The long-term objective of the proposed work involves the establishment of the relationship between the synthesis and characteristics of the cell wall matrix polymers (polyuronides) and the morphogenetic development of Mucor.
The specific aims will focus on (i) the physico-chemical characterization of the matrix polymers in hyphal and yeast-like cell walls, (ii) characterization of the matrix polymer precursors synthesized within the cytoplasmic endomembrane system and comparison to the matrix polymers in the wall, (iii) characterization of the synthesis or activation of the enzymes involved in the synthesis of matrix polymers, and (iv) comparison of these parameters to be studies in Mucor rouxii, Mucor racemosus and the coy (conditional yeast) mutant of M. racemosus. The methodology to be employed in the proposed work will involve (1) the sequential extraction of matrix polymers from isolated cell wall fractions with LiCl followed by KOH (2) the resolution of polyuronides by DEAE-Sephalex chromatography, (3) analysis of the sugar composition of polymers by gas-liquid chromatography, (4) molecular weight determination of polymers by sedimentation equilibrium centrifugation, (5) analysis ok polymers by methylene-induced circular dichroism activity, (6) preferential labelling of the cell wall polymers with 14C-D-glucose in order to locate the polymers synthesized within the cytoplasmic endomembrane system, (7) pulse-chase the labelled polymers from the cytoplasmic endomembrane system on to the cell wall, (8) immunological testing of the antigenicity of the precursor polymers against the cell wall polymers in an attempt to establish immunological cross-reactions, (9) electron microscopic autoradiographic pulse-chase of the precursor polymers on to the cell wall, (10) characterize the synthesis or activation of the enzymes involved in polymer synthesis by using 14C- labelled GDP mannose, UDP-glucuronic acid, UDP-galactose and GDP-fucose to determine the mannosyl, glucuronosyl, galactosyl and fucosyl transferase activities. The study of cell wall matrix polymers in Mucor dimorphism is worth pursuing since yeast-hyphal dimorphism and its control has direct relevance to medical mycology.
