Biochemical characterization of the Pythium ultimum NAD-GDH and genetic analysis of associated genomic regions in Pythium ultimum and Achlya klebsiana
| dc.contributor.author | Barker, Douglas Shaw | en_US |
| dc.date.accessioned | 2007-05-18T20:01:33Z | |
| dc.date.available | 2007-05-18T20:01:33Z | |
| dc.date.issued | 1999-10-01T00:00:00Z | en_US |
| dc.degree.discipline | Microbiology and Medical Genetics | en_US |
| dc.degree.level | Doctor of Philosophy (Ph.D.) | en_US |
| dc.description.abstract | 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. | en_US |
| dc.format.extent | 12621165 bytes | |
| dc.format.extent | 184 bytes | |
| dc.format.mimetype | application/pdf | |
| dc.format.mimetype | text/plain | |
| dc.identifier.uri | http://hdl.handle.net/1993/1868 | |
| dc.language.iso | eng | en_US |
| dc.rights | open access | en_US |
| dc.title | Biochemical characterization of the Pythium ultimum NAD-GDH and genetic analysis of associated genomic regions in Pythium ultimum and Achlya klebsiana | en_US |
| dc.type | doctoral thesis | en_US |