The objectives of the proposal are to develop and assess the specifications and sensitivities of several different DNA-based methods for detecting different types of mutation in man.
The research objective is to develop and assess the specifications and sensitivities of several different deoxyribonucleic acid (DNA) based methods for detecting different types of mutation in man. Results to date in development of the techniques and their assessment follow.
Identification of suitable lymphoblastoid (LB) lines. Cell lines from normal and DNA repair deficient individuals have been made and genotyping of one line from a normal human subject, has demonstrated that this line is suitable for both minisatellite and in particular for human leucocyte antigen (HLA)A2 mutation analysis. A suitable line from an xeroderma pigmentosum (XP) individual has also been identified.
Mutation experiments. Experiments using the normal and XP cells have been undertaken at Sussex, using gamma and ultraviolet B (VB) irradiation and the alkylating agents ethyl methanesulphonate and ethyl nitrosourea.
Molecular analysis. Minisatellite analysis of the repeat unit structures of ORI cells at D1S8 and D16S309 have been determined, and small pool polymerase chain reaction (PCR) used as the primary method of mutational screening for length change minisatellite mutations in untreated and ethyl methanesulphonate (EMS) treated ORI DNA. A detailed Class I and Class II HLA genotypes for ORI has been established using monoclonal antibodies with small numbers of cells. Experiments to improve the sensitivity of polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) in detecting mutations in target DNA sequences have been investigated. No shift bands were visualised, therefore, an alternative, the Long Linker Arms (LLA) technique has been set up and validated using 22 known hprt mutations. Restriction site polymorphism (RSP) analysis has revealed variability between cell lines in the apparent spontaneous mutation frequency at restriction sites.
Several molecular assays for measuring mutation will be developed:
i) Loss of heterozygosity as a result of mitotic recombination is a common type of alteration in the carcinogenic process. The HLA-A locus provides a suitable system for detection of such alterations. Mutations at this locus are detected with monoclonal antibodies and the nature of the mutations characterised by molecular procedures;
ii) Minisatellite repeats occur at various loci throughout the genome, and at any one locus the repeat length is highly variable between individuals. Alterations in repeat length can be detected with great sensitivity using PCR techniques;
iii) Mutations at restriction sites render the mutated site resistant to digestion by an appropriate restriction enzyme. Exhaustive digestion with restriction enzyme cleaves all normal molecules whereas mutated molecules remain intact. They are then specifically amplified using PCR and the nature of the mutations is analysed. These complementary techniques use powerful molecular methods in entirely different ways to detect different types of genetic alterations, namely mitotic recombination, repeat length deletions and point mutations. The results will be compared to a widely used clonal assay for induced mutant frequency, which detects mutations at the X-linked hprt gene.
In order to compare the specificities and sensitivities of the different techniques, lymphoblastoid cell lines will be treated with different mutagens and the cells or DNA from them will be analysed in the participating laboratories. Subsequently lymphocytes from normal, repair-deficient and exposed individuals will be used, again with the same material being analysed in the participating laboratories. Again, the results will be compared to the hprt mutant frequency, for which a considerable data-base is already available.
These procedures have the potential to provide : (i) means for mutogenecity testing in any cell type in any organ of a laboratory animal; (ii) new methods for biomonitoring using different tissues including sperm; (iii) new ways to study mutagenic mechanisms.
Call for proposalData not available
Funding SchemeCSC - Cost-sharing contracts
2300 RA Leiden
LE1 7RH Leicester