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

Matthew Kinney, Aric Wiest, Michael Plamann and Kevin McCluskey. Fungal Genetics Stock Center, School of Biological Sciences, University of Missouri-Kansas City

Fungal Genetics Reports 56:6-7

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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; Table 1).

Table 1. Identification of cosmids that complement un-10

^a. Not Done. The pSV50 does not encode hygromycin resistance.

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.

Table 2. Identification of PCR products that complement un-10

^aData from two different replicates

^bFewer 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).  

N. crassa FGSC 2342           GVPVEVVDTIKDTVINFAREPKGDRFVIITTTEPVGATAVP

N. crassa 2489                GVPVEVVDTIKDTVINFAWEPKGDRFVIITTTEPVGATAVP

Podospora anserina            GVPVEVVDTIKDTVINFAWEPKGDRFVTITTTEPVGATAVP

Chaetomium globosum           GVPVEVVDTIKDTVINFAWEPKGDRFVIITTPEPVGAVAVA

Gibberella zeae               GVPVEVVDTIKDTVINFAWEPKGDRFLIITTVTPTGEVAVQ

Magnaporthe grisea            GVPVEVVDTIKDTVINFAWEPKGDRFAIISTTEPVGVTAVA

Aspergillus niger             EFPVEVVE-LKDAVTAFAWEPFGTHFALISSNDPQLGTPAS

Candida albicans              DIPVEKLE-LKDVVVNFAWEPNTERFITISRLDDGNPNPAI

Saccharomyces cerevisiae      DIPVEKVE-LKDSVFEFGWEPHGNRFVTISVHEVADMNYAI

Ustilago maydis               NTPVEVID-LKEVVLNFAWEPKSDRFAIFSANDQS-LGSPN

Drosophila yakuba             DIPVEKVE-LKDSVFEFGWEPHGNRFVTISVHEVADMNYAI

Homo sapiens                  GVPVEVVDSLKDTVINFAWEPNGGRFVAITTGEAPSGAAVL

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.

Acknowledgement:  The FGSC is supported by award number 0235887 from the National Science Foundation and receives additional funds from award 5P01GM068087-04 from the National Institutes of Health.

References:

Evans, D.R.H. , C. Rasmussen, P.J. Hanic-Joyce, G.C. Johnston, R.A. Singer and C.A. Barnes.  1995. Mutational Analysis of the Prt1 Protein Subunit of Yeast Translation Initiation Factor 3. Mol. Cellular Biol. 15:4525–4535

Galagan, James E., et al.  2003. The genome sequence of the filamentous fungus Neurospora crassa.  Nature 422: 859 - 868.

Hanic-Joyce, P.J ., R.A. Singer and G.C. Johnston. 1987. Molecular characterization of the yeast PRT1 gene in which mutations affect translation initiation and regulation of cell proliferation

J. Biol. Chem. 262:2845-2851

Inoue, H., and T. Ishikawa. 1970. Macromolecule synthesis and germination of conidia in temperature- sensitive mutants of Neurospora crassa. Jpn. J. Genet. 45:357-369

Margolin, B.S., Freitag, M. and Selker, E.U.. 2000. Improved plasmids for gene targeting at the his-3 locus of Neurospora crassa by electroporation:correction. Fungal Genet. Newsl. 47:112

McCluskey, K.,  S. A. Walker, R. L. Yedlin, D. Madole and M. Plamann, 2007. Complementation of un-16 and the development of a selectable marker for transformation of Neurospora crassa. Fungal Genet. Newsl. 54:9-11

Vollmer S.J. and C. Yanofsky, 1986. Efficient cloning of genes of Neurospora crassa. Proc Natl Acad Sci U S A. 83:4869-4873.

Wiest, A., M. Plamann and K. McCluskey, 2008. Identification of the Neurospora crassa mutation un-4 as the mitochondrial inner membrane translocase subunit tim16. Fungal Genet. Report 55:37-39.

Wilson, C. FUNGAL GENETICS STOCK CENTER CATALOGUE OF STRAINS 3^rd Edition 1990. Supplement to Fungal Genet. Newsl. No. 37

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