A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.
Hildur V. Colot,Gyungsoon Park,Gloria E. Turner,Carol S. Ringelberg,Christopher M. Crew,Liubov Litvinkova,Richard L. Weiss,Katherine A. Borkovich,Jay C. Dunlap +8 more
TL;DR: This study describes a method for rapidly creating knockout mutants in which it makes use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics.
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Abstract: The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.
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Neurospora crassa: looking back and looking forward at a model microbe.
TL;DR: The origins of Neurospora as a model microbe, the immediate scientific impacts from work in this filamentous fungus, and how the introduction of other model organisms redirected the focus of neurospora research are discussed.
Rediscovery by Whole Genome Sequencing: Classical Mutations and Genome Polymorphisms in Neurospora crassa
Kevin McCluskey,Aric Wiest,Igor V. Grigoriev,Anna Lipzen,Joel Martin,Wendy Schackwitz,Scott E. Baker,Scott E. Baker +7 more
TL;DR: Although some strains carry multiple unique nonsynonymous, nonsense, or frameshift mutations, the combined power of limiting the scope of the search based on genetic markers and of using a comparative analysis among the eighteen genomes provides strong support for the association between mutation and phenotype.
Allorecognition upon Fungal Cell-Cell Contact Determines Social Cooperation and Impacts the Acquisition of Multicellularity.
A. Pedro Gonçalves,Jens Heller,Jens Heller,Elise A. Span,Gabriel Rosenfield,Hung P. Do,Javier Palma-Guerrero,Natalia Requena,Michael A. Marletta,Michael A. Marletta,N. Louise Glass,N. Louise Glass +11 more
TL;DR: An allorecognition surveillance system based on kind discrimination that inhibits cooperative behavior in fungi by blocking cell fusion upon contact is revealed, contributing to fungal immunity by preventing formation of chimeras between genetically non-identical colonies.
A Nonhomologous End-Joining Mutant for Neurospora sitophila Research
TL;DR: This paper showed that deletion of N. sitophila mus-51 increases the efficiency of targeted-transgene integration, presumably by disrupting NHEJ in a second species of Neurospora crassa.
Identification of allorecognition loci in Neurospora crassa by genomics and evolutionary approaches
Jiuhai Zhao,Pierre Gladieux,Elizabeth A. Hutchison,Joanna A Bueche,Charles Hall,Fanny Perraudeau,N. Louise Glass +6 more
TL;DR: The evolutionary history of candidate het loci in the filamentous fungus Neurospora crassa are identified, characterized, and inferred, representing a compelling case of balancing selection functioning on multiple alleles across multiple loci potentially involved in allorecognition.
References
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Stephen F. Altschul,Thomas L. Madden,Alejandro A. Schäffer,Jinghui Zhang,Zheng Zhang,Webb Miller,David J. Lipman +6 more
TL;DR: A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original.
Functional profiling of the Saccharomyces cerevisiae genome.
Guri Giaever,Angela M. Chu,Li Ni,Carla Connelly,Linda Riles,Steeve Veronneau,Sally Dow,Ankuta Lucau-Danila,Keith Anderson,Bruno André,Adam P. Arkin,Anna Astromoff,Mohamed El Bakkoury,Rhonda Bangham,Rocío Benito,Sophie Brachat,Stefano Campanaro,Matt Curtiss,Karen Davis,Adam M. Deutschbauer,K. D. Entian,Patrick Flaherty,Françoise Foury,David J. Garfinkel,Mark Gerstein,Deanna Gotte,Ulrich Güldener,Johannes H. Hegemann,Svenja Hempel,Zelek S. Herman,Daniel F. Jaramillo,Diane E. Kelly,Steven L. Kelly,Peter Kötter,Darlene LaBonte,David C. Lamb,Ning Lan,Hong Liang,Hong Liao,Lucy Y. Liu,Chuanyun Luo,Marc Lussier,Rong Mao,Patrice Menard,Siew Loon Ooi,José L. Revuelta,Christopher J. Roberts,Matthias Rose,Petra Ross-Macdonald,Bart Scherens,Greg Schimmack,Brenda Shafer,Daniel D. Shoemaker,Sharon Sookhai-Mahadeo,Reginald Storms,Jeffrey N. Strathern,Giorgio Valle,Marleen Voet,Guido Volckaert,Ching Yun Wang,Teresa R. Ward,Julie Wilhelmy,Elizabeth A. Winzeler,Yonghong Yang,Grace Yen,Elaine M. Youngman,Kexin Yu,Howard Bussey,Jef D. Boeke,Michael Snyder,Peter Philippsen,Ronald W. Davis,Mark Johnston +72 more
TL;DR: It is shown that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment, and less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal Growth in four of the tested conditions.
Functional Characterization of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis
Elizabeth A. Winzeler,Daniel D. Shoemaker,Anna Astromoff,Hong Liang,Keith Anderson,Bruno André,Rhonda Bangham,Rocío Benito,Jef D. Boeke,Howard Bussey,Angela M. Chu,Carla Connelly,Karen Davis,Fred S. Dietrich,Sally Dow,Mohamed El Bakkoury,Françoise Foury,Stephen H. Friend,Erik Gentalen,Guri Giaever,Johannes H. Hegemann,Ted Jones,Michael T. Laub,Hong Liao,Nicole Liebundguth,David J. Lockhart,Anca Lucau-Danila,Marc Lussier,Nasiha M'Rabet,Patrice Menard,Michael Mittmann,Chai Pai,Corinne Rebischung,José L. Revuelta,Linda Riles,Christopher J. Roberts,Petra Ross-Macdonald,Bart Scherens,Michael Snyder,Sharon Sookhai-Mahadeo,Reginald Storms,Steeve Veronneau,Marleen Voet,Guido Volckaert,Teresa R. Ward,Robert W. Wysocki,Grace Yen,Kexin Yu,Katja Zimmermann,Peter Philippsen,Mark Johnston,Ronald W. Davis +51 more
TL;DR: A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome), finding that 17 percent were essential for viability in rich medium.
Molecular Bases for Circadian Clocks
TL;DR: It used to be that research in chronobiology moved biochemical functions [transcriptional activators], the along at a gentlemanly pace, but by mid 1997 the word in determining what the authors perceive as time was PASWCCLK.
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The genome sequence of the filamentous fungus Neurospora crassa
James E. Galagan,Sarah E. Calvo,Katherine A. Borkovich,Eric U. Selker,Nick O. Read,David B. Jaffe,William Fitzhugh,Li-Jun Ma,Serge Smirnov,Seth Purcell,Bushra Rehman,Timothy Elkins,Reinhard Engels,Shunguang Wang,Cydney B. Nielsen,Jonathan Butler,Matthew G. Endrizzi,Dayong Qui,Peter Ianakiev,Deborah Bell-Pedersen,Mary Anne Nelson,Margaret Werner-Washburne,Claude P. Selitrennikoff,John A. Kinsey,Edward L. Braun,Alex Zelter,Alex Zelter,Ulrich Schulte,Gregory O. Kothe,Gregory Jedd,Werner Mewes,Chuck Staben,Edward M. Marcotte,David Greenberg,Alice Roy,Karen Foley,Jerome Naylor,Nicole Stange-Thomann,Robert Barrett,Sante Gnerre,Michael Kamal,Manolis Kamvysselis,Evan Mauceli,Cord Bielke,Stephen Rudd,Dmitrij Frishman,Svetlana Krystofova,Carolyn G. Rasmussen,Robert L. Metzenberg,David D. Perkins,Scott Kroken,Carlo Cogoni,Giuseppe Macino,David E. A. Catcheside,Weixi Li,Robert J. Pratt,Stephen A. Osmani,Colin P.C. DeSouza,Louise Glass,Marc J. Orbach,J. Andrew Berglund,Rodger B. Voelker,Oded Yarden,Michael Plamann,Stephan Seiler,Jay C. Dunlap,Alan Radford,Rodolfo Aramayo,Donald O. Natvig,Lisa A. Alex,Gertrud Mannhaupt,Daniel J. Ebbole,Michael Freitag,Ian T. Paulsen,Matthew S. Sachs,Eric S. Lander,Chad Nusbaum,Bruce W. Birren +77 more
TL;DR: A high-quality draft sequence of the N. crassa genome is reported, suggesting that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes.
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