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

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.     

High Frequency Repeat-Induced Point Mutation (Rip) Is Not Associated with Efficient Recombination in Neurospora

Duplicated DNA sequences in Neurospora crassa are efficiently detected and mutated during the sexual cycle by a process named repeat-induced point mutation (RIP). Linked, direct duplications have previously been shown to undergo both RIP and deletion at high frequency during premeiosis, suggesting a relationship between RIP and homologous recombination. We have investigated the relationship between RIP and recombination for an unlinked duplication and for both inverted and direct, linked duplications. RIP occurred at high frequency (42-100%) with all three types of duplications used in this study, yet recombination was infrequent. For both inverted and direct, linked duplications, recombination was observed, but at frequencies one to two orders of magnitude lower than RIP. For the unlinked duplication, no recombinants were seen in 900 progeny, indicating, at most, a recombination frequency nearly three orders of magnitude lower than the frequency of RIP. In a direct duplication, RIP and recombination were correlated, suggesting that these two processes are mechanistically associated or that one process provokes the other. Mutations due to RIP have previously been shown to occur outside the boundary of a linked, direct duplication, indicating that RIP might be able to inactivate genes located in single-copy sequences adjacent to a duplicated sequence. In this study, a single-copy gene located between elements of linked duplications was inactivated at moderate frequencies (12-14%). Sequence analysis demonstrated that RIP mutations had spread into these single-copy sequences at least 930 base pairs from the boundary of the duplication, and Southern analysis indicated that mutations had occurred at least 4 kilobases from the duplication boundary.     

Action of repeat-induced point mutation on both strands of a duplex and on tandem duplications of various sizes in Neurospora.

In Neurospora crassa, DNA sequence duplications are detected and altered efficiently during the sexual cycle by a process known as RIP (repeat-induced point mutation). Affected sequences are subjected to multiple GC-to-AT mutations. To explore the pattern in which base changes are laid down by RIP we examined two sets of strains. First, we examined the products of a presumptive spontaneous RIP event at the mtr locus. Results of sequencing suggested that a single RIP event produces two distinct patterns of change, descended from the two strands of an affected DNA duplex. Equivalent results were obtained using an exceptional tetrad from a cross with a known duplication flanking the zeta-eta (zeta-eta) locus. The mtr sequence data were also used to further examine the basis for the differential severity of C-to-T mutations on the coding and noncoding strands in genes. The known bias of RIP toward CpA/TpG sites in conjunction with the sequence bias of Neurospora accounts for the differential effect. Finally, we used a collection of tandem repeats (from 16 to 935 bp in length) within the mtr gene to examine the length requirement for RIP. No evidence of RIP was found with duplications shorter than 400 bp while all longer tandem duplications were frequently affected. A comparison of these results with vegetative reversion data for the same duplications is consistent with the idea that reversion of long tandem duplications and RIP share a common step.     

Recurrent locus-specific mutation resulting from a cryptic ectopic insertion in Neurospora

New mutations are found among approximately 20% of progeny when one or both parents carry eas allele UCLA191 (eas(UCLA), easily wettable, hydrophobin-deficient, linkage group II). The mutations inactivate the wild-type allele of cya-8 (cytochrome aa3 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+ that was inserted ectopically at eas when the UCLA191 mutation occurred. As expected for RIP, eas(UCLA)-induced cya-8 mutations occur during nuclear proliferation prior to karyogamy. When only one parent is eas(UCLA), the new mutations arise exclusively in eas(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(UCLA) allele is associated with a long paracentric inversion. A discontinuity is present in eas(UCLA) DNA. The eas promoter is methylated in cya-8 progeny of eas(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.     

Genetic analysis of wild-isolated Neurospora crassa strains identified as dominant suppressors of repeat-induced point mutation

Repeat-induced point mutation (RIP) in Neurospora results in inactivation of duplicated DNA sequences. RIP is thought to provide protection against foreign elements such as retrotransposons, only one of which has been found in N. crassa. To examine the role of RIP in nature, we have examined seven N. crassa strains, identified among 446 wild isolates scored for dominant suppression of RIP. The test system involved a small duplication that targets RIP to the easily scorable gene erg-3. We previously showed that RIP in a small duplication is suppressed if another, larger duplication is present in the cross, as expected if the large duplication competes for the RIP machinery. In two of the strains, RIP suppression was associated with a barren phenotype--a characteristic of Neurospora duplications that is thought to result in part from a gene-silencing process called meiotic silencing by unpaired DNA (MSUD). A suppressor of MSUD (Sad-1) was shown not to prevent known large duplications from impairing RIP. Single-gene duplications also can be barren but are too short to suppress RIP. RIP suppression in strains that were not barren showed inheritance that was either simple Mendelian or complex. Adding copies of the LINE-like retrotransposon Tad did not affect RIP efficiency.     

Recurrence of Repeat-Induced Point Mutation (Rip) in Neurospora Crassa

Duplicate DNA sequences in the genome of Neurospora crassa can be detected and mutated in the sexual phase of the life cycle by a process termed RIP (repeat-induced point mutation). RIP occurs in the haploid nuclei of fertilized, premeiotic cells before fusion of the parental nuclei. Both copies of duplications of gene-sized sequences are affected in the first generation at frequencies of approximately 50-100%. We investigated the extent to which sequences altered by RIP remain susceptible to this process in subsequent generations. Duplications continued to be sensitive to RIP, even after six generations. The fraction of progeny showing evidence of RIP decreased rapidly, however, apparently as a function of the extent of divergence of the duplicated sequences. Analysis of the stability of heteroduplexes of DNA altered by RIP and their native counterpart indicated that linked duplications diverged further than did unlinked duplications. DNA methylation, a common feature of sequences altered by RIP, did not seem to inhibit the process. A sequence that had become resistant to RIP was cloned and reintroduced into Neurospora in one or more copies to investigate the basis of the resistance. The altered sequence regained its methylation in vegetative cells, indicating that the methylation of sequences altered by RIP observed in vegetative cells is a consequence of the mutations. Duplication of the sequence restored its sensitivity to RIP suggesting that resistance to the process was due to loss of similarity between the duplicated sequences. Consistent with this, we found that the resistant sequence did not trigger RIP of the native homologous sequences of the host, even when no other partner was available. High frequency intrachromatid recombination, which is temporally associated with RIP, was more sensitive than RIP to alterations in the interacting sequences.     

Titration of repeat-induced point mutation (RIP) by chromosome segment duplications in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Repeat-induced point mutation (RIP) is a hypermutational process that alters duplicated DNA sequences in Neurospora crassa. In previous studies, five of six large (>100 kb) chromosome segment duplications (Dp’s) examined were shown to dominantly suppress RIP in smaller (<5 kb) duplications. The suppressor duplications were >270 kb, whereas the lone non-suppressor duplication was ∼117 kb. We have now screened another 33 duplications and found 29 more suppressors and four more non-suppressors. All 22 suppressor duplications whose size could be estimated were >270 kb, whereas two newly identified non-suppressor duplications examined were 140–154 kb. RIP was suppressed in a subset of crosses heterozygous for more than one ordinarily non-suppressor duplication. These results strengthen the hypothesis that large duplications titrate out the RIP machinery and suggest the “equivalence point” for the titration is close to 300 kb. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This article is dedicated to the memory of Robert L. Metzenberg.     

Cytogenetics of an intrachromosomal transposition in Neurospora

Knowledge of intrachromosomal transpositions has until now been primarily cytological and has been limited to Drosophila and to humans, in both of which segmental shifts can be recognized by altered banding patterns. There has been little genetic information. In this study, we describe the genetic and cytogenetic properties of a transposition in Neurospora crassa. In Tp(IRIL)T54M94, a 20 map unit segment of linkage group I has been excised from its normal position and inserted near the centromere in the opposite arm, in inverted order. In crosses heterozygous for the transposition, about one-fifth of surviving progeny are duplications carrying the transposed segment in both positions. These result from crossing over in the interstitial region. There is no corresponding class of progeny duplicated for the interstitial segment. The duplication strains are barren in test crosses. A complementary deficiency class is represented by unpigmented, inviable ascospores. Extent of the duplication was determined by duplication-coverage tests. Orientation of the transposed segment was determined using Tp x Tp crosses heterozygous for markers inside and outside the transposed segment, and position of the insertion relative to the centromere was established using quasi-ordered half-tetrads from crosses x Spore killer. Quelling was observed in the primary transformants that were used to introduce a critical marker into the transposed segment by repeat-induced point mutation (RIP).     

Collateral damage: Spread of repeat-induced point mutation from a duplicated DNA sequence into an adjoining single-copy gene inNeurospora crassa

Repeat-induced point mutation (RIP) is an unusual genome defense mechanism that was discovered inNeurospora crassa. RIP occurs during a sexual cross and induces numerous G : C to A : T mutations in duplicated DNA sequences and also methylates many of the remaining cytosine residues. We measured the susceptibility of theerg-3 gene, present in single copy, to the spread of RIP from duplications of adjoining sequences. Genomic segments of defined length (1, 1.5 or 2 kb) and located at defined distances (0, 0.5, 1 or 2 kb) upstream or downstream of theerg-3 open reading frame (ORF) were amplified by polymerase chain reaction (PCR), and the duplications were created by transformation of the amplified DNA. Crosses were made with the duplication strains and the frequency oferg-3 mutant progeny provided a measure of the spread of RIP from the duplicated segments into theerg-3 gene. Our results suggest that ordinarily RIP-spread does not occur. However, occasionally the mechanism that confines RIP to the duplicated segment seems to fail (frequency 0.1–0.8%) and then RIP can spread across as much as 1 kb of unduplicated DNA. Additionally, the bacterialhph gene appeared to be very susceptible to the spread of RIP-associated cytosine methylation.     

Chromosome segment duplications in Neurospora crassa and their effects on repeat-induced point mutation and meiotic silencing by unpaired DNA

The size and extent of four Neurospora crassa duplications, Dp(AR17), Dp(IBj5), Dp(OY329), and Dp(B362i), was determined by testing the coverage of RFLP markers. The first three duplications were all > approximately 350 kb and have been shown in earlier studies to act as dominant suppressors of repeat-induced point mutation (RIP) in gene-sized duplications, possibly via titration of the RIP machinery. Dp(B362i), which is only approximately 117 kb long, failed to suppress RIP. RIP suppression in gene-sized duplications by large duplications was demonstrated using another test gene, dow, and supposedly applies generally. Crosses homozygous for Dp(AR17) or Dp(IBj5) were as barren as heterozygous crosses. Barrenness of the heterozygous but not the homozygous crosses was suppressible by Sad-1, a semidominant suppressor of RNAi-dependent meiotic silencing by unpaired DNA. A model is proposed in which large duplications recessively suppress semidominant Sad-1 mutations. The wild-isolated Sugartown strain is hypothesized to contain a duplication that confers not only dominant suppression of RIP but also a barren phenotype, which is linked (9%) to supercontig 7.118 in LG VII.     

In vivo levels of S-adenosylmethionine modulate C:G to T:A mutations associated with repeat-induced point mutation in Neurospora crassa

In Neurospora crassa, the mutagenic process termed repeat-induced point mutation (RIP) inactivates duplicated DNA sequences during the sexual cycle by the introduction of C:G to T:A transition mutations. In this work, we have used a collection of N. crassa strains exhibiting a wide range of cellular levels of S-adenosylmethionine (AdoMet), the universal donor of methyl groups, to explore whether frequencies of RIP are dependent on the cellular levels of this metabolite. Mutant strains met-7 and eth-1 carry mutations in genes of the AdoMet pathway and have low levels of AdoMet. Wild type strains with high levels of AdoMet were constructed by introducing a chimeric transgene of the AdoMet synthetase (AdoMet-S) gene fused to the constitutive promoter trpC from Aspergillus nidulans. Crosses of these strains against tester duplications of the pan-2 and am genes showed that frequencies of RIP, as well as the total number of C:G to T:A transition mutations found in randomly selected am(RIP) alleles, are inversely correlated to the cellular level of AdoMet. These results indicate that AdoMet modulates the biochemical pathway leading to RIP.     

A factor in a wild isolated <Emphasis Type="BoldItalic">Neurospora crassa</Emphasis> strain enables a chromosome segment duplication to suppress repeat-induced point mutation

Repeat-induced point mutation (RIP) is a sexual stage-specific mutational process of Neurospora crassa and other fungi that alters duplicated DNA sequences. Previous studies from our laboratory showed that chromosome segment duplications (Dps) longer than ~300 kbp can dominantly suppress RIP, presumably by titration of the RIP machinery, and that although Dps <200 kbp did not individually suppress RIP, they could do so in homozygous and multiply heterozygous crosses, provided the sum of the duplicated DNA exceeds ~300 kbp. Here we demonstrate suppression of RIP in a subset of progeny carrying the normally sub-threshold 154 kbp Dp(R2394) from a cross of T(R2394) to the wild isolated Carrefour Mme. Gras strain (CMG). Thus, the CMG strain contains a factor that together with Dp(R2394) produces a synthetic RIP suppressor phenotype. It is possible that the factor is a cryptic Dp that together with Dp(R2394) can exceed the size threshold for titration of the RIP machinery and thereby causes RIP suppression.     

Tandem duplications of the histidine operon observed following generalized transduction in Salmonella typhimurium

Unstable merodiploid transductants may be observed among the progeny of certain generalized transductional crosses between complementing mutations in the histidine operon of Salmonella typhimurium. In the presence of a functional recombination system, these transductants are unstable and they segregate His^? clones of both parental genotypes. The properties of these His⁺ transductants suggest that they contain tandem duplications of a region of DNA which includes the histidine operon, such that each copy of the duplication contains one of the two complementing mutations involved in the transduction. Transductional duplications have been observed from 14 pairs of his mutations, but only with complementing pairs of parental mutations. The length of duplicated material may be quite large: two duplications were found to include genetic markers ten minutes removed from the histidine operon on the Salmonella chromosomal map.These transductants appear to arise in a subpopulation of recipient cells which contain pre-existing tandem duplications of the histidine operon. As much as 0.01 to 0.1% of the cell population appears to be tandemly duplicated for a chromosomal region which includes the histidine operon.     

Repeat-induced point mutation (RIP) in crosses with wild-isolated strains of Neurospora crassa: evidence for dominant reduction of RIP

Seventy-one wild-isolated strains of Neurospora crassa were examined for their ability to support repeat-induced point mutation (RIP) in the erg-3 locus. RIP was exceptionally inefficient but detectable in crosses with the strain FGSC 430 from Adiopodoume, Ivory Coast. We could find no consistent differences in ascospore yields when wild isolates identified as "low-RIP" or "high-RIP" strains were crossed with strains bearing the segmental duplication Dp(IIIR > [I; II])AR17. This suggested that RIP may not be responsible for the barren phenotype of crosses involving segmental duplication strains.     

DNA methylation associated with repeat-induced point mutation in Neurospora crassa.

Repeat-induced point mutation (RIP) is a process that efficiently detects DNA duplications prior to meiosis in Neurospora crassa and peppers them with G:C to A:T mutations. Cytosine methylation is typically associated with sequences affected by RIP, and methylated cytosines are not limited to CpG dinucleotides. We generated and characterized a collection of methylated and unmethylated amRIP alleles to investigate the connection(s) between DNA methylation and mutations by RIP. Alleles of am harboring 84 to 158 mutations in the 2.6-kb region that was duplicated were heavily methylated and triggered de novo methylation when reintroduced into vegetative N. crassa cells. Alleles containing 45 and 56 mutations were methylated in the strains originally isolated but did not become methylated when reintroduced into vegetative cells. This provides the first evidence for de novo methylation in the sexual cycle and for a maintenance methylation system in Neurospora cells. No methylation was detected in am alleles containing 8 and 21 mutations. All mutations in the eight primary alleles studied were either G to A or C to T, with respect to the coding strand of the am gene, suggesting that RIP results in only one type of mutation. We consider possibilities for how DNA methylation is triggered by some sequences altered by RIP.     

Merodiploidy in ESCHERICHIA COLI-SALMONELLA TYPHIMURIUM Crosses: The Role of Unequal Recombination between Ribosomal RNA Genes

Previous workers have shown that intergeneric crosses between Salmonella typhimurium and Escherichia coli produce a high proportion of merodiploid recombinants among the viable progeny. We have examined the unequal cross-over event that was responsible for a number of intergeneric merodiploids. The merodiploids that we studied were all heterozygous for the metB-argH interval and were the products of intergeneric conjugal crosses. We found that when the S. typhimurium donor had its transfer origin closely linked to metB and argH, all recombinants examined were merodiploid, and they generally arose as F-prime factors. Many of these F-prime factors had been created by recombination between flanking rrn genes in the donor. When the S. typhimurium Hfr transfer origin was more distant from the selected markers, quite different results were obtained. Depending on the donor, 19-47% of the recombinants that acquired the donor argH+ or metB+ genes were merodiploid for these loci, but none of the recombinants were F-prime. A majority of the merodiploids had a novel (nonparental) rrn gene, indicating that unequal recombination between nonidentical rrn genes was a prevalent mechanism for establishing the merodiploidy. Both tandem and nontandem duplications were found. Some of the merodiploids duplicated E. coli genes in addition to acquiring S. typhimurium genes. Some merodiploids contained the oriC region from each parent. Of a total of 118 intergeneric merodiploids characterized from all donors, 48 different genotypes were observed, and 38 of the 48 had one or more nonparental rrn operons.     

Spontaneous Mutation at the Mtr Locus of Neurospora: The Spectrum of Mutant Types

We have isolated 135 strains of Neurospora which have mutations at the mtr locus resulting from independent spontaneous events. mtr is the structural gene for the neutral amino acid permease. The mutants have been characterized by their reversion behavior (both spontaneous and induced) and by hybridization studies of restriction digests of their DNA. About half of the mutants (54%) appear to result from single base-pair substitutions. Thirty-four percent have deletions, including some which extend into neighboring genes. Most of the remaining mutants have insertions. Several of the insertions are tandem duplications of 400-1000 bp and these mutants are unstable, reverting to mtr+ with a high frequency. When tandem-duplication mutants go through a cross, they are modified: the mutant progeny are fully stable. This modification is probably due to RIP (repeat-induced point mutation). This process has an important bearing on the comparison of germinal to somatic mutation.     

The Neurospora Transposon Tad Is Sensitive to Repeat-Induced Point Mutation (Rip)

RIP (repeat-induced point mutation) efficiently mutates repeated sequences in the sexual phase of the Neurospora crassa life cycle. Nevertheless, an active LINE-like retrotransposon, Tad, was found in a N. crassa strain from Adiopodoume. The possibility was tested that Tad might be resistant to RIP, or that the Adiopodoume strain might be incompetent for RIP. Tad elements derived from the Adiopodoume strain were found to be susceptible to RIP. In addition, strains lacking active Tad elements, including common laboratory strains and strains representing seven species of Neurospora, were found to have sequences closely related to Tad but with numerous mutations of the type resulting from RIP (G:C to A:T). Even the Adiopodoume strain showed Tad-like elements with mutations characteristic of RIP. Results of crossing of an Adiopodoume transformant with progeny of Adiopodoume suggest that the Adiopodoume strain is proficient at RIP. We conclude that Tad is an old transposable element that has been inactivated by RIP in most strains. Finding relics of RIP in both heterothallic and homothallic species of Neurospora implicates RIP across the genus.     

Escape from repeat-induced point mutation of a gene-sized duplication in Neurospora crassa crosses that are heterozygous for a larger chromosome segment duplication

In Neurospora crassa the ability of an ectopic gene-sized duplication to induce repeat-induced point mutation (RIP) in its target gene was suppressed in crosses that were heterozygous for another larger chromosome segment duplication. Specifically, the frequency of RIP in the erg-3 gene due to a 1.3-kb duplication was reduced if the chromosome segment duplications Dp(IIIR > [I;II]) AR17, Dp(VIR > IIIR) OY329, or Dp(IVR > VII) S1229 were present in either the same or the other parental nucleus of the premeiotic dikaryon. We suggest that the larger duplications act as sinks to titrate the RIP machinery away from the smaller duplication. In contrast, RIP efficiency was relatively unaffected in comparably unproductive interspecies crosses with N. intermedia and N. tetrasperma. These findings offer a novel explanation for the observed persistence of the transposable element Tad in only a subset of Neurospora strains.     

Stability of large segmental duplications in the yeast genome

The high level of gene redundancy that characterizes eukaryotic genomes results in part from segmental duplications. Spontaneous duplications of large chromosomal segments have been experimentally demonstrated in yeast. However, the dynamics of inheritance of such structures and their eventual fixation in populations remain largely unsolved. We analyzed the stability of a vast panel of large segmental duplications in Saccharomyces cerevisiae (from 41 kb for the smallest to 268 kb for the largest). We monitored the stability of three different types of interchromosomal duplications as well as that of three intrachromosomal direct tandem duplications. In the absence of any selective advantage associated with the presence of the duplication, we show that a duplicated segment internally translocated within a natural chromosome is stably inherited both mitotically and meiotically. By contrast, large duplications carried by a supernumerary chromosome are highly unstable. Duplications translocated into subtelomeric regions are lost at variable rates depending on the location of the insertion sites. Direct tandem duplications are lost by unequal crossing over, both mitotically and meiotically, at a frequency proportional to their sizes. These results show that most of the duplicated structures present an intrinsic level of instability. However, translocation within another chromosome significantly stabilizes a duplicated segment, increasing its chance to get fixed in a population even in the absence of any immediate selective advantage conferred by the duplicated genes.