Specificity of repeat-induced point mutation (RIP) in Neurospora: sensitivity of non-Neurospora sequences, a natural diverged tandem duplication, and unique DNA adjacent to a duplicated region.

Abstract

The process designated RIP (repeat-induced point mutation) alters duplicated DNA sequences in the sexual cycle of Neurospora crassa. We tested whether non-Neurospora sequences are susceptible to RIP, explored the basis for the observed immunity to this process of a diverged tandem duplication that probably arose by a natural duplication followed by RIP (the Neurospora zeta-eta region), and investigated whether RIP extends at all into unique sequences bordering a duplicated region. Bacterial sequences of the plasmid pUC8 and of a gene conferring resistance to hygromycin B were sensitive to RIP in N. crassa when repeated in the genome. When the entire 1.6-kb zeta-eta region was duplicated, it was susceptible to RIP, but was affected by it to a lesser extent than other duplications. Only three of 62 progeny from crosses harboring unlinked duplications of the region showed evidence of changes. We attribute the low level of alterations to depletion of mutable sites. The stability of the zeta-eta region in strains having single copies of the region suggests that the 14% divergence of the tandem elements is sufficient to prevent RIP. DNA sequence analysis of unduplicated pUC8 sequences adjacent to a duplication revealed that RIP continued at least 180 bp beyond the boundary of the duplication. Three mutations occurred in the 200-bp segment of bordering sequences examined.

Microsatellite evolution: polarity of substitutions within repeats and neutrality of flanking sequences

“…Again, it can be speculated that the mismatch-repair systems involved in ¢lling in such loops may di¡er in terms of ¢delity compared to DNA polymerases, leading to an elevated substitution rate at the border between microsatellites and £anking sequence. Interestingly, our observations are analogous to RIPing (repeat-induced point mutation) in Neurospora (Foss et al 1991), and it is possible that other mechanisms than loop repair give an increased mutability at the border between repeat and unique sequence at meiotic recombination. In this context we note that various repetitive DNAs, including microsatellites, have been found to frequently be associated with synaptonemal complexes at recombination (Kmiec & Holloman 1986;Pearlman et al 1992).…”

Organization of paramutagenicity in R-stippled maize.

“…In a minority of cases, copy numberdependent silencing is partially heritable following reduction in copy number by meiotic segregation (e.g., GORING et al 1991;MATZKE and MATZKE 1991). In fungi, duplicated sequences are recognized and methylated/inactivated with high specificity and efficiency both in Neurospora (SELKER et al 1987;FOSS et al 1991) and in Ascobolus (FAUGERON et al 1990;BARRY et al 1993).…”

Inactivation of the Neurospora crassa gene encoding the mitochondrial protein import receptor MOM19 by the technique of "sheltered RIP".

“…There was little difference between the transformants and control strains when grown on medium that does or does not contain fpa (Figure 7, panels B and C, respectively). The ability of cDNA clones to rescue the mutant phenotype is significant since it proves that extension of RIP from duplicated mom-I9 sequence into neighboring genes (FOSS et al 1991) is not responsible for the phenotypes observed.…”