Academic journal article Genetics

Recurrent Locus-Specific Mutation Resulting from a Cryptic Ectopic Insertion in Neurospora

Academic journal article Genetics

Recurrent Locus-Specific Mutation Resulting from a Cryptic Ectopic Insertion in Neurospora

Article excerpt

ABSTRACT

New mutations are found among ~20% of progeny when one or both parents carry eas allele UCLA191 (eas^sup UCLA^, easily wettable, hydrophobin-deficient, linkage group II). The mutations inactivate the wild-type allele of cya-8 (cytochrome aa^sub 3^ deficient, linkage group VII), resulting in thin, "transparent" mycelial growth. Other eas alleles fail to produce cya-8 mutant progeny. The recurrent cya-8 mutations are attributed to repeat-induced point mutation (RIP) resulting from a duplicated copy of cya-8^sup +^ that was inserted ectopically at eas when the UCLA191 mutation occurred. As expected for RIP, eas^sup UCLA^-induced cya-8 mutations occur during nuclear proliferation prior to karyogamy. When only one parent is eas^sup UCLA^, the new mutations arise exclusively in eas^sup UCLA^ nuclei. Mutation of cya-8 is suppressed when a long unlinked duplication is present. Stable cya-8 mutations are effectively eliminated in crosses homozygous for rid, a recessive suppressor of RIP. The eas^sup UCLA^ allele is associated with a long paracentric inversion. A discontinuity is present in eas^sup UCLA^ DNA. The eas promoter is methylated in cya-8 progeny of eas^sup UCLA^, presumably by the spreading of methylation beyond the adjoining RIP-inactivated duplication. These findings support a model in which an ectopic insertion that created a mutation at the target site acts as a locus-specific mutator via RIP.

THE easily wettable gene was discovered when allele UCLA191 was obtained following mutagenesis of Neurospora crassa wild-type 74-OR8-1a using ethyl methanesulfonate (SELITRENNIKOFF 1976). The gene has been of interest because of its physiological role in the cell, and this first allele has attracted special attention because of its curious genetic properities, described below. Whereas wild-type conidia are completely covered with a thin layer of hydrophobic rodlets, rodlets are absent in eas mutants (BEEVER and DEMPSEY 1978). Conidia of the mutant are hydrophilic, entering instantly into water suspension, in contrast to wild-type conidia, which are hydrophobic (SELITRENNIKOFF 1976). Strains containing eas^sup UCLA^ are normal in growth rate, viability, and fertility. Linear growth of a typical strain continued at a normal rate through eight serial transfers on minimal medium at 34° in 30-cm growth tubes (method of RYAN et al. 1943), with no sign of senescence or stop-start growth when the experiment was terminated at 30 days.

Cloning and sequencing of eas^sup +^ was accomplished inadvertently. A circadian-clock-controlled gene originally called ccg-2 (allele JD105; BELL-PEDERSON et al. 1992) and a blue-light-induced gene originally called bli-7 (LAUTER et al. 1992) were identified independently using messenger RNAs that were obtained under completely different conditions. Both of these mutations were first indicated to be eas alleles by DNA sequence similarity to the rodletless gene in Aspergillus (STRINGER et al. 1991). Mutant alleles of ccg-2 and bli-7 both resembled eas^sup UCLA^ phenotypically and bothmapped at the same locus (BELL-PEDERSON et al. 1992; LAUTER et al. 1992).

The eas^sup +^ gene encodes a cysteine-rich hydrophobic protein that is similar to hydrophobins identified in other fungi (WESSELS et al. 1991; reviewed by WESSELS 2000). In Neurospora, the hydrophobic rodlets of powdery wild-type conidia no doubt promote aerial dispersal in nature. In contrast, the hydrophilic rodlet-deficient conidia of easmutants stick together and do not become airborne. Scoring of eas on agar slants is readily accomplished using a "tap test" to determine whether conidia shake loose or remain stuck together.

Experiments described in this study were initiated when an unexpected class of sparse-growing ("transparent") progeny was discovered in crosses parented by easily wettable allele UCLA191 (symbolized here as eas^sup UCLA^). The anomalous progeny were suggested to result from recurrent mutation of the cya-8 gene, which is unlinked to eas, by virtue of repeat-induced point mutation (RIP; SELKER 1990). …

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