Identification of the Neurospora crassa mutation un-10 as a point mutation in a gene encoding eukaryotic translation initiation factor 3 , subunit

Inoue and Ishikawa defined a set of non-remediable, temperature-sensitive "unknown" mutants in Neurospora crassa (Inoue and Ishikawa,1970). To this day, the actual gene altered in many of these "unknown" mutants has not been determined. In order to add value to the Fungal Genetics Stock Center collection, we continue to define the genetic defects associated with these temperature-sensitive mutations (McCluskey et al., 2007, Wiest et al., 2008).

Identification of the Neurospora crassa mutation un-10 as a point mutation in a Identification of the Neurospora crassa mutation un-10 as a point mutation in a gene encoding eukaryotic translation initiation factor 3, subunit B. gene encoding eukaryotic translation initiation factor 3, subunit B.

Abstract Abstract
The Neurospora crassa temperature-sensitive mutant known as un-10 has been shown by a map-based complementation approach to be a single nucleotide change in the open reading frame of the eukaryotic translation initiation factor 3b (NCU02208.3).

Authors Authors
Matthew Kinney, Aric Wiest, Michael Plamann, and Kevin McCluskey This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol56/iss1/3 Identification of the Neurospora crassa mutation un-10 as a point mutation in a gene encoding eukaryotic translation initiation factor 3, subunit B. The Neurospora crassa temperature-sensitive mutant known as un-10 has been shown by a map-based complementation approach to be a single nucleotide change in the open reading frame of the eukaryotic translation initiation factor 3b (NCU02208.3). ________________________________________________________________________ Inoue and Ishikawa defined a set of non-remediable, temperature-sensitive "unknown" mutants in Neurospora crassa (Inoue and Ishikawa,1970). To this day, the actual gene altered in many of these "unknown" mutants has not been determined. In order to add value to the Fungal Genetics Stock Center collection, we continue to define the genetic defects associated with these temperature-sensitive mutations (McCluskey et al., 2007, Wiest et al., 2008. Using a complementation-based approach, we have identified the mutation in un-10 as a missense mutation in the eIF3b open reading frame. Building on the demonstration by T. Schmidhauser, that cosmids from the pSV50 cosmid library (Vollmer and Yanofsky, 1986) complement the un-10 mutation in strain FGSC 2342 (Wilson, 1990), we had cosmids 10E12, 11D2, 16C5, and 23C1 end-sequenced. Based on this sequence data, the mutation in FGSC 2342 was predicted to be on contig 10 between bases 68,000 and 92,000 (Galagan, et al, 2003). We selected overlapping cosmids spanning this region and tested their ability to complement the un-10 mutation in FGSC 2342 using electroporation-based transformation (Margolin et. al, 2000;  Complementation was successful with cosmids 25:D10, 75:A9 and 107:D10 but not 66:B1. There were four open reading frames in the region common to these overlapping cosmid clones: NCU02205.3, NCU02206.3, NCU02207.3 and NCU02208.3. We amplified copies of the genomic DNA for these open reading frames and used them to transform strain 2342 (Table 2). Only PCR product from NCU02208.3 complemented the un-10 mutation.

PCR Product
Colonies at 37ºC (per ug DNA) a NCU02205 0 0 NCU02206 0 <1 b NCU02207 0 <1 NCU02208 10 13 No DNA 0 0 Table 2. Identification of PCR products that complement un-10 a Data from two different replicates b Fewer than one transformant colony per microgram of DNA DNA sequence obtained directly from PCR amplified genomic DNA from strain 2342 showed a single T to C transition at position 1411, resulting in a tryptophan to arginine change in amino acid residue 471. This tryptophan residue is conserved among most fungi ( Figure 1) and even higher eukaryotes. The orthologous gene in Saccharomyces cerevisae, PRT1, has alleles which confer a temperature-sensitive phenotype (Hanic-Joyce et al, 1987). None of these corresponds to the mutation in Neurospora (Evans et al., 1995 Figure 1. Alignment of the amino acid sequence from eIF3b among sequenced fungi. Neurospora sequence is shown from amino acid 452 to 493. Position 471is highlighted in white.
The demonstration that un-10 is in the eIF3b gene adds value to the strains carrying this mutation. The ability to study both the interactions of subunits of the initiation factor 3 complex and the ability to use a temperature-sensitive mutation to control protein production are significant benefits of the identification of this mutation. Further, the high degree of conservation of the tryptophan residue at position 471 opens the possibility that this mutation can generate a temperature-sensitive growth phenotype when introduced into other organisms.