A second form of the single-strand specific endonuclease of Neurospora crassa which is associated with a double-strand exonuclease

1976 ◽  
Vol 54 (11) ◽  
pp. 971-980 ◽  
Author(s):  
M. J. Fraser ◽  
R. Tjeerde ◽  
K. Matsumoto
Keyword(s):  
Neurospora Crassa ◽  
Heat Stability ◽  
Single Strand ◽  
Dna Unwinding ◽  
Endonuclease Activity ◽  
Wild Type ◽  
Double Stranded Rna ◽  
Heat Stable ◽  
Double Stranded Dna ◽  
Heating Up

A second form of single-strand specific endonuclease, which is stable to heating up to 74 °C and does not bind strongly to phosphocellulose, has been partially purified from extracts of mycelia of wild-type Neurospora crassa. The endonuclease is associated with an equally heat-stable exonuclease which degrades linear but not circular double-stranded DNA and does not attack double-stranded RNA. The exonuclease probably also degrades single-stranded DNA. Both endonuclease and exonuclease activities are inhibited by 0.1–0.5 mM ATP. The exonuclease is preferentially inhibited by a variety of agents and preferentially inactivated by trypsin. A DNA-unwinding activity has also been detected in the nuclease preparation. Protease(s) present in the nuclease preparation destroy the DNA-unwinding and exonuclease activities on incubation at 37 °C, but do not affect the endonuclease activity. However, the heat-stability and chromatographic properties of the endonuclease are affected by this treatment. The altered properties of the endonuclease are very similar to those of the single-strand specific endonuclease which has been previously described. The combined nuclease activities of the unaltered preparation make up a putative recombination nuclease of N. crassa.

Specific inhibitors of Neurospora endo-exonuclease

1989 ◽  
Vol 67 (9) ◽  
pp. 632-641 ◽  
Author(s):  
Zafer Hatahet ◽  
Murray J. Fraser
Keyword(s):  
Neurospora Crassa ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Exonuclease Activity ◽  
Endonuclease Activity ◽  
Average Molecular Mass ◽  
Strand Breaks ◽  
Heat Stable ◽  
First Order Kinetics ◽  
Pbr322 Dna

A heat-stable, trypsin-sensitive cytosolic inhibitor of Neurospora crassa endo-exonuclease has been purified 300-fold in 90% yield from crude extracts of mycelia. On electrophoresis in sodium dodecyl sulfate – polyacrylamide gels, it showed a relatively broad band corresponding to polypeptides with an average molecular mass of about 24 kDa. The protein inhibited the single strand specific endonuclease activity of endo-exonuclease noncompetitively, but the inhibition was not complete (maxima of 70–95%) in the presence of excess inhibitor. The inhibition of the double strand exonuclease activity was competitive and complete when inhibitor was present in excess. In addition, the inhibitor completely blocked the formation of site-specific double strand breaks and nicking by endo-exonuclease in linearized pBR322 DNA. The ribonuclease activity of endo-exonuclease was also inhibited by this protein. The inhibition of Neurospora endo-exonuclease was very specific. Although inhibition was seen also for an immunochemically related nuclease derived from mycelia of Aspergillus nidulans, no inhibition was observed for two other nucleases of Neurospora, nor for a wide variety of commercially available nucleases. A bound form of inhibitor was also partially purified from the cytosol of Neurospora in association with inactive, but trypsin-activable endo-exonuclease. Activity gel analysis showed that this was an endo-exonuclease–inhibitor complex. On heating at 80 °C, inhibitor was released from the complex with the same first order kinetics as inactivation of the trypsin-activable endo-exonuclease and in approximately equivalent amounts. The bound inhibitor was also 24 kDa in size, but completely inhibited both the endo- and exonuclease activities of endo-exonuclease. It was found unexpectedly that heat-inactivated endo-exonuclease stimulated the exonuclease activity of the active enzyme without affecting the endonuclease activity. The stimulated exonuclease activity was also completely blocked by inhibitor.Key words: Neurospora crassa endo-exonuclease, inhibitor, constitutive, cytosolic.

scholarly journals Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling

2006 ◽  
Vol 80 (11) ◽  
pp. 5168-5178 ◽  
Author(s):  
Washington B. Cárdenas ◽  
Yueh-Ming Loo ◽  
Michael Gale ◽  
Amy L. Hartman ◽  
Christopher R. Kimberlin ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Binding Activity ◽  
Wild Type ◽  
Antagonist Activity ◽  
Double Stranded Rna ◽  
Beta Interferon ◽  
Double Stranded Dna ◽  
Dsrna Binding ◽  
Alpha Beta

ABSTRACT The Ebola virus (EBOV) VP35 protein blocks the virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical for the induction of alpha/beta interferon (IFN-α/β) expression. However, the mechanism(s) by which this blockage occurs remains incompletely defined. We now provide evidence that VP35 possesses double-stranded RNA (dsRNA)-binding activity. Specifically, VP35 bound to poly(rI) · poly(rC)-coated Sepharose beads but not control beads. In contrast, two VP35 point mutants, R312A and K309A, were found to be greatly impaired in their dsRNA-binding activity. Competition assays showed that VP35 interacted specifically with poly(rI) · poly(rC), poly(rA) · poly(rU), or in vitro-transcribed dsRNAs derived from EBOV sequences, and not with single-stranded RNAs (ssRNAs) or double-stranded DNA. We then screened wild-type and mutant VP35s for their ability to target different components of the signaling pathways that activate IRF-3. These experiments indicate that VP35 blocks activation of IRF-3 induced by overexpression of RIG-I, a cellular helicase recently implicated in the activation of IRF-3 by either virus or dsRNA. Interestingly, the VP35 mutants impaired for dsRNA binding have a decreased but measurable IFN antagonist activity in these assays. Additionally, wild-type and dsRNA-binding-mutant VP35s were found to have equivalent abilities to inhibit activation of the IFN-β promoter induced by overexpression of IPS-1, a recently identified signaling molecule downstream of RIG-I, or by overexpression of the IRF-3 kinases IKKε and TBK-1. These data support the hypothesis that dsRNA binding may contribute to VP35 IFN antagonist function. However, additional mechanisms of inhibition, at a point proximal to the IRF-3 kinases, most likely also exist.

Intercellular transfer of beta-glucuronidase in chimeric mice

1979 ◽  
Vol 40 (1) ◽  
pp. 21-31
Author(s):  
K. Herrup ◽  
R.J. Mullen
Keyword(s):  
Gene Product ◽  
Heat Stability ◽  
Wild Type Allele ◽  
Chimeric Mice ◽  
Wild Type ◽  
Glucuronidase Activity ◽  
Heat Stable ◽  
Dark Staining ◽  
Activity Loss ◽  
Light Staining

Chimeric mice were produced by aggregating 2 embryos, each of which was homozygous for a different structural allele of the enzyme beta-glucuronidase. The alleles used were Gusb, the ‘wild-type’ allele, and Gush, an allele whose gene product shows decreased activity in all tissues as well as decreased heat stability. Staining of untreated adult chimeric livers for glucuronidase activity revealed a mosaic of high (Gusb) and low (Gush) activity cells. The boundaries between cells of different activity were sharp and revealed no diffusion of enzyme or reaction product. Treating sections at 73 degrees c before staining led to a decay of staining activity in normal (non-chimeric) Gush/Gush and Gusb/Gusb tissue. The rates of activity loss under the conditions used differed by 10-fold. Of the 2 genotypes in the chimera, the dark-staining, Gusb, cells decayed in a fashion similar to that of the Gusb/Gusb control. The light-staining, Gush, cells of the chimera lost their staining a a unique fashion. Within 20 min they quickly lost a majority of their staining activity but that which remained was relatively heat stable. The second Gusb-like phase of the decay, seen both photographically and photometrically, suggests that Gusb gene product has been transferred to cells of Gush/Gush genotype.

scholarly journals REGULATION OF PHOSPHATE METABOLISM IN NEUROSPORA CRASSA: ISOLATION OF MUTANTS DEFICIENT IN THE REPRESSIBLE ALKALINE PHOSPHATASE

Genetics ◽  
1974 ◽  
Vol 78 (2) ◽  
pp. 645-659
Author(s):  
Mary K Gleason ◽  
Robert L Metzenberg
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Linkage Group ◽  
Neurospora Crassa ◽  
Heat Stability ◽  
Phosphate Metabolism ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Group V ◽  
Suppressor Mutations

ABSTRACT Mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase, but, unlike nuc-1 and nuc-2 mutants, are able to make the repressible acid phosphatase and the repressible phosphate permease under conditions of derepression (phosphate deprivation). The new mutants, called pho-2, map in Linkage Group V, and are unlinked to the putative control mutants, nuc-1, nuc-2-pconc, and pregc. Three of the pho-2 mutants do not make detectable amounts of repressible alkaline phosphatase, but the fourth makes about 1% of the level found in wild type. The small amount of alkaline phosphatase made by this strain appears to be qualitatively similar or identical to the wild-type enzyme, as judged by electrophoretic mobility, heat stability, and titration with specific antibody to the wild-type enzyme. Several revertants of this strain have been examined in the same way, and the alkaline phosphatase of these strains also appears to be qualitatively normal. Reversion events can occur at, or near, the pho-2 locus, but also occur in at least two unlinked sites (suppressor mutations). One suppressor maps very close to nuc-1.

scholarly journals Glu-370 in the Large Subunit Influences the Substrate Binding, Allosteric, and Heat Stability Properties of Potato ADP-glucose Pyrophosphorylase

2016 ◽  
Author(s):  
Ayse B Seferoglu ◽  
Seref Gul ◽  
Ugur M Dikbas ◽  
Kaan Koper ◽  
Ibrahim Baris ◽  
...  
Keyword(s):  
Computational Analysis ◽  
Large Subunit ◽  
Heat Stability ◽  
Starch Biosynthesis ◽  
Enzyme Kinetic ◽  
Wild Type ◽  
Binding Properties ◽  
Allosteric Enzyme ◽  
Heat Stable ◽  
Adp Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. In this study, we showed that the conversion of Glu to Gly at position 370 in the LS of AGPase alters the heterotetrameric stability along with the binding properties of substrate and effectors of the enzyme. Kinetic analyses revealed that the affinity of the LSE370GSSWT AGPase for glucose-1-phosphate is 3-fold less than for wild type (WT) AGPase. Additionally, the LSE370GSSWT AGPase requires 3-fold more 3-phosphogyceric acid to be activated. Finally, the LSE370GSSWTAGPase is less heat stable compared with the WT AGPase. Computational analysis of the mutant Gly-370 in the 3D modeled LS AGPase showed that this residue changes charge distribution of the surface and thus affect stability of the LS AGPase and overall heat stability of the heterotetrameric AGPase. In summary, our results show that LSE370 intricately modulate the heat stability and enzymatic activity of the AGPase.

Ultrastructure and Morphology of the Neurospora Crassa Cell Wall Mutants, Slime And Slime X, Using Tem and Sem

1976 ◽  
Vol 34 ◽  
pp. 54-55
Author(s):  
Karen S. Howard ◽  
H. D. Braymer ◽  
M. D. Socolofsky ◽  
S. A. Milligan
Keyword(s):  
Cell Wall ◽  
Neurospora Crassa ◽  
Wild Type ◽  
Morphological Comparison ◽  
Small Areas ◽  
Solid Media ◽  
Thin Sectioning ◽  
X Cells ◽  
Mutant Cells ◽  
Isolated Cell

The recently isolated cell wall mutant slime X of Neurospora crassa was prepared for ultrastructural and morphological comparison with the cell wall mutant slime. The purpose of this article is to discuss the methods of preparation for TEM and SEM observations, as well as to make a preliminary comparison of the two mutants.TEM: Cells of the slime mutant were prepared for thin sectioning by the method of Bigger, et al. Slime X cells were prepared in the same manner with the following two exceptions: the cells were embedded in 3% agar prior to fixation and the buffered solutions contained 5% sucrose throughout the procedure.SEM: Two methods were used to prepare mutant and wild type Neurospora for the SEM. First, single colonies of mutant cells and small areas of wild type hyphae were cut from solid media and fixed with OSO4 vapors similar to the procedure used by Harris, et al. with one alteration. The cell-containing agar blocks were dehydrated by immersion in 2,2-dimethoxypropane (DMP).

scholarly journals Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

2007 ◽  
Vol 28 (3) ◽  
pp. 897-906 ◽  
Author(s):  
Thomas J. Pohl ◽  
Jac A. Nickoloff
Keyword(s):  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Homology Search ◽  
Wild Type ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
In The Wild ◽  
Break Induced Replication

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

scholarly journals DIRECT INDUCTION IN WILD-TYPE NEUROSPORA CRASSA OF MUTANTS (qa-1C) CONSTITUTIVE FOR THE CATABOLISM OF QUINATE AND SHIKIMATE

Genetics ◽  
1972 ◽  
Vol 72 (3) ◽  
pp. 411-417
Author(s):  
C W H Partridge ◽  
Mary E Case ◽  
Norman H Giles
Keyword(s):  
Neurospora Crassa ◽  
Single Gene ◽  
Ultra Violet ◽  
Wild Type ◽  
Color Test ◽  
Catabolic Enzymes ◽  
Direct Induction

ABSTRACT A color test has been developed for the selection and identification of mutants in Neurospora crassa, constitutive for the three normally inducible enzymes which convert quinate to protocatechuate. By this means seven such mutants have been recovered after ultra violet irradiation of wild type and have been shown to be allelic (or very closely linked) to the qa-1C mutants previously obtained by other means. Thus, the regulation of the synthesis of these three catabolic enzymes is indicated to be under the control of a single gene, qa-1+.

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