scholarly journals High-Throughput Production of Gene Replacement Mutants in Neurospora crassa

2011 ◽  
pp. 179-189 ◽  
Author(s):  
Gyungsoon Park ◽  
Hildur V. Colot ◽  
Patrick D. Collopy ◽  
Svetlana Krystofova ◽  
Christopher Crew ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
High Throughput ◽  
Gene Replacement

A targeted-replacement system for identification of signals for de novo methylation in Neurospora crassa

1994 ◽  
Vol 14 (11) ◽  
pp. 7059-7067
Author(s):  
V P Miao ◽  
M J Singer ◽  
M R Rountree ◽  
E U Selker
Keyword(s):  
Dna Methylation ◽  
Neurospora Crassa ◽  
De Novo ◽  
Gene Replacement ◽  
Position Effects ◽  
Efficient Gene ◽  
Bona Fide ◽  
Replacement System ◽  
Methylation Signal ◽  
De Novo Methylation

Transformation of eukaryotic cells can be used to test potential signals for DNA methylation. This approach is not always reliable, however, because of chromosomal position effects and because integration of multiple and/or rearranged copies of transforming DNA can influence DNA methylation. We developed a robust system to evaluate the potential of DNA fragments to function as signals for de novo methylation in Neurospora crassa. The requirements of the system were (i) a location in the N. crassa genome that becomes methylated only in the presence of a bona fide methylation signal and (ii) an efficient gene replacement protocol. We report here that the am locus fulfills these requirements, and we demonstrate its utility with the identification of a 2.7-kb fragment from the psi 63 locus as a new portable signal for de novo methylation.

scholarly journals High-Throughput Construction of Gene Deletion Cassettes for Generation of Neurospora crassa Knockout Strains

2010 ◽  
pp. 33-40 ◽  
Author(s):  
Patrick D. Collopy ◽  
Hildur V. Colot ◽  
Gyungsoon Park ◽  
Carol Ringelberg ◽  
Christopher M. Crew ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
High Throughput ◽  
Gene Deletion

scholarly journals Osmoregulation and Fungicide Resistance: the Neurospora crassa os-2 Gene Encodes a HOG1 Mitogen-Activated Protein Kinase Homologue

2002 ◽  
Vol 68 (2) ◽  
pp. 532-538 ◽  
Author(s):  
Yan Zhang ◽  
Randy Lamm ◽  
Christian Pillonel ◽  
Stephen Lam ◽  
Jin-Rong Xu
Keyword(s):  
Map Kinase ◽  
Neurospora Crassa ◽  
Fungicide Resistance ◽  
Point Mutations ◽  
Gene Replacement ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
High Osmolarity ◽  
Mitogen Activated Protein ◽  
Sib Selection

ABSTRACT Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway.

scholarly journals Use of gene replacement transformation to elucidate gene function in the qa gene cluster of Neurospora crassa.

Genetics ◽  
1992 ◽  
Vol 130 (4) ◽  
pp. 729-736
Author(s):  
M E Case ◽  
R F Geever ◽  
D K Asch
Keyword(s):  
Neurospora Crassa ◽  
Gene Cluster ◽  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Genetic Recombination ◽  
Constitutive Expression ◽  
Quinic Acid ◽  
Gene Replacement ◽  
Growth Characteristics ◽  
Repressor Molecule

Abstract Gene replacement by transformation, employing selective genetic recombination techniques, has been used to delete or disrupt the qa-x, qa-y and qa-1S genes of the qa gene cluster of Neurospora crassa. The growth characteristics of the strain carrying the deletion of the qa-y gene support earlier evidence that this gene encodes a quinic acid permease. The strain containing the deletion of the qa-1S gene (delta qa-1S) was examined with respect to quinic acid induction and carbon catabolite repression. The delta qa-1S strain exhibits constitutive expression of the qa genes supporting earlier evidence that the qa-1S gene codes for a repressor. Several of the qa genes continued to be expressed at high levels even in the presence of glucose in the delta qa-1S strain, which indicates that transcription of these genes is not being affected directly by a repressor molecule in the presence of glucose.

scholarly journals A targeted-replacement system for identification of signals for de novo methylation in Neurospora crassa.

1994 ◽  
Vol 14 (11) ◽  
pp. 7059-7067 ◽  
Author(s):  
V P Miao ◽  
M J Singer ◽  
M R Rountree ◽  
E U Selker
Keyword(s):  
Dna Methylation ◽  
Neurospora Crassa ◽  
De Novo ◽  
Gene Replacement ◽  
Position Effects ◽  
Efficient Gene ◽  
Bona Fide ◽  
Replacement System ◽  
Methylation Signal ◽  
De Novo Methylation

Transformation of eukaryotic cells can be used to test potential signals for DNA methylation. This approach is not always reliable, however, because of chromosomal position effects and because integration of multiple and/or rearranged copies of transforming DNA can influence DNA methylation. We developed a robust system to evaluate the potential of DNA fragments to function as signals for de novo methylation in Neurospora crassa. The requirements of the system were (i) a location in the N. crassa genome that becomes methylated only in the presence of a bona fide methylation signal and (ii) an efficient gene replacement protocol. We report here that the am locus fulfills these requirements, and we demonstrate its utility with the identification of a 2.7-kb fragment from the psi 63 locus as a new portable signal for de novo methylation.

scholarly journals A high-throughput, restriction-free cloning and screening strategy based on ccdB-gene replacement

2014 ◽  
Vol 13 (1) ◽  
pp. 38 ◽  
Author(s):  
Bjarte Lund ◽  
Hanna-Kirsti Leiros ◽  
Gro Elin Bjerga
Keyword(s):  
High Throughput ◽  
Gene Replacement ◽  
Screening Strategy ◽  
Ccdb Gene

scholarly journals Comparison and Validation of Putative Pathogenicity-Related Genes Identified by T-DNA Insertional Mutagenesis and Microarray Expression Profiling inMagnaporthe oryzae

2017 ◽  
Vol 2017 ◽  
pp. 1-7
Author(s):  
Ying Wang ◽  
Ying Wáng ◽  
Qi Tan ◽  
Ying Nv Gao ◽  
Yan Li ◽  
...  
Keyword(s):  
High Throughput ◽  
Expression Profiling ◽  
Insertional Mutagenesis ◽  
Target Gene ◽  
Gene Replacement ◽  
Knockout Mutants ◽  
Target Gene Replacement ◽  
Microarray Expression Profiling ◽  
Microarray Expression ◽  
Dna Insertional Mutagenesis

High-throughput technologies of functional genomics such as T-DNA insertional mutagenesis and microarray expression profiling have been employed to identify genes related to pathogenicity inMagnaporthe oryzae. However, validation of the functions of individual genes identified by these high-throughput approaches is laborious. In this study, we compared two published lists of genes putatively related to pathogenicity inM. oryzaeidentified by T-DNA insertional mutagenesis (comprising 1024 genes) and microarray expression profiling (comprising 236 genes), respectively, and then validated the functions of some overlapped genes between the two lists by knocking them out using the method of target gene replacement. Surprisingly, only 13 genes were overlapped between the two lists, and none of the four genes selected from the overlapped genes exhibited visible phenotypic changes on vegetative growth, asexual reproduction, and infection ability in their knockout mutants. Our results suggest that both of the lists might contain large proportions of unrelated genes to pathogenicity and therefore comparing the two gene lists is hardly helpful for the identification of genes that are more likely to be involved in pathogenicity as we initially expected.

scholarly journals Directed replacement of mt A by mt a-1 effects a mating type switch in Neurospora crassa.

Genetics ◽  
1994 ◽  
Vol 138 (1) ◽  
pp. 75-81 ◽  
Author(s):  
S Chang ◽  
C Staben
Keyword(s):  
Neurospora Crassa ◽  
Mating Type ◽  
Single Copy ◽  
Gene Replacement ◽  
Type Locus ◽  
Sexual Activities ◽  
Sexual Process ◽  
Type Switch ◽  
Type Gene ◽  
Necessary And Sufficient

Abstract To test the functions of a mating type genes, we developed an efficient strategy to select transformants of Neurospora crassa in which resident A mating type DNA was replaced by cloned DNA from the mt a idiomorph. Cloned a idiomorphic DNA could specify all functions, including fertility, of a mating type, but only when it replaced A DNA at the mating type locus. Only the mt a-1 region of the a idiomorph was necessary in order to specify a mating type. Gene replacement events involved the homologous sequences flanking the unique mating type idiomorphic DNA, resulting in apparently isogenic a and A strains. These isogenic strains were fertile when crossed with one another, indicating that no determinants outside the transforming DNA are necessary for fertility as a and that no host sequences of A strains interfere with fertility as a. One a replacement strain bore a duplication of the transforming mt a-1 and hph DNA. The duplication strain had unexpected properties. Although mating type segregated 1:1 in crosses of this strain to A, the duplicated regions were efficiently altered during the sexual process to generate a single copy in the progeny. No progeny were recovered that had undergone RIP (repeat induced point mutation) sufficient to inactivate the mt a-1 gene. We infer that the mt a-1 gene is necessary and sufficient to specify a mating type identity in all vegetative and sexual activities. Mt a-1 may also play an essential role in ascosporogenesis after fertilization.

scholarly journals Suppressors of Meiotic Silencing by Unpaired DNA

2019 ◽  
Vol 5 (1) ◽  
pp. 14 ◽  
Author(s):  
Hua Xiao ◽  
Thomas Hammond ◽  
Patrick Shiu
Keyword(s):  
Gene Silencing ◽  
Neurospora Crassa ◽  
High Throughput ◽  
High Throughput Screen ◽  
Meiotic Silencing ◽  
Single Pass

Meiotic silencing by unpaired DNA (MSUD) is a gene silencing process that occurs within meiotic cells of Neurospora crassa and other fungi. We have previously developed a high-throughput screen to identify suppressors of this silencing pathway. Here, a list of MSUD suppressor candidates from a single pass of the first 84 plates of the Neurospora knockout library is provided.

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