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 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.

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 Adenosine kinase-deficient mutant of Neurospora crassa

1982 ◽  
Vol 152 (3) ◽  
pp. 1292-1294
Author(s):  
J M Magill ◽  
P Dalke ◽  
T S Lyda ◽  
C W Magill
Keyword(s):  
Neurospora Crassa ◽  
Resistant Mutant ◽  
Adenosine Kinase ◽  
Deficient Mutant ◽  
Wild Type ◽  
Purine Nucleotides ◽  
Mutant Strains

Tubercidin-resistant mutant strains of Neurospora crassa were isolated, and at least one appeared to be deficient in adenosine kinase. No significant differences in [8-14C]adenosine labeling of purine nucleotides or nucleosides were found between the wild type and the adenosine kinase-deficient strains.

Premeiotic disruption of duplicated and triplicated copies of the Neurospora crassa am (glutamate dehydrogenase) gene

1989 ◽  
Vol 15 (5) ◽  
pp. 327-334 ◽  
Author(s):  
J. R. S. Fincham ◽  
I. F. Connerton ◽  
E. Notarianni ◽  
K. Harrington
Keyword(s):  
Neurospora Crassa ◽  
Glutamate Dehydrogenase ◽  
Dehydrogenase Gene

Polyene antibiotic affinities for the sterols of resistant and sensitive strains of Neurospora crassa

1977 ◽  
Vol 23 (1) ◽  
pp. 113-115 ◽  
Author(s):  
D. Johnson ◽  
R. Subden
Keyword(s):  
Candida Albicans ◽  
Neurospora Crassa ◽  
Resistant Mutant ◽  
Wild Type ◽  
Polyene Antibiotic ◽  
Polyene Antibiotics ◽  
Mutant Strains ◽  
Resistant Strains

Ergosterol, the principle sterol of many wild-type Neurospora and other Ascomycetes, had a greater affinity for polyene antibiotics than did lichesterol or eburicol, the sterols of some resistant mutant strains. The affinity was demonstrated by comparing the sterols extracted from sensitive and resistant strains of Neurospora crassa and Candida albicans for protection against polyene inhibition of sensitive N. crassa and for their ability to alter specific polyene absorption maxima.

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