The Gene cloning and biochemical and genetic analysis of the ribonuclease HI from Mycobacterium smegmatis
Date
2017
Authors
Dawes, Stephanie Susan
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Abstract
Two members of the genus Mycobacteria are the causative agents of tuberculosis
and leprosy, and many others are opportunistic pathogens in immune-compromised
individuals. An alarming :resurgence of tuberculosis in First World countries, coupled
with the high toll of mortality that this disease exacts in the Developing World, has led
the World Health Organisation to declare tuberculosis a global emergency. Relatively
little is known, however, about the molecular biology of the mycobacteria Elucidation
of mechanisms involved in the regulation of 'fundamental processes in this genus, such as
DNA replication, are expected to provide a basis for understanding mechanisms of
pathogenesis in the mycobacteria. RNase Hl, encoded by rnhA, plays a central role m
DNA replication in Escherichia coli by fixing initiation of DNA replication to one origin.
The presence of a similar activity in the mycobacteria was therefore investigated by
probing a genetically manipulable. member of the mycobacteria, Ai. smegmatis, for a
homologue of the mhA gene. Degenerate primers based on blocks of amino acids
conserved in other bacterial RNase HI homologues had been shown to amplify an
internal portion ofmhA fromM. smegmatis (Mizrahi et al., 1993). This peR generated
probe was used in this study to isolate the entire gene from M smegma tis, which was
cloned as two overlapping fragments and sequenced. The deduced amino acid sequence
ofM smegmatis rnhA coded for an RN~"e HI of 159 amino adds which showed 50%
identity to E. coli RNase HI. A recombinant form afM smegma/is RNase III was
expressed as part of a fusion protein to the maltose binding protein to facilitate in vitro
biochemical characterisation of the protein. The recombinant RNase fIT exhibited
hydrolytic activity specific for the RNA strand of an RNADNA hybrid and generated a
similar distribution of hydrolysis products to that of E. coli RNase Hl,
Description
A dissertation submitted to the Faculty of science,
University of Witwatersrand, Johannesburg,
for the Degree of Doctor of Philosophy,
June, 1998