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cDNA and genomic clones of Neurospora calmodulin were obtained by PCR. Characterization revealed an open reading frame encoding a predicted protein of 149 amino acids, showing 85% identity to the human calmodulin protein sequence. Comparison of the cDNA and genomic sequence reveals the position of five introns, organized differently than is found in calmodulin genes from higher eukaryotes.

SEVERAL peptides are synthesised on mitochondrial chloramphenicol-sensitive ribosomes1. In the presence of cycloheximide (∼ 200 µg ml −1), amino acids are incorporated exclusively into mitochondrial proteins in Neurospora crassa2 and the flight muscle of Locusta migratoria3, leading to the generally accepted idea that mitochondrially synthesised proteins remain in the mitochondria. It is possible, however, that the high concentration of cycloheximide used in those experiments could inhibit any potential export of such proteins to other parts of the cell, particularly since the drug has been shown to affect several vital cellular processes independently of its effect on protein synthesis; for example, it stimulates the catabolism of nucleic acids and decreases intracellular ATP (ref. 4). This may explain some published indications of export of mitochondrially synthesised proteins in experiments in which cycloheximide was not used, such as the continued synthesis of myelin proteolipid in crude mitochondrial fractions of rat brain homogenates treated with RNase5; and the cytoplasmic location of the mutation in a line of Zea mays responsible for the susceptibility of its root plasma membrane K+-stimulated ATPase to the toxin of Helminthosporium maydis race T.6 We describe here observations on the incorporation of radioactivity into membranes of the post-mitochondrial supernatant of homogenates of N. crassa which had been pulsed with radioactive amino acids in the presence of cycloheximide (25 µg ml −1) then grown for 2h in drug- and label-free medium. We have been unable to account for this solely on the basis of mitochondrial fragmentation. Of the membranes present in the postmitochondrial supernatant, we have investigated in detail a microsomal fraction enriched for endoplasmic reticulum, as a first step in determining the nature of this incorporation.

The four mitochondrial proteins of Neurospora crassa labeled by amino acid incorporation in vivo in the presence of cycloheximide have been characterized with regard to their solubility properties in acidic chloroform-methanol. The labeled proteins are present in pulse-labeled whole mitochondria, pulse-chase labeled whole mitochondria, and a purified preparation of cytochrome oxidase. The labeled proteins in each of these preparations are similar with respect to molecular weight, as determined by dodecylsulfate · polyacrylamide-gel electrophoresis. In addition, proteins of similar molecular weight in each preparation exhibit identical solubility properties in acidic chloroform-methanol. The possibility of the mitochondrially synthesized subunits of cyto-chrome oxidase being representative of total mitochondrial protein synthesis is discussed.

Two methods of analysis of the fine structure of genes are discussed. These methods yield data that support the concept of linear organization within a gene. This organization is apparent in both genetic and complementation maps. Experiments designed to give information concerning the mechanism of complementation are described. These data indicate that it is possible to simulate complementation in vivo. In this case complementation in vivo in Neurospora crassa is characterized by the ability of two ad-4 mutants growing together as a heterocaryon to produce functional adenylosuccinase, which neither mutant can make when grown alone. The simulated complementation occurs in vitro by mixing partially purified and presumably differentially defective forms of mutant adenylosuccinase. Maximum efficiency of complementation in vitro is obtained when attempting to modify or interchange-SH and-S-S-bonds of the protein. Theoretically, such a treatment could result in the formation of aggregates, hybrid molecules, or ...

When Neurospora mycelium is transferred from a medium containing sucrose to one containing acetate as sole source of carbon, a preferential synthesis of many Krebs cycle, glyoxylate cycle, and associated enzymes occurs. Respiration was inhibited during preferential enzyme synthesis in the following ways. (i) The amount of aeration (shaking) was reduced, (ii) cyanide was added to the culture, (iii) the carbon source, acetate, was removed, (iv) a mutant strain was starved of its Krebs cycle intermediates, and (v) respiration was inhibited by mutation. The effect of this respiratory inhibition on the synthesis of a number of enzymes was measured. It was found that the synthesis of nicotinamide adenine dinucleotide (NAD)-linked glutamate dehydrogenase and phosphoenolpyruvate carboxykinase was significantly less inhibited under conditions of respiratory inhibition than was the synthesis of Krebs cycle, glyoxylate cycle, and most other cell proteins synthesized during the adaptation period. This differential inhibition of enzyme synthesis was almost certainly not due to differential repression by regulatory metabolic end product effectors. Inhibition of mitochondrial respiration under these conditions most likely results in a limitation of the energy supply of the cell. Thus, it is suggested that the inhibition of synthesis of most proteins after inhibition of mitochondrial respiration results from a lack of energy in a utilizable form. Possible reasons to account for the relative insensitivity of NAD-linked glutamate dehydrogenase and phosphoenolpyruvate carboxykinase to inhibition under these conditions are discussed.

Krebs cycle enzymes were repressed in Neurospora during growth on sucrose as carbon source compared with growth on acetate. In mitochondrial, cytochrome-deficient strains grown on sucrose, this glucose or catabolite repression was reduced. In mutant strains in which protein synthesis and mitochondrial respiration were inhibited, catabolite repression was enhanced. These data support the hypothesis that ATP or the cellular energy level is involved in catabolite repression. The interrelationships between energy metabolism, the energy distribution in the cell and catabolite repression are discussed. The addition of casamino acids to a culture growing on sucrose or acetate, did not cause repression of any of the Krebs cycle enzymes measured, in spite of the fact that many of the constituent amino acids can be considered as biosynthetic products of the Krebs cycle.

The kinetics of derepression of isocitrate lyase, malate synthase, phosphoenolpyruvate carboxykinase, citrate synthase, fumarate hydratase, and malate dehydrogenase were studied following transfer of mycelium grown on a medium containing sucrose to one containing acetate as sole source of carbon. The maximum rate of isocitrate lyase synthesis was proportional to the number of isocitrate lyase structural genes. Comparisons of the repressed levels of isocitrate lyase and malate synthase on sucrose medium and the derepressed levels in acetate medium showed an over-all coordination between the levels of these two enzymes. A similar relationship existed between the levels of fumarate hydratase, citrate synthase and malate dehydrogenase. The Krebs cycle enzymes also showed coordinate repression on sucrose medium under conditions of enhanced catabolite repression. Other results, however, showed that neither for the glyoxylate shunt enzymes nor for the Krebs cycle enzymes was this coordination invariable. Under conditions in which some of the Krebs cycle enzymes assumed different roles no coordination was observed. It is suggested that this type of coordination, which is frequently observed in the pathways of central carbon metabolism in eucaryotes, results from the metabolic repressors of each enzyme being in equilibrium or in a constant ratio to one another. The coordination does not arise because the structural genes of each enzyme belong to the same operon.

A spontaneous, single-gene mutation responsible for a total lack of invertase activity in Neurospora crassa is described. The mutation is believed to lie in the structural gene for invertase, since an immunologically cross-reacting protein is made by the mutant strain. In addition, there was no evidence for a defect in regulation of invertase activity or synthesis by the following criteria. (i) The invertaseless condition was recessive in heterokaryons; (ii) no invertase inhibitor was found in mutant extracts by mixing experiments; and (iii) none of the several sugars able to induce activity in wild-type strains was able to induce activity in the mutant strain. It was also discovered that most of the wild-type enzyme (55 to 75%) cannot be washed free from the rapidly sedimenting cell debris. This finding provided additional support for the hypothesis that Neurospora invertase is located within or about the cell wall.

A new strain of Neurospora crassa which exhibits a rhythm of conidiation when growing along an agar surface in a growth tube is described. The rhythm has been shown to be circadian for it meets the following criteria: A) the period under constant environmental conditions in the dark is about 24 hours (22.7 hours at 25°); B) the period is relatively temperature-independent (Q10 is between 0.95 and 1.21 for temperature range of 18 to 35°); C) the rhythm persists in continuous darkness at constant temperature for a minimum of 14 days without damping out; and D) the phase of the rhythm can be shifted by a single brief exposure to light. The sensitivity of this strain to light has been demonstrated further by the entrainment of the rhythm to a period of 24.0 hours using a suitable light-dark regime, and by the inhibition by light of the appearance of a rhythm; i.e., continuous conidiation occurs when the strain is subjected to continuous light. The new strain is compared to 2 other strains of Neurospora which also express a rhythm, patch and clock.

Chloramphenicol, an inhibitor of mitochondrial protein synthesis, shortened the period length of the circadian rhythm in the Timex strain of Neurospora crassa by 2 hours. Both the l(+) threo and d(−) threo optical isomers had the same effect on the period of the rhythm, whereas only the d(−) threo isomer significantly inhibited mitochondrial protein synthesis. Tetracycline, another inhibitor of mitochondrial protein synthesis, did not change the period of the circadian rhythm. The effect of chloramphenicol on the circadian rhythm is, therefore, presumably not directly related to inhibition of mitochondrial protein synthesis, suggesting that chloramphenicol has other effects.

The effects of cycloheximide on cytoplasmic protein synthesis in Neurospora are reversible. In these experiments, the products of mitochondrial protein synthesis were labeled in vivo in the presence of cycloheximide; analyses were made of the size distribution and enzyme association of these products as the cells recovered from cycloheximide inhibition. In cells harvested immediately after a 20-min cycloheximide pulse, four protein species of high specific activity were resolved in polyacrylamide gels of mitochondrial protein electro-phoresed in the presence of 1% sodium dodecylsulfate. Only small amounts of labeled protein were found to co-purify with cytochrome oxidase isolated from these cells. In cells harvested after two to three hours of recovery from cycloheximide inhibition, three of the four original labeled peaks remain in whole mitochondrial protein. These peaks correspond precisely in gel mobility to the three highest-molecular-weight protein species associated with cytochrome oxidase, species which are heavily labeled in enzyme preparations from recovery cultures. These gel patterns can be obtained only when the preparations are treated with phenylmethylsulfonyl fluoride, an inhibitor of protease activity. The results obtained are discussed in terms of an assembly process in which the major stable products of mitochondrial protein synthesis become integrated into the cytochrome oxidase complex only when cytoplasmically-made components are available for assembly.

Publisher Summary The enzyme adenylosuccinate adenosine monophosphlyase (AMP-lyase) from Neurospora crassa is a bifunctional enzyme and catalyzes two nonsequential reactions in purine biosynthesis. The early reaction is the conversion of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide (succino-AICAR) to 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR) and fumaric acid. The other reaction is the terminal step in adenosine monophosphate (AMP) biosynthesis, the cleaving of adenylosuccinate (AMP-S) to adenylic acid and fumaric acid. The two reactions are similar in that they both involve the breakage of a carbon–nitrogen bond to yield fumaric acid. The principal assay of adenylosuccinate-AMP-lyase is a spectrophotometric assay that takes advantage of the slightly different absorption spectra of AMP-S and adenylic acid. A qualitative assay can be performed by separating adenylic acid and AMP-S by paper chromatography after the completion of the reaction. In vitro , the enzyme exists in three forms that can be separated by ultracentrifugation or by moving boundary electrophoresis.

Chloramphenicol, an inhibitor of mitochondrial protein synthesis, shortened the period length of the circadian rhythm in the Timex strain of Neurospora crassa by 2 hours. Both the l(+) threo and d(-) threo optical isomers had the same effect on the period of the rhythm, whereas only the d(-) threo isomer significantly inhibited mitochondrial protein synthesis. Tetracycline, another inhibitor of mitochondrial protein synthesis, did not change the period of the circadian rhythm. The effect of chloramphenicol on the circadian rhythm is, therefore, presumably not directly related to inhibition of mitochondrial protein synthesis, suggesting that chloramphenicol has other effects.

Two kinetically distinct states of succinate oxidase have been detected in the mitochondria of Neruospora crassa . One state has a K m for succinate of 4.1 × 10 −3 m , and the other has a K m for succinate of 3.5 × 10 −4 m . The high K m state was found in freshly extracted mitochondria from either 20- or 72-hr mycelium. However, the succinate oxidase activity in mitochondria from 20-hr mycelium rapidly deteriorated in vitro, leaving a stable residual activity with the lower K m for succinate. Adenosine triphosphate (ATP) plus Mg 2+ stabilized the high K m state in these preparations. The high K m state of succinate oxidase was further characterized by a two- to threefold increase in activity over the p H range 6.6 to 8.0 and by classical competitive inhibition by fumarate and malonate. By contrast, the low K m state of succinate oxidase showed a relatively flat response to p H over the range 6.6 to 8.0 and a nonclassical pattern of inhibition by fumarate and malonate, as shown by nonlinear plots of reciprocal velocity versus reciprocal substrate concentration in the presence of inhibitor or reciprocal velocity versus inhibitor concentration at fixed substrate concentrations. The relationship of mycelial age to the in vitro stability of succinate oxidase is considered with reference to probable changes in the relative pool sizes of extra- and intramitochondrial ATP in response to changes in the rate of glycolysis.

The glyoxylate shunt enzymes, isocitrate lyase and malate synthase, were present at high levels in mycelium grown on acetate as sole source of carbon, compared with mycelium grown on sucrose medium. The glyoxylate shunt activities were also elevated in mycelium grown on glutamate or Casamino Acids as sole source of carbon, and in amino acid-requiring auxotrophic mutants grown in sucrose medium containing limiting amounts of their required amino acid. Under conditions of enhanced catabolite repression in mutants grown in sucrose medium but starved of Krebs cycle intermediates, isocitrate lyase and malate synthase levels were derepressed compared with the levels in wild type grown on sucrose medium. This derepression did not occur in related mutants in which Krebs cycle intermediates were limiting growth but catabolite repression was not enhanced. No Krebs cycle intermediate tested produced an efficient repression of isocitrate lyase activity in acetate medium. Of the two forms of isocitrate lyase in Neurospora , isocitrate lyase-1 constituted over 80% of the isocitrate lyase activity in acetate-grown wild type and also in each of the cases already outlined in which the glyoxylate shunt activities were elevated on sucrose medium. On the basis of these results, it is concluded that the synthesis of isocitrate lyase-1 and malate synthase in Neurospora is regulated by a glycolytic intermediate or derivative. Our data suggest that isocitrate lyase-1 and isocitrate lyase-2 are the products of different structural genes. The metabolic roles of the two forms of isocitrate lyase and of the glyoxylate cycle are discussed on the basis of their metabolic control and intracellular localization.

Neurospora mitochondrial structural protein associates with Neurospora malate dehydrogenase (EC 1.1.1.37) as evidenced by fluorimetric titration, enzyme inhibition, immunodiffusion-precipitin reaction, and solubility tests. Association of the structural protein with horse-heart myoglobin is revealed by sedimentation and immunodiffusion-precipitin tests. Evidence of association specificity of the structural protein for other proteins is presented. Fluorimetric titration data indicate that 1 mole of either of the coenzyme nucleotides, ATP or NADH, is bound per mole of structural protein.

THE mi-1 (poky) strain of Neurospora crassa is a relatively stable, respiration-deficient mutant, which exhibits cyto-plasmically-inherited reduction of growth rate and aberrations in the mitochondrial eytochrome system. In young cultures of mi-1, the cells accumulate up to sixteen times the amount of cytochrome c present in wild-type Neurospora, and cytochromes b and a are not detectable spectroscopically in these same cells1. In sexual crosses the mi-1 mutation is transmitted only through the cytoplasm of the protoperithecial parent, and the pleiotropic mi-1 phenotype is caused by an alteration in a cytoplasmic gene2, presumably in the mitochondrial DNA.

Timex, a strain of Neurospora crassa which exhibits a circadian rhythm of conidia formation in growth-tube cultures, has been found to differ from wild-type strains by two genes. One gene, inv , is responsible for an invertase deficiency, whereas the second gene, bd , is of unknown function. Both genes map independently from other genes known to induce Neurospora rhythmicity. The inv gene is not essential for the timex phenotype because bd strains express that phenotype on certain media. Although inv strains do exhibit some rhythmicity of their own, the rhythmicity apparently is not a direct result of the invertase deficiency, since there is no correlation between invertase level and rhymicity in 29 strains tested. Of the 29 strains tested, 20 exhibited some rhythmicity in growth-tube cultures, suggesting that morphological manifestations of rhythmicity in Neurospora may result from the function or the loss of function of numerous genes, or both. There was no correlation in these strains between rhythmicity and (i) genetic background; (ii) geographical origin; or (iii) nutritional requirements.