Heather M. Hood

Enabling a Community to Dissect an Organism: Overview of the Neurospora Functional Genomics Project

A consortium of investigators is engaged in a functional genomics project centered on the filamentous fungus Neurospora, with an eye to opening up the functional genomic analysis of all the filamentous fungi. The overall goal of the four interdependent projects in this effort is to accomplish functional genomics, annotation, and expression analyses of Neurospora crassa, a filamentous fungus that is an established model for the assemblage of over 250,000 species of non yeast fungi. Building from the completely sequenced 43-Mb Neurospora genome, Project 1 is pursuing the systematic disruption of genes through targeted gene replacements, phenotypic analysis of mutant strains, and their distribution to the scientific community at large. Project 2, through a primary focus in Annotation and Bioinformatics, has developed a platform for electronically capturing community feedback and data about the existing annotation, while building and maintaining a database to capture and display information about phenotypes. Oligonucleotide-based microarrays created in Project 3 are being used to collect baseline expression data for the nearly 11,000 distinguishable transcripts in Neurospora under various conditions of growth and development, and eventually to begin to analyze the global effects of loss of novel genes in strains created by Project 1. cDNA libraries generated in Project 4 document the overall complexity of expressed sequences in Neurospora, including alternative splicing alternative promoters and antisense transcripts. In addition, these studies have driven the assembly of an SNP map presently populated by nearly 300 markers that will greatly accelerate the positional cloning of genes.

Genetic association of a GABAA receptor γ2 subunit variant with severity of acute physiological dependence on alcohol

Abstract. The ultimate goal of quantitative trait locus (QTL) mapping is to identify the genes affecting complex traits. Using animal models, we recently identified QTLs on mouse Chromosomes (Chrs) 1, 4, and 11 affecting genetic predisposition to acute alcohol withdrawal. Among mice derived from the C57BL/6J (B6) and DBA/2J (D2) inbred strains, the locus identified on Chr 11 (∼20 cM) accounted for 12% of the genetic variability in withdrawal liability. Candidate genes in proximity to this QTL encode the γ2, α1, and α6 subunits of GABAA receptors. The present studies identify a polymorphism between the B6 and D2 strains in the γ2 subunit gene, Gabrg2, and expand genotypic analysis to their BXD recombinant inbred strains. This polymorphism predicts a difference in amino acid sequence (Ala-11 vs. Thr-11) within the extracellular amino-terminal region of the γ2 subunit. Analysis using BXD strain means for acute alcohol withdrawal severity suggests that the γ2 subunit polymorphism is genetically correlated with alcohol withdrawal severity. This is the first report that QTL mapping for an alcohol-related trait has successfully led to the identification of a polymorphic candidate gene product that is genetically associated with the trait.

Characterization of Chromosome Ends in the Filamentous Fungus Neurospora crassa

Telomeres and subtelomere regions have vital roles in cellular homeostasis and can facilitate niche adaptation. However, information on telomere/subtelomere structure is still limited to a small number of organisms. Prior to initiation of this project, the Neurospora crassa genome assembly contained only 3 of the 14 telomeres. The missing telomeres were identified through bioinformatic mining of raw sequence data from the genome project and from clones in new cosmid and plasmid libraries. Their chromosomal locations were assigned on the basis of paired-end read information and/or by RFLP mapping. One telomere is attached to the ribosomal repeat array. The remaining chromosome ends have atypical structures in that they lack distinct subtelomere domains or other sequence features that are associated with telomeres in other organisms. Many of the chromosome ends terminate in highly AT-rich sequences that appear to be products of repeat-induced point mutation, although most are not currently repeated sequences. Several chromosome termini in the standard Oak Ridge wild-type strain were compared to their counterparts in an exotic wild type, Mauriceville. This revealed that the sequences immediately adjacent to the telomeres are usually genome specific. Finally, despite the absence of many features typically found in the telomere regions of other organisms, the Neurospora chromosome termini still retain the dynamic nature that is characteristic of chromosome ends.

Genomewide Search for Epistasis in a Complex Trait: Pentobarbital Withdrawal Convulsions in Mice

The well-documented difference in pentobarbital withdrawal severity between DBA/2J and C57BL/6J mice offers the opportunity to study how differences between allelic variants influence pentobarbital withdrawal via their additive and/or dominance effects and to identify modifier loci that also influence the trait via gene-gene interactions (a form of epistasis). Previous work in our laboratory identified seven provisional quantitative trait loci (QTLs) for pentobarbital withdrawal using BXD recombinant inbred strains. To date, only one of these QTLs has been confirmed, Pbwl. We hypothesized that other loci that act epistatically may also influence genetic variance in pentobarbital withdrawal severity. Using Epistat, a program developed to carry out full-genome searches for epistasis, we identified six provisional epistatic interactions (p < .002) between the provisional QTLs and modifier loci elsewhere in the genome. Verification testing of these interactions using 404 B6D2F2 mice provided supporting evidence that a QTL on chromosome 11 contributes to genetic variance in pentobarbital withdrawal, but only in the presence of a modifier allele on distal chromosome 1 (p = .0004). This modifier is in the same genomic vicinity as loci detected for a variety of withdrawal and seizure phenotypes.

Reconstruction and Validation of a Genome-Scale Metabolic Model for the Filamentous Fungus Neurospora crassa Using FARM

The filamentous fungus Neurospora crassa played a central role in the development of twentieth-century genetics, biochemistry and molecular biology, and continues to serve as a model organism for eukaryotic biology. Here, we have reconstructed a genome-scale model of its metabolism. This model consists of 836 metabolic genes, 257 pathways, 6 cellular compartments, and is supported by extensive manual curation of 491 literature citations. To aid our reconstruction, we developed three optimization-based algorithms, which together comprise Fast Automated Reconstruction of Metabolism (FARM). These algorithms are: LInear MEtabolite Dilution Flux Balance Analysis (limed-FBA), which predicts flux while linearly accounting for metabolite dilution; One-step functional Pruning (OnePrune), which removes blocked reactions with a single compact linear program; and Consistent Reproduction Of growth/no-growth Phenotype (CROP), which reconciles differences between in silico and experimental gene essentiality faster than previous approaches. Against an independent test set of more than 300 essential/non-essential genes that were not used to train the model, the model displays 93% sensitivity and specificity. We also used the model to simulate the biochemical genetics experiments originally performed on Neurospora by comprehensively predicting nutrient rescue of essential genes and synthetic lethal interactions, and we provide detailed pathway-based mechanistic explanations of our predictions. Our model provides a reliable computational framework for the integration and interpretation of ongoing experimental efforts in Neurospora, and we anticipate that our methods will substantially reduce the manual effort required to develop high-quality genome-scale metabolic models for other organisms.

Fine mapping of a sedative-hypnotic drug withdrawal locus on mouse chromosome 11

We have established that there is a considerable amount of common genetic influence on physiological dependence and associated withdrawal from sedative‐hypnotic drugs including alcohol, benzodiazepines, barbiturates and inhalants. We previously mapped two loci responsible for 12 and 9% of the genetic variance in acute alcohol and pentobarbital withdrawal convulsion liability in mice, respectively, to an approximately 28‐cM interval of proximal chromosome 11. Here, we narrow the position of these two loci to a 3‐cM interval (8.8 Mb, containing 34 known and predicted genes) using haplotype analysis. These include genes encoding four subunits of the GABAA receptor, which is implicated as a pivotal component in sedative‐hypnotic dependence and withdrawal. We report that the DBA/2J mouse strain, which exhibits severe withdrawal from sedative‐hypnotic drugs, encodes a unique GABAA receptor γ2 subunit variant compared with other standard inbred strains including the genetically similar DBA/1J strain. We also demonstrate that withdrawal from zolpidem, a benzodiazepine receptor agonist selective for α1 subunit containing GABAA receptors, is influenced by a chromosome 11 locus, suggesting that the same locus (gene) influences risk of alcohol, benzodiazepine and barbiturate withdrawal. Our results, together with recent knockout studies, point to the GABAA receptor γ2 subunit gene (Gabrg2) as a promising candidate gene to underlie phenotypic differences in sedative‐hypnotic physiological dependence and associated withdrawal episodes.

Recommendations for assigning symbols and names to Neurospora crassa genes now that its genome has been sequenced.

Originally, Neurospora crassa genes were named for their mutant phenotypes or natural variant properties. Genes are now increasingly named on the basis of cross-species sequence similarity. These names may also be supported by predicted or experimentally identified molecular function. As a consequence, N. crassa gene nomenclature in practice is frequently no longer adequately covered by the established conventions (Perkins et al. 2001). Here we provide additional nomenclature guidelines relevant to these new circumstances, and some general guidelines for providing information on the identity of N. crassa genes in scientific communications. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol55/iss1/7 32 Fungal Genetics Reports The Neurospora crassa colonial temperature sensitive 2, 4 and 5 (cot-2, cot-4 and cot-5) genes encode regulatory and structural proteins required for hyphal elongation and branching Zipi Resheat-Eini, Alex Zelter, Rena Gorovits, Nick D. Read and Oded Yarden* Department of Plant Pathology and Microbiology, The Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, 76100, Israel and Fungal Cell Biology Group, Institute of Cell and Molecular Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, UK. *corresponding author, email: Oded.Yarden@huji.ac.il Fungal Genetics Reports 55:32-36 The morphology and the genetic defects of the Neurospora crassa colonial temperature-sensitive-2, -4 and -5 mutants were analyzed. cot-2 is allelic to gs-1 and encodes a component of the glucan synthesis process. cot-4 encodes the catalytic subunit of a type 2B phosphatase and is allelic to calcineurin (cna-1). cot-5 encodes a homologue of the S. cerevisiae ALG2 manosyltransferase-encoding gene, a component of the dolichol pathway. A group of five non-allelic Neurospora crassa colonial temperature sensitive (cot) mutants was described by Garnjobst and Tatum (1967). The cot-1 gene was found to encode a Ser/Thr protein kinase (Yarden et al. 1992) which is the founding member of the NDR kinase family. The nature of the cot-3 defect has also been analyzed and the cot-3 gene was found to encode protein elongation factor 2 (Propheta et al. 2001). In order to expand our understanding of the genetic defects that can confer abnormal hyphal elongation/branching patterns, we have performed morphological and genetic analyses of the three remaining cot mutants isolated by Garnjobst and Tatum. We found that even though they all exhibit compact temperaturesensitive macroscopic colonial features, their microscopic hyphal morphology and branching patterns differ. Furthermore, the genetic defects involved in conferring their phenotypes include both regulatory as well as structural factors, all of which are required for maintaining proper hyphal elongation and branching patterns. Confocal microscopic examination, using the membrane-selective dye FM4-64 (as described by Hickey et al. 2005) of Neurospora crassa wild-type (74-OR23-1A; FGSC987), cot-1 (FGSC 4065), cot-2 (FGSC 1512), cot-3 (FGSC 1517), cot4 (FGSC 3600) and cot-5 (FGSC 1362), revealed significant morphological differences between the different strains (Fig. 1). As the morphological features of cot-1 and cot-3 have been studied in depth (Collinge and Trinci, 1974; Collinge et al. 1978; Propheta et al. 2001), we focused on the quantification of the observed differences on cot-2, cot-4 and cot-5. Hyphal extension rates of cot-2 cot-4 and cot-5 were measured on a standard solid medium at permissive and restrictive conditions. All of the mutants exhibited a significant reduction (75 to 99%) in elongation rates (to 0.15±0.03 mm/h, 0.27±0.05 mm/h and negligible elongation for cot-2, cot-4 and cot-5, respectively) and an increase in branching rates when cultured at the restrictive temperature. Even though the mutants phenotypes are clearly temperature sensitive, we found that their branching rates were significantly higher (60 to 160%) even at the permissive temperatures (Table 1). For the most part, the hyperbranching patterns observed are lateral, rather than apical (Watters et al. 2000). Nonetheless, some apical/dichotomous branching was evident in the cot-5 strain (regardless of temperature; Fig. 1j-k). Figure 1. Morphology of wild type and colonial temperature sensitive strains of N. crassa at permissive (24 C) and restrictive (37 C) temperatures. Fungi were stained with FM4-64 and imaged using a confocal microscope. (a) cot-1 grown at 24 C; (b) cot-1, 37 C; (c) wild type, 37 C; (d) cot-2, 24 C; (e) cot-2, 37 C; (f) cot-3, 24 C; (g) cot-3, 37 C; (h) cot4, 24 C; (i) cot-4, 37 C; (j) cot-5, 24 C; (k) cot-5, 37 C. Bars are 50 um. Published by New Prairie Press, 2017