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The art and design of genetic screens: filamentous fungi

Nature Reviews Genetics volume 3, pages 683–697 (2002)Cite this article

Key Points

  • Filamentous fungi represent a billion years of evolutionary divergence and show a developmental complexity that allows the design of new screens.

  • The relationship between genes and proteins was first clearly established using auxotrophic mutants of Neurospora crassa.

  • The fungi that have been most useful for genetic analysis are haploid and reproduce asexually, as well as sexually.

  • Methods used to mutagenize fungi include radiation, chemicals, and plasmid or transposon insertion.

  • Growth over a wide temperature range allows isolation of temperature-sensitive mutants with mutations in essential genes.

  • The filamentous hypha and the regular distribution of its nuclei lends itself to screens for genes that are required for mitosis and nuclear migration.

  • Fungi develop a wide range of multicellular structures, composed of several cell types, for vegetative growth, asexual and sexual reproduction. These lend themselves to visual screens for genes that are involved in many developmental pathways. Mutants that are unable to produce sexual spores are particularly relevant to studies on meiosis.

  • N. crassa is an important model for studying circadian rhythm. Mutants can be identified by a race-tube assay in which the distance between bands of conidiospores is a measure of circadian day length.

  • Quelling is a post-transcriptional gene-silencing mechanism that is analogous to co-suppression in plants and RNA interference in Caenorhabditis elegans. Quelling-defective mutants are selected in N. crassa using a conidiospore colour assay.

  • Genes are generally cloned using transformation to screen available genomic libraries for sequences that complement a mutation of interest. Techniques for insertional mutagenesis have been developed that allow recovery of genes tagged by the inserted sequence.

Abstract

In the 1940s, screens for metabolic mutants of the filamentous fungus Neurospora crassa established the fundamental, one-to-one relationship between a gene and a specific protein, and also established fungi as important genetic organisms. Today, a wide range of filamentous species, which represents a billion years of evolutionary divergence, is used for experimental studies. The developmental complexity of these fungi sets them apart from unicellular yeasts, and allows the development of new screens that enable us to address biological questions that are relevant to all eukaryotes.

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Figure 1: Morphological variation of filamentous fungi.
Figure 2: ropy mutants of Neurospora crassa.
Figure 3: Race-tube assay to detect circadian rhythm mutants in Neurospora crassa.
Figure 4: Fluorescence in situ hybridization analysis of meiotic mutants of Coprinus cinereus.

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Acknowledgements

We thank numerous colleagues for figures, preprints and advice, and we apologize to those whose work was not cited due to the breadth of this topic and our space limitations. We thank B. Metzenberg, R. Davis, R. Morris, J. Hamer, J. Clutterbuck, S. Crosthwaite, D. Bell-Pedersen, S. Gold, S. Gurr, M. Riquelme, A. Radford and R. Aramayo for valuable discussions; E. Selker for sharing unpublished results; and R. Morris, M. Celerin, D. Maillet, J. Loros, M. Riquelme, R. Kahmannn, J. Kämper, N. Talbot, M. Momony, H. Wosten, L. Lugones and N. Raju for figures. Work in the Zolan lab is supported by the National Institutes of Health, and the research of L.A.C. is supported by the Biotechnology and Biological Sciences Research Council.

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Authors and Affiliations

  1. Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK

    Lorna Casselton

  2. Department of Biology, Indiana University, 1001 East Third Street, Bloomington, 47405, Indiana, USA

    Miriam Zolan

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  1. Lorna Casselton
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  2. Miriam Zolan
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Correspondence to Lorna Casselton.

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DATABASES

LocusLink

AS3

BMAL1

Clk

CLOCK

CRY1

CRY2

Cyc

LIS1

Msh5

Per

Tim

<i>Saccharomyces</i> Genome Database

BUB1

BUB3

LEU1

TRP4

FURTHER INFORMATION

Fungal Genetics Stock Center

Fungal genome sequencing programmes

Nature Reviews Genetics focus on 'The art and design of genetic screens'

Neurospora crassa genome database

Glossary

CONDITIONAL LETHAL

A mutation that inhibits growth under some conditions, such as high or low temperature or in the absence of a specific growth supplement, but allows growth under other conditions.

GENE CONVERSION

A non-reciprocal recombination process that results in an alteration of the sequence of a gene to that of its homologue.

SAPROPHYTIC

An organism that obtains nutrition from dead or decaying plant or animal tissue.

KINESIN

A motor protein that is involved in organelle transport towards the plus end of microtubules.

DYNEIN

A multisubunit motor enzyme that is involved in the transport of organelles to the minus end of microtubules.

DYNACTIN

A multisubunit complex that is required for activating cytoplasmic dynein.

PAS DOMAIN PROTEINS

A family of proteins that are related by the presence of a conserved 300 amino-acid sequence that promotes dimerization. PAS is an acronym for the Drosophila melanogaster and mammalian proteins PER, ARNT and SIM that originally defined this family of transcriptional regulatory proteins.

CO-SUPPRESSION

The phenomenon whereby an endogenous plant gene is silenced owing to the presence of a homologous transgene.

RNA INTERFERENCE

(RNAi). The process by which double-stranded RNA specifically silences the expression of homologous genes through degradation of their cognate mRNA.

SEMI-RANDOM, TWO-STEP PCR

(ST-PCR). A procedure that is used to isolate unknown genomic DNA that flanks a known insert. One primer that binds to the known sequence and a degenerate primer with a non-degenerate 5′ end are used to amplify products. A second round of PCR uses a second primer in the known sequence and a primer to the non-degenerate 5′ end of the degenerate primer. This process is repeated until a single PCR product is obtained.

REPLICATIVE PLASMID

A plasmid molecule that contains in its sequence an origin for DNA replication and can replicate autonomously after transformation into host cells.

PROTOPLAST

A cell from which the cell wall has been removed by enzymatic digestion.

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Casselton, L., Zolan, M. The art and design of genetic screens: filamentous fungi. Nat Rev Genet 3, 683–697 (2002). https://doi.org/10.1038/nrg889

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