?recurrence of repeat-induced point mutation (rip) in ... · recurrence of rip in neurospora 70 1...

Copyright 0 1991 by the Genetics Society of America

?Recurrence of Repeat-Induced Point Mutation (RIP) in Neurosporu crassa

Edward B. Cambareri,’ Michael J. Singer and Eric U. Selker Department of Biology and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

Manuscript received May 18, 1990 Accepted for publication December 20, 1990

ABSTRACT 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 =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.

T HE size and structure of eukaryotic genomes suggest that D N A sequence duplications are

common in evolution. Duplications can result by mis- takes in DNA replication, recombination, or DNA repair, or by the repeated insertion into the genome of transposable elements, viral DNAs or transforming sequences. When a duplication occurs, selective con- straint may be reduced allowing divergence so that, ultimately, novel gene products may be produced. On the other hand, gene conversion, unequal crossing over, and other processes of concerted evolution tend to preserve identity among members of gene families. One wonders whether organisms use special mechanisms to speed the onset of divergence. The RIP (“rearrangement induced premeiotically” or “repeat- induced point mutation”) process of Neurospora crassa may be an example of such a mechanism (SELKER et al. 1987; SELKER and GARRETT 1988; CAMBARERI et al. 1989; SELKER 1990b). This process riddles duplicated sequences with transition mutations during the sexual stage of the life cycle. As a step toward under- standing the evolutionary significance of RIP, we investigated the recurrence of this process and its effect on recombination between repeated sequences.

Inmunology, University of Kansas Medical School, Kansas City, Kansas ’ Present address: Department of Microbiology, Molecular Genetics and

66103.

C;enetics 127: 699-710 (April, 1991)

RIP was discovered (SELKER et al. 1987) as a result of a detailed analysis of progeny from crosses of strains transformed with PES 174, a plasmid built to investigate the control of D N A methylation in Neurospora (SELKER, JENSEN and RICHARDSON 1987). The transformation host contained sequences homologous only to an approximately 6-kb segment of the plasmid, referred to as j u n k (see companion study by FOSS et al. 199 1). A key observation was that in transformants harboring single copies of PES1 74-either integrated by homologous recombination, generating a local duplication of Junk, or integrated by nonhomologous recombination, resulting in unlinked duplications of punk-these sequences were specifically subject to radical alterations during the sexual phase of the life cycle (SELKER et al. 1987). Primary sequence alterations were initially detected by changes in the position of restriction sites. Whether the duplications were linked or unlinked, in every example studied, both copies of the duplicated sequences were affected. The linked duplication never survived a cross unaltered. Altera- tions of unlinked duplications appeared at a frequency of roughly 50% (SELKER et al. 1987; E. B. CAMBARERI and E. U. SELKER, unpublished data), and seemed less severe than those of the linked duplication. Analysis of meiotic products arising from a single diploid nu- cleus indicated that the changes occurred prior t o

700 E. B. Cambareri, M. J. Singer and E. U. Selker

premeiotic DNA synthesis, and thus prior to karyogamy (SELKER et al. 1987). Analysis of asci originating from the same fertilization event demonstrated that changes occur after fertilization. Apparently RIP is confined to dikaryotic cells of the ascogenous tissue, which go through eight or more cell divisions (see PERKINS and BARRY 1977).

Additional studies verified that sequence duplications per se trigger RIP (SELKER and GARRETT 1988; FINCHAM et al. 1989), and demonstrated that the process results exclusively in G:C to A:T mutations (CAMBARERI et ai. 1989). Analysis of an example of the linked duplication offlank that seemed radically altered by RIP on the basis of hybridization data, revealed that approximately 50% of the G:C pairs had mutated. About 10% of the G:C pairs had mutated in a segment of the flank sequences from an unlinked duplication that appeared on the basis of Southern hybridizations to be lightly altered by RIP. In both cases, the two strands were affected similarly. Never- theless, the mutations did not occur randomly. For example, in the segment of the linked duplication, cytosines in 264% of CpA dinucleotides, but only 218, = I 3 and 2 5 % of the CpT, CpG and CpC dinucleotides, respectively, were lost.

Sequences altered by RIP are typically, but not invariably, found methylated at cytosines. This methylation, which may be unrelated to the mechanism of RIP, is stable through numerous rounds of DNA replication, and does not depend on the continued presence of sequence duplications (see SELKER 1990b). Experiments using sequences from the heavily methylated {-7 region (SELKER and STEVENS 1985), a diverged tandem duplication that is thought to be a relic of RIP (GRAYBURN and SELKER 1989), suggest that RIP can cause de novo methylation (SELKER, JEN- SEN and RICHARDSON 1987). A similar experiment using a sequence that became methylated after exposure to RIP in the laboratory is described here. A model to account for how RIP might cause methylation has recently been presented (SELKER 1990a).

It seems likely that RIP depends on homologous pairing to detect repeated sequences in the haploid nuclei of the ascogenous tissue. One reason for think- ing this is that the two components of an unlinked duplication are either both altered, or they are both not altered (SELKER and GARRETT 1988; FINCHAM et al., 1989). Interestingly, a high rate of intrachromatid homologous recombination is temporally correlated with RIP (SELKER et al. 1987; BUTLER and METZEN- BERG 1989). In a cross of transformant T-ES174-1, which bears a tandem repeat of the flank sequences separated by amf and pUC8 sequences, recombination between the flank sequences, resulting in loss of the interstitial sequences, occurred at a frequency of ~ 6 8 % (SELKER et al. 1987).

To gain insight into the evolutionary significance of RIP, and to gather clues to its mechanism, we investigated the activity of RIP and intrachromatid recombination through seven generations. We addressed the following questions: (1) Are duplicated sequences susceptible to multiple cycles of RIP? (2) Can a sequence altered by RIP trigger mutation of an unaltered homologous sequence? (3) Does linkage influence the extent of alterations by RIP? (4) Is the resistance to RIP exhibited by sequences altered by this process due to loss of mutable G:C pairs, loss of similarity between the related sequences, or DNA methylation? ( 5 ) T o what extent are linked duplicated sequences susceptible to premeiotic intrachromatid recombination after they are altered by RIP?

MATERIALS AND METHODS

A set of single-copy pES174 transformants (T-ES174-1, T-ES174-3 and T-ES174-8) of N. crassa strain N24 ( i d , arn132, A), served as starting material for this study (SELKER, JENSEN and RICHARDSON 1987). The 13.5-kb plasmid pES174 includes the N. crussa urn (glutamate dehydrogenase) gene, pUC8 sequences, the 1.6-kb l-7 region, and 6 kb of sequences adjacent to l-7 (“Junk”). All crosses of the transformants were with a strain equivalent to N24, but of opposite mating type (N36: inl, ~ ~ ~ 1 3 2 , a). Depending on their mating types, progeny were crossed with either N24 or N36. Transformants obtained with pEC24 were crossed with Fsp l , Fsp2, a (N202) orfl, a (N40). The plasmid pEC24 contains a copy of theflank region exposed to RIP that was isolated from LG2;1:1, the second generation isolate of T- ES174-1 (CAMBARERI et al. 1989). All crosses were carried out at 25” on VOCEL (1956) minimal medium modified, reducing the ammonium nitrate concentration 10-fold (Russo, SOMMER and CHAMBERS 1985) and containing 0. I % sucrose. Random ascospores were collected, germinated, and tested for Am and mating type using standard techniques (DAVIS and DESERRES 1970). DNA isolation was performed using stationary liquid cultures of Neurospora as previously described (SELKER, JENSEN and RICHARDSON 1987). DNA samples (0.5 rg) were digested using 10 X unit excess of restriction endonucleases in buffers supplied by the manufacturer. Digests were fractionated by electropho- resis through 1 .O% agarose gels and transferred by blotting to Zetabind (AMF) nylon membranes. Southern hybridizations were performed as previously described (SELKER, JEN- SEN and RICHARDSON 1987), and the membranes were stripped and reprobed as suggested by the manufacturer. Theflank region was probed in most of the hybridizations (including all those illustrated in Figures 1, 2 and 3) using a 4.8-kb BamHI-EcoRI fragment of PES1 74 that includes most of the nativeflank region (see Figure 5A in the companion study by Foss et al. 199 1). For the hybridizations illustrated in Figure 4, a gel-purified fragment from the homologous region of pEC24 was used.

Cotransformation of N. crussa strain N24 with pEC24 (CAMBARERI et ul. 1989), which includesflank sequences of strain LC,; 1 : 1 (illustrated in Figure l), and pESZOO, which includes a gene conferring hygromycin B resistance in N . crassa (STABEN et al. 1989). was performed using 0.5 rg of pES200 and 2.0 pg of pEC24.

DNA sequence divergence between the native flank sequences, in the 4.8-kb BamHI-EcoRI fragment of PES174 used as the hybridization probe, and homologous sequences

Recurrence of RIP in Neurospora 70 1

altered by RIP, isolated from progeny of the transformants, was estimated from the melting behavior of heteroduplexes detected by Southern hybridization. To do this we applied the formula T, = 81.5 + 16.6 logM + 0.41(G + C) - 0.67(%mismatch), where T, is the melting temperature in degrees Celsius, M is the salt concentration (molar), and G + C is the mole percentage of guanine plus cytosine (SCHILD- KRAUT and LIFSON 1965; DAVIS and HYMAN 197 1). It should be noted that the multiplier used to relate mismatch and T, (0.67) is open to dispute (THOMAS and DANCIS 1973). In a previous study we estimated the T,of the nativeflank region to be c9 1 in 0.165 M salt (CAMBARERI et al. 1989). From this we estimate that under the conditions that our Southern blots were washed (0.087 M Na+), the T, of the nativeJ2ank would be ~ 8 6 O .

RESULTS

Duplicated sequences are sensitive to multiple cycles of RIP. To investigate the recurrence of RIP, we followed the duplicated DNA of several single- copy PES 174 transformants through seven generations. The transformants used were T-ESl74-l, which resulted from homologous integration of the plasmid generating a linked duplication of the z6 kb _flank region, and T-ES174-3 and T-ES174-8, both of which resulted from integration at chromosomal sites unlinked to the native copy offlank (SELKER, JENSEN and RICHARDSON 1987; B. JENSEN, K. HAACK and E. SELKER, unpublished results). The transformation host, N24, lacked the l-q region and had a deletion of the entire am region (aml32; KINSEY and HUNG 198 1). Thus only the flank region was duplicated in these single-copy trasformants. T-ESl74-3 was chosen because the duplicated flank sequences of this strain alone were not methylated after RIP (SELKER et at. 1987; B. JENSEN, K. HAACK and E. SELKER, unpublished results), perhaps due to a chromosomal position effect. T-ES174-8 was chosen because its behavior in crosses appeared typical. The am+ gene was used to follow the transforming DNA through a series of crosses with am132 strains. In preliminary experiments, evidence of RIP was observed at a frequency of 100% among Am+ progeny from crosses of T-ESl74-1 and roughly 50% among first generation progeny of both T-ESl74-3 and T-ESl74-8 (SELKER et at. 1987). We obtained equivalent results in the present study. All of twelve Am+ progeny of T-ES174-1 (panel LGI (linked, generation one) in Figure 1) and two of six Am+ progeny of T-ES174-3 (left side of panel UG, (unlinked, generation one) in Figure 2) and three of six Am+ progeny of T-ESl74-8 (right side of panel UG1 in Figure 2) revealed changes in digests with the isoschizomers Sau3a and MboI. Sau3a and MboI both cleave the sequence GATC when it is unmethylated, but Sau3a fails to cut if the C is methylated. Thus changes in MboI digestion products are indicative of primary sequence alterations, and differences between digestion products of these isoschizomers are diagnostic of methylation.

We selected isolates 1 : 1 and 1:2 from the first generation cross of the linked duplication (LG1) strain to represent progeny exhibiting relatively major or minor alterations by RIP, respectively. These two strains were crossed with N36 and six Am+ progeny were selected arbitrarily from each cross for analysis (Figure 1). Five of the six progeny of 1 : 1 (labeled P: 1 in panel LG2), and all six progeny of 1:2 (labeled P:2 in panel LG2) exhibited changes. Thus duplications are not immune to RIP after one passage through the sexual cycle. That one isolate (LG2;1:2) did not show alterations suggested, however, that RIP was less ef- ficient than in the first generation. This possibility was tested by following the sequences through five more generations.

Frequency of RIP decreases in successive crosses and appears correlated with extent of alterations. We carried two strains with the linked duplication through five successive generations. Our intention was to carry out one series of crosses using isolates that showed major changes, starting with LGI; 1: 1 (referred to as the “radical” series and illustrated on the left half of panels LG2-LG7; Figure l), and a second series using isolates that showed relatively mi- nor changes, starting with LGl; 1:2 (referred to as the “conservative” series and illustrated on the right half of panels LG,-LG,; Figure 1). Strain LG2; 1:l was selected to parent LG3 for the radical series. lnterest- ingly, no change was observed among six Am+ progeny from this strain (LG2; 1: 1 = LG3; P: l), nor in their successive progeny in the next three generations. In the seventh generation, one of six Am+ progeny from this series showed change. Thus, the flank sequences of LG2; 1 : 1 were markedly resistant to RIP, exhibiting only one change (LG7, 1:4) in five generations. The methylation state of the progeny does not seem clearly correlated with the relative frequency of RIP. Heavy methylation preceded apparent stabiliza- tion by one or two generations (compare LG2 with LG3-6).

In LG3 of the the conservative series, we selected LG,; 2:6 (= LG3; P:6) as the Am+ parent. Five out of six of its progeny showed additional alterations, in striking contrast to the lack of alterations seen among LGs progeny of LG2; 1: 1 (Figure 1). Curiously, one Am+ product of LG3; P:6 (isolate LG3; 6:4) showed almost complete loss of methylation. The same result was observed in a second Southern hybridization using DNA isolated a second time from this isolate (data not shown). It should be noted, however, that these blots were probed with the nativeflank sequences, and we know that RIP can cause sequences to change to an extent such that hybridization to the native sequences is very weak. T o continue the conservative series, we selected LG3; 6:6 (= LG4; P:6). Three of six of its progeny showed alterations in LG4 (Figure 1). In the

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FIGURE 1 .-Recurrence of RIP in linked duplication. Transformant T-ES174-1, in which plasmid pES174 integrated by homologous recombination in the ~ 6 - k b "flank" region shared by the plasmid and the host genome, generating a tandem duplication offlank, served as the starting material for a series of seven crosses with strain N36 (ink am,,^; a). DNA samples from random progeny (labeled n:l, n:2, n:3 , etc., for first, second, third, etc., isolates from a cross of strain n of the previous generation) containing the transforming DNA were digested separately with Sau3A (S) and Mbol (M). The digests were fractionated along with molecular weight standards (kb ladder; BRL) and probed with a 4.8-kb BamHI- EcoRl fragment from theflank region. The autoradiograms are labeled LC", for linked generation n, where n denotes the generation number starting with the cross of T-ES174- 1 (T: 1). For generations 2-7, two isolates from the previous generation were chosen as the parents (P:n, where n refers to the isolate number from the previous generation), and six random Am+ progeny from each were analyzed for changes (right and left halves of the gels, respectively). For the series illustrated on the left side of the panels, the "radical" series, the isolate that seemed most changed was selected to parent the next generation,

- 0 3 while for the series illustrated on the right side of the panels, the "conservative" series, an isolate that seemed minimally altered was selected.

Recurrence of RIP in Neurospora 703

next three generations, 216, 216 and 116 Am+ progeny, respectively, showed further signs of RIP (Figure 1). Thus the sequences became resistant to RIP, as in the radical series, but our selection bias appeared successful in prolonging the mutation process. RIP of unlinked sequences appears less severe. In

a parallel series of backcrosses, theflank sequences of transformants T-ESl74-3 and T-ES 174-8 were followed through seven generations. These two series of crosses were qualitatively different from the crosses involving the linked duplication in that the duplicated sequences could segregate. Roughly half of the progeny in the T-ES174-3 and T-ESl74-8 series received a fresh (native) copy offlank from strain N36, as is evident for example in the progeny of P:5 in UG5 (Figure 2). Because of the relatively minor changes that occurred in the crosses of these strains, it was not generally clear from the restriction patterns of the progeny DNA whether or not the unlinked flank sequences had in fact segregated. T o parent each new generation, we tried to choose isolates that displayed new restriction fragments. While the results generated from these crosses are not directly comparable to those from crosses of strains with the linked duplication, similarities are apparent.

In the backcross series stemming from T-ES 174-3 (left half of panels UGI-G7), evidence of RIP was detected in one or more progeny from every generation except UGs. Only nine of 56 progeny showed alterations over the entire course of the study, however, and none were complex. Five of the changes occurred in generations 1-3, and four occurred in generations 4-7 (Figure 2). As we had observed previously, methylation was rare among progeny derived from T-ESl74-3 (SELKER et al., 1987; K. R. HAACK and E. U. SELKER, unpublished data). As expected, more methylation was associated with RIP in the crosses starting with T-ES 174-8 (right half of panels UGI-UG7; Figure 2). The T-ES174-8 series did not yield a higher fraction of progeny with alterations, however. Overall, only 6 of 56 progeny showed alterations over the course of the study. In UG5, three of the progeny in the T-ESl74-8 series showed loss of an MboI fragment of x500 bp, but this was presumably just due to segregation of the duplicate copies offlank. The progeny that lost the 500-bp fragment regained a set of unmethylated flank fragments, presumably from the other parent (N36). With the exception of UG5; 5:3, which showed increased methylation, methylation differences (detected using Sau3A) cosegre- gated with the 2500 bp MboI band. No changes were observed in the final two generations of the T-ESl74- 8 series, possibly because the strain chosen for UG6 had a fresh copy offlank from N36. The divergence between two copies of a sequence altered by RIP can be less than that between an altered sequence and its

unaltered precursor (GRAYBURN and SELKER 1989). If resistance to RIP is due to loss of sequence similarity rather than to loss of substrate (e.g., G:C pairs), the observed result would be expected. The possibility that a sequence altered by RIP interacts better with altered, than with unaltered, homologous sequence was addressed in an experiment described below.

Extent of divergence by RIP. The use of restriction enzymes to assess sequence alterations is obviously limited by the number of available sites within the sequence of interest. T o estimate the extent of sequence divergence of successive isolates, we assessed the degree of mismatch of hybrids formed in Southern hybridizations between the original flank sequence and the corresponding altered sequences. To do so, membranes with genomic DNA that had been allowed to hybridize with radioactively labeled native flank under relatively low stringency conditions were washed at a series of increasing temperatures. After each successive wash, remaining hybrids were detected by autoradiography. We then estimated the approximate degree of divergence between the flank regions based on the melting behavior observed.

The melting behavior of duplexes between the 4.8- kb BamHI-EcoRI fragment of pESl74 used as the hybridization probe and DNA samples from the primary transformants, or from the representatives of each generation that were used as parents in the subsequent generations (Figures 1 and 2), are illustrated in Figure 3. Panels A and B show, respectively, results for strains from the radical and conservative series starting with the linked duplication of T-ES 174- 1. In the first few generations, the two series show marked differences in sequence divergence. In the radical series, the first generation isolate (LGI; 1:l; Figure 3A, lane 1) exhibited loss of most hybridization after washing at 75". Evidence of comparable divergence in the conservative series was not seen until the third generation (panel B, lane 3). Assuming that a change in melting temperature of 1 O corresponds to 1.5% sequence mismatch, loss of bands between 70" and 75 O would reflect melting of heteroduplexes hav- ing 16-24% mismatches (see MATERIALS AND METH- ODS). The second generation isolate of the radical series (LG,; 1: 1; Figure 3A, lane 2) exhibited some loss of hybridization by washing at 65" (second band from top in lane 2, panel A), suggestive of >31% mismatch. The 70" wash released most remaining hybrids, suggesting that most of the flank sequences had diverged 24-32%. This estimate corresponds well with results from studies using isolatedflank sequences from this isolate that suggested that ~ 5 0 % of the G:C pairs had mutated by this point (CAMBARERI et al. 1989). The isolates from generations 3-6 of this series did not show any sign of further divergence, consistent with the conclusion that the region was resistant to


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- 1.8 FIGURE 2.-Recurrence of RIP in two unlinked duplications. Transformants T-ESl74- 3 (T:3) and T-ES174-8 (T8), in which pES174 integrated by non-homologous recombination at sites unlinked to the native copy of theflank region, served as the starting material for a series of seven crosses with strain N36. DNA samples were analyzed forflank sequences as described for Figure 1. The autoradiograms are labeled UG., for unlinked generation n, where n denotes the generation starting with the cross of original transformants. In each

- 0 5 generation, six random Am+ progeny bearing transforming DNA from T-ESl74-3 (left ' halves of autoradiograms) and six bearing transforming DNA from T-ESl74-8 (right halves

of autoradiograms) were analyzed for changes. Parents and progeny are designated as described in Figure 1.


further RIP. Interestingly, the instance of RIP observed in generation 7 was associated with reduced stability of one MboI fragment (lowest band visible after 60" wash (Figure 3A, lane 7).

In the conservative series of isolates from the linked duplication (series B in Figure 3), RIP apparently did not result in great sequence divergence until the third generation. The MboI fragments in DNA from generations 3, 4 and 5 displayed similar stability; the probe melted off at =70°, corresponding to ~ 2 4 % divergence. A second apparent burst of mutation apparently occurred in the sixth generation. The flank sequences of the 6th and 7th generation strains released the homologous native sequences at ~ 6 5 " , which would correspond to >31% sequence divergence, overall.

Progressive washes using DNA from the series of isolates originating with transformants T-ES 174-3 and T-ESl74-8 were also carried out. The results, shown in panels C and D of Figure 3, respectively, reveal considerably less sequence divergence than detected with the linked duplication. Very little release of probe was observed at 65" or 70". Several bands were lost at 75", however, suggesting that some regions diverged >16%.

Premeiotic intramolecular recombination and RIP differ in their requirements for sequence homology. We reported previously that intramolecular recombination between the two copies of the flank region of transformant T-ES 174-1 occurred frequently in crosses, resulting in loss of the am gene (SELKER et al. 1987). Crosses of T-ES174-1 (am+) X N36 (um132) produced only ~ 1 6 % am+ progeny, suggesting that the gene was lost from about two-thirds of the chromosomes that entered the cross with the tandem duplication. Analysis of DNA from progeny of individual asci arising from a single fertilization event indicated that the deficiency of Am+ progeny was due to intrachromatid recombination (rather than gene inactivation, or unequal interchromosomal exchanges), and that it occurred at the same stage as RIP-that is between fertilization and karyogamy (SELKER et al. 1987). Tetrads exhibiting intrachromatid recombination only, both intrachromatid recombination and RIP, or RIP only were seen. Those exhibiting both recombination and RIP could have resulted from RIP followed by intrachromatid recombination, or else from the simultaneous operation of these processes. DNA pairing of some sort is probably required for both RIP and intrachromatid recombination. To investigate whether sequences altered by RIP remain good substrates for intrachromatid recombination, we scored the transmission of Am+ in crosses between an Am- strain and T-ES174-1, and their derivatives LGI; 1:2, LG,; 6:6 and LG,; 3: 1 (Table 1).

Approximately 16% of the progeny of the original transformant were Am+, corresponding to an intrachromatid recombination frequency of ~ 6 7 % , as observed previously (SELKER et al. 1987). In striking contrast, the first, third, and seventh generation progeny of T-ESl74-1 all showed Mendelian, or nearly Mendelian segregation of am. Of the strains tested, only LGI; 1:2, which showed very mild alterations from RIP (Figures 1 and 3) produced significantly fewer than 50% Am+ progeny. Approximately 46% of the progeny of this strain were Am+, corresponding to an apparent intrachromatid recombination frequency of ~ 8 % . In the same cross, theflank sequences exhibited RIP in all six progeny examined. Appar- ently the relatively minor sequence alterations or the DNA methylation resulting from the first generation of RIP (or both) effectively prevented recombination but not additional RIP. FAUGERON, RHOUNIM and ROSSIGNOL (1990) have also found that DNA methylation does not prevent premeiotic inactivation of duplicated genes in Ascobolus.

Basis of resistance to RIP: One could imagine a number of possible explanations for the resistance to RIP showed by DNA sequences altered by this process: (1) The mutations could destroy specific RIP initiation sites or required higher order structures in the DNA. (2) Resistance could reflect depletion of the substrate for the process (ie., C:G pairs in favorable sequence contexts). (3) Resistance could be due to loss of potential for pairing as a result of the divergence of the homologous sequences. (4) It was also conceiv- able that DNA methylation was responsible for resistance to RIP, although the results presented in Figures 1 and 2 rendered this possibility unlikely. T o further investigate the basis for the resistance to RIP of sequences altered by the process, we tested whether the resistance to RIP of the flank sequences in strain LG2; 1 : 1 was due to sequence divergence of the duplicate elements. T o do this, we transformed a wild-type N . crassa strain with pEC24, which includes flank sequences from strain LG2;l: 1 (CAMBARERI et al. 1989). These sequences were resistant to RIP, as indicated by the fact that they showed no apparent alteration through the subsequent four generations (Figure 1). The idea was to test whether presence of two or more identical copies of this altered sequence in a strain would lead to reactivation of RIP. We also wished to determine whether the introduced altered flank sequences could trigger RIP in the nativeflank sequences of the host. Transformants were analyzed by Southern hybridization to determine copy number. As is typical in Neurospora transformants, the multiple-copy transformants seemed to have one or more clusters of pEC24 sequences (data not shown). Rep- resentative single- and multiple-copy transformants were crossed to N202 (Fsp l , Fsp2, a) or N40 ($, a)

E. B. Cambareri, M. J. Singer and E. U. Selker 706

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FIGURE 3.-DNA sequence divergence of duplicated sequences through seven generations of exposure to RIP. DNA samples from the primary transformants described in Figures 1 and 2 (lane 0). and from the representatives of each generation used as parents in the subsequent generations (lanes 1-7) were digested with MboI, and then fractionated and probed for theflank region as described in Figure 1. Sequence divergence was assessed by making successive exposures from the blots after washing in 50 mM NaCI, 20 mM sodium phosphate (pH 7.0). 1 mM EDTA, 0.1% SDS at the temperatures indicated. The two series of crosses of strains with the linked duplication offlank (Figure 1) are illustrated in panels A (radical series) and B (conservative series), and the series of crosses stemming from transformants T-ES174-3 and T- ESI 74-8. each with an unlinked duplication offlank, are illustrated in panels C and D, respectively.

TABLE 1

Intramolecular recombination in crosses of T-ES174-1 and its derivatives

Strains" Am+ progeny Am- progeny %Am+ progeny

T-ES 1 74- I 182 926 16.4' LG,;l:2 490 576 46' LG3;6:6 21 1 243 46.4 LG,;3: I 496 435 53.2

All strains were crossed with N36 (am,,z; inl; a ) except LG7;3: 1 which was crossed with N24 (am,,z; i d ; A). The Am- strain was used as the female in each cross. ' x' test identifies these as significantly different from 50% ( P <

0.01).

and progeny of crosses were analyzed to determine the frequency and extent of alterations by RIP.

Evidence of RIP was observed in the transforming DNA of three out of four random progeny from the multiple-copy transformant T-EC24-6 and in the transforming DNA of all of five random progeny from the multiple-copy transformant T-EC24-9. Results from one tetrad from each of the transformants are shown in panel A of Figure 4. We conclude that sequence divergence was largely responsible for resistance to RIP of the flank sequences in strain LC,; 1: 1. The high frequency of RIP in these transformants presumably reflects close proximity of some or all copies of the introduced DNA. In the single- copy transformant T-EC24-11. no changes were ob-

FIGURE 4.-Reactivation of RIP bv duDlication of RIP-resistant sequences. The RIP-resistant flank sequences of

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LGn: 1.

707

I . ~ ~ _, . l (Figure l), cloned in the plasmid pEC24 (CAMBARERI et al. 1989), were introduced inti N24, generating multiple-copy t&nsformants T-EC24-6 (T-6 in panel A) and T-EC24-9 (T-9 in panel A), and the single-copy transformant T-EC24-11. The transformants were crossed to N202 and progeny were scored for alterations in theflank sequences by analysis of genomic DNA using Sau3A (S) and MboI (M), as described in Figure 1 using a probe for theflank region of of LC,; 1 : 1 (see MATERIALS AND METHODS). Results from one tetrad (1 -4) from each multiple-copy transformant are shown in panel A. The exposure shown was not sufficient to detect the native copies offlank unner the conditions of the hybridization. The singlecopy transformant (which failed to exhibit RIP) was crossed with LGz;l: 1 to build strain LGz;l:l/EC24-11 (P:2 in panel B), which received a copy of the same alteredflank DNA from each parent. This duplication strain was crossed with N40 (P: 1 in panel B) and thirteen random progeny (lanes A-M) were analyzed for flank. Solid arrows indicate bands diagnostic of the flank sequences from grandparent LGz; 1 : 1, and the open arrow indicates a band unique to grandparent T-EC24-11. The open and filled stars mark the old and new positions, respectively, of an MboI restriction fragment altered by RIP in isolate F.

served among 4 1 progeny from independent asci (data not shown). This suggested that the altered flank sequences of strain LG2; 1: 1 were unable to interact with the native flank sequences. T o address the possibility that the single introduced copy had somehow integrated in a way that precluded any pairing, we crossed this transformant with the strain from which the transforming sequences were originally isolated (LG2;l: l), and analyzed their progeny by Southern hybridization to identify a strain that had the altered sequences from both parents (data not shown). Such a strain was found (LG2;1: I/EC24-11) and then crossed to test if provision of an identical copy of the transforming sequences would trigger RIP. Analysis of progeny from the duplication strain revealed a clear sign of RIP (lane F, Figure 4B). The frequency of RIP in this strain (1/20) was very low, suggesting that, at least for unlinked duplications, loss of sequence similarity between homologous sequences is not the only factor that can render a sequence resistant to RIP. Since we know that more than half of the G:C pairs in the sites that appeared most mutable (CpA dinucleotides) were already affected in LG,; 1 : 1 (CAM- BARERI et al. 1989), it seems likely that depletion of the substrate for RIP was responsible for the low frequency of RIP observed when this sequence was

present as an unlinked duplication. Similar results were obtained using a duplication of the l-7 region, which is thought to represent an ancestral case of RIP (GRAYBURN and SELKER 1989; Foss et al. 199 1). ln- terestingly, the introduced flank sequences became methylated, de novo, unlike what is found in transfor- mations using a native copy offlank (SELKER, JENSEN and RICHARDSON 1987). The high degree of methylation of the transforming sequences did not seem to prevent them from engaging in RIP, consistent with the lack of correlation observed between the methylation state and the frequency of RIP in the backcross series (Figures 1 and 2).

DISCUSSION

We have analyzed the recurrence of two premeiotic processes that are triggered by DNA duplications in N . crassa: RIP and intrachromatid recombination. Sequences altered by RIP remained sensitive to the process, although the frequency of RIP (the fraction of progeny showing evidence of RIP) decreased considerably within just a few generations. The frequency appeared to depend on the extent of previous alterations. In the series of crosses using progeny that seemed to have changed minimally, the frequency of RIP remained higher than in the series using progeny


that seemed to have changed most. Results of restriction analyses and melting studies on heteroduplexes demonstrated that the closely linked copies of the Junk sequences diverged considerably further from the original sequence than did the unlinked copies of the same sequences. We can not rule out the possibility that after some number of generations, the unlinked duplication would have reached the level of divergence exhibited by the linked duplication.

There are several possible explanations for finding that the unlinked duplications did not degenerate as much as did the linked duplication. It is possible that disruption of theflank region as a result of integration of the transforming DNA T-ESl74-3 and T-ESl74-8 affected the outcome of the series of crosses. This does not seem likely, however, since integration of the transforming DNA in T-ES174-3 occurred close to the BamHI site near the edge of theflank region (B. JENSEN personal communication), and since we have not observed a higher frequency of RIP in PES1 74 transformants in which thejank region was not inter- rupted. Distance between the unlinked copies of the jlank sequences, which clearly affects the frequency of RIP in the first generation, is probably the most important factor in limiting the divergence of unlinked sequences by RIP. One might speculate that pairing of the duplicate sequences, which probably is required for RIP, is both more frequent and more stable if the two partners are closely linked. The higher level of mutation in the linked sequences may be attributable, in part, to the fact that they were allowed to diverge in parallel. As a consequence of the site preference of RIP (cytosines immediately pre- ceding adenines are most frequently mutated, for example; CAMBARERI et al. 1989), the most mutable sites become scarce after less than 25% sequence divergence. Two copies of a sequence that are inde- pendently exposed to a heavy dose of RIP may end up more similar to each other than either is to their common ancestor. The {-q region of N . crassa, which is a diverged tandem duplication of a 794 bp segment that shows all the hallmarks of RIP, provides an illus- tration of this. The { and q repeats differ from their apparent ancestor at 127 and 140 positions, respectively, but differ from each other at only 11 3 positions (GRAYBURN and SELKER 1989). Thus, the segregation of the unlinked copies of the junk sequences in our study could have played a role in limiting the divergence of these sequences. As noted above, no RIP was observed in the T-ES174-8 series after generation five, when a fresh copy of thejank region entered.

Premeiotic intrachromatid recombination appeared more sensitive than RIP in the duplicated sequences. Looping out of the am gene by recombination between the neighboring repeated sequences dropped from a frequency of 267% in the first generation to

<lo% in the second generation. In contrast, RIP was still very active in the second generation; all six Am+ progeny analyzed from the cross exhibited evidence of RIP. In broad terms, RIP and intrachromatid recombination can be regarded as alternative premeiotic processes. Intrachromatid recombination eliminates the duplication, thereby preventing RIP, while the alterations resulting from RIP render a tandem duplication resistant to subsequent recombination between the repeated sequences. Nevertheless, it is worth noting that in a previous survey, two of six completely Am- asci from independent perithecia of T-ES174-1 X N36 showed evidence of RIP in the single remaining copy offlank. Finding two cases in which both RIP and recombination occurred in a small sample raises the possibility that the two processes can occur simultaneously. The difference in frequency between RIP and recombination in the second and subsequent generations suggests that the requirements for interaction of the DNA duplexes are different for the two processes. Recombination may require greater similarity of the interacting sequences than does RIP. It is also possible that some other consequence of RIP, such as DNA methylation, or destruction of hypothetical recombinators, is responsible for the apparent differential decline in the frequencies of the processes. In any case, the sensitivity of intrachromatid recombination to the divergence resulting from RIP suggests that RIP might prevent recombination between repeated sequences in meiosis that would lead to chromosome rearrangements. Sim- ilarly, RIP might prevent gene conversion that would tend to homogenize repeated sequences.

The eventual resistance to RIP of the linked duplication was largely due to loss of sequence similarity between the interacting sequences. A sequence that was resistant to RIP after two passages through the sexual cycle was still a reasonably good substrate for RIP when multiple identical copies of it were in the cell. This is consistent with results of experiments in which the {-q region of N . crassa, a diverged tandem duplication that shows the hallmarks of RIP (SELKER and STEVENS 1985; GRAYBURN and SELKER 1989), was reduplicated (Foss et al. 199 1). Introduction into Neurospora of one or more copies of the flank sequences that had become resistant to RIP did not trigger alterations in the homologous native sequences, consistent with the conclusion that acquired resistance to RIP is due to sequence divergence.

The connection, or connections, between RIP and DNA methylation remain largely unknown. While the nature of the mutations is consistent with the notion that they occur by methylation followed by deamina- tion, mechanisms not involving methylation are also possible (see SELKER 1990b). It is important to bear in mind that the methylation frequently found asso-


ciated with sequences altered by RIP is detected in vegetative cells, which are not active for RIP. Our finding from transformation experiments that a sequence left methylated by RIP can direct its methylation de novo in vegetative cells indicates that the mutations can themselves invite methylation. This is consistent with the previous observation that sequences from the {-7 region, which is thought to represent a natural relic of RIP (GRAYBURN and SELKER 1989), direct their de novo methylation (SELKER, JENSEN and RICHARDSON 1987). As discussed more fully elsewhere (SELKER 1990a), these observa- tions provide a clue that methylation is the default state for sequences in eukaryotes capable of cytosine methylation.

Although the Neurospora genome contains very little repeated DNA (KRUMLAUF and MARZLUF 1980), it does include some that must be resistant, or immune, to RIP. The results of our study suggest that if allowed to partially diverge, homologous genes would become resistant to RIP. This cannot account for all Neurospora repeated sequences, however. The tandemly arranged genes encoding the large RNA molecules of the ribosome provide a prominent example of a relatively homogeneous gene family that must be resistant, or immune, to RIP. Interestingly, BUTLER and METZENBERG (1 989) have found that the copy number of these genes change dramatically during the same period of the life cycle in which both intrachromatid recombination between the pair of

$flank sequences, and RIP occur. One wonders if the high level of premeiotic recombination in the rDNA somehow precludes RIP or if RIP operates efficiently only in gene-sized duplications. It also seems possible that inhibition of RIP in the rDNA is related to the suppression of meiotic interchromosomal recombination that has been observed in the rDNA region of many organisms, including N. crassa (see RUSSELL, PETERSEN and WAGNER 1988). The approximately 100 5s rRNA genes of Neurospora probably escape RIP in another way. These genes are dispersed in the Neurospora genome (SELKER et al. 198 1 ; METZEN- BERG et al., 1985), and probably evade RIP because of their very short (<I50 bp) region of sequence homology.

What is the advantage to an organism of a process that could destroy any gene that had become duplicated? Considering the contribution of gene duplications to evolution, such as in the generation of multi- gene families, RIP would seem to constitute an evolutionary obstacle for Neurospora. On the other hand, one of the major stumbling blocks to models for evolution of related genes is the acquisition of sufficient sequence divergence to escape the homogenizing influence of gene conversion and unequal exchanges between tandemly repeated sequences (WALSH 1987).

Presumably a threshold of divergence is reached, after which a significant level of recombination does not occur. RIP provides a tool to swiftly achieve such divergence.

The authors thank J. Foss, K. HAACK, B. JENSEN and M. LYNCH for stimulating discussions and J. FOS and V. MIAO for comments on the manuscript. This work was supported by National Science Foundation grant DCB 8718163 and U.S. Public Health Services grant GM-35690 from the National Institutes of Health. Part of this work was done during the tenure of an Established Investiga- torship of the American Heart Association (to E.U.S.).

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Communicating editor: P. J. PUKKILA

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