As an application for the Physician Scientist Award, the ultimate goal of this proposal is to obtain the research background and skills necessary to become an independent biomedical investigator, with particular emphasis on protein chemistry and recombinant DNA methodology. A program for didactic study and laboratory experience has been outlined for Phase I. The area of the research project for Phase II is the biosynthesis of alginic acid by mucoid Pseudomonas aeruginosa isolated from cystic fibrosis patients, specifically focusing on the enzyme that catalyzes epimerization of mannuronate residues to guluronic acid. The almost exclusive association of the mucoid phenotype with cystic fibrosis and the deleterious effect P. aeruginosa infections have on the respiratory function of these patients makes this topic a pertinent area of interest.
The specific aims are to purify and characterize the polymannuronate 5'-epimerase from mucoid strains of P. aeruginosa, and to clone the structural gene for this enzyme and characterize its regulation of expression. Strains of mucoid P. aeruginosa with known amounts of mannuronate and guluronate will be used. Epimerase activity will be assayed by the amount of 3H released from labeled polymannuronic acid. An HPLC method will be developed to measure guluronic acid and mannuronic acid concentrations in the alginate produced. The epimerase will be purified using ion exchange celluloses and affinity chromatography. It will be characterized by SDS-Page, HPLC gel-filtration and partial amino acid sequencing. DNA from mucoid strains of P. aeruginosa will be isolated by cesium chloride density gradient centrifugation, digested with Eco Rl, ligated into lambda gtll, and introduced into E. coli. The correct clone will be identified by using both specific antibody against purified epimerase and DNA probes. A restriction map of the structural gene will be prepared and DNA sequencing will be accomplished using Sanger chain termination method. The regulatory gene will be identified by digesting the structural gene and flanking DNA with restriction enzymes, and recloning it into a pMMN66 vector. Initial steps to determine the regulation of expression of structural genes in alginate production will be to study the expression of the epimerase in E. coli and P. aeruginosa.
| Steele, M I; Marshall, C M; Lloyd, R E et al. (1995) Reovirus 3 not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease. Hepatology 21:697-702 |
| Steele, M I; Lorenz, D; Hatter, K et al. (1992) Characterization of the mmsAB operon of Pseudomonas aeruginosa PAO encoding methylmalonate-semialdehyde dehydrogenase and 3-hydroxyisobutyrate dehydrogenase. J Biol Chem 267:13585-92 |
