DNA sequence duplications trigger gene inactivation in Neurospora crassa.

Selker, Eric U.; Garrett, Philip W.

doi:10.1073/pnas.85.18.6870

Cited by 223 publications

(138 citation statements)

References 11 publications

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“…An ideal host strain should be deleted or severely disrupted for the gene in question to prevent obtaining a wild-type gene by homologous recombination and to eliminate any possible interference by mutant proteins present in a simple missense mutant. The repeatinduced point mutation (Rip) procedure (Selker and Garrett, 1988) was used to obtain a completely disrupted nit-2 gene. In N. crassa, when two or more copies of a DNA sequence are present, both copies will be subjected to multiple GC to AT mutations with a high frequency during meiosis, resulting in extensive damage to the endogenous gene.…”

Section: Creation Of a Nit-2 Rip Mutantmentioning

confidence: 99%

“…nit-2 gene disruption by repeat-induced point mutation A 6 kb fragment containing the entire coding region and promoter of nit-2 was obtained from plasmid pNit2 (Fu and Marzluf, 1990b) and subcloned into the pDE vector in order to use the rip phenomenon to disrupt this gene (Selker and Garrett, 1988). The resulting vector, pDN2, was transformed into Neurospora strain Y234M723 to obtain strain T2, which contained two copies of the nit-2 gene.…”

Section: Dna Manipulationsmentioning

confidence: 99%

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Two distinct protein–protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa

Pan

Feng

Marzluf

1997

Molecular Microbiology

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SummaryNitrogen metabolism is a highly regulated process in Neurospora crassa. The structural genes that encode nitrogen catabolic enzymes are subject to nitrogen metabolite repression, mediated by the positive-acting NIT2 protein and by the negative-acting NMR protein. NIT2, a globally acting factor, is a member of the GATA family of regulatory proteins and has a single Cys 2 / Cys 2 zinc finger DNA-binding domain. The negativeacting NMR protein interacts via specific proteinprotein binding with two distinct regions of the NIT2 protein, a short alpha-helical motif within the NIT2 DNA-binding domain and a second motif at its carboxy terminus. Deletions of segments of NIT2 throughout most of its length result in truncated proteins, which are still functional for activating gene expression; most of these mutant NIT2 proteins still allow proper nitrogen repression of nitrate reductase synthesis. In contrast, deletions or certain amino acid substitutions within the zinc finger and the carboxy-terminal tail result in a loss of nitrogen metabolite repression. Those mutated forms of NIT2 that are insensitive to nitrogen repression have also lost one of the NIT2-NMR protein-protein interactions. These results provide compelling evidence that the specific NIT2-NMR interactions have a regulatory function and play a central role in establishing nitrogen metabolite repression.

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Section: Creation Of a Nit-2 Rip Mutantmentioning

confidence: 99%

Section: Dna Manipulationsmentioning

confidence: 99%

Two distinct protein–protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa

Pan

Feng

Marzluf

1997

Molecular Microbiology

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“…The N. crassa nit-3 mutant strain 14789A carries a point mutation in the 3Ј end of the nit-3 proteincoding region (16) and could undergo homologous recombination with the transforming mutant nit-3 gene and therefore was not suitable as the host for mutant promoter analysis. RIP was used to obtain a mutant strain damaged throughout the entire promoter and coding regions of the nit-3 gene (18). Furthermore, to avoid the position effects when transforming DNAs integrate at various ectopic locations in the genome, it is preferable to target each construct to the same genomic location.…”

Section: Methodsmentioning

confidence: 99%

Binding affinity and functional significance of NIT2 and NIT4 binding sites in the promoter of the highly regulated nit-3 gene, which encodes nitrate reductase in Neurospora crassa

Chiang

Marzluf

1995

J Bacteriol

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In the filamentous fungus Neurospora crassa, both the global-acting regulatory protein NIT2 and the pathway-specific regulatory protein NIT4 are required to turn on the expression of the nit-3 gene, which encodes nitrate reductase, the first enzyme in the nitrate assimilatory pathway. Three NIT2 binding sites and two NIT4 binding sites have been identified in the 1.3-kb nit-3 promoter region via mobility shift and footprinting experiments with NIT2-␤-galactosidase and NIT4-␤-Gactosidase fusion proteins. Quantitative mobility shift assays were used to examine the affinity of individual NIT2 binding sites for the native NIT2 protein present in N. crassa nuclear extracts. In vivo analysis of nit-3 promoter 5 deletion constructs and individual NIT2 and NIT4 binding-site deletions or mutations revealed that all of the NIT2 and NIT4 binding sites are required for the full level of expression of the nit-3 gene. A cluster of two NIT2 and two NIT4 binding sites located more than 1 kb upstream of the translational start site is required for nit-3 expression, and one NIT2 binding site and one NIT4 site, which are immediately adjacent to each other, are of particular functional importance. A significant NIT2-NIT4 protein-protein interaction might occur upon their binding to nearby sites.The filamentous fungus Neurospora crassa is able to utilize secondary nitrogen sources such as nitrate, purines, and various amino acids when preferred primary nitrogen sources such as ammonia, glutamine, or glutamate are not available (14,15). A series of unlinked structural genes which encode various nitrogen catabolic enzymes must be turned on by global-acting as well as pathway-specific regulatory factors for secondary nitrogen source assimilation. When nitrate is present as the sole nitrogen source, nitrogen catabolic repression is lifted, and nitrate serves as an inducer for expression of nitrate assimilation pathway genes, nit-3 and nit-6, which encode nitrate reductase and nitrite reductase, respectively. Expression of nit-3 and nit-6 is turned on by the global positive-acting nitrogen regulatory factor NIT2 and the pathway-specific regulatory protein NIT4.NIT2 is a 110-kDa regulatory protein with a single Cys-X 2 -Cys-X 17 -Cys-X 2 -Cys-type zinc finger DNA binding motif (5, 6). Three NIT2 binding sites have been identified in the nit-3 promoter; one is located near the start site for the nit-3 gene, whereas the other two binding sites are far upstream, approximately 1 kb away from the nit-3 gene (7). NIT2 DNA binding is dependent on core GATA sequence elements (2). NIT4 is a 120-kDa positive-acting regulatory protein with a single GAL4-like Zn(II)Cys 6 -type zinc finger DNA binding motif (20). Two NIT4 binding sites have also been identified in the nit-3 promoter region (4). In addition to the proximal NIT2 binding site, a cluster of two NIT2 and two NIT4 binding sites is located far upstream of the nit-3 gene, as diagrammed in Fig. 1.In this study, we report the results of quantitative in vitro electrophoretic mobility shift assa...

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“…Many early studies focused on mutation and mutability (De Serres & Kolmark 1958; De Serres & Brockman 1999) and, for example, the reference genome strain is called “Oak Ridge” reflecting its origins as a model organism for the study of low-dose radiation at the Atomic Energy Commission (a predecessor to the current US Department of Energy) Oak Ridge National Laboratory (Kafer 1982). As a model for cell biology and physiology, Neurospora has allowed significant impact in understanding of genome defence through vegetative gene silencing (Fulci & Macino 2007), meiotic gene silencing (Shiu et al 2001; Shiu & Metzenberg 2002), mutation of repeated DNA during meiosis (Selker & Garrett 1988; Cambareri et al 1991), and overall epigenetic gene regulation (Cambareri et al 1996; Selker et al 2003; Aramayo & Selker 2013). As a model for molecular physiology, it has been one of the leading models for analysis of the endogenous circadian rhythm (Baker et al 2012).…”

Section: Introductionmentioning

confidence: 99%

“…While most transforming vectors for Neurospora integrated into the nuclear genome, a system for transformation based on autonomous replication in host mitochondria was developed (Stohl & Lambowitz 1983). Neurospora researchers were able to generate targeted mutation of genes by use of the endogenous Repeat Induced Point Mutation process (Selker & Garrett 1988; Cambareri et al 1991). Other targeting systems, including targeting to the his-3 locus (Aramayo & Metzenberg 1996), overcame some of the problems with multiple integrations found with earlier systems.…”

Section: Introductionmentioning

confidence: 99%

Diverse data supports the transition of filamentous fungal model organisms into the post-genomics era

McCluskey

Baker

2017

Mycology

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Filamentous fungi have been important as model organisms since the beginning of modern biological inquiry and have benefitted from open data since the earliest genetic maps were shared. From early origins in simple Mendelian genetics of mating types, parasexual genetics of colony colour, and the foundational demonstration of the segregation of a nutritional requirement, the contribution of research systems utilising filamentous fungi has spanned the biochemical genetics era, through the molecular genetics era, and now are at the very foundation of diverse omics approaches to research and development. Fungal model organisms have come from most major taxonomic groups although Ascomycete filamentous fungi have seen the most major sustained effort. In addition to the published material about filamentous fungi, shared molecular tools have found application in every area of fungal biology. Similarly, shared data has contributed to the success of model systems. The scale of data supporting research with filamentous fungi has grown by 10 to 12 orders of magnitude. From genetic to molecular maps, expression databases, and finally genome resources, the open and collaborative nature of the research communities has assured that the rising tide of data has lifted all of the research systems together.

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DNA sequence duplications trigger gene inactivation in Neurospora crassa.

Cited by 223 publications

References 11 publications

Two distinct protein–protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa

Two distinct protein–protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa

Binding affinity and functional significance of NIT2 and NIT4 binding sites in the promoter of the highly regulated nit-3 gene, which encodes nitrate reductase in Neurospora crassa

Diverse data supports the transition of filamentous fungal model organisms into the post-genomics era

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