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Title: Characterisation of cDNA clones for the am gene of Neurospora crassa
Author: Lindsay, Carol E.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1991
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ICR 170, an acridine half-mustard, has been shown to induce predominantly frameshift addition mutations in the am genes of Neurospora crassa (Burns et al., 1986) and other eukaryotic genes. The related compound ICR 191 produces both addition and deletion mutations in prokaryotic DNA. The introduction of the am gene into a prokaryote such as E.coli, followed by the induction of frameshift mutations using ICR 170 should provide an insight into the mechanism by which frameshift mutations are induced in eukaryotic and prokaryotic DNA and their different responses to induction of frameshift mutations by acridine half-mustards. In order to study the mechanism by which mutations are induced in prokaryotic and eukaryotic DNA, it is necessary to have a system which will allow the identification of frameshift mutations in a copy of the N.crassa am gene which has been transformed into E.coli. This study was an attempt to set up such a system. This work involved the production of complementary DNA to the am gene with the intention of expressing the am gene product, NADP-dependent glutamate dehydrogenase, in an E.coli strain which is auxotrophic for glutamate. Expression of the am gene product should complement the E.coli mutation and revert the E.coli strain to glutamate prototrophy. In this study, optimum conditions for producing cDNA from Neurospora crassa mRNA were determined and cDNA libraries were produced both from total Neurospora crassa mRNA and from N.crassa mRNA which had been selected by its ability to hybridise to the am genomic sequence. Potential cDNA clones to the am gene were isolated and characterised. 19 clones from the cDNA library constructed using total N.crassa mRNA showed homology to the am sequence in colony blots. Characterisation of these clones identified them as am cDNA sequence in colony blots. Characterisation of these clones identified them as am cDNA clones which contained only the 3' end of the am coding sequence. Since all 19 clones represented the same area of the am coding sequence, it is likely that some form of structural constraint in the mRNA prevented full length cDNA molecules for am being formed. 1328 cDNA clones were identified as homologous to the am genomic sequence within the cDNA library constructed from am-selected N.crassa nRNA. This represented a 90-fold increase in the representation of cDNA clones to the am gene using the selected mRNA.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available