Graduate Thesis Or Dissertation
 

Coliphage T3 S-adenosylmethionine hydrolase : properties of the cloned gene and consequences of its expression in Escherichia coli

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  • To begin the development of a research tool for use in the in vivo study of S-adenosylmethionine (SAM) metabolism, I attempted to locate, isolate, clone and elicit the expression of the SAM hydrolase gene (E.C. 3.3.1.2) of the coliphage T3. The gene was isolated and cloned into the M13mp8 cloning vector. Southern blots, restriction digests, identification of SAMase activity, and analyses of the purified proteins and the end products of SAMase digestion verified these clones as producing the authentic enzyme. The nucleotide sequence of the SAMase gene sequence revealed an open reading frame capable of encoding a 17 kilodalton protein. Within this sequence is a second translation initiation site that defines a 14 kd peptide identical to the carboxy terminal 82% of the larger protein. The sequence itself is quite dissimilar to that of the homologous 0.3 gene of the related phage T7 except for the highly conserved RNase III RNA transcript processing sites that bracket these genes in both viruses. The SAMase gene was cloned into a variety of plasmids to determine the effect high-level expression might have on E. coli. It was expressed in viable cells at levels sufficient to inhibit DNA methylation and spermidine biosynthesis. The cells maintained a normal intracellular concentration of SAM by inducing SAM synthetase (MATase) activity. Unregulated SAMase expression from the strong P[subscript tac] promoter or moderate SAMase activity in MATase defective cells inhibited cell division (possibly by inducing the SOS response) and killed the bacteria. Under these conditions, SAMase likely degrades SAM as it is produced and before it can be used by processes essential to cell division and cell viability. These results confirm the potential of SAMase as a powerful tool for the study of in vivo SAM metabolism and form a basis for further studies in E. coli and other organisms.
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