scholarly journals Hybridization in vitro between Wild-Type and Mutant Forms of Glutamate Dehydrogenase from Neurospora crassa

1966 ◽  
Vol 99 (2) ◽  
pp. 9C-11C ◽  
Author(s):  
Alan Coddington
Keyword(s):  
Neurospora Crassa ◽  
Glutamate Dehydrogenase ◽  
Wild Type ◽  
Mutant Forms

Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism

1981 ◽  
Vol 1 (2) ◽  
pp. 158-164
Author(s):  
N S Dunn-Coleman ◽  
E A Robey ◽  
A B Tomsett ◽  
R H Garrett
Keyword(s):  
Neurospora Crassa ◽  
Glutamate Dehydrogenase ◽  
Nitrogen Metabolism ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Glutamate Synthase ◽  
Wild Type ◽  
Mutant Strains ◽  
Synthase Regulation ◽  
Glutamate Biosynthesis ◽  
Glutamine Limitation

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.

scholarly journals Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism.

1981 ◽  
Vol 1 (2) ◽  
pp. 158-164 ◽  
Author(s):  
N S Dunn-Coleman ◽  
E A Robey ◽  
A B Tomsett ◽  
R H Garrett
Keyword(s):  
Neurospora Crassa ◽  
Glutamate Dehydrogenase ◽  
Nitrogen Metabolism ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Glutamate Synthase ◽  
Wild Type ◽  
Mutant Strains ◽  
Synthase Regulation ◽  
Glutamate Biosynthesis ◽  
Glutamine Limitation

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.

scholarly journals Functional characterization of an N-terminally-truncated mitochondrial porin expressed in Neurospora crassa

2017 ◽  
Vol 63 (8) ◽  
pp. 730-738 ◽  
Author(s):  
Sabbir R. Shuvo ◽  
Uliana Kovaltchouk ◽  
Abdullah Zubaer ◽  
Ayush Kumar ◽  
William A.T. Summers ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Outer Membrane ◽  
Functional Characterization ◽  
Helical Structure ◽  
Terminal Segment ◽  
Wild Type ◽  
Mitochondrial Porin ◽  
N Terminus

Mitochondrial porin, which forms voltage-dependent anion-selective channels (VDAC) in the outer membrane, can be folded into a 19-β-stranded barrel. The N terminus of the protein is external to the barrel and contains α-helical structure. Targeted modifications of the N-terminal region have been assessed in artificial membranes, leading to different models for gating in vitro. However, the in vivo requirements for gating and the N-terminal segment of porin are less well-understood. Using Neurospora crassa porin as a model, the effects of a partial deletion of the N-terminal segment were investigated. The protein, ΔN2-12porin, is assembled into the outer membrane, albeit at lower levels than the wild-type protein. The resulting strain displays electron transport chain deficiencies, concomitant expression of alternative oxidase, and decreased growth rates. Nonetheless, its mitochondrial genome does not contain any significant mutations. Most of the genes that are expressed in high levels in porin-less N. crassa are expressed at levels similar to that of wild type or are slightly increased in ΔN2-12porin strains. Thus, although the N-terminal segment of VDAC is required for complete function in vivo, low levels of a protein lacking part of the N terminus are able to rescue some of the defects associated with the absence of porin.

scholarly journals REGULATION OF PHOSPHATE METABOLISM IN NEUROSPORA CRASSA: IDENTIFICATION OF THE STRUCTURAL GENE FOR REPRESSIBLE ACID PHOSPHATASE

Genetics ◽  
1976 ◽  
Vol 84 (2) ◽  
pp. 183-192
Author(s):  
Robert E Nelson ◽  
John F Lehman ◽  
Robert L Metzenberg
Keyword(s):  
Acid Phosphatase ◽  
Neurospora Crassa ◽  
Structural Gene ◽  
Group Iv ◽  
Structural Genes ◽  
Wild Type ◽  
Linkage Groups ◽  
Michaelis Constant ◽  
The Right

ABSTRACT A mutant of Neurospora crassa with an altered repressible acid phosphatase has been isolated. The enzyme is much more thermolabile than that of wild type, and has an increased Michaelis constant. Tests of allelic interactions (in partial diploids) and in vitro mixing experiments were consistent with the mutation being in the structural gene for the enzyme. This gene, pho-3, was found to be located in the right arm of Linkage Group IV (LG IV). Thus, pho-3 and the structural gene for repressible alkaline phosphatase, pho-2 (LG V), map in separate linkage groups and cannot be part of the same operon. Neither of these structural genes is linked to the known regulatory genes, nuc-1 (LG I), nuc-2 (LG II), and preg (LG II).

scholarly journals STP and Tip Are Essential for Herpesvirus Saimiri Oncogenicity

1998 ◽  
Vol 72 (2) ◽  
pp. 1308-1313 ◽  
Author(s):  
S. Monroe Duboise ◽  
Jie Guo ◽  
Sue Czajak ◽  
Ronald C. Desrosiers ◽  
Jae U. Jung
Keyword(s):  
Cell Culture ◽  
Experimental Infection ◽  
Interleukin 2 ◽  
Common Marmoset ◽  
Herpesvirus Saimiri ◽  
Deletion Mutants ◽  
Wild Type ◽  
Common Marmosets ◽  
Mutant Forms

ABSTRACT Mutant forms of herpesvirus saimiri (HVS) subgroup C strain 488 with deletions in either STP-C488 or Tip were constructed. The transforming potentials of the HVS mutants were tested in cell culture and in common marmosets. Parental HVS subgroup C strain 488 immortalized common marmoset T lymphocytes in vitro to interleukin-2-independent growth, but neither of the deletion mutants produced such growth transformation. Wild-type HVS produced fatal lymphoma within 19 to 20 days of experimental infection of common marmosets, while HVS ΔSTP-C488 and HVS ΔTip were nononcogenic. Virus was repeatedly isolated from the peripheral blood of marmosets infected with mutant virus for more than 5 months. These results demonstrate that STP-C488 and Tip are not required for replication or persistence, but each is essential for transformation in cell culture and for lymphoma induction in common marmosets.

scholarly journals Structure of papain-like protease from SARS-CoV-2 and its complexes with non-covalent inhibitors

2020 ◽  
Author(s):  
Jerzy Osipiuk ◽  
Saara-Anne Azizi ◽  
Steve Dvorkin ◽  
Michael Endres ◽  
Robert Jedrzejczak ◽  
...  
Keyword(s):  
Peptidase Activity ◽  
Wild Type ◽  
Clinical Value ◽  
Structure Based Drug Design ◽  
Replication Studies ◽  
Main Protease ◽  
Covalent Inhibitors ◽  
Viral Proliferation ◽  
Mutant Forms

ABSTRACTThe number of new cases world-wide for the COVID-19 disease is increasing dramatically, while efforts to contain Severe Acute Respiratory Syndrome Coronavirus 2 is producing varied results in different countries. There are three key SARS-CoV-2 enzymes potentially targetable with antivirals: papain-like protease (PLpro), main protease (Mpro), and RNA-dependent RNA polymerase. Of these, PLpro is an especially attractive target because it plays an essential role in several viral replication processes, including cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex (RTC), and disruption of host viral response machinery to facilitate viral proliferation and replication. Moreover, this enzyme is conserved across different coronaviruses and promising inhibitors have already been discovered for its SARS-CoV variant. Here we report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the enzyme from SARS-CoV-2 in both wild-type and mutant forms. These efforts include the first structures of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors, determined at 1.60–2.70 Angstroms. This collection of structures provides fundamental molecular and mechanistic insight to PLpro, and critically, illustrates details for inhibitors recognition and interactions. All presented compounds inhibit the peptidase activity of PLpro in vitro, and some molecules block SARS-CoV-2 replication in cell culture assays. These collated findings will accelerate further structure-based drug design efforts targeting PLpro, with the ultimate goal of identifying high-affinity inhibitors of clinical value for SARS-CoV-2.

Transformation of Neurospora crassa with the cloned am (glutamate dehydrogenase) gene

1984 ◽  
Vol 4 (1) ◽  
pp. 117-122
Author(s):  
J A Kinsey ◽  
J A Rambosek
Keyword(s):  
Neurospora Crassa ◽  
Glutamate Dehydrogenase ◽  
Plasmid Vector ◽  
Wild Type ◽  
Mendelian Segregation ◽  
Dehydrogenase Gene ◽  
Mutant Strains ◽  
Colonial Morphology ◽  
Relationship Of ◽  
Lambda Dna

We used DNA containing the am gene of Neurospora crassa, cloned in the lambda replacement vector lambdaL-47 (this clone is designated lambdaC-10), and plasmid vector subclones of this DNA to transform am deletion and point mutant strains. By means of subcloning, all sequences required for transformation to am prototrophy and expression of glutamate dehydrogenase have been shown to reside on a 2.5-kilobase BamHI fragment. We also characterized several am+ strains that were obtained after transformation with lambdaC-10. These strains showed Mendelian segregation of the am+ gene, although less than 50% of the transformed strains showed the normal linkage relationship of am with inl. In all cases tested, the strains had incorporated lambda DNA as well. The lambda DNA also showed a Mendelian segregation pattern. In one case, the incorporation of am DNA in a novel position was associated with a mutagenic event producing a strain with a very tight colonial morphology. In all cases in which the am+ gene had become the resident of a new chromosome, glutamate dehydrogenase was produced to only 10 to 20% of the wild-type levels.

scholarly journals Methylation of Histone H3 Lysine 36 Is Required for Normal Development in Neurospora crassa

2005 ◽  
Vol 4 (8) ◽  
pp. 1455-1464 ◽  
Author(s):  
Keyur K. Adhvaryu ◽  
Stephanie A. Morris ◽  
Brian D. Strahl ◽  
Eric U. Selker
Keyword(s):  
Neurospora Crassa ◽  
Growth And Development ◽  
Histone H3 ◽  
Normal Growth ◽  
Wild Type ◽  
Set Domain ◽  
Amino Terminal ◽  
Histone H3 Gene ◽  
Type Gene

ABSTRACT The SET domain is an evolutionarily conserved domain found predominantly in histone methyltransferases (HMTs). The Neurospora crassa genome includes nine SET domain genes (set-1 through set-9) in addition to dim-5, which encodes a histone H3 lysine 9 HMT required for DNA methylation. We demonstrate that Neurospora set-2 encodes a histone H3 lysine 36 (K36) methyltransferase and that it is essential for normal growth and development. We used repeat induced point mutation to make a set-2 mutant (set-2 RIP1 ) with multiple nonsense mutations. Western analyses revealed that the mutant lacks SET-2 protein and K36 methylation. An amino-terminal fragment that includes the AWS, SET, and post-SET domains of SET-2 proved sufficient for K36 HMT activity in vitro. Nucleosomes were better substrates than free histones. The set-2 RIP1 mutant grows slowly, conidiates poorly, and is female sterile. Introducing the wild-type gene into the mutant complemented the defects, confirming that they resulted from loss of set-2 function. We replaced the wild-type histone H3 gene (hH3) with an allele producing a Lys to Leu substitution at position 36 and found that this hH3 K36L mutant phenocopied the set-2 RIP1 mutant, confirming that the observed defects in growth and development result from inability to methylate K36 of H3. Finally, we used chromatin immunoprecipitation to demonstrate that actively transcribed genes in Neurospora crassa are enriched for H3 methylated at lysines 4 and 36. Taken together, our results suggest that methylation of K36 in Neurospora crassa is essential for normal growth and development.

scholarly journals Amino acid substitutions in mutant forms of histidinol dehydrogenase from Neurospora crassa

1967 ◽  
Vol 105 (1) ◽  
pp. 39-44 ◽  
Author(s):  
D J Bennett ◽  
E H Creaser
Keyword(s):  
Amino Acid ◽  
Neurospora Crassa ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Temperature Sensitive Mutants ◽  
Nad Oxidoreductase ◽  
Histidinol Dehydrogenase ◽  
Mutant Forms ◽  
Two Temperature

Amino acid changes in the enzyme l-histidinol dehydrogenase (l-histidinol–NAD oxidoreductase, EC 1.1.1.23) have been determined between the wild-type Neurospora crassa and two temperature-sensitive mutants. Comparison was made between amino acid analyses of peptides of differing electrophoretic and chromatographic mobilities resulting from tryptic and chymotryptic digestion of protein from wild-type and mutant K26, and wild-type and mutant K445 strains, respectively. The analyses demonstrate the substitution of aspartic acid for alanine in mutant K26, and leucine for histidine in mutant K445. The effects of the resulting changes in polarity and charge are discussed in relation to the catalytic functioning of the proteins.

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