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R Brambl

Publications and source records attributed to R Brambl.

35 records · Page 2Linked to original sources
PubMed1986

Heat shock protects germinating conidiospores of Neurospora crassa against freezing injury.

Germinating conidiospores of Neurospora crassa that were exposed to 45 degrees C, a temperature that induces a heat shock response, were protected from injury caused by freezing in liquid nitrogen and subsequent thawing at 0 degrees C. Whereas up to 90% of the control spores were killed by this freezing and slow thawing, a prior heat shock increased cell survival four- to fivefold. Survival was determined by three assays: the extent of spore germination in liquid medium, the number of colonies that grew on solid medium, and dry-weight accumulation during exponential growth in liquid culture. The heat shock-induced protection against freezing injury was transient. Spores transferred to normal growth temperature after exposure to heat shock and before freezing lost the heat shock-induced protection within 30 min. Spores subjected to freezing and thawing stress synthesized small amounts of the heat shock proteins that are synthesized in large quantities by cells exposed to 45 degrees C. Pulse-labeling studies demonstrated that neither chilling the spores to 10 degrees C or 0 degrees C in the absence of freezing nor warming the spores from 0 degrees C to 30 degrees C induced heat shock protein synthesis. The presence of the protein synthesis inhibitor cycloheximide during spore exposure to 45 degrees C did not abolish the protection against freezing injury induced by heat shock. Treatment of the cells with cycloheximide before freezing, without exposure to heat shock, itself increased spore survival.

Cycloheximide↗
PubMed1985

Altered gene expression during cold acclimation of spinach.

Exposure of spinach (Spinacia oleracea L.) plants to a constant 5 degrees C induced a greater tolerance to extracellular freezing. The metabolic basis of this cold acclimation response in plants is not understood. In this study we tested the hypothesis that cold acclimation derives from altered gene transcription. We found that exposure of plants to low temperature resulted in a rapid and stable change in the translatable poly(A)+ RNA populations extracted from leaves, as determined by a cell-free in vitro translation assay. The initial appearance of mRNAs for two high molecular weight translation products correlated with an increase in freezing tolerance. Cold acclimation of plants for 8 days resulted in further qualitative changes in mRNA populations. At least four additional mRNAs increased in concentration upon continued exposure of spinach to 5 degrees C, whereas three other mRNAs present in 20 degrees C-grown leaves decreased. We also tested the possibility that the low temperature-induced mRNAs might encode heat shock proteins. We studied heat shock-induced protein synthesis by in vivo labeling techniques and found that spinach synthesized at least eight distinctive heat shock proteins during exposure to 40 degrees C. Most polypeptides induced by exposure to low temperature, however, appeared not to be heat shock proteins. Thus, the change in mRNAs induced by low temperature is a separate response from that induced by high temperature.

Cold Temperature↗
PubMed1985

Heat shock response of Neurospora crassa: protein synthesis and induced thermotolerance.

At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.

Cell Survival↗
PubMed1984

Three subunit proteins of membrane enzymes in mitochondria of Neurospora crassa contain a pantothenate derivative.

Three proteins of the inner mitochondrial membrane of Neurospora crassa were found to be covalently modified with a derivative of pantothenic acid. One of these proteins is a subunit of cytochrome c oxidase and two are subunits of the ATPase-ATP synthase. Cells of a pantothenate auxotroph of N. crassa were labeled with [14C]pantothenic acid, and mitochondrial proteins containing radiolabeled pantothenate were detected by electrophoresis of detergent-solubilized mitochondria. Mitochondria from cells that were colabeled with [14C]pantothenate and [3H]leucine were reacted with specific antisera against the cytochrome c oxidase and F1-ATPase enzyme complexes. Electrophoresis of the labeled subunits of these isolated complexes showed that the [14C]pantothenate-associated peptides corresponded to [3H]leucine-labeled subunit 6 of cytochrome c oxidase and two [3H]leucine-labeled subunits (tentatively identified as subunits 8 and 11) of the ATPase-ATP synthase. Pantothenate modification of these enzyme subunits, which are synthesized on extramitochondrial ribosomes, may contribute to their transport and assembly into mitochondria, or it may participate in the catalytic activity of the assembled enzymes.

ATP Synthetase Complexes↗
PubMed1984

Ribosomal RNA contents of maize genotypes with different ribosomal RNA gene numbers.

Ribosomal RNA (rRNA) contents were determined in 16 maize genotypes whose individual rRNA gene numbers varied from 5000 to 23,000 per 2C nucleus. Analytical polyacrylamide gel electrophoresis of total RNA showed that no obvious relation existed between rRNA gene number and rRNA content. Only two of nine common inbred lines contained more rRNA than W-23, the inbred with the lowest rRNA gene number. Two of four lines with altered protein content (due to long-term experimental selection) had rRNA contents significantly reduced from those of W-23. A line with an apparent duplication of the nucleolus organizer region of chromosome 6 (called 2-NOR) was expected to possess an elevated quantity of rRNA because it possesses a larger nucleolus; however, we produced a 2-NOR isogenic version and found no difference in rRNA content. The rRNA genes in maize are distributed throughout the NOR-heterochromatin and the NOR-secondary constriction portions of the NOR. The absence of an obvious correlation between rRNA gene number and cellular rRNA content may reflect the presence of a large number of rRNA genes in an inactive state, at least during the stage of growth examined in these experiments.

Gene Expression Regulation↗
PubMed1981

Cytochrome c oxidase in cytochrome c oxidase-deficient mutant strains of Neurospora crassa.

Three mutant strains of Neurospora crassa, previously characterized as cytochrome c oxidase deficient, were found to possess this respiratory enzyme. The mitochondrial cytochrome contents of logarithmic phase cells of wild type (74A) and the respiration-deficient strains mi-3, cya-4-23, and cya-5-34 were investigated with low temperature difference spectrophotometry; two of the strains were found to possess cytochrome a. Cytochrome c oxidase catalytic activity measurements performed on isolated mitochondria showed that all three mutant strains contained significant amounts of active enzyme which was subsequently found to be active in vivo. However, activity of the cyanide-insensitive, alternate electron transport pathway was responsible for most of the oxygen consumed by the logarithmic phase cells of these mutant strains. Electrophoretic analysis of the polypeptide subunits of cytochrome c oxidase radiolabeled in vivo during logarithmic phase growth showed that, in contrast to previous reports, the respiration-deficient strains appeared to contain all seven subunits of the enzyme.

Electron Transport Complex IV↗
PubMed1981

Mitochondrial biogenesis during fungal spore germination: respiration and cytochrome c oxidase in Neurospora crassa.

The germination of conidiospores of wild-type Neurospora crassa was found to be dependent upon the function of the cytochrome-mediated electron transport pathway. The cyanide-insensitive alternate oxidase did not contribute significantly to the respiration of these germinating spores. The dormant spores contained all of the cytochrome components and a catalytically active cytochrome c oxidase required for the activity of the standard respiratory pathway, and these preserved components were responsible for the accelerating rates of oxygen uptake which began immediately upon suspension of the spores in an incubation medium. Mitochondria of the dormant spores contained all of the subunit peptides of the functional cytochrome c oxidase; nevertheless, de novo synthesis of these subunits began at low rates in the first stages of germination. Reactivation of the respiratory system of germinating N. crassa spores seems not to be dependent initially upon the function of either the mitochondrial or cytoplasmic protein-synthesizing systems. The respiratory activity of spores of three mutant cytochrome c oxidase-deficient strains of N. crassa also was found to depend upon the function of the cytochrome electron transport pathway; the dormant and germinating spores of these strains contained a catalytically active cytochrome c oxidase. Cytochrome c oxidase may be present in the dormant and germinating spores of these strains as the result of a developmental-phase-specific synthesis of and requirement for the enzyme.

Electron Transport↗
PubMed1980

Mitochondrial biogenesis during fungal spore germination. Purification, properties and biosynthesis of cytochrome c oxidase from Botryodiplodia theobromae.

1. Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was purified from the mycelial fungus Botryodiplodia theobromae by sodium cholate and ammonium sulfate solubilization, ammonium sulfate fractionation, and DEAE-cellulose chromatography. The purified enzyme contained 6--7 nmol heme a/mg of protein. The specific activity of the purified enzyme was 2.1--2.3 . 10(3) k (min-1) per mg of protein with 15 mumol ferrocytochrome c and at pH 5.9 and optimal phosphate and Tween 80 concentrations (65 mM and 0.1%, respectively). The Km for ferrocytochrome c was determined to be 1.2--1.3 . 10(-5) M, while at infinite substrate concentration the enzyme catalyzed the oxidation of about 60 mumole of ferrocytochrome c/min per mg of protein. Sedimentation behavior and kinetic evidence suggest that the purified enzyme exists as aggregates of the single molecule. The purified B. theobromae cytochrome c oxidase with its 428 nm Soret absorption maximum may be similar if not identical to oxygenated forms of the enzyme from other fungal species. 2. The purified enzyme was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis to consist of seven polypeptides with the following respective molecular weights: I, 41 000; II, 28 000; III, 19 000; IV, 14 800; V, 12 800; VI, 11 500; and VII, 9300. Biosynthesis studies showed that the three highest molecular weight polypeptides of the enzyme were synthesized on mitochondrial ribosomes, and the four smaller polypeptides were products of cytoplasmic ribosomes.

Cytochrome c Group↗
PubMed1978

In vitro translation of polyadenylate-containing RNAs from dormant and germinating spores of the fungus Botryodiplodia theobromae.

Polyadenylated RNA isolated by oligodeoxythymidylate-cellulose chromatography from spores of the fungus Botryodiplodia theobromae was translated in a cell-free protein-synthesizing system derived from wheat embryo. Reaction conditions which would yield efficient and accurate in vitro translation of the spore RNA were established. Dual isotopically labeled mixtures of in vitro translation products from germinated and dormant spore polyadenylated RNA, as well as polyadenylated RNAs from intermediate stages of germination, produced qualitatively similar gel electrophoresis patterns, with polypeptides of 10,000 to 55,000 molecular weight. Proteins synthesized in vivo and extracted from germinating spores at three different stages possessed a greater size range, with molecular weights up to 85,000, although the in vitro synthesis apparently did yield the lower-molecular-weight proteins which were synthesized in vivo. Tryptic digest patterns of proteins translated in vitro from polyadenylated RNA of dormant and germinated spores were found to be identical in positions in only 40% of the spots. Furthermore, a dual-label comparison by isoelectric focusing of proteins translated from polyadenylated RNA of germinated and dormant spores also showed qualitative and quantitative differences among the in vitro translation products. We conclude that there are differences between the in vitro translation products of mRNA from dormant and germinated spores and that the mRNA preserved in the dormant spores contains genetic information which is qualitatively different from that of the germinated spores.

Cell-Free System↗
PubMed1978

Mitochondrial biogenesis during fungal spore germination: effects of the antilipogenic antibiotic cerulenin upon Botryodiplodia spores.

Germination of spores of the fungus Botryodiplodia theobromae was inhibited by the antilipogenic antibiotic cerulenin. The spores remained viable in the presence of the antibiotic, however, and after prolonged incubation they were able to overcome the inhibition. Cerulenin inhibition of germination was reversed by Tween 40 and Tween 60 (derivatives of palmitate and stearate, respectively), but not by representatives of a range of free fatty acids or their soaps. Cerulenin abolished incorporation of [14C]acetate into sterols and triglycerides and reduced its incorporation into fatty acids by 69%. Cyanide-sensitive oxygen consumption by spores incubated in the presence of cerulenin was greatly reduced throughout germination, and the activity of cytochrome c oxidase was no more than 13% of the activity in untreated spores, even after prolonged incubation. However, low-temperature difference spectra of mitochondrial extracts showed that the cerulenin-treated spores accumulated a threefold excess of cytochrome a, whereas the cellular concentrations of cytochroms c and b were identical to those of untreated spores. Cerulenin treatment sharply reduced the rates of whole spore protein and RNA synthesis. Cerulenin had no effects upon mitochondrial morphology which could be discerned with an electron microscope.

Antifungal Agents↗
PubMed1977

Mitochondrial biogenesis during fungal spore germination: respiratory cytochromes of dormant and germinating spores of Botryodiplodia.

The mitochondrial respiratory cytochrome contents of dormant and germinating conidia of Botryodiplodia theobromae were examined. Oxidized versus reduced difference spectra at 77 degrees K of whole mitochondria from physiologically mature germinated spores showed a typical a-band pattern for cytochromes c, b, and a, with absorption maxima at 549, 554 + 559, and 604 nm, respectively, whereas the difference spectrum of the counterpart mitochondrial fraction from dormant spores showed no cytochrome a bands. However, a fraction prepared from dormant spore mitochondria by detergent extraction and (NH4)2SO4 fractionation contained readily detectable quantities of cytochromes c and b (as shown by the a and Soret absorption bands), but it did not contain the a or Soret bands of cytochrome a observed in a counterpart preparation from germinated spores. The pyridine hemochromogen preparation from the dormant spore mitochondria contained no material that is spectroscopically characteristic of a-type heme and protoheme. These results suggest that cytochrome a is not present as a functional molecule in dormant spores. The first spectroscopically detectable cytochromes were observed in whole mitochondria at 210 min of spore germination, and the amount of each of the cytochromes increased with cell growth. A precursor of the heme porphyrin, delta-[4-14C]aminolevulinic acid, was first incorporated (at accelerating rates) into acid-insoluble spore material at 180 min of germination, which appears to be the approximate time of organization of new mitochondria in these spores.

Aminolevulinic Acid↗
PubMed1975

Characteristics of developing mitochondrial genetic and respiratory functions in germinating fungal spores.

Spores of the fungus Botryodiplodia theobromae began a cyanide-sensitive oxygen consumption immediately upon exposure to a liquid medium, and spore germination and respiration were not affected by ethidium bromide, D-threochloramphenicol, and acriflavin until later during germ tube emergence. These inhibitors of the mitochondrial genetic system all inhibited total cell protein synthesis to the same intermediate degree from the outset of incubation. When spores were incubated in water under non-germinating conditions, protein synthesis and oxygen uptake proceeded at initial rates almost identical to those seen in spores germinating in the presence of the three mitochondrial system inhibitors. Although the spores respired at rapid rates from the onset of incubation, no cytochrome absorption peaks could be observed in mitochondrial fractions prepared from ungerminated spores; they were readily observed in germinated spores, however. When the spores were germinated in the presence of inhibitors of the mitochondrial system, an excess of cytochrome c was observed in the near absence of cytochromes a and b. The results indicate that the ungerminated spores of this organism contain a preserved, potentially functional aerobic respiratory system which requires cycloheximide-sensitive ribosome activity to become functional when the spores are inoculated into a liquid medium.

Acriflavine↗