Patel, Sejal; (1996) A novel thermostable restriction modification system. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A thermostable restriction modification system has been isolated from a thermophilic bacterium. The isolate designated Rot34Al was thought to be the extreme thermophile T. filiformis. The strain grows optimally at 70[degrees]C and was isolated from a thermal site in Rotorua, New Zealand. The endonuclease named in accordance to the rules proposed by Smith and Nathans (1973), recognises the sequence GAA/TTC which is a hitherto unknown specificity and has not been reported from either mesophilic or thermophilic sources. The endonuclease was purified and characterised. The molecular weight (Mr) of TfiI endonuclease, estimated under denaturing conditions was 37 000. The Mr of the native form of 7TfiI endonuclease, as estimated by gel filtration was approximately 75 000 i.e., TfiI endonuclease is a dimer in its active form. The optimal pH for TfiI endonuclease activity was determined to be pH 8.0 although TfiI endonuclease exhibits activity over a broad pH range. The enzyme is remarkably thermostable, surviving at room temperature for several weeks and having a half life of greater than one hour at 65°C. Other characteristics of TfiI endonuclease have been determined such as ability to cleave single stranded DNA and it's salt requirement. Two kinds of "star activity" were observed for TfiI endonuclease; the indiscriminate endonucleolytic activity exhibited in certain buffers and the relaxed specificity exhibited by buffers containing Mn2+. The TfiI methylase was partially purified and a single step method was developed to separate the methylase from the endonuclease. The site at which the TfiI methylase incorporates methyl groups into DNA was determined. Genomic libraries were created in both plasmid and phage vectors and strategies for screening the libraries are discussed.

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