The erg-3 (sterol Δ14,15-reductase) gene of Neurospora crassa: generation of null mutants by repeat-induced point mutation and complementation by proteins chimeric for human lamin B receptor sequences

Abstract

Null mutations were generated in the erg-3 gene of Neurospora crassa by repeat-induced point mutation (RIP). The mutants were viable, lacked ergosterol, were resistant to the steroidal glycoside a-tomatine and were sensitive to the phytoalexins pisatin and biochanin A. RIP was frequently associated with silencing of the hph gene located adjacent to the duplicated erg-3 sequence. The silencing of hph was reversible in the two cases examined and appeared to be due to the spread of cytosine methylation associated with RIP. The erg-3 mutant could be complemented by transformation with recombinant genes that encode proteins chimeric for amino acid sequences from the transmembrane (TM) domain of human lamin B receptor (LBR). This indicates that the LBR T M domain possesses A14n15-red~cta~e activity.

Chromosome Segment Duplications in Neurospora crassa and Their Effects on Repeat-Induced Point Mutation and Meiotic Silencing by Unpaired DNA

“…Strains made previously in our laboratory: The Dp1.3 ec hph A and a strains have been described (Prakash et al 1999;Bhat et al 2003). The transgene Dp1.3 ec hph contains a 1.3-kb HindIII fragment of the LG IIIR gene ergosterol-3 (erg-3; encoding the sterol biosynthetic enzyme sterol C-14 reductase) and is tagged with the bacterial hph gene, which confers resistance to hygromycin.…”

Lamin B receptor

“…97 The authors speculate that LBR may be involved in cholesterol biosynthesis in the NE, or might be a receptor for cell cycle signaling sterol molecules. Another investigation 98 demonstrated complementation of mutations in the erg-3 gene in N. crassa (a C-14 sterol reductase) by the C-terminal region of human LBR or by TM7SF2. They also speculated on a possible role of sterols in cell cycle changes of the NE.…”

Repeat-Induced Point Mutation (RIP) in Crosses with Wild-Isolated Strains of Neurospora crassa: Evidence for Dominant Reduction of RIP

“…It is located in linkage group (LG) IIIR and encodes the ergosterol biosynthetic enzyme sterol C-14 reductase (Perkins et al, 1982;Papavinasasundaram and Kasbekar, 1994). Prakash et al (1999) constructed strains in which a tagged duplicate copy of a 1.3-kb fragment of erg-3, designated Dp 1.3 ec hph, was introduced ectopically to target RIP to erg-3. RIP-induced erg-3 mutants are viable and have altered sensitivities to isoflavonoids and to the steroidal glycoside ␣-tomatine (Sengupta et al, 1995).…”