Improving the efficiency of gene replacements in Neurospora crassa:
a first step towards a large-scale functional genomics project
Robert J. Pratt and Rodolfo Aramayo
*
Department of Biology, College of Science, Texas A&M University, Room 415, Building BSBW, College Station, TX 77843-3258, USA
Received 9 January 2002; accepted 9 May 2002
Abstract
Here, we report the use of the mating type heterokaryon incompatibility system as a counterselection to increase the probability
of identifying gene replacements in Neurospora crassa. We compared the frequencies of gene replacements observed among
transformants obtained by using plasmids with or without the mat a-1
þ
gene (hereby called ‘‘Toxic Gene’’) placed adjacent to
disruption cassettes. On an average, we were 20Â more likely to identify a correct gene replacement by incorporating the toxic gene
in our constructs. Using this strategy, we constructed strains containing a deletion of the inl (1
L
-myo-inositol-1-phosphate synthase)
gene. Finally, we demonstrated that we were able to remove the transformation marker (the hygromycin B phosphotransferase-
thymidine kinase gene fusion [hph
þ
::tk
þ
]) from the genome by using a strategy similar to the ‘‘URA-blaster’’ strategy used in yeast,
which we call ‘‘tk-blaster.’’ Ó 2002 Elsevier Science (USA). All rights reserved.
Keywords: Neurospora crassa; Gene replacement; Homologous integration; Toxic gene; Mating type; Heterokaryon incompatibility; hygromycin B
phosphotransferase–thymidine kinase gene fusion; Counterselection; Marker eviction;
L
-myo-inositol-1-phosphate synthase
1. Introduction
The upcoming completion of the sequence of Neu-
rospora crassa marks the beginning of a new science of
genomics in filamentous fungi. The next logical step is
functional genomics, which is the process of determining
the function of each gene in the genome. Establishing
gene function is not trivial. Traditionally, gene function
has been determined by defining the phenotype resulting
from gene mutation. In fungi like N. crassa, as well as in
other model organisms, mutagenesis has been routinely
performed by treatment of cells with chemicals or irra-
diation followed by genetic selection or screening, under
a variety of environmental and/or nutritional condi-
tions. Aclear advantage of this traditional or ‘‘forward
genetics’’ method is that one can easily saturate the ge-
nome with mutations. Unfortunately, not every mutated
gene will have a phenotype that will facilitate its cloning
(e.g., by complementation of the mutant phenotype with
a wild-type genomic DNAlibrary). More troublesome is
the fact that unless special care is taken to identify del-
eterious mutations, mutations in lethal genes will never
be recovered. This is an especially sensitive issue in the
case of N. crassa due to the lack of a vegetative diploid
stage for this organism. Therefore, traditional forward
genetics can be very time consuming, complicated and
can be only applicable on a genomic scale with a sig-
nificant amount of work.
In contrast to the traditional ‘‘forward genetics’’, the
process of ‘‘reverse genetics’’ can overcome these limi-
tations and can be applied at the genome level. Reverse
genetics requires having the DNAsequence on hand.
The information provided by this DNAsequence will
then be used in a series of formal logical steps that will
culminate with the construction of a strain carrying the
desired tagged mutation. For any given gene, one must:
(1) Identify a known or a putative gene by computer
analysis. (2) Mutagenize the coding region of the gene by
either ‘‘random’’ or ‘‘targeted’’ mutagenesis. (3) Intro-
duce the mutated DNAfragment into wild-type cells by
transformation. (4) Select for the cells that incorporated
the transforming DNAinto the genome (i.e., identify
‘‘true transformants’’ from ‘‘abortive transformants’’).
Fungal Genetics and Biology 37 (2002) 56–71
www.academicpress.com
*
Corresponding author. Fax: 979-845-2891.
E-mail address: raramayo@mail.bio.tamu.edu (R. Aramayo).
1087-1845/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
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