Multiple active varieties of Neurospora glutamate dehydrogenase formed by hybridization between two inactive mutant proteins in vivo and in vitro

Coddington, Alan; Fincham, J. R. S.; Sundaram, T. K.

doi:10.1016/s0022-2836(66)80160-2

Cited by 52 publications

(20 citation statements)

References 14 publications

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“…As expected, none was active in the glutamate synthesis assay, but a total of 8 showed activity with 0n15 M glutamate, stimulated in two cases (am#$ -'" and am#$ -($ ) by preincubation at 35 mC for 30 min with 0n15 M succinate (system S of Coddington et al, 1966). None of the hycanthone-induced mutants showed this potential activity.…”

Section: (I) More Mutants With Conformationally Disturbed Gdhsupporting

confidence: 69%

“…In some experiments, extracts were incubated at pH 8n5 and 35 mC with 0n15 M succinate, a pretreatment which had been found to activate the rather stably inactive GDH of am"* (' system S ' of Coddington et al, 1966). Activity in the direction of glutamate synthesis was assayed in a system (' system A ') containing the same Tris buffer, 10 mM disodium αketoglutarate, 33 mM NH % Cl and 7.0i10 −& M NADPH.…”

Section: (Ii) Preparation Of Mycelial Extracts and Enzyme Assaysmentioning

confidence: 99%

“…They are all complemented in heterocaryons by mutants which are GDH-negative for other reasons : failure to bind NADP (am", Ashby et al, 1974) or presumed failure to form stable hexamers (am"%, . This interallelic complementation appears to be due to conformational normalization within hybrid hexamers (Coddington et al, 1966, Watson & Wootton, 1978. Thus conformationally abnormal mutant forms of GDH can be more or less normalized either by cooperative binding of the substrate glutamate or its analogue succinate, or by complementation in hybrid hexamers.…”

Section: Introductionmentioning

confidence: 99%

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The Neurospora am gene and NADP-specific glutamate dehydrogenase: mutational sequence changes and functional effects – more mutants and a summary

et al. 2000

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A further series of mutant am alleles, encoding potentially active NADP-specific glutamate dehydrogenase (GDH) and capable of complementation in heterocaryons, have been characterized with respect to both GDH properties and DNA sequence changes. Several mutants previously studied, and some of their same-site or second-site revertants, have also been sequenced for the first time. We present a summary of what is known of the properties of all am mutants that have been defined at the sequence level.

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Section: (I) More Mutants With Conformationally Disturbed Gdhsupporting

confidence: 69%

Section: (Ii) Preparation Of Mycelial Extracts and Enzyme Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Neurospora am gene and NADP-specific glutamate dehydrogenase: mutational sequence changes and functional effects – more mutants and a summary

et al. 2000

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“…This opens the possibility of using transformation or mutagenesis to place a known DNA sequence into an intractable N. crassa gene, thus providing a convenient handle for the subsequent molecular cloning of that gene. 4 2.2 DISCUSSION am gene information is on a 2.5-kb BamHI fragment. Using plasmid subclones of X-C10, we have demonstrated that all sequences required for transformation and expression of the am gene are contained on a 2.5-kb BamHI fragment.…”

Section: Methodsmentioning

confidence: 99%

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

Kinsey¹,

Rambosek²

1984

Mol. Cell. Biol.

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We used DNA containing the am gene of Neurospora crassa, cloned in the X replacement vector XL-47 (this clone is designated XC-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 XC-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 X DNA as well. The X 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.Case et al. (1, 2) and Schweizer et al. (13) have developed an efficient transformation procedure for Neurospora crassa utilizing the cloned qa-2 gene and a qa-2 aro-9 doublemutant strain as recipient.This. was a significant step towards facilitating molecular genetics studies with N. crassa. Until now, qa-2 has been the only system available to study N. crassa transformation in detail. Recently, however, the am gene of N. crassa, which codes for NADP-specific glutamate dehydrogenase (GDH), has been cloned on a 9.1-kilobase (kb) HindIlI fragment (7) in the replacement vector, XL-47 (9). This X-chimeric clone (herein referred to as XC10), which was isolated with a synthetic 17-mer as a probe, was used to transform an am deletion mutant strain to prototrophy (7) by the procedure of Schweizer et al. (13).In this communication, we report the transformation of both deletion and point mutant strains with this clone, as well as with subclones containing the am gene, made in the plasmid vector pUC8 (15). By this technique, all sequences required for transformation of am auxotrophs to prototrophy and for the expression of GDH were shown to be contained on a 2.5-kb BamHI fragment of the original clone. This fragment has also recently been shown by DNA sequencing to contain all of the coding sequences of the am gene (Kinnaird and Fincham, personal communication).We also characterized several transformants obtained when am mutant strains are transformed with XC1O DNA. MATERIALS AND METHODSStrains used. The am mutant strains were from our stock collection and have been previously described (8, 12). The alcoy strain of Perkins et al. (11) was employed in preliminary mapping of transformed am genes. The bacterial strain JM83 and plasmid vector pUC8 (15) were obtained from Bethesda Research Laboratories, Inc.Construction of plasmid subclones. Figure 1 shows the N. crassa DNA insert for each of the constructed subclones as well as a restriction m...

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“…Complementation of an excess of am with am 19 results in an activated hybrid enzyme with normal electrophoretic properties. If the proportions of subunits are varied so that am 19 is in excess, the hybrid enzyme is electrophoretically abnormal and is only gradually activated (66). Recently, the mutation in am 9 was shown to be the substitution of a methionine for lysine (67,68).…”

Section: Glutamic Dehydrogenasementioning

confidence: 99%

Protein Complementation

Zabin¹,

Villarejo²

1975

Annu. Rev. Biochem.

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Multiple active varieties of Neurospora glutamate dehydrogenase formed by hybridization between two inactive mutant proteins in vivo and in vitro

Cited by 52 publications

References 14 publications

The Neurospora am gene and NADP-specific glutamate dehydrogenase: mutational sequence changes and functional effects – more mutants and a summary

The Neurospora am gene and NADP-specific glutamate dehydrogenase: mutational sequence changes and functional effects – more mutants and a summary

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

Protein Complementation

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