Green fluorescent protein (GFP) as a direct biosensor for mutation detection: elimination of false-negative errors in target gene expression.
A new method was developed to determine the mutagenic efficacy of a suspected mutagen by employing green fluorescent protein (GFP) as a direct biosensor for mutation detection. Alterations in target gene (AcGFP1) expression after mutagen [(+/-)-7p,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)] treatment were measured to detect the mutagenic efficacy of the carcinogen. In contrast to mutagen treatment of the entire plasmid or cell culture, the target AcGFP1gene devoid of the plasmid backbone was exposed to BPDE (10-500 microM) to eliminate the need for an additional fusion gene. Shuttle vectors (pAcGFP-N1) were religated to the AcGFP1 gene with BPDE adducts (0-8.59 microM) and replicated in the eukaryotic host. This approach eliminated false-negative errors in target gene expression that arose from BPDE adduct formation in the residual plasmid backbone rather than in the AcGFP1 gene. Determination of the BPDE-AcGFP1 adducts allowed the quantitative mutagenic effect of the BPDE adducts on AcGFP1 gene expression to be monitored. The results obtained with flow cytometry and confocal microscopy validate our method and demonstrate efficient and direct use of GFP as a biosensor for mutation detection.
Tak
Yu Kyung
YK
Research Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University, San 56-1, Gwanak-gu, Seoul-151-742, Republic of Korea.
Naoghare
Pravin K
PK
Lee
Kyeong-Hee
KH
Park
Soon-Sup
SS
Song
Joon Myong
JM
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000378:metabolism / Q000633:toxicity; D000818:Animals; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002417:Cattle; D002460:Cell Line; D004252:DNA Mutational Analysis / Q000379:methods*; D005188:False Negative Reactions; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008969:Molecular Sequence Data; D009153:Mutagens / Q000378:metabolism / Q000633:toxicity; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation
D009153:Mutagens; D049452:Green Fluorescent Proteins; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide