Biochemical characterization of the Pythium ultimum NAD-GDH and genetic analysis of associated genomic regions in Pythium ultimum and Achlya klebsiana

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Barker, Douglas Shaw
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Investigation of an antisense gene pair previously identified in the oomycete 'Achlya klebsiana' and of a NAD-specific glutamate dehydrogenase (NAD-GDH) and putative heat shock 70 stress response protein gene ('hsp70') in the oomycete 'Pythium ultimum' strain 471 were conducted. The transcriptionally active nature of the ' A. klebsiana' antisense gene pair in both eukaryotic and prokaryotic host cell lines was confirmed although the specific identities and compositions of the transcripts produced were not. The 'P. ultimum' NAD-GDH in crude cell extracts was characterized, and shown to be similar to the previously characterized NAD-GDH of 'Pythium debaryanum', but less so to the NAD-GDH of 'A. klebsiana'. Biochemical characterization of the NAD-GDH included analyses of enzyme instability at several temperatures (counteracted with high concentrations of glycerol); pH optima of both the reductive and oxidative reactions of NAD-GDH (8.8 and 7.2, respectively); induction by L-glutamate; and allosteric activation(of up to 14 fold) by micromolar concentrations of NADP+. As concentrations of NADP + are increased, Km decreased over 10 fold and Vmax increased over 2 fold with _-ketoglutarate as substrate, while Km decreased 30 fold and Vmax increased 1.3 fold with L-glutamate as substrate. Partial sequencing of the putative ' hsp70' gene showed it to have strong homology to other 'hsp70 ' genes, and also to possess consensus sequences corresponding to transcriptional promoter regions (CCAAT boxes, TATAAT boxes, CTF/NF1 binding sites, heat shock elements) in the putative 5' untranslated region. The gene appears to consist of a single open reading frame, which is almost identical to another putative 'hsp70' gene identified in 'A. klebsiana' approximately 7.8 kb away from the ' hsc70':'nad-gdh' antisense gene pair.