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The kinetic parameters of [8-(14)C]adenosine transport by a general nucleoside uptake system were studied in germinated conidia of the ad 8 strain of Neurospora crassa. The apparent K(m) for adenosine uptake by this system was found to be 6.2 muM. The apparent K(i) values for other nucleosides competing with adenosine for uptake were measured by using Dixon plots. Nucleosides which were efficient competitive inhibitors of adenosine transport were found to inhibit severely the rate of growth of strain ad-8 on adenosine-supplemented medium. Xanthosine and thymidine did not inhibit [8-(14)C]adenosine uptake as severely as other nucleosides, nor did they cause significant inhibition of ad-8 growth rate on adenosine.

Two new mutant genes in Neurospora crassa prevent the formation of free macroconidia from proconidial chains. These genes, called conidial separation-1 and conidial separation-2, are phase-specific, playing no role in either the sexual life cycle or other aspects of the asexual life cycle. A cell-wall-associated autolytic activity was found to increase in wild-type cultures at the time of active formation of free conidia from proconidial chains; no such increase was detected in mutant cultures. It appears that the products of these genes are both essential for and unique to macroconidiation.

In nutrient medium containing 3.22 M ethylene glycol or glycerol, conidia of Neurospora crassa grow as single cells, without forming the germ tubes characteristic of normal morphological germination. Ethylene glycol is more effective than glycerol in producing this response. After growth in ethylene glycol medium for a suitable time, the cells are easily disrupted by an abrupt decrease in osmotic pressure. Osmotic disruption yields intact nuclei and mitochondria, although mitochondrial fractions obtained in this way show significantly reduced concentrations of cytochromes c + c(1), as compared to those observed for comparable fractions obtained from vegetative hyphae. Cell cultures gradually adapted to lower concentrations of the glycol show a much higher degree of synchrony in the formation of germ tubes than do untreated conidia.

The level of polysomes in ungerminated conidia of Neurospora crassa depends on the method used to collect spores. Spores harvested and exposed to hydration contain 30% of their ribosomes as polysomes, whereas those not exposed to hydration contain only 3% of their ribosomes as polysomes. During the germination process, the percentage of the ribosomes which sediment as polysomes increases rapidly to a level of approximately 75% during the first 15 to 30 min of germination. This rapid increase has been shown to require a carbon source. During the first 30 min of germination, spores synthesize ribosomal ribonucleic acid (RNA) and heterogeneously sedimenting RNA, i.e., presumptive messenger RNA.

The inactivation of Neurospora crassa conidia by galactosaminoglycan isolated from cultures of this organism was followed by measuring colony-forming ability and ability to take up radiolabeled metabolites. When kinetic data on the loss of transport function and on killing were analyzed by use of target theory, it appeared that few "hits" are required for inactivation. However, studies with radio-labeled galactosaminoglycan mucopolysaccharides showed that cells receiving a single lethal hit have approximately 10(5) galactosaminoglycan molecules bound to them.

The NH(2)-terminal amino acid composition of the soluble and ribosomal proteins from Neurospora crassa mycelia and conidia was determined by the dinitrophenyl method. A nonrandom distribution of NH(2)-terminal amino acids was observed in the complex protein mixtures. Glycine, alanine, and serine accounted for 75% of the NH(2)-terminal amino acids, and glycine appeared most frequently in mature proteins of mycelia. The appearance of phenylalanine as one of the major NH(2)-termini in crude conidial fraction suggests that the composition of proteins may vary in different developmental stages.

The cell wall of Neurospora crassa contains bound enzymes that can digest its structural polymers. These enzymes are not present at the same levels at all stages of growth. The levels of these autolytic enzymes vary and generally show some relationship to the process of branching. These enzymes were removed from the cell wall by beta-mercaptoethanol extraction and were tested for activity against isolated cell wall fractions. Such studies, as well as autolytic studies, showed that enzymes acting on the protein portion of the cell wall (proteases) are more prominent than enzymes that act on the glucan portion (glucanases) of the cell wall. Comparative studies between the wild type and a spreading colonial mutant spco-1 showed that earlier and higher frequency of branching in spco-1 was correlated with a greater amount of these enzymes bound to the cell walls. It is concluded from these observations that autolytic enzymes acting on the protein and glucan portion of the cell walls occur as wall-bound and participate in the process of branching in Neurospora.

In Neurospora crassa, evidence has recently been obtained for a cluster of four closely-linked genes controlling the inducible enzymes catalyzing the first three reactions in the catabolism of quinic acid. Three of these genes appear to be the structural genes for the three enzymes. The fourth gene, designated qa-1, has been interpreted as having a regulatory function, since qa-1 mutants are pleiotropic types, are noninducible for the three enzymes, and form heterocaryons which complement mutants in the structural genes. The present studies were undertaken to elucidate further the nature of the regulatory role of the qa-1 locus. A number of constitutive (qa-1(C)) mutants have been obtained from certain qa-1 mutants as revertants selected for their ability to grow on quinic acid as a sole source of carbon. These qa-1(C) mutants produce high levels of all three enzymes in the absence of an inducer, map within (or very close to) the qa-1 locus, and produce a catabolic dehydroquinase (EC 4.2.1.10) which is indistinguishable, on the basis of thermolability tests, from that of wild type. In addition, when grown in the absence of an inducer, heterocaryons between wild-type (qa-1(+)) and different qa-1(C) mutants exhibit markedly different levels of constitutivity (from 5 to 50% of the particular parental qa-1(C) mutant) for catabolic dehydroquinase, one of the enzymes under qa-1 control. These overall results are interpreted as supporting the hypothesis that the qa-1(+) gene product (presumably a multimeric protein) plays only a positive regulatory role in initiating synthesis of the three quinate catabolic enzymes.

Four extranuclear mutants, [exn-1], [exn-2],[exn-4], and [stp-C], were obtained from N-methyl-N'-nitro-N-nitrosoguanidine-treated conidia and mycelium of Neurospora crassa. The three exn mutants grow with a pronounced lag from conidia and ascospores and are female fertile, whereas [stp-C] has a stop-start growth phenotype and is female sterile. The mitochondria from all four mutants are deficient in cytochromes a+a(3) and b, but contain an excess of cytochrome c. On the basis of growth and fertility, nuclear suppressors and complementation in heteroplasmons, 16 of the extranuclear mutants now available in Neurospora can be divided into three groups. Group I consists of 8 female-fertile variants with both poky-like growth and cytochrome defects. Their slow growth is suppressed by the nuclear factor, f, but not by a second nuclear suppressor, su-1([mi-3]). They complement with group III mutants in mixed cytoplasmons. Group II is represented by a single variant, [mi-3]. It is phenotypically modified by the su-1([mi-3]) factor, but not by f. Its unique cytochrome spectrum shows a deficiency of cytochrome a, but c and b are present. It complements in heteroplasmons with group I and III mutants. Group III included 7 female-sterile variants with stopper growth phenotypes and the same cytochrome defects as group I. Group III mutants complement both with group I and II isolates, but they are unaffected by either f or su-1.

A colonial mutant of Neurospora crassa, previously shown to be altered in the structure of glucose-6-P dehydrogenase [a reduced nicotinamide adenine dinucleotide phosphate (NADPH) producing reaction], contained only 40% as much NADPH in extracts as did the wild type. A partial revertant strain, when grown at 23 C, had the same total NADPH content as the wild type, but, at 34 C, had lower levels of NADPH as well as a colonial morphology. A revertant with complete wild-type morphology had wild-type levels of NADPH. Two different colonial mutants, which have also been reported to be altered in NADPH-generating reactions, were found to have a lower content of NADPH, whereas other colonial mutants had wild-type levels. The wild-type strain, when grown under conditions in which it contained a lower total content of NADPH, had a morphology similar to that of a colonial mutant. The evidence indicates that lowered NADPH content leads to a dramatic alteration in the morphology of Neurospora, but not necessarily vice versa. The possible pleiotropic effects of the NADPH deficiency are discussed.

Through the use of a mutant deficient in ornithine-delta-transaminase (OTA), it is shown that this enzyme normally has no obligate or even major biosynthetic role in Neurospora. The pathways of ornithine and proline synthesis proceed wholly independently of each other in OTA-less strains. It is probable that OTA functions as an enzyme of arginine catabolism. With mutants affected in OTA, ornithine transcarbamylase, and the synthesis of ornithine, it was demonstrated that exogenous and endogenous ornithine are utilized in different ways. Exogenous ornithine is destined mainly for catabolism, whereas endogenous ornithine is destined mainly for biosynthesis. It is suggested that this distinction depends upon differences in the intracellular location or origin of the two sources of ornithine.