| Genetic toxicity (Genotoxic) refers to the property which can directly or indirectly damage cell DNA. Mutagen is the compound that has genetic toxicity and can change the genetic material and induce mutation. Nowadays, the chemical mutagen has become a large class of hazard factors to the environment and human health. The preventions of these chemical pollutants have become the main means of preventing cancer.At present, people have proposed all kinds of test method for evaluation of chemical mutagen mutagenic toxicity, such as Ames test, micronucleus and chromosome test etc. With the development of science and technology, some of these conventional evaluation methods have shortcomings:low sensitivity, time-consuming, complex operation and high cost that effect the evaluation of chemical mutagen toxicity. This topic using biomolecular technology, combined with microbial sensor techniques, using recombinant yeast as host cell, by affect the chemical mutagens action makes the yeast cell this characteristic color as signal conversion of the logo, then after a series of measuring systems for converting signals were collected and analyzed the final evaluation of mutagenic intensity toxic effect of chemical mutagen.Mutagenic chemical mutagens are mainly divided into two categories:direct chemical mutagens and indirect chemical mutagens, the former has a direct damage effects on DNA, the latter itself without damage effect on DNA, but it can enter the body through some reactions into has genetic toxicity effect of substances. In the 96 hole plate are respectively on direct and indirect chemical mutagen for high throughput screening of construction of recombinant yeast cells using this topic.1. The development of the recombinant yeast cell W303-1A/RNR3-Lac Z to screen direct chemical mutagens.OBJECTIVE:To establish the recombinant Lac Z yeast cell regulated by RNR3 to screen direct chemical mutagens. METHOD:RNR3 promoter was amplified using polymerase chain reaction (PCR) from yeast genome and inserted into the yeast report vector of pMP206/ERE to construct yeast Lac Z gene report vector regulated by RNR3. This vector was transformed into the yeast cell of W303-1A to construct the recombinant Lac Z gene yeast cell regulated by RNR3. The yeast cell was treated by several kinds of chemical mutagens to observe the expression of RNR3 and Excel was used to analyze the data. RESULTS:It was found that the actinomycin D, mitomycin C, aphidicolin and ethidium bromide could not induce RNR3 expression; and methyl methanesulfonate, chlorambucil, cisplatin,4-nitro-N-oxidation of quinoline, bleomycin, phleomycin,5-fluorouracil, camptothecin and hydroxyurea mutagen can induce RNR3 expression which exhibited a dose effect relationship. CONCLUSION:the recombinant yeast can be used for rapid screening of many direct chemical mutagens.2. The development of the recombinant yeast cell W303-lA/RNR3-Lac Z/GPDV5-CYP1A1 to screen indirect chemical mutagens.OBJECTIVE:To establish the recombinant Lac Z yeast cell regulated by RNR3 with the expression of CYP1A1 gene to screen indirect chemical mutagens. METHOD:CYP1A1 gene was amplified using polymerase chain reaction (PCR) from GV142 plasmid and inserted into the yeast expression vector of pGPDV5 to construct pGPDV5-CYP1A1 yeast expression vector. This vector was transformed into the yeast cell of W303-lA/RNR3-Lac Z to construct the recombinant Lac Z gene yeast cell regulated by RNR3 with the expression of CYP1A1 gene. Detect the expression of CYP1A with the kits of Promega company. The yeast cell was treated by 2-Aminofluorene to study the activity of CYP1A and Excel was used to analyze the data. RESULTS:DNA sequencing indicates that pGPDV5-CYP1A1 yeast expression vector has been constructed successfully and identified by PCR, but the activity of CYP1A1 is low and the yeast cell treated by 2-Aminofluorene did not exhibit a dose effect relationship with the expression of Lac Z gene. CONCLUSION:The expression of CYP1A1 is insufficient, some factors may block the process of transcription or translation which leads to the recombinant yeast can not screen the indirect chemical mutagens very well, the reason reminds to be further studied. |
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