Date of Award

1987

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Michael Orlowski

Abstract

Mitochondria from aerobic hyphae of Mucor racemosus displayed a relatively elongated morphology with well developed cristae and stained in positive contrast. Mitochondria from anaerobic yeast cells were more circular in form, displayed less distinct but well recognizable cristae, and stained in negative contrast. Tween 80, ergosterol or cerulenin did not alter mitochondrial morphology when added to cultures. Mitochondria from cells grown in the presence of chloramphenicol displayed a morphology intermediate between those of yeast and hyphae and stained in negative contrast. Mitochondria were isolated by isopycnic centrifugation in Percoll. A single band in hyphal preparations displayed cyanide-sensitive respiration and was observed to contain a homogeneous population of mitochondria with distinct cristae. The corresponding band from anaerobic yeast preparations contained mitochondria with less distinct cristae and some additional membrane-bounded structures that were more compact and densely stained than the recognizable mitochondria. The staining and morphological characteristics of these structures were dependent on the osmolarity of the isolation media. Two-dimensional polyacrylamide gel electrophoresis revealed that mitochondria prepared from aerobic hyphae contained fewer overall but several more intensely-stained proteins than mitochondria from anaerobic yeasts. A 9.76-Kb nuclear rDNA repeat unit from M. racemosus was cloned in E. coli plasmids in four overlapping pieces and mapped. The 26S rRNA gene was sequenced in its entirety. The 5$\sp\prime$ and 3$\sp\prime$ ends of the gene were identified upon comparison of the present nucleotide sequence with that reported for the 26S rRNA gene from Saccharomyces cerevisiae. The Mucor gene contained 3469 bp, showing 79% homology with that of Saccharomyces. A 106-bp segment, not present in the Saccharomyces gene, differed from the typical GC-rich sequences found inserted in the equivalent gene from higher eukaryotes. All of the thirty known methylation sites in Saccharomyces 26S rRNA were conserved in Mucor and 750 bp surrounding these sites showed 98% homology with those from Saccharomyces. The gene from Mucor was shown to possess about 800 bp more than that from E. coli, due to insertions of varying lengths. When these specific sequences were compared in Mucor and Saccharomyces they displayed a much lower (58%) homology than that found over the rest of the gene (85%).

Pages

208

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