Genetic and molecular analysis of the temperature-sensitive mutant un-17 carrying a mutation in the gene encoding poly(A)-polymerase in Neurospora crassa

Abstract

The un-17 mutant was originally isolated as an irreparable temperature-sensitive (ts) mutant in Neurospora crassa. Early experiments showed that cells of this mutant immediately stopped growing and died when the temperature of the culture was shifted from a permissive temperature (25°C) to non-permissive temperature (35°C). This ts phenotype is suppressed by addition of cycloheximide or in some conditions of growth repression. Even at the permissive temperature, it shows a female sterile phenotype and is deficient in production of exocellular superoxide dismutase SOD4 (EC 1.15.1.1). By searching for a DNA fragment that complements the ts phenotype of the un-17 mutant from a N. crassa genome library, we found the un-17 gene. The cloned un-17 gene encodes a homolog of the Saccharomyces cerevisiae poly(A) polymerase (PAP). The un-17 mutant had a one-base substitution mutation in the gene. The cloned un-17 genes from the wild-type strain and the un-17 mutant were introduced into both the un-17 mutant and wild-type strain. The un-17 mutant introduced by un-17 DNA from the wild-type strain showed recovery of both the ts and female sterile phenotypes. Moreover, the purified product derived from the wild-type strain showed PAP activity in vitro. These findings indicate that the un-17 mutant carries a ts mutation in the gene encoding PAP.

Functional analysis of the Fusarium graminearum phosphatome

“…In this study, we found that Fg06103 and Fg02043 may be essential in F. graminearum (Table S3), but their orthologs are nonessential in A. nidulans (Son & Osmani, ), suggesting that other unidentified phosphatases may share similar functions with the Fg06103 and Fg02043 orthologs in A. nidulans . Similarly, the ortholog of Fg01918 is essential in N. crassa (Tanaka et al ., ), but ΔFg01918 of F. graminearum grew as well as the wild‐type strain. Thus, it would be interesting to further characterize these phosphatases with species‐specific functions in the pathogenic fungi.…”

Neurospora mrc1 homologue is involved in replication stability and is required for normal cell growth and chromosome integrity in mus-9 and mus-21 mutants

“…Immunoprecipitation and Western blotting were carried out as described (Kawabata et al, 2007;Tanaka et al, 2007;Wakabayashi et al, 2010). Conidia (1 Â 10 8 ) were incubated in minimal liquid medium (20 ml) for 12 h at 30°C with or without 0.76 mg/ml HU.…”

ATM and ATR homologes of Neurospora crassa are essential for normal cell growth and maintenance of chromosome integrity

“…Immunoprecipitation and Western blotting were carried out as described by Kawabata et al (2007) and Tanaka et al (2007). Conidia (1 Â 10 8 ) that had been incubated in minimal liquid medium (20 ml) for 6 h at 25°C were treated with 1 ll/ml MMS for 3 h at 25 or 37°C.…”