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Title: The DNA specificity of type I restriction and modification enzymes
Author: Thorpe, Peter Harold
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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A deletion analysis of the hsdS gene of EcoKI was initiated to provide information on the roles of the conserved and variable regions. The aim was to correlate phenotype with an analysis of protein products, but attempts to overexpress the hsdS gene of EcoKI in a soluble form were unsuccessful, and the HsdS subunit could not be purified. Another approach to studying the interaction of TRDs with their DNA targets is to compare the amino acid sequences of those TRDs that specify the same DNA target. Areas of sequence which are similar within these TRDs may reflect a similarity of DNA recognition function. From amongst the limited number of TRDs available, there are several sequence alignments of TRDs which specify the same DNA target. A method based upon the Polymerase Chain Reaction (PCR) was developed to amplify new variable regions, which encode TRDs, from wild-type bacteria. In a DNA hybridisation screen of members of the ECOR collection of wild-type Escherichia coli, Barcus et al. (1995) found that almost half contained hsd genes. The conserved regions of the hsd genes were used to design primers that would amplify 5' variable regions of members of a given type I family. Nine IA family and four IB family 5' variable regions were amplified and their DNA sequences determined. The information derived from these sequences illustrates both the evolutionary diversity of the hsdS genes, and the flexible nature of the TRDs as independent target recognising domains. The sequencing of the N-terminal TRD from ECOR17 shares 28% identity with those of EcoKI and StySPI.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available