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Title: Nucleotide excision repair of the plasmid borne NFA2 gene in Saccharomyces cerevisiae
Author: Klungsupya, P.
Awarding Body: University of Wales Swansea
Current Institution: Swansea University
Date of Award: 2001
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The role of transcription in repair of UV-induced CPD was investigated in the Saccharomyces cerevisiae MFA2 gene. MFA2 mutants were constructed for cloning into a yeast artificial chromosome (YAC) by deletion of either the 22nt TATA box sequences (T2ΔMFA2) or 55nt Mcm1 binding site (MΔMFA2) from its promoter. In vivo transcription analysis using eGFP reporter system reveals a 10-fold reduction in transcription activity for the T2ΔMFA2 a cells compared to the WTMFA2 a cells. This reduced transcription level is very close to the level found in α cells where MFA2 is inactive. A 7-fold reduction in transcription activity was found for MΔMFA2 in both a and α cells. This result indicates the deletion of the MBS eliminates Mcm1/α2-mediated MFA2 repression in α cells dramatically reduced MFA2 expression in a cells. Among 13 CPDs found in the promoter region, three CPDs are increasingly induced following the deletion of the TATA box and MBS sequences. However, their repair rates are not affected. Analysis of NER was performed on individual CPDs in both the transcribed (TS) and non-transcribed (NTS) strands by comparison between the YAC-borne MFA2 and the endogenous gene for each construct. The endogenous MFA2 in YAC strains harbouring the WTMFA2 and MΔMFA2 constructs, exhibit an identical repair profile. This suggests no genetic variation occurs within the isogenic strain harbouring the three promoter element deletion constructs. The repair results on the TS indicate the fastest rate with t50% of 1.5 hr for some CPDs in the transcription region of the transcribed strand of the WTMFA2.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available