In vivo reporter gene mutation and micronucleus assays in gpt delta mice treated with a flame retardant decabromodiphenyl ether

Abstract DNA polymerase zeta (Polζ) is a heterotetramer composed of the catalytic subunit Rev3l, Rev7 and two subunits of Polδ (PolD2/Pol31 and PolD3/Pol32), and this polymerase exerts translesion DNA synthesis (TLS) in yeast. Because Rev3l knockout results in embryonic lethality in mice, the functions of Polζ need further investigation in vivo. Then, we noted the two facts that substitution of leucine 979 of yeast Rev3l with methionine reduces Polζ replication fidelity and that reporter gene transgenic rodents are able to provide the detailed mutation status. Here, we established gpt delta mouse knocked in the constructed gene encoding methionine instead of leucine at residue 2610 of Rev3l (Rev3l L2610M gpt delta mice), to clarify the role of Polζ in TLS of chemical-induced bulky DNA adducts in vivo. Eight-week-old gpt delta mice and Rev3l L2610M gpt delta mice were treated with benzo[a]pyrene (BaP) at 0, 40, 80, or 160 mg/kg via single intraperitoneal injection. At necropsy 31 days after treatment, lungs were collected for reporter gene mutation assays. Although the gpt mutant frequency (MF) was significantly increased by BaP in both mouse genotypes, it was three times higher in Rev3l L2610M gpt delta than gpt delta mice after treatment with 160 mg/kg BaP. The frequencies of G:C base substitutions and characteristic complex mutations were significantly increased in Rev3l L2610M gpt delta mice compared with gpt delta mice. The BaP dose–response relationship suggested that Polζ plays a central role in TLS when protective mechanisms against BaP mutagenesis, such as error-free TLS, are saturated. Overall, Polζ may incorporate incorrect nucleotides at the sites opposite to BaP-modified guanines and extend short DNA sequences from the resultant terminal mismatches only when DNA is heavily damaged.

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Oxidative DNA damage and reporter gene mutation in the livers of gpt delta rats given non-genotoxic hepatocarcinogens with cytochrome P450-inducible potency

Cancer Science ◽

10.1111/j.1349-7006.2010.01705.x ◽

2010 ◽

Vol 101 (12) ◽

pp. 2525-2530 ◽

Cited By ~ 16

Author(s):

Masako Tasaki ◽

Takashi Umemura ◽

Yuta Suzuki ◽

Daisuke Hibi ◽

Tomoki Inoue ◽

...

Keyword(s):

Dna Damage ◽

Cytochrome P450 ◽

Gene Mutation ◽

Reporter Gene ◽

Oxidative Dna Damage ◽

Gpt Delta

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New homozygous gpt delta transgenic rat strain improves an efficiency of the in vivo mutagenicity assay

Genes and Environment ◽

10.1186/s41021-021-00195-1 ◽

2021 ◽

Vol 43 (1) ◽

Author(s):

Kenichi Masumura ◽

Tomoko Ando ◽

Akiko Ukai ◽

Sho Fujiwara ◽

Shigeo Yokose ◽

...

Keyword(s):

Bone Marrow ◽

Gene Mutation ◽

Point Mutations ◽

Chromosome 1 ◽

Transgenic Rat ◽

Target Tissue ◽

Sequencing Analysis ◽

Rat Strain ◽

Packaging Efficiency

Abstract Background Gene mutation assays in transgenic rodents are useful tools to investigate in vivo mutagenicity in a target tissue. Using a lambda EG10 transgene containing reporter genes, gpt delta transgenic mice and rats have been developed to detect point mutations and deletions. The transgene is integrated in the genome and can be rescued through an in vitro packaging reaction. However, the packaging efficiency is lower in gpt delta rats than in mice, because of the transgene in gpt delta rats being heterozygous and in low copy number. To improve the packaging efficiency, we herein describe a newly developed homozygous gpt delta rat strain. Results The new gpt delta rat has a Wistar Hannover background and has been successfully maintained as homozygous for the transgene. The packaging efficiency in the liver was 4 to 8 times higher than that of existing heterozygous F344 gpt delta rats. The frequency of gpt point mutations significantly increased in the liver and bone marrow of N-nitroso-N-ethylurea (ENU)- and benzo[a]pyrene (BaP)-treated rats. Spi− deletion frequencies significantly increased in the liver and bone marrow of BaP-treated rats but not in ENU-treated rats. Whole genome sequencing analysis identified ≥ 30 copies of lambda EG10 transgenes integrated in rat chromosome 1. Conclusions The new homozygous gpt delta rat strain showed a higher packaging efficiency, and could be useful for in vivo gene mutation assays in rats.

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The in vivo Pig-a gene mutation assay is applied to study the genotoxicity of procarbazine hydrochloride in Sprague-Dawley rats

Fundamental Toxicological Sciences ◽

10.2131/fts.3.167 ◽

2016 ◽

Vol 3 (4) ◽

pp. 167-175 ◽

Cited By ~ 2

Author(s):

Jiang Pu ◽

Yuanyuan Deng ◽

Xiaoyan Tan ◽

Gaofeng Chen ◽

Cong Zhu ◽

...

Keyword(s):

Gene Mutation ◽

Sprague Dawley ◽

Sprague Dawley Rats ◽

Mutation Assay ◽

Gene Mutation Assay

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Sir Proteins, Rif Proteins, and Cdc13p BindSaccharomyces Telomeres In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5600 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5600-5608 ◽

Cited By ~ 71

Author(s):

Brenda D. Bourns ◽

Mary Kate Alexander ◽

Andrew M. Smith ◽

Virginia A. Zakian

Keyword(s):

Reporter Gene ◽

Transcriptional Activation ◽

Binding Proteins ◽

Position Effect ◽

Initiation Site ◽

Telomeric Sequence ◽

Transcriptional Activation Domain ◽

Amino Terminal ◽

Sir Proteins

ABSTRACT Although a surprisingly large number of genes affect yeast telomeres, in most cases it is not known if their products act directly or indirectly. We describe a one-hybrid assay for telomere binding proteins and use it to establish that six proteins that affect telomere structure or function but which had not been shown previously to bind telomeres in vivo are indeed telomere binding proteins. A promoter-defective allele of HIS3 was placed adjacent to a chromosomal telomere. Candidate proteins fused to a transcriptional activation domain were tested for the ability to activate transcription of the telomere-linked HIS3 gene. Using this system, Rif1p, Rif2p, Sir2p, Sir3p, Sir4p, and Cdc13p were found to be in vivo telomere binding proteins. None of the proteins activated the same reporter gene when it was at an internal site on the chromosome. Moreover, Cdc13p did not activate the reporter gene when it was adjacent to an internal tract of telomeric sequence, indicating that Cdc13p binding was telomere limited in vivo. The amino-terminal 20% of Cdc13p was sufficient to target Cdc13p to a telomere, suggesting that its DNA binding domain was within this portion of the protein. Rap1p, Rif1p, Rif2p, Sir4p, and Cdc13p activated the telomeric reporter gene in a strain lacking Sir3p, which is essential for telomere position effect (TPE). Thus, the telomeric association of these proteins did not require any of the chromatin features necessary for TPE. The data support models in which the telomere acts as an initiation site for TPE by recruiting silencing proteins to the chromosome end.

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