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1.
G. Stauder H. Riesemann W.M. Joester K.E. Joester 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,741(3)
A third DNA polymerase ‘C’ with low molecular weight was isolated and purified 3700-fold from ground hyphae of Neurospora crassa WT 74 A, which shows similarities to β- and γ-polymerases from higher eukaryotes: preference for poly(rA)(dT) as a template/primer, inhibition by p-chloromercuribenzoate, resistance against N-ethylmaleimide up to 10 mmol/l, and molecular weight of about 40 000. This polymerase elutes as a distinct peak from DEAE-cellulose at 0.60 mol/l KCl and has an optimum for K+ at 2–20 mmol/l, for Mn2+ at 0.8 mmol/l, for Mg2+ at 4.0 mmol/l, the pH optimum is 8.0. Its Km is 1.5 μmol/l using dTTP as substrate. The enzyme activity described here is free of endonuclease but contains detectable amounts of exonuclease. 相似文献
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As a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved,
in Neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere
with the activity of well-known signal transducing molecules in vivo. We tested the influence of these compounds on the induction
of the al-3 gene, a key step in light-induced carotenoid biosynthesis. This approach has implicated protein kinase C (PKC) as a component
of the light transduction machinery. The conclusion is based on the effects of specific inhibitors (calphostin C and chelerythrine
chloride) and activators of PKC (1,2-dihexanoyl-sn-glycerol). During vegetative growth PKC may be responsible for desensitization to light because inhibitors of the enzyme
cause an increase in the total amount of mRNA transcribed after illumination. PKC is therefore proposed here to be an important
regulator of transduction of the blue light signal, and may act through modification of the protein White Collar-1, which
we show to be a substrate for PKC in N. crassa.
Received: 4 December 1998 / Accepted: 21 May 1999 相似文献
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Peter J. Russell Richard C. Petersen Sheryl Wagner 《Molecular & general genetics : MGG》1988,211(3):541-544
Summary The genetic segregation of ribosomal DNA (rDNA) in Neurospora crassa was analyzed by exploiting restriction fragment length polymorphisms in the nontranscribed spacer (NTS) sequences of nine laboratory wild-type strains and wild-collected strains. In an analysis of random spore progeny from seven crosses, and of ordered tetrads from two of those crosses the rDNA was shown to be inherited in a simple, stable Mendelian fashion, exhibiting an approximately 1:1 ratio of the two parental rDNA types. No meiotic recombinants were detected among the progeny, indicating that non-sister-chromatid crossing over is highly suppressed in the rDNA region. The basis for this suppression of meiotic recombination is not known. 相似文献
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S. Ito Y. Matsui A. Toh-e T. Harashima H. Inoue 《Molecular & general genetics : MGG》1997,255(4):429-437
Genes belonging to the ras superfamily encode low-molecular-weight GTP/GDP-binding proteins that are highly conserved in wide variety of organisms.
We used the polymerase chain reaction (PCR) to isolate a novel member of the ras superfamily from the filamentous fungus Neurospora crassa and obtained a mammalian Krev-1 homolog. We named the gene krev-1 and analyzed its structure and function. The krev-1 gene encodes a polypeptide of 225 amino acids, which is nearly 60% homologous to the mammalian Krev-1 p21. The krev-1 gene product (KREV1) is functionally analogous to mammalian Krev-1 p21 and Rsr1p/Bud1p, a Krev-1 homolog from the yeast Saccharomyces cerevisiae. GAL1-driven expression of KREV1 in a wild-type yeast strain resulted in a random budding pattern, as did its mammalian counterpart
Krev-1 p21. We disrupted the krev-1 gene by RIP (repeat-induced point mutation), but the krev-1 disruptants showed no abnormalities. By in vitro mutagenesis, we constructed several mutant krev-1 genes (G21V, A68T, and D128A) which mimic constitutively active mutants of Ha-ras, and the krev-1 (K25N) mutant which is analogous to a dominant-negative mutant of Ha-ras. Each mutant gene was introduced into the wild-type strain and the phenotypes were analyzed. We could not observe any difference
in vegetative growth between these transformants. When each strain was used as the female in mating tests, the development
of perithecia from protoperithecia was inhibited in all cases. The results indicate that the krev-1 gene may be involved in sexual cycle progression.
Received: 28 January 1997 / Accepted: 3 April 1997 相似文献
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Celedonio González Nelida Brito George A. Marzluf 《Molecular & general genetics : MGG》1995,249(4):456-464
Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3
– null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors. 相似文献
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H. Prokisch O. Yarden M. Dieminger M. Tropschug I. B. Barthelmess 《Molecular & general genetics : MGG》1997,256(2):104-114
The function of Neurospora crassa calcineurin was investigated in N. crassa strains transformed with a construct that provides for the inducible expression of antisense RNA for the catalytic subunit
of calcineurin (cna-1). Induction of antisense RNA expression was associated with reduced levels of cna-1 mRNA and of immunodetectable CNA1 protein and decreased calcineurin enzyme activity, indicating that a conditional reduction
of the target function had been achieved in antisense transformants with multiple construct integrations. Induction conditions
caused growth arrest which indicated that the cna-1 gene is essential for growth of N. crassa. Growth arrest was preceded by an increase in hyphal branching, changes in hyphal morphology and concomitant loss of the
distinctive tip-high Ca2+ gradient typical for growing wild-type hyphae. This demonstrates a novel and specific role for calcineurin in the precise
regulation of apical growth, a common form of cellular proliferation. In vitro inhibition of N. crassa calcineurin by the complex of cyclosporin A (CsA) and cyclophilin20, and increased sensitivity of the induced transformants
to the calcineurin-specific drugs CsA and FK506 imply that the drugs act in N. crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin. The finding that exposure of growing wild-type mycelium to these drugs leads to a phenotype
very similar to that of the cna-1 antisense mutants is consistent with this idea.
Received: 18 February 1997 / Accepted: 20 April 1997 相似文献
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A. Kana-uchi C. T. Yamashiro S. Tanabe T. Murayama 《Molecular & general genetics : MGG》1997,254(4):427-432
In order to study the role of signal transduction pathways in the regulation of morphology in Neurospora crassa, we cloned and characterized a ras homologue, termed NC-ras2. The predicted protein product of this gene is composed of 229 amino acid residues and contains all the consensus sequences
shared by the ras protein family. The gene is located in linkage group V. An NC-ras2 disruptant showed morphological characteristics very similar to those of the smco7 mutant, which also maps to linkage group V. Nucleotide sequence analysis revealed that the smco7 mutant harbored a single base deletion in the NC-ras2 gene, which is predicted to result in the truncation of the protein product. Introduction into the smco7 mutant of an NC-ras2 clone yielded stable transformants with a wild-type phenotype. The smco7 mutant exhibited very slow hyphal growth and the rate of conidial formation was approximately one two-hundredth of wild type.
The smco7 mutation causes both the changes in the pattern of hyphal growth and the defects in cell wall synthesis. Both the diameter
and the length of the apical compartment were shorter in the hyphae of the smco7 mutant. These results suggest that NC-ras2 is identical to smco7, and that the signal transduction pathway mediated by the NC-ras2 protein regulates the apical growth of hyphae, cell wall
synthesis, and conidial formation in N. crassa.
Received: 1 October 1996 / Accepted: 9 December 1996 相似文献
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Ian F. Connerton 《Molecular & general genetics : MGG》1990,223(2):319-323
Summary Repeat-induced point mutation (RIP) has been used to generate new mutations in the previously uncharacterised gene for malate synthase in Neurospora crassa. Molecular clones carrying the am (NADP-glutamate dehydrogenase) gene and the malate synthase gene from either N. crassa or Aspergillus nidulans have been introduced into Neurospora as ectopic duplicate copies by transformation, selecting for the am function in a deletion host. A number of meiotic progeny derived from these transformants were unable to use acetate as sole carbon source, yielded no detectable malate synthase activity and demonstrated extensive cytosine methylation of their duplicated sequences. The new locus has been designated acu-9 and has been assigned to linkage group VII. 相似文献
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The period length of the circadian conidiation rhythm was examined in a mutant strain of Neurospora crassa, un-18, that is temperature sensitive for mycelial growth. The un-18 mutant showed a temperature-sensitive phenotype with respect to both mycelial growth and the period length of the conidiation
rhythm. Below 22° C, the un-18 mutation did not affect the period length, but at temperatures between 22° C and 32° C, the period length of the un-18 mutant was ∼2 h longer than that of the wild-type strain. The un-18
+ gene was cloned and was found to encode the second-largest subunit of RNA polymerase I, which is involved in the synthesis
of rRNA. These results indicate that a defect in ribosome synthesis, which must result in a lower rate of protein synthesis,
lengthens the period of the circadian conidiation rhythm in Neurospora.
Received: 17 October 1997 / Accepted: 26 April 1998 相似文献
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Summary Purrtins can be utilized as a secondary nitrogen source by Neurospora crassa during conditions of nitrogen limitation. The expression of purine catabolic enzymes is governed by the nitrogen regulatory circuit and requires induction by uric acid. The major positive-acting nitrogen regulatory gene, nit-2, turns on the expression of the purine catabolic enzymes, which may also be subject to negative regulation by a second control gene, nmr. We have cloned alc, the structural gene which encodes allantoicase, an inducible enzyme of the purine degradative pathway. The identity of the alc clone was confirmed by restriction fragment length polymorphism analysis and by repeat-induced mutation. The alc gene is transcribed to give a single messenger RNA, approximately 1.2 kb in length. The negative-acting nmr gene affects the expression of alc in the expected manner. Both the nit-2 and the nmr control genes affect alc mRNA levels and allantoicase enzyme activity in both the induced and nitrogen-repressed conditions. 相似文献
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粗糙脉孢菌是一种重要的模式生物,在遗传调节机制、昼夜节律运行以及真菌光应答反应研究中起重要的作用.本综述主要介绍粗糙脉孢菌光受体WC-1和VVD的结构与功能,以及它们参与调节昼夜节律和光适应机制方面的研究进展.在该真菌中,所有已知的光应答反应都受蓝光调节,由光受体WC-1和VVD介导.WC-1是该真菌的转录因子,介导最初的光反应过程,产生VVD等多种光反应蛋白,而VVD通过负反馈机制抑制WC-1的转录作用.此外,vvd基因已经用于构建在哺乳动物中表达的光调节基因元件. 相似文献
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Mutants that show reduced DNA methylation were identified in a mutant screen based on the assumptions that (i) the nucleoside
analog 5-azacytidine (5-azaC) promotes the formation of potentially lethal DNA-methyltransferase adducts; (ii) reduction in
DNA methyltransferase will decrease the sensitivity of cells to 5-azaC; and (iii) this potential selective advantage will
be enhanced in mutants that are deficient in the repair of 5-azaC-induced DNA damage. Of fifteen potential repair mutants
screened for sensitivity to 5-azaC, five (mus-9, mus-10, mus-11, mus-18, and uvs-3) showed moderately increased sensitivity and two (mus-20, mei-3) showed highly increased sensitivity. A mus-20 mutation was used to isolate three non-complementing methylation mutants. The mutations, named dim-1 (defective in methylation), reduced female fertility, reduced methylation by 40–50%, and altered patterns of methylation. In wild-type strains
hypomethylation perse fails to alter methylation specificity. We demonstrate a growth-phase-dependent change in methylation patterns, detectable
only in hypomethylated DNA from dim
+ cultures. This may represent a growth-phase-dependent change in the relative amounts of distinct species of methyltransferase,
one of which may be encoded by the dim-1 gene.
Received: 3 January 1998 / Accepted: 26 March 1998 相似文献
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Fabiana M. de Almeida Beatriz M. Bonini Daniela Beton Joo A. Jorge Hctor F. Terenzi Aline M. da Silva 《Protein expression and purification》2009,65(2):185-189
Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80–150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 °C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 °C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. KM was 42 mM, and Vmax was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence. 相似文献
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