Dan O. McClary

An alternate respiratory pathway in Candida albicans.

Usual concentrations of antimycin A, rotenone and EDTA, individally or in combination, reduced aerobic growth rate and cell yield of Candida albicans to about half its normal level and to about the levels of previously-described acetate-negative, cytochrome-complete and aa3-deficient variants which were little affected by the inhibitors. Anaerobic conditions (not affected by antimycin A) reduced growth rate and cell yield of all cultures-including that of a nonrespiring aa3, b-deficient mutant-to low, equal levels. Antimycin A but not rotenone prevented growth of the normal strain on ethanol medium. Cyanide and antimycin A blocked most of the respiration of the normal strain and cytochrome-complete variant, but did not affect that of the cytochrome aa3-deficient mutant. Rotenone and EDTA did not affect respiration of any of the cultures. SHAM blocked cyanide- and antimycin A-insensitive respiration and prolonged the lag phases of the three respiring cultures, especially in the presence of antimycin A, but alone increased oxygen-uptake rate of the cytochromecomplete cultures while curtailing that of the cytochrome aa3-deficient mutant. Resting cells, especially wild-type, grown in medium containing antimycin A exhibited lowered oxygen-uptake rate, which was increased upon the addition of cyanide or antimycin A. Antimycin A stimulated, but cyanide inhibited, respiration of cytochrome-complete cultures grown in the presence of rotenone but did not affect that of the cytochrome aa3-deficient mutant. SHAM inhibited respiration of all antimycin A- or rotenone-grown cultures. The high rate of respiration of C. albicans in the presence of inhibitors for three sites of electron transport in the conventional oxidative pathway, the inhibition of this respiration by SHAM and its loss by the absence of cytochrome b, indicate an alternate oxidative pathway in this organism which crosses the conventional one at cytochrome b.

The distribution of chromatin in budding yeast cells.

Summary and conclusionsA critical examination of well fixed yeast cells killed at different states in the growth cycle shows that the distribution of chromatin in the yeast cell varies depending on the stage in the growth cycle. Our data support the view (1) that the spindle is intravacuolar, (2) that mitosis occurs on the spindle, (3) that the chromosomes spin out after mitosis to extend into the vacuole, (4) that a large nucleolus appears in the nuclear vacuole of rapidly growing wellnourshed cells, and (5) that in the presence of adequate amounts of phosphate and otherwise favorable conditions the chromosomes are covered with metaphosphate.

Selection and characterization of acriflavine-induced mutants of Candida albicans

Acriflavine-treated cells of C. albicans plated on a medium containing glucose as the principal carbon source gave rise to numerous colonies, most of which grew when replica-plated onto a similar medium containing sodium acetate substituted for glucose. Of the small fraction of colonies from the glucose medium which failed to replicate, some were found to be true petites, deficient for cytochromes a and b; others possessed complete cytochrome spectra, like those of their wild-type parents, but, nevertheless, respired at reduced rates on both fermentable and non-fermentable substrates. The role of the conventional cytochrome system in a wild-type culture was indicated by the strong inhibition of its respiration by cyanide, azide and antimycin A.

Growth, respiration and cytology of acetate-negative mutants ofCandida albicans

A number of acriflavine-induced mutants ofCandida albicans, characterized by their inability to grow on acetate as a source of energy, were screened for their cytochrome absorption spectra. Three mutants with different spectra, along with their parent, were selected for comparative studies, of their growth, respiratory activities and cellular structure. The spectrum of one of the mutants was the same as that of the wild-type, but the growth rate and yield of cells on glucose medium were only about 60% of the wild-types; those of a second mutant deficient in cytochromes aa3 were 50%, and those of a third mutant deficient in cytochromes aa3 and b were less than 5% of those of the wild-type. The cytochrome-complete mutant and the wild-type showed respiratory activity both on glucose and ethanol well above the endogenous, the cytochrome aa3-deficient mutant showed only endogenous respiration, and the cytochrome aa3, b-deficient mutant no respiration at all. Electron microscopy of the wild-type cells revealed discrete, regular ovoidal, cristate mitochondria spaced near the periphery of the protoplasm; the cytochrome-complete mutant showed an abundance of large, cristate, but morphologically irregular mitochondria; the cytochrome aa3-deficient mutant had fewer but still large, cristate, somewhat irregular mitochondria; and the cytochrome aa3, b-deficient mutant only a few simple vesicles without discernible cristae.

Growth inhibition of Candida albicans by folate pathway inhibitors. Their potential in the selection of auxotrophs

Growth studies were conducted on C. albicans in a glucose — salts — biotin (GSB) medium in the presence of folate inhibitors. Sulfanilamide inhibited growth which was restored by PABA or tetrahydrofolate (THF). Aminopterin inhibited growth to about the same level as did sulfanilamide, but this inhibition was not reversed with PABA nor THF, singly or in combination. Inhibition by combined sulfanilamide and aminopterin was synergistic, reducing growth by more than 90% for 48 h. The sulfanilamide component of the combined inhibition was reversed by PABA or THF to the level of that of aminopterin alone. Cytochrome synthesis was not affected by the inhibitors, but marked increases occurred in α-ketoglutarate, malate, isocitrate, and pyruvate dehydrogenases, especially in the presence of both inhibitors. The pyrimidines in combination with sulfanilamide were as inhibitory as was the combination of aminopterin and sulfanilamide, but they had no effect when added alone or in combination with aminopterin. Unlike the pyrimidines, the purines stimulated about a 50% recovery from inhibition by either of the inhibitors. Growth inhibition by combined sulfanilamide and aminopterin was overcome by about 50% by the addition of the THF-mediated end-produits: deoxythymidylate, adenine, histidine and methionine. The use of GSB medium containing adenine, histidine, methionine and the folate inhibitors but without deoxythymidylate resulted in thymineless death of prototrophic cells providing a method for the selection of auxotrophic mutants.

The cytology of yeasts

Introduction ..................................................................................................................................................................... 168 The Cell Wall ............................................................................................................................................................... 169 Physical Structure ......................................................................................................................................... 169 Chemical Structure ...................................................................................................................................... 170 Bud and Birth Scars ................................................................................................................................... 174 The Cytoplasm ............................................................................................................................................................ 176 The Cytoplasmic Membrane ............................................................................................................ 176 The Endoplasmic Reticulum ............................................................................................................ 177 Cytoplasmic Bodies ...................................................................................................................................... 178 Mitochondria .......................................................................................................................................... 181 Ribosomes ................................................................................................................................................... 187 The Vacuole ..................................................................................................................................................................... 189 The Nucleus .................................................................................................................................................................. 193 Methods for Identification .................................................................................................................. 193 Non-specific Methods ..................................................................................................................... 193 The Feulgen Technique ............................................................................................................ 193 The Giemsa Stain .............................................................................................................................. 194 Electron Microscopy ........................................................................................................................ 195 Mundkur s Freeze-Drying and Cytochemical Techniques ..................... 196 Methods Involving Living Cells ...................................................................................... 196 Concepts of the Yeast Nucleus ........................................................................................................ 197 The Nuclear Membrane ............................................................................................................ 197 The Nuclear Vacuole Concept ............................................................................................ 198 The Extravacuolar-amitotic Concept ........................................................................... 202 The Extravacuolar-mitotic Concept .............................................................................. 204 Summary and Conclusions .............................................................................................................................. 211 Literature Cited ............................................................................................................................................................ 214

Selection and fusion of auxotrophic protoplasts of Candida albicans

Auxotrophic mutants of C. albicans obtained by the method described by Henson and McClary (1979) were conditioned in a tris buffered EDTA-dithiothreitol solution then converted to protoplasts by suspension in osmotically stabilized buffer containing β-glucuronidase. Complementary protoplasts were mixed in an osmotically stabilized polyethylene glycol solution and at appropriate times were plated respectively in osmotically stabilized minimal and complete agar media. From colony counts resulting from growth on the respective media, the proportion of fused complementary protoplasts (prototrophic colonies) to the total viable number of colony forming units was determined. Stability tests of selected colonies from the minimal and complete agar revealed multiple revertants, but the numbers declined to low frequencies upon repeated selective plating and isolation. Acridine orange staining of cultures thus stabilized revealed various sizes of cells with their numbers of nuclei (DNA-staining regions) varying from one to five, such that it was not determined whether the prototrophic cultures were monokaryons, heterokaryons or a mixture of the two.

The relationship between the oxidation and fermentation of galactose in the course of adaptation by Saccharomyces cerevisiae.

Abstract The onset and degree of aerobic fermentation of galactose during adaptation was found to be dependent upon the preliminary accumulation of reserves and intermediates and eventual saturation of the respiratory systems. Also, the oxidation of galactose resulted in the accumulation of fructose phosphates indicating that the oxidative breakdown of galactose also proceeds to some extent through the Embden-Meyerhof scheme. The accumulation of metabolic compounds in the cells increased at an even greater rate following the start of fermentation. An important implication of these results is that fermentation by whole cells is not a reliable criterion for measuring accurately the activity of the galactose-adaptive enzyme, and experiments using this method to demonstrate that adaptive enzyme synthesis is autocatalytic in nature are subject to error.

The oxidation of galactose in the presence of 2,4-dinitrophenol as a measure of galactose adaptation in Saccharomyces cerevisiae☆☆☆

Abstract The degree of oxidation of galactose in the presence of 2,4-dinitrophenol was used as a measure of galactose adaptation in Saccharomyces cerevisiae . The results indicated that the rate of synthesis of the limiting adaptive enzyme in the galactozymase system assumes a linear or hyperbolic form. An important implication of this finding is that it permits an explanation to be made of the kinetics of galactokinase synthesis in yeast without assuming the existence of extrachromosomal hereditary factors. A comparison of the Q O 2 and Q CO 2 N 2 at the same stages of galactose adaptation demonstrated further that the oxidative phase is not a pre-adaptive oxidation of galactose, but is also dependent upon the formation of the adaptive enzyme.