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Characterization of mutations induced by N-hydroxy-2-aminofluorene and N-acetoxy-2-acetylaminofluorene in Chinese hamster ovary AS52 cells.

紀錄類型:	書目-語言資料,手稿 : Monograph/item
正題名/作者:	Characterization of mutations induced by N-hydroxy-2-aminofluorene and N-acetoxy-2-acetylaminofluorene in Chinese hamster ovary AS52 cells./
作者:	Beranek, David T.
面頁冊數:	1 online resource (133 pages)
附註:	Source: Dissertations Abstracts International, Volume: 51-08, Section: B.
Contained By:	Dissertations Abstracts International51-08B.
標題:	Biochemistry. -
電子資源:	click for full text (PQDT)
ISBN:	9798206771084

Characterization of mutations induced by N-hydroxy-2-aminofluorene and N-acetoxy-2-acetylaminofluorene in Chinese hamster ovary AS52 cells.
Beranek, David T.

Characterization of mutations induced by N-hydroxy-2-aminofluorene and N-acetoxy-2-acetylaminofluorene in Chinese hamster ovary AS52 cells. - 1 online resource (133 pages)

Source: Dissertations Abstracts International, Volume: 51-08, Section: B.

Thesis (Ph.D.)--University of Arkansas for Medical Sciences, 1989.

Includes bibliographical references

Chinese hamster ovary AS52 cells (CHO-AS52) are deficient in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene. However, a single copy of the plasmid, pSV2gpt, which contains the analogous Escherichia coli guanine phosphoribosyl transferase (gpt) gene, has been integrated into the genome of these cells. CHO-AS52 cells were treated with 5.1 to 20.2 μM N-hydroxy-2-aminofluorene and 0.5 and 1.0 μM N-acetoxy-2-acetylaminofluorene, and the 6-thioguanine resistant gpt mutants were characterized. Mutation frequencies from cells treated with 20.2 μM N-hydroxy-2-aminofluorene and 1.0 μM N-acetoxy-2-acetylaminofluorene were 3 and 20-fold higher than solvent controls, respectively. Thirty-four mutant colonies were isolated, their DNA extracted, and a 500 base-pair (bp) region of genomic DNA containing the entire E. coli gpt gene was amplified by the polymerase chain reaction (PCR). PCR-amplified fragments were analyzed by electrophoresis on a 1% agarose gel and visualized with ethidium bromide staining. Sixteen of 17 N-hydroxy-2-aminofluorene mutants, 14 of 14 N-acetoxy-2-acetylaminofluorene mutants and 3 of 3 solvent control mutants produced an amplified product that migrated at 500 bp on agarose gel electrophoresis. No amplification occurred with DNA from the remaining N-hydroxy-2-aminofluorene mutant. Sequence analysis of these mutants indicated that N-hydroxy-2-aminofluorene and N-acetoxy-2-acetylaminofluorene induced minor DNA sequence modifications including base pair substitutions, primarily G:C → A:T transitions, as well as insertions and deletions. Mutational hot spots were observed at base pair location 165-174 for N-hydroxy-2-aminofluorene and base pair location 286-294 for N-acetoxy-2-acetylaminofluorene. Analysis of total RNA isolated from CHO-AS52 mutants indicated that mRNA expression was inhibited by most mutations; however, two mutants showed enhanced RNA expression. These data indicate that the majority of mutations induced in the CHO-AS52 cell gpt gene by these aromatic amine derivatives involve minor DNA modifications, primarily at G-C base pairs and that these modifications affect mRNA expression in mutant cells.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2024

Mode of access: World Wide Web

ISBN: 9798206771084Subjects--Topical Terms:

582831
Biochemistry.
Subjects--Index Terms:

aminofluoreneIndex Terms--Genre/Form:

554714
Electronic books.

Characterization of mutations induced by N-hydroxy-2-aminofluorene and N-acetoxy-2-acetylaminofluorene in Chinese hamster ovary AS52 cells.
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504 $a Includes bibliographical references
520 $a Chinese hamster ovary AS52 cells (CHO-AS52) are deficient in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene. However, a single copy of the plasmid, pSV2gpt, which contains the analogous Escherichia coli guanine phosphoribosyl transferase (gpt) gene, has been integrated into the genome of these cells. CHO-AS52 cells were treated with 5.1 to 20.2 μM N-hydroxy-2-aminofluorene and 0.5 and 1.0 μM N-acetoxy-2-acetylaminofluorene, and the 6-thioguanine resistant gpt mutants were characterized. Mutation frequencies from cells treated with 20.2 μM N-hydroxy-2-aminofluorene and 1.0 μM N-acetoxy-2-acetylaminofluorene were 3 and 20-fold higher than solvent controls, respectively. Thirty-four mutant colonies were isolated, their DNA extracted, and a 500 base-pair (bp) region of genomic DNA containing the entire E. coli gpt gene was amplified by the polymerase chain reaction (PCR). PCR-amplified fragments were analyzed by electrophoresis on a 1% agarose gel and visualized with ethidium bromide staining. Sixteen of 17 N-hydroxy-2-aminofluorene mutants, 14 of 14 N-acetoxy-2-acetylaminofluorene mutants and 3 of 3 solvent control mutants produced an amplified product that migrated at 500 bp on agarose gel electrophoresis. No amplification occurred with DNA from the remaining N-hydroxy-2-aminofluorene mutant. Sequence analysis of these mutants indicated that N-hydroxy-2-aminofluorene and N-acetoxy-2-acetylaminofluorene induced minor DNA sequence modifications including base pair substitutions, primarily G:C → A:T transitions, as well as insertions and deletions. Mutational hot spots were observed at base pair location 165-174 for N-hydroxy-2-aminofluorene and base pair location 286-294 for N-acetoxy-2-acetylaminofluorene. Analysis of total RNA isolated from CHO-AS52 mutants indicated that mRNA expression was inhibited by most mutations; however, two mutants showed enhanced RNA expression. These data indicate that the majority of mutations induced in the CHO-AS52 cell gpt gene by these aromatic amine derivatives involve minor DNA modifications, primarily at G-C base pairs and that these modifications affect mRNA expression in mutant cells.
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