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At least 19 recordsLinked to original sources
PubMed1976

The mitochondrial ribosomes of Neurospora crassa. II. Comparison of the proteins from Neurospora crassa mitochondrial ribosomes with ribosomal proteins from Neurospora cytoplasm, from rat liver mitochondria and from bacteria.

1. It has been shown by Datema et al. (Datema, R., Agsteribbe, E. and Kroon, A.M. (1974) Biochim. Biophys. Acta 335, 386--395) that Neurospora mitochondria isolated in a Mg2+-containing medium (or after homogenization of the mycelium in this medium and subsequent washing of the mitochondria in EDTA-containing medium) possess 80-S ribosomes; mitochondria homogenized and isolated in EDTA medium yield 73-S ribosomes. The ribosomal proteins of the subunits of 80-S and 73-S ribosomes were compared by two-dimensional electrophoresis. The protein patterns of the large, as well as of the small subunits are very similar but not completely identical; the most conspicuous difference is that the large subunit of 80 S contains about eight more proteins than the large subunit of 73 S. 2. The contamination by Neurospora cytoplasmic 77-S ribosomes in the 80-S preparations, if present, is only minor. 3. Neurospora cytoplasmic ribosomes contain 31 proteins in the large, and 21 proteins in the small subunit. 4. Neurospora 80- mitochondrial ribosomes contain 39 proteins in the large, and 30 proteins in the small subunit 30 proteins. 5. Rat liver mitochondrial ribosomes contain 40 proteins in the large and at least 30 proteins in the small subunit. About 50% of these proteins has an isoelectric point below pH 8.6. 6. The pattern of Paracoccus denitrificans is very similar to that of other bacterial ribosomes, the large subunit contains 29, the small subunit 18 proteins.

Animals↗
PubMed1976

Biogenesis of mitochondrial membranes in Neurospora crassa during cellular differentiation: ultrastructural changes accompanying differentiation.

The ultrastructural characteristics of Neurospora cells during dedifferentiation and redifferentiation of conidiospores into vegetative cells have been determined. This germination process occurs between 2 and 5 h after inoculation; by 3-5 h, approximately 50% of the cells have germinated. The cells enter the exponential phase of dry-weight gain between 4 and 5 h after inoculation. Several unusual structures are observed in Neurospora cells during germination. Whorled structures are frequently seen in the cytoplasm during germination, and occasionally at other times. They appear to be derived from the cytoplasmic membrane. Whorled structures of different appearance were observed in the mitochondria between 2 and 4 h after inoculation. Their number was related to the level of metabolizable carbohydrate, and was higher in 15% glucose-than in 2% sucrose-supplemented medium, and very low in medium containing 15% mannitol, or 2% sucrose+13% 2-deoxyglucose, or no added carbohydrate. The mitochondrial inclusions were osmiophilic and could be removed by treatment with 90% aqueous acetone in the cold, indicating that they were composed at least in part of lipid. The strong dependence of the number of mitochondrial inclusions on time and on carbohydrate supplementation, suggests that there is a physiological basis for these structures and that they reflect changes occurring in the mitochondria at times significant to cellular differentiation.

Carbohydrate Metabolism↗
PubMed1970

In vitro assembly of Neurospora assimilatory nitrate reductase from protein subunits of a Neurospora mutant and the xanthine oxidizing or aldehyde oxidase systems of higher animals.

In vitro assembly or complementation of a hybrid assimilatory nitrate reductase was attained by mixing a preparation of nitrate-induced N. crassa mutant nit-1 specifically with acid-treated (pH 2.5) bovine milk or intestinal xanthine oxidase, rabbit liver aldehyde oxidase, or chicken liver xanthine dehydrogenase. The complementation reaction specifically required induced nit-1, the only nitrate reductase mutant of Neurospora that lacked xanthine dehydrogenase and was unable to use hypoxathine or nitrate as a sole nitrogen source. The complementing activities of the above acid-treated enzymes correspond to their xanthine or aldehyde oxidizing activity profiles on sucrose density gradients. The resulting soluble, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductases are the same as the Neurospora wild type enzyme in sucrose density gradient profile, molecular weight, substrate affinities, and sensitivity to inhibitors and temperature. By analogy to a similar in vitro complementation of nitrate reductase in mixtures of induced nit-1 and individual nonalleic Neurospora mutants, or uninduced wild type, the complemented nitrate apparently consists of an inducible protein subunit (possessing inducible NADPH-cytochrome c reductase) furnished by nit-1 and a subunit from the acid-treated xanthine or aldehyde oxidizing system which can substitute for the constitutive component furnished by the other mutants or uninduced wild type. The data suggest that Neurospora nitrate reductase and the xanthine oxidizing system and aldehyde oxidase of animals, all of which are molybdenum-containing enzymes catalyzing the reduction of nitrate to nitrite, share a highly similar protein subunit.

Aldehydes↗
PubMed1991

Specificity of repeat-induced point mutation (RIP) in Neurospora: sensitivity of non-Neurospora sequences, a natural diverged tandem duplication, and unique DNA adjacent to a duplicated region.

The process designated RIP (repeat-induced point mutation) alters duplicated DNA sequences in the sexual cycle of Neurospora crassa. We tested whether non-Neurospora sequences are susceptible to RIP, explored the basis for the observed immunity to this process of a diverged tandem duplication that probably arose by a natural duplication followed by RIP (the Neurospora zeta-eta region), and investigated whether RIP extends at all into unique sequences bordering a duplicated region. Bacterial sequences of the plasmid pUC8 and of a gene conferring resistance to hygromycin B were sensitive to RIP in N. crassa when repeated in the genome. When the entire 1.6-kb zeta-eta region was duplicated, it was susceptible to RIP, but was affected by it to a lesser extent than other duplications. Only three of 62 progeny from crosses harboring unlinked duplications of the region showed evidence of changes. We attribute the low level of alterations to depletion of mutable sites. The stability of the zeta-eta region in strains having single copies of the region suggests that the 14% divergence of the tandem elements is sufficient to prevent RIP. DNA sequence analysis of unduplicated pUC8 sequences adjacent to a duplication revealed that RIP continued at least 180 bp beyond the boundary of the duplication. Three mutations occurred in the 200-bp segment of bordering sequences examined.

Base Sequence↗
PubMed1991

Molecular comparison of the negative-acting nitrogen control gene, nmr, in Neurospora crassa and other Neurospora and fungal species.

In Neurospora crassa, the expression of unlinked structural genes which encode nitrogen catabolic enzymes is subject to genetic and metabolic regulation. The negative-acting nmr regulatory gene appears to play a role in nitrogen catabolite repression. Using the N. crassa nmr gene as a probe, homologous sequences were identified in a variety of other filamentous fungi. The polymerase chain reaction was used to isolate the nmr-like gene from the exotic Mauriceville strain of N. crassa and from the two related species, N. intermedia and N. sitophila. Sequence comparisons were carried out with a 1.7-kb DNA segment which includes the entire coding region of nmr plus 5' and 3' noncoding sequences. The size of the nmr coding region was identical in all three Neurospora species. Approximately 30 nucleotide base substitutions were found in the coding region of the nmr gene of each of the sister species when compared to the standard N. crassa sequence. However, most of the base changes occurred in third codon positions and were silent. The NMR proteins of N. sitophila and of N. intermedia display only three and four amino acid substitutions, respectively, from the N. crassa protein. Two regions of high variability, which include deletions and insertions of bases, were found in the 5' and 3' noncoding regions of the gene.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and expression in Saccharomyces cerevisiae and Neurospora crassa of the invertase gene from Neurospora crassa.

A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7. Saccharomyces cerevisiae suco and N. crassa inv strains transformed with pNC2 were able to grow on sucrose-based media and expressed invertase activity. Saccharomyces cerevisiae suco (pNC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N. crassa, although S. cerevisiae suc+ did not. The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI-restricted wild type N. crassa DNA. Plasmid pNC2 transformed N. crassa Inv- to Inv+ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events). It appears therefore that the cloned DNA piece encodes the N. crassa invertase enzyme. A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter.

Blotting, Southern↗
PubMed1973

Regulation of exocellular proteases in Neurospora crassa: role of Neurospora proteases in induction.

Cells of Neurospora crassa strain 74A, grown on sucrose for 12 h and transferred to a medium containing protein as sole carbon source, would not produce exocellular protease in significant amounts. When a filtrate from a culture induced to make protease by normal growth on a medium containing protein as principal carbon source was added to an exponential-phase culture in protein medium, exocellular protease was made in amounts similar to those made during normal induction. The material in the culture filtrate that participated in the induction process was identified as protease by its heat lability, molecular weight, and the dependence of induction rate on units of proteolytic activity added to the exponential-phase culture. Induction of the formation of exocellular protease by exponential-phase cells appears to require a protein substrate, added proteolytic activity, and protein synthesis. The protease produced by induced exponential-phase cells was as efficient in promoting induction as normally induced enzyme, whereas constitutive intracellular enzyme was only 50% as efficient. The bacterial protease thermolysin was able to induce exocellular protease at 90.7% of the rate observed with added N. crassa exocellular protease.

Chromatography↗
PubMed1985

Carbamoyl-phosphate synthetases from Neurospora crassa. Immunological relatedness of the enzymes from Neurospora, bacteria, yeast, and mammals.

Neurospora crassa contains two carbamoyl-phosphate synthetases: a mitochondrial enzyme (CPS-A) which supplies carbamoyl phosphate for arginine biosynthesis, and a nuclear enzyme whose product is used for the synthesis of pyrimidines. We have prepared antiserum against a highly purified preparation of the large subunit of CPS-A and have used the antiserum to demonstrate that the large subunit is, like most mitochondrially localized proteins, initially synthesized as a higher molecular weight precursor. The CPS-A antiserum cross-reacts with the nuclear enzyme, allowing us to identify the product of the complex N. crassa pyr-3 genetic locus as a protein with a subunit molecular weight of 180,000. Finally, we have found that the CPS-A antiserum also cross-reacts with carbamoyl-phosphate synthetases from bacteria, yeast, and mammals. The immunological relatedness of carbamoyl-phosphate synthetases from such diverse species suggests that the protein sequences required for carbamoyl phosphate production have been highly conserved during the course of evolution.

Animals↗
PubMed1983

Construction of a shuttle vector for the filamentous fungus Neurospora crassa.

We have constructed a recombinant plasmid, pALS-1, that replicates autonomously in both Neurospora and Escherichia coli. pALS-1 consists of the mitochondrial plasmid from Neurospora strain P405-Labelle, the Neurospora qa-2+ gene, and E. coli plasmid pBR325. pALS-1 transforms the Neurospora qa-2+ gene at frequencies 5- to 10-fold higher than those for plasmids that transform mainly by integration. When E. coli was transformed with DNA from Neurospora transformants, we recovered not only pALS-1 but also a smaller plasmid, pALS-2, which had undergone deletion of most and possibly all Labelle sequences, and the immediately flanking sequences in pBR325. pALS-2 also appears to replicate autonomously in Neurospora, but less efficiently than does pALS-1. Southern blots show that free pALS-1 and pALS-2 are present in nuclear and cytosolic (supernatant from high-speed centrifugation) fractions of Neurospora transformants and that small, variable proportions of the plasmids also can be detected in mitochondria. pALS-1 and pALS-2 constitute putative shuttle vectors for Neurospora.

DNA, Circular↗
PubMed1985

Plasmid recovery from transformants and the isolation of chromosomal DNA segments improving plasmid replication in Neurospora crassa.

The efficient recovery of plasmid DNA from Neurospora crassa transformants is described. Lithium acetate-treated spores were transformed with plasmid DNA and grown in mass in liquid culture. The resulting mycelial growth was harvested and plasmid DNA was extracted and used to transform E. coli to ampicillin resistance. Although at low frequency, routine recovery of plasmid pSD3 which carries the Neurospora qa-2+ gene and pBR322 sequences has been demonstrated. About 10% of the recovered plasmids carried deletions and transformed Neurospora at a higher frequency. The liquid culture procedure was also used in attempts to isolate autonomously replicating sequences (ars). In order to select for a stable vector which contains an ars sequence, a clone bank containing a selectable marker (qa-2+) and Neurospora chromosomal BamHI fragments was constructed and used to transform Neurospora. Several plasmids isolates resulting from a screening of the clone bank showed an improvement in the efficiency of recovery from Neurospora transformants. The properties of one such isolated plasmid, pJP102, suggest that it may contain an ars sequence. Some potential applications of these results for cloning in Neurospora and other filamentous fungi are discussed.

DNA Replication↗
PubMed1982

Comparison of the induction of specific locus mutations in wild-type and repair-deficient strains of Neurospora crassa.

A comparison of mutation induction between wild-type and excision repair-deficient strains has shown that, after treatment with four of the five mutagens tested, an enhanced recovery of induced mutants was found in the excision repair-deficient strains. In this sense we have confirmed for Neurospora Ames' (1977) observations with Salmonella. Furthermore, genetic analysis of the mutants induced in Neurospora in both wild-type and excision repair-deficient strains has shown that in some cases the enhanced recovery of mutants can be attributed to the recovery of a spectrum of genetic alterations in the excision repair-deficient strains that is qualitatively different from that found in the wild-type strain. This qualitatively different spectrum appears to arise as a result of a preferential increase in frameshift mutations. Thus, it appears that in excision repair-deficient strains of Neurospora genetic lesions are processed differently than in wild-type strains both to give enhanced yield as well as a selective increase in frameshift mutations. In this regard, the apparent difference between Salmonella and Neurospora data with regard to the characterization of the genetic effects of chemical carcinogens is most likely attributed to a difference in the genetic background of the strains. The present data with Neurospora suggest that this difference not only results in an enhanced recovery of revertants in Salmonella strains carrying the uvrB mutation but also that a different spectrum of genetic damage was detected from that which would have been observed in the standard wild-type strain G46. Observations from the present experiments with Neurospora have important implications not only for comparative mutagenesis, where the effects of the same mutagen are studied in different laboratory organisms, but also for risk estimation since the spectrum of genetic damage produced by a given agent may not be uniform in our genetically heterogeneous human population.

Adenine↗
PubMed1990

Characterization of eukaryotic DNA polymerases: aphidicolin resistant mutants of Neurospora with altered DNA polymerase.

Sucrose density gradient analysis of Neurospora cell free extract showed at least three distinct peaks of enzyme activity; of these, a high molecular weight enzyme was identified as DNA polymerase alpha because of its sensitivity to aphidicolin and to NEM. DNA polymerase mutants of Neurospora crassa were isolated by their resistance to aphidicolin, a specific inhibitor of the eukaryotic DNA polymerase alpha. Some mutants showed an increase in the specific activity of the enzyme. One mutant (E-2-4-1) characterized in detail showed the presence of DNA polymerase which was resistant to inhibitory action of aphidicolin in an in vitro assay. Another mutant (C-3) showed changes in the pH optimum of the enzyme activity. Genetic characterization of the mutants provided evidence for the dominance of the aphr allele controlling aphidicolin resistance and its Mendelian segregation. Some of the aphidicolin resistant mutants were found to be UV-sensitive. Neurospora wild-type and mutant genomic DNA digest was found to hybridize with a cloned yeast DNA polymerase gene. The nick translated yeast DNA polymerase gene was used to screen a genomic cosmid library of Neurospora. A putative clone containing Neurospora DNA polymerase gene has been identified. Further molecular characterization of the Neurospora DNA polymerase gene and enzyme is in progress.

Aphidicolin↗
PubMed1987

Potassium-proton symport in Neurospora: kinetic control by pH and membrane potential.

Active transport of potassium in K+-starved Neurospora was previously shown to resemble closely potassium uptake in yeast, Chlorella, and higher plants, for which K+ pumps or K+/H+-ATPases had been proposed. For Neurospora, however, potassium-proton cotransport was demonstrated to operate, with a coupling ratio of 1 H+ to 1 K+ taken inward so that K+, but not H+, moves against its electrochemical gradient (Rodriguez-Navarro et al., J. Gen. Physiol. 87:649-674). In the present experiments, the current-voltage (I-V) characteristic of K+-H+ cotransport in spherical cells of Neurospora has been studied with a voltage-clamp technique, using difference-current methods to dissect it from other ion-transport processes in the Neurospora plasma membrane. Addition of 5-200 microM K+ to the bathing medium causes 10-150 mV depolarization of the unclamped membrane, and yields a sigmoid I-V curve with a steep slope (maximal conductance of 10-30 microS/cm2) for voltages of -300 to -100 mV, i.e., in the normal physiologic range. Outside that range the apparent I-V curve of the K+-H+ symport saturates for both hyperpolarization and depolarization. It fails to cross the voltage axis at its predicted reversal potential, however, an effect which can be attributed to failure of the I-V difference method under reversing conditions. In the absence of voltage clamping, inhibitors-such as cyanide or vanadate-which block the primary proton pump in Neurospora also promptly inhibit K+ transport and K+-H+ currents. But when voltage clamping is used to offset the depolarizing effects of pump blockade, the inhibitors have no immediate effect on K+-H+ currents. Thus, the inhibition of K+ transport usually observed with these agents reflects the kinetic effect of membrane depolarization rather than any direct chemical action or the cotransport system itself. Detailed study of the effects of [K+]o and pHo on the I-V curve for K+-H+ symport has revealed that increasing membrane potential systematically decreases the apparent affinity of the transporter for K+, but increases affinity for protons (Km range: for [K+]o, 15-45 microM; for [H+]o, 10-35 nM). This behavior is consistent with two distinct reaction-kinetic models, in which (i) a neutral carrier binds K+ first and H+ last in the forward direction of transport, or (ii) a negatively charged carrier (-2) binds H+ first and K+ last.

Carrier Proteins↗
PubMed1982

Binding and screening by cations and the effect on exogenous NAD(P)H oxidation in Neurospora crassa mitochondria.

1. The uncoupled oxidation of exogenous NADH by mitochondria from Neurospora crassa has a pH optimum at 7.0. In the presence of EDTA (1 mM) the optimum is at pH 6.5; maximal inhibition (65%) occurs at pH 7.2. This is comparable to the results with higher plant mitochondria. 2. The corresponding pH optima for NADPH oxidation are 7.75 (control), 7.0 (+ EDTA) and 8.0 (effect of EDTA), respectively. NADPH oxidation is completely inhibited by EDTA at pH 8.0. These pH optima are all about 1 higher than observed in mitochondria from higher plants. 3. The inhibition of NADH oxidation by EDTA is shown to be due to the removal of Mg2+ bound to the mitochondrial membranes. 4. It is shown that 9-aminoacridine can be used to monitor the surface potential of the membranes of Neurospora mitochondria. 5. Cations stimulate NADH oxidation by Neurospora mitochondria in a manner consistent with the theory of the diffuse layer. Quantitatively, the results suggest that Neurospora mitochondria contain fewer charges per mass of protein than Jerusalem artichoke (Helianthus tuberosus) mitochondria but more than mitochondria from Arum maculatum spadices. 6. A good correlation is found between the effect of La3+ on the fluorescence of 9-aminoacridine in the presence of mitochondria and on the oxidation of NADH by the mitochondria. La3+ has different effects on mitochondria from Neurospora, Jerusalem artichoke tubers and Arum spadices. THe results indicate that the fluorescence of 9-aminoacridine can be used to monitor binding sites on biological membranes.

Aminacrine↗