a lack of personal integrity, such features were not a dominant feature of our participants’ accounts. These findings suggest that the dichotomies of hot/cold spots and care/convenience are not always experienced in opposition when embedded within family practices. Hence, this study furthers understandings of family meals, temporality and the distinction between hot and cold spots.

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Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽

10.1093/genetics/166.2.661 ◽

2004 ◽

Vol 166 (2) ◽

pp. 661-668

Author(s):

Mandy Kim ◽

Erika Wolff ◽

Tiffany Huang ◽

Lilit Garibyan ◽

Ashlee M Earl ◽

...

Keyword(s):

Dna Sequences ◽

Deinococcus Radiodurans ◽

Genetic System ◽

Base Change ◽

Base Substitution ◽

Wild Type ◽

Base Pairs ◽

E Coli ◽

Radiation Induced ◽

Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

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Intratumor Heterogeneity in Uveal Melanoma BAP-1 Expression

Cancers ◽

10.3390/cancers13051143 ◽

2021 ◽

Vol 13 (5) ◽

pp. 1143

Author(s):

Gustav Stålhammar ◽

Hans E. Grossniklaus

Keyword(s):

Gene Expression ◽

Uveal Melanoma ◽

Tumor Size ◽

Hot Spots ◽

Malignant Tumors ◽

Intratumor Heterogeneity ◽

Prognostic Information ◽

Size Category ◽

Cold Spots ◽

Metastatic Risk

Malignant tumors are rarely homogenous on the morphological, genome, transcriptome or proteome level. In this study, we investigate the intratumor heterogeneity of BAP-1 expression in uveal melanoma with digital image analysis of 40 tumors. The proportion of BAP-1 positive cells was measured in full tumor sections, hot spots, cold spots and in scleral margins. The mean difference between hot spots and cold spots was 41 percentage points (pp, SD 29). Tumors with gene expression class 1 (associated with low metastatic risk) and 2 (high metastatic risk) had similar intratumor heterogeneity. Similarly, the level of intratumor heterogeneity was comparable in tumors from patients that later developed metastases as in patients that did not. BAP-1 measured in any tumor region added significant prognostic information to both American Joint Committee on Cancer (AJCC) tumor size category (p ≤ 0.001) and gene expression class (p ≤ 0.04). We conclude that there is substantial intratumor heterogeneity in uveal melanoma BAP-1 expression. However, it is of limited prognostic importance. Regardless of region, analysis of BAP-1 expression adds significant prognostic information beyond tumor size and gene expression class.

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The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination.

Genetics ◽

10.1093/genetics/122.4.727 ◽

1989 ◽

Vol 122 (4) ◽

pp. 727-736

Author(s):

C E Bauer ◽

J F Gardner ◽

R I Gumport ◽

R A Weisberg

Keyword(s):

Attachment Site ◽

Strand Exchange ◽

Segregation Pattern ◽

Base Substitution ◽

Base Pairs ◽

Multiple Mutation ◽

Attachment Sites ◽

Dna Strands ◽

Substitution Mutations ◽

Overlap Region

Abstract Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.

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