THE ISOLATION AND CHARACTERIZATION OF MUTANTS DEFECTIVE IN NITRATE ASSIMILATION IN NEUROSPORA CRASSA

ABSTRACT The isolation and characterization of mutants altered for nitrate assimilation in Neurospora crassa is described, The mutants isolated can be subdivided into five classes on the basis of growth tests that correspond to the growth patterns of existing mutants at six distinct loci. Mutants with growth characteristics like those of nit-2, nit-3 and nit-6 are assigned to those loci on the basis of noncomplementation and lack of recombination. Mutants that, from their growth patterns, appear to lack the molybdenum-containing cofactor for both nitrate reductase and xanthine dehydrogenase subdivide into three loci (nit-7, nit4 and nit-9), all of which are genetically distinct from nit-1. nit-9 is a complex locus consisting of three complementation groups and thus appears similar to the cnxABC locus of Asperillus nidulans. Extensive complementational and recombinational analyses reveal that nit-4 and nit-5 are alleles of the same locus, and two new alleles of that locus have been isolated. The results indicate that, as in A. nidulans, nitrate assimilation in N. crassa requires at least four loci (nit-1,7,8 and 9) to produce the molybdenum co-factor for nitrate reductase (and xanthine dehydrogenase), one locus (nit-3) to code for the nitrate reductase apoprotein, one locus (nit-6) to code for the nitrite reductase approtein and only one locus (nit-4/5) for the regulation of induction of the pathway by nitrate and nitrite.

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Biochemical Characterization of Molybdenum Cofactor-free Nitrate Reductase from Neurospora crassa

Journal of Biological Chemistry ◽

10.1074/jbc.m113.457960 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14657-14671 ◽

Cited By ~ 12

Author(s):

Phillip Ringel ◽

Joern Krausze ◽

Joop van den Heuvel ◽

Ute Curth ◽

Antonio J. Pierik ◽

...

Keyword(s):

Nitrate Reductase ◽

Neurospora Crassa ◽

Biochemical Characterization ◽

Nitrate Assimilation ◽

Molybdenum Cofactor ◽

Site Formation ◽

Epr Spectrum ◽

Redox Active

Nitrate reductase (NR) is a complex molybdenum cofactor (Moco)-dependent homodimeric metalloenzyme that is vitally important for autotrophic organism as it catalyzes the first and rate-limiting step of nitrate assimilation. Beside Moco, eukaryotic NR also binds FAD and heme as additional redox active cofactors, and these are involved in electron transfer from NAD(P)H to the enzyme molybdenum center where reduction of nitrate to nitrite takes place. We report the first biochemical characterization of a Moco-free eukaryotic NR from the fungus Neurospora crassa, documenting that Moco is necessary and sufficient to induce dimer formation. The molybdenum center of NR reconstituted in vitro from apo-NR and Moco showed an EPR spectrum identical to holo-NR. Analysis of mutants unable to bind heme or FAD revealed that insertion of Moco into NR occurs independent from the insertion of any other NR redox cofactor. Furthermore, we showed that at least in vitro the active site formation of NR is an autonomous process.

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THE CONTROL OF ALDEHYDE OXIDASE AND XANTHINE DEHYDROGENASE ACTIVITIES BY THE CINNAMON GENE IN DROSOPHILA MELANOGASTER

Canadian Journal of Genetics and Cytology ◽

10.1139/g79-051 ◽

1979 ◽

Vol 21 (4) ◽

pp. 457-471 ◽

Cited By ~ 22

Author(s):

Michael M. Bentley ◽

John H. Williamson

Keyword(s):

Drosophila Melanogaster ◽

Aldehyde Oxidase ◽

Heat Stability ◽

Xanthine Dehydrogenase ◽

Wild Type ◽

Eye Color ◽

Isolation And Characterization ◽

Heat Labile ◽

Complementation Groups

The isolation and characterization of 16 alleles of the cinnamon (cin, 1-0.0) locus in Drosophila melanogaster are described. The effects of cin on viability and the maternal effect of cin+ on eye color have been separated from each other as well as from the deficiency for aldehyde oxidase (AO) and xanthine dehydrogenase (XDH) activities. These 16 alleles have been assigned to four complementation groups based on analysis of AO and XDH activities in all heteroallelic female combinations. Zygotic complementation for lethality and eye color has been characterized and allows the ordering of cin alleles in a consistent pattern for the ability to produce viable zygotes and/or complement for the eye color phene. Several complementing cin combinations were analyzed for heat stability of AO. In all cases, AO from allelic heterozygotes was more heat labile than wild-type AO. One cin allele, cin13, produces heat labile AO in combination with cin+ from Oregon-R, hence exhibiting a "dominant" heat stability phenotype.

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