Item – Theses Canada

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OCLC number
1032917013
Link(s) to full text
LAC copy
Author
Jia, Xuming.
Title
Creation and improvement of a yeast 'RNR3-lacZ' genotoxicity testing system.
Degree
M. Sc. -- University of Saskatchewan, 2012
Publisher
Saskatoon : University of Saskatchewan, 2012.
Description
1 online resource
Notes
Includes bibliographical references.
Unrestricted.
I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.
Abstract
A variety of environmental toxicants can damage DNA and thereby produce congenital malformation and cancer. In order to evaluate the genotoxic effects of environmental agents, numerous genotoxicity testing systems have been developed. These tests either directly assess the genetic alterations (e.g., the Ames test) or indirectly measure the cellular response to DNA damage (e.g., SOS Chromotest). With the knowledge obtained from studying molecular mechanisms of DNA damage and signal transduction in 'Saccharomyces cerevisiae', a sensitive and stable genotoxicity testing system was developed based on the induction of 'a S. cerevisiae RNR3-lacZ' reporter gene expression in response to a broad range of DNA-damaging agents and agents that interfere with DNA synthesis. The tested agents include known carcinogenic and genotoxic agents, ranging from DNA alkylating agents, oxidative chemicals to ionizing radiation as well as some known non-genotoxic agents. All the tested known genotoxic agents were able to induce 'RNR3-lacZ' expression at a sub-lethal dose. In particular, a potent colon carcinogen, 1, 2-dimethyl hydrazine, was not detected as a mutagen by a standard Ames test, but was able to induce 'RNR3-lacZ' expression. In contrast. both non-mutagenic and non-genotoxic chemicals tested were unable to induce 'RNR3-lacZ' expression. The sensitivity and inducibility of three well-characterized yeast DNA damage-inducible genes have been compared and it was found that 'RNR3' is more sensitive than 'RNR2' and 'MAG1'. The effects of agent dose, post-treatment incubation time and cell growth stage on 'RNR3-lacZ' expression were also determined and optimized. In order to create a stable and user-friendly testing system, the 'RNR3-lacZ' cassette was integrated into the yeast genome to demonstrate that its inducibility is indistinguishable from that of the plasmid-based studies. Although the sensitivity of 'RNR3-lacZ' testing is comparable to that of the Ames test and SOS Chromotest, it was reasoned that the sensitivity might be further improved by using yeast strains defective in certain DNA repair pathways. Hence, several deletion mutant strains from different repair pathways were used as host strains for the 'RNR3-lacZ' test. It was found that the 'mag1' null mutation specifically enhanced the sensitivity of 'RNR3-lacZ' test in response to DNA alkylating agents such as methyl methanesulfonate and ethyl methanesulfonate, while the 'rad2' null mutation enhanced the sensitivity of this system in response to ultraviolet (UV) radiation and a UV mimetic agent 4-nitroquinoline 1-oxide. In the 'rad2' null mutant, 'RNR3-lacZ' induction was also more sensitive to MMS than that in wild type strain at low dosed. In summary, it appears that the enhancement of 'RNR3-lacZ' induction is agent and repair specific, although inactivation of the nucleotide excision repair pathway may affect a broad range of testing agents which can be incorporated into the 'RNR3-lacZ' test.
Other link(s)
library.usask.ca