The M26 Hotspot of Schizosaccharomyces pombe Stimulates Meiotic Ectopic Recombination and Chromosomal Rearrangements

Corresponding author: Jeffrey B. Virgin, Department of Pathology, Wayne State University, 540 East Canfield Ave., Detroit, MI 48201, jvirgin{at}med.wayne.edu (E-mail).

Communicating editor: L. S. SYMINGTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination). Recombination hotspots are important elements in controlling meiotic allelic recombination. We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination. Ectopic recombination was reduced 10–1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S. pombe. The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination. Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35–60% of recombination events and was stimulated 12-fold by M26. These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.

REPETITIVE DNA sequences, such as ribosomal and transfer RNA genes, transposable elements, multigene families, and repeats associated with centromeric and telomeric DNA, are dispersed throughout the genome of every species. Recombination between dispersed repetitive DNA (ectopic recombination) is a source of genetic variation, through both nonreciprocal recombination (gene conversion) and reciprocal recombination (crossing over). Combined with selective pressures, ectopic gene conversion contributes to the spread or elimination of mutations within gene families (MUNZ et al. 1982 ; MURTI et al. 1994 and references therein). Crossing over during ectopic recombination leads to chromosomal rearrangements and interferes with meiotic chromosome segregation (JINKS-ROBERTSON et al. 1997 ). Viable carriers of chromosomal rearrangements typically produce inviable or abnormal offspring due to aneuploidy created during meiotic segregation (PERKINS 1974 ; GARDNER and SUTHERLAND 1996 ). Several human disease phenotypes have been attributed to chromosomal rearrangements induced by ectopic recombination between homologous sequences (VNENCAK-JONES and PHILLIPS 1990 ; YEN et al. 1990 , YEN et al. 1991 ; LOPES et al. 1996 ). Therefore, meiotic ectopic recombination is a potentially important etiologic factor in reproductive and hereditary disorders.

Ectopic recombination has features in common with homologous recombination between sequences at the same chromosomal position (allelic recombination). Both types of recombination occur between homologous sequences and therefore could potentially share similar mechanisms of recombination initiation, strand transfer, and resolution of intermediates. Ectopic and allelic recombination have been compared in fungi using either naturally repeated sequences or artificially dispersed repeated sequences. With both natural and artificial repeats, the frequencies of both ectopic and allelic recombination are increased during meiosis relative to mitosis (MINET et al. 1980 ; JINKS-ROBERTSON and PETES 1986 ; SZANKASI et al. 1986 ; KUPIEC and PETES 1988A ). With artificial repeats, ectopic and allelic intragenic recombination show a similar frequency of associated crossing over (JINKS-ROBERTSON and PETES 1986 ; LICHTEN et al. 1987 ; GOLDMAN and LICHTEN 1996 ), similar DNA heteroduplex formation (LICHTEN et al. 1990 ; NAG and PETES 1990 ), and similar phenotypes in recombination mutants (STEELE et al. 1991 ). These common features suggest considerable overlap in the mechanisms.

With natural repeats, ectopic recombination is much less frequent than allelic recombination. The frequency of meiotic intergenic conversion between nonallelic tRNA genes in the fission yeast Schizosaccharomyces pombe is reduced 50–200-fold relative to intragenic recombination between allelic tRNA genes (THURIAUX 1985 ; GYSLER-JUNKER et al. 1991 ). Likewise, in the budding yeast Saccharomyces cerevisiae meiotic ectopic gene conversion between dispersed Ty retrotransposons is 100-fold less frequent than allelic Ty gene conversion (KUPIEC and PETES 1988A ). In contrast, ectopic recombination with artificial repeats in S. cerevisiae varies from equal to allelic recombination to 20-fold reduced (JINKS-ROBERTSON and PETES 1986 ; LICHTEN et al. 1987 ; HABER et al. 1991 ; GOLDMAN and LICHTEN 1996 ). Reciprocal ectopic recombination between natural repeats resulting in a chromosomal rearrangement is a rare event in S. pombe and S. cerevisiae. Only one translocation has been reported in S. pombe, which resulted from crossing over during ectopic recombination between nonallelic tRNA genes (SZANKASI et al. 1986 ). Similarly, rearrangements associated with S. cerevisiae Ty reciprocal ectopic recombination are very rare (BREILMANN et al. 1985 ; KUPIEC and PETES 1988A , KUPIEC and PETES 1988B ). This contrasts with the frequent crossing over associated with ectopic recombination between artificial repeats (JINKS-ROBERTSON and PETES 1986 ; LICHTEN et al. 1987 ; HABER et al. 1991 ; GOLDMAN and LICHTEN 1996 ). These results suggest some differences between natural and artificial repeats in the control of ectopic recombination.

Recombination hotspots are important in controlling the levels of meiotic allelic recombination (reviewed in LICHTEN and GOLDMAN 1995 ). Hotspots are sites on the chromosome where the frequency of homologous recombination is increased. There is evidence that hotspots are initiation sites in allelic recombination. If active in ectopic recombination, hotspots could lead to an increased frequency of chromosomal rearrangements. The ade6-M26 mutation in S. pombe creates a meiotic recombination hotspot that is ideal for a comparison of hotspot activity in ectopic and allelic recombination. The M26 mutation is a G to T tranversion near the 5' end of the ade6 gene. In heteroallelic crosses with different ade6 mutations, M26 induces a 5–20-fold increase in recombinant frequencies relative to a control mutation, M375, located only three base pairs (bp) away from M26 (GUTZ 1971 ; reviewed in SMITH 1994 ). The M26 mutation creates the heptanucleotide sequence 5'ATGACGT (M26 is the first T in the sequence) that is necessary but not sufficient for hotspot activity (SCHUCHERT et al. 1991 ; PONTICELLI and SMITH 1992 ; VIRGIN et al. 1995 ). Transplacements of 300–3000-bp DNA fragments containing the ade6-M26 allele to novel chromosomal positions show hotspot activity in some, but not all, integrations (PONTICELLI and SMITH 1992 ; VIRGIN et al. 1995 ). These chromosomal context effects appear to be mediated by alterations in chromatin structure induced by integrations of large DNA fragments. When site-directed mutagenesis of 1 or 2 bp is used to create the M26 heptamer in novel genomic locations, hotspot activity is maintained at all sites tested (FOX et al. 1997 ).

Previously, M26 has been shown to induce a threefold increase in ectopic recombination between the two copies of an artificially constructed ade6 duplication at the endogenous ade6 locus (SCHUCHERT and KOHLI 1988 ). We have used artificially dispersed copies of the ade6 gene to study meiotic ectopic recombination among all three chromosomes in S. pombe. As a means of comparing the mechanisms of ectopic and allelic recombination, we wanted to determine if the chromosomal context effects on M26 hotspot activity were similar in the two types of recombination. In addition, we wanted to determine if a recombination hotspot could increase the frequency of chromosomal rearrangements.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

S. pombe strains and crosses:
Construction of strains has been previously reported (VIRGIN et al. 1995 ). Strain genotypes are listed in Table 1. Growth of S. pombe strains and heterothallic matings were performed as previously described (PONTICELLI et al. 1988 ; PONTICELLI and SMITH 1992 ). Strains were grown in liquid yeast extract medium or plated on yeast extract agar (YEA) supplemented with 0.1 mg/ml uracil. Crosses were plated on EMM2 minimal medium (NURSE 1975 ) with appropriate nutritional supplements to assay total viable spores and ade6⁺ recombinants. Each cross was performed on multiple days with independent starting cultures. For recombinants and total spores >50 colonies each were counted, except in crosses where the recombinant frequency was very low. In the latter crosses, 1–5 x 10⁶ spores were plated and 4–>50 colonies were counted.

To determine the recipient locus in the ectopic recombination events, we crossed recombinants to tester strains carrying ade6⁺ alleles at the different integration sites (see RESULTS). These matings were performed by cross-stamping strains on either malt extract agar or EMM2. Spores were harvested from the cross-stamp areas and transferred to microtiter wells with 200 µl 0.5% glusulase (DuPont, Wilmington, DE) in water. After incubation overnight at 30°, 5 µl of a 1:10 dilution from each well was spotted on YEA. Ade^- segregants were detected as red papillae among the white Ade⁺ spore colonies. In some cases, Ade^- papillae were obscured by overgrowth of the Ade⁺ cells. Therefore, we retested all recombinants that produced no red papillae in either test cross by mating on sporulation agar and plating the spores from individual crosses onto a single YEA plate. When detected, Ade^- red colonies comprised ~50% of spore colonies.

Cloning and analysis of integration sites:
We have previously integrated copies of the ade6 gene at different chromosomal sites, some at random and some at the ura4 locus by homologous transplacement (VIRGIN et al. 1995 ). The plasmid used for the random integrations, pJV8, contained a 2.9-kb DNA fragment with the complete ade6 transcriptional unit with the M26 mutation and the ura4 gene for positive selection of transformants in the multiple cloning site of pUC19 (Figure 1). After linearization with NarI, the plasmid was used to transform strain GP981 (h^- ade6-D1 ura4-D18 leu1-32), in which the ura4 and ade6 genes have been deleted. Therefore, pJV8 lacks homology to any genomic sequence in GP981 and should integrate at random. The integration sites were initially unknown and designated zzzX. Two of those integrations (zzz7 and zzz15) have been further characterized and studied in ectopic recombination. Although the integration sites have been mapped (see below), for consistency the zzz allele designations have been retained (referred to as zX in the text). After integration of pJV8 we replaced the M26 allele by homologous transplacement with either the M375 or the 469 allele to create substrates for heteroallelic recombination (VIRGIN et al. 1995 ).

View larger version (17K): In this window In a new window Download PPT slide   Figure 1. Integration sites and structures. (A) Genomic map of the different loci studied. Heavy vertical lines represent the three S. pombe chromosomes. The relative chromosome lengths and centromere positions () are taken from MUNZ et al. 1989 . The integration sites are placed on the map according to sequence data (see MATERIALS AND METHODS) and HOHEISEL et al. 1993 and MUNZ et al. 1989 . The solid arrow indicates the direction of transcription of the ade6 gene relative to cenIII (GRIMM et al. 1994 ). Dashed arrows indicate the inferred orientations of the ade6 genes at the different integration sites based on the pattern of viable translocations (see RESULTS). The ura4 integrations include both orientations, with ade6 transcription directed toward the centromere [ura4(3.0) and ura4(5.9)] and away from the centromere [ura4(Sac) and ura4(Spe)]. (B) The plasmid pJV8 was linearized with NarI prior to transformation of GP981 (h- ade6-D1 ura4-D18 leu1-32) to Ura+. Independent random integrations, designated z7 and z15, were isolated. (C) Plasmids were constructed with different lengths of S. pombe genomic DNA containing the ade6 gene, together with several hundred base pairs of bacterial plasmid DNA, inserted into the EcoRV site of the ura4 gene. These constructs were integrated into the chromosomal ura4 locus by homologous transplacement. See text for discussion of allele designations. For details of construction, see VIRGIN et al. 1995 . Solid arrows, ade6 gene; open boxes, ura4 gene; heavy solid lines, bacterial plasmid DNA; thin solid line, S. pombe genomic DNA. Arrowheads indicate the direction of transcription. Numbers below the lines represent distances in kilobase pairs. For clarity, portions of the plasmid are not drawn to scale. E, EcoRV; N, NarI; P, PvuII; Sc, SacI; Se, SpeI.

Genomic DNA from strains carrying randomly integrated pJV8 was digested with BglII or ClaI and religated. Circular DNA molecules containing the integrated plasmid and flanking genomic DNA were isolated by transformation of Escherichia coli DH5 followed by selection for ampicillin-resistant colonies. The DNA sequence was determined for 200–300 bp around the plasmid-chromosomal integration site junctions by cycle sequencing (Perkin-Elmer, Norwalk, CT) with the primers 5'-CTTAACTATGCGGCATCA-3' and 5'TTAATCGCCTTGCAGCAC-3', which flank the NarI site of pBluescript. Sequences were analyzed on an ABI Prism 377 (ABI Adv. Biotechnologies, Inc., Columbia, MD). In addition, integration structures and recombinant structures were verified by Southern blotting using the Genius non-radioactive blotting and hybridization system (Boehringer-Mannheim, Indianapolis), according to instructions supplied by the manufacturer.

The junction sequences indicated that the integrations occurred by a nonhomologous DNA end-joining reaction, with a few common bases between the plasmid and genomic DNA near the junctions (data not shown). The genomic DNA sequences at the integration sites were matched to GenBank and EMBL database entries (data not shown). One end of the z7 integration sequence matches the puc1 gene on the long arm of chromosome II (FORSBURG and NURSE 1994 ). The other junction of the z7 integration site does not match puc1 or any other sequence in the database. We have determined by a combination of Southern blotting and PCR assays that a deletion of chromosomal DNA from the puc1 locus of ~2 kb is associated with the z7 integration (data not shown). Both junctions of the z15 integration site match the 2F7 cosmid (GenBank accession no. Z50142) near the telomere of the long arm of chromosome I (HOHEISEL et al. 1993 ). Deletions of ~100 bp of plasmid DNA and ~600 bp of chromosomal DNA are associated with the integration. To ensure that the small deletions of chromosomal DNA associated with the z7 and z15 integrations were not due to cloning artifacts, we used a PCR assay to compare the chromosomal junctions and the cloned junctions. Both plasmid and chromosomal DNA were amplified with primers specific to the pJV8-chromosomal junction sequences. The sizes of the amplified fragments were identical between the reactions with chromosomal DNA and plasmid DNA as template (data not shown), indicating that the junction sequences in the cloned DNA accurately reflect the chromosomal junctions at the different integration sites. Although the integration junctions vary slightly in the amount of pJV8 DNA lost from the ends, the random integrations are identical in the region of the ade6 marker alleles used in the recombination assays.

The remaining ade6 integrations were created at the ura4 locus by homologous transplacement. The integrations contain different amounts of transplaced genomic DNA from around the ade6 gene or have different cointegrated plasmid DNA (Figure 1). Details of their structure have been reported (VIRGIN et al. 1995 ). Briefly, either a 3.0-kb or a 5.9-kb DNA fragment containing ade6 was integrated, together with several hundred bp of pBR322 DNA, into the EcoRV site of the ura4 gene. Three ade6 alleles, M26, M375, or 469, were integrated for each length of ade6 DNA. These integrations were structured with ade6 transcription in the opposite direction of ura4 transcription. Alternatively, a 3.0-kb DNA fragment containing ade6 was inserted into either the SacI or SpeI restriction site in the multiple cloning site of pBluescript II, followed by integration into ura4 accompanied by several hundred bp of pBluescript DNA. In this case the M26, M375, and 469 alleles were integrated with ade6 transcription in the same direction as ura4 transcription (Figure 1). For clarity the allele designations used here include an abbreviation signifying either the length of ade6 DNA integrated for the set of integrations with pBR322 DNA [ura4(3.0) or ura4(5.9)] or the restriction site of the plasmid DNA into which the ade6 gene is inserted for the integrations with pBluescript DNA [ura4(Sac) or ura4(Spe)]. These different integrations into the same site of the ura4 gene show chromosomal context effects with respect to M26 hotspot activity in allelic recombination (VIRGIN et al. 1995 ). In comparing results of ectopic and allelic recombination between the random integrations, the ura4 transplacements, and the endogenous ade6 locus, some differences are expected to result from the different chromosomal contexts, including effects of the cointegrated plasmid DNA (VIRGIN et al. 1995 ). However, M26 hotspot activity was determined by the frequency of an M26 x 469 cross compared to the frequency of an M375 x 469 cross in the same chromosomal context, and therefore the influence of chromosomal context on hotspot activity was studied as an independent variable.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Experimental approach:
To compare M26 hotspot activity in meiotic ectopic and allelic recombination, we performed multiple pair-wise crosses with strains carrying a single copy of the ade6 gene, either at the endogenous ade6 locus, or integrated at a novel chromosomal site (Figure 1). At each site three different alleles, M26, M375, or 469, were inserted separately. Each allele is a single-bp mutation that creates a translation termination codon and an adenine auxotroph. Except for the different single-bp mutations, the structures at each chromosomal site are identical for all three alleles. Strains carrying either the M26 hotspot or M375 control mutation were crossed with strains carrying a 469 test allele. These markers are separated by a distance of ~1300 bp (SZANKASI et al. 1988 ). Intragenic recombinants were detected as adenine prototrophs among spore colonies. M26 hotspot activity is the ratio of the recombinant frequency of an M26 x 469 cross divided by the recombinant frequency of an M375 x 469 cross involving the same loci. Heteroallelic crosses were performed between strains with ade6 alleles at different chromosomal sites (ectopic recombination) or at the same site (allelic recombination).

Allelic recombination and hotspot activity:
To study the effects of chromosomal context on allelic recombination, we performed heteroallelic crosses with artificially integrated ade6 alleles at multiple chromosomal sites. For the random integrations of pJV8 (z7 and z15), the ade6 alleles are surrounded by plasmid DNA on each end (Figure 1). Allelic recombinant frequencies have been reported previously (VIRGIN et al. 1995 ). The same crosses were performed independently as controls in the current study (shown on the diagonals in Table 2 and Table 3), with less than twofold variation between the results of the two studies. In the ade6-M375 x ade6-469 crosses there is up to an 80-fold variation in the allelic recombinant frequencies for crosses with the same set of marker alleles at the different integration sites (Table 2). However, most of the variation is attributable to the markedly elevated recombination in the ura4-147(Sac) integration. Excluding this integration, there is a sixfold range in recombinant frequencies. At the ura4 locus, several different ade6 integration structures were studied, and the recombinant frequencies varied up to ~80-fold for different structurally related integrations, consistent with previous findings (VIRGIN et al. 1995 ).

The M26 hotspot showed chromosomal context dependence in allelic recombination. M26 was active at the natural ade6 locus, where it was originally created (GUTZ 1971 ), and at the ura4-108(5.9) and ura4-142(Spe) integrations (Table 4; VIRGIN et al. 1995 ), with 6–22-fold induction of intragenic recombination relative to M375. It was inactive at all other integration sites tested. As discussed previously, these and other studies indicate that chromosomal context is an important determinant of recombinant frequency and recombination hotspot activity (VIRGIN et al. 1995 ; FOX et al. 1997 ).

Ectopic recombination and hotspot activity:
To determine if ectopic recombination is subject to the same chromosomal context effects as allelic recombination, we crossed strains with ade6 integrations at different chromosomal positions. Ectopic recombination is much less frequent than allelic recombination. The ectopic recombinant frequencies in the ade6-M375 x ade6-469 crosses reflect recombination without the influence of the M26 hotspot. The frequencies vary over a nearly 300-fold range (from 2.7 x 10^-4 to 10^-6 recombinants per viable spore), and most of the frequencies were reduced 100–1000-fold relative to allelic recombination (Table 2). The greatest reduction relative to allelic recombination was 3000-fold [ura4-147(Sac) x ade6-469], and the least reduction was ~3-fold [z15-2 x ura4-152(Spe)]. In all but three crosses [z15-2 x ura4-152(Spe), z15-2 x ura4-145(Sac), and z15-3 x ura4-147(Sac)] the ectopic recombinant frequency was less than 10% of either corresponding allelic recombinant frequency.

The pattern of M26 hotspot activity was the same in ectopic and allelic recombination. As discussed above, M26 is active in allelic recombination at the natural ade6 locus and in the ura4-108(5.9) and ura4-142(Spe) integrations (Table 2 Table 3 Table 4; VIRGIN et al. 1995 ). It was active in these same integrations in ectopic recombination with all other loci tested, with hotspot values ranging from 7–22-fold (Table 4). Likewise, in integrations where M26 is inactive in allelic recombination, it was also inactive in ectopic recombination. In some cases of ectopic recombination, hotspot values of 2–3-fold were observed. These small increases were inconsistent and of doubtful significance. These results imply a similar mechanism of M26 action in ectopic and allelic recombination.

To further compare M26 hotspot activity in ectopic and allelic recombination, we determined the locus that was converted to ade6⁺ for some of the crosses. GUTZ 1971 has shown by tetrad dissection that in allelic recombination at the endogenous ade6 locus, the M26 allele is predominantly a recipient of wild-type information in conversion events, whereas the M375 allele shows parity in the direction of information transfer. We wanted to compare the direction of information transfer in ectopic recombination with the previous results from allelic recombination. However, with the low frequencies of ectopic recombination, tetrad dissection is impractical. As an alternative method of determining the direction of information transfer, we used test crosses to determine the location of the ade6⁺ allele in the recombinants. Recombinants were crossed with two different tester strains, each with an ade6⁺ allele at one of the parental integration sites (Figure 2). With recombinants resulting from nonreciprocal recombination, only one of these two test crosses is expected to generate red Ade^- spore colonies, because one test cross will have ade6⁺ alleles at the same locus in both parents (the Ade⁺ recombinant and the Ade⁺ tester strain). Thus, in test crosses that segregate Ade^- spores, the Ade⁺ tester strain identifies the locus that was the donor in the original cross (Figure 2A). Using this assay, we determined which allele was a recipient in a subset of the crosses.

View larger version (26K): In this window In a new window Download PPT slide   Figure 2. Segregation patterns in test crosses. The possible segregation patterns for the first (reductional) meiotic division are shown for test crosses between Ade+ recombinants from an original cross (e.g., Table 2 and Table 3) and Ade+ tester strains (Table 1). The two chromatids are shown as a single bar and the second (equational) meiotic division is omitted. In the examples shown, recombinants originated from a cross between a strain carrying an ade6-M26 allele at the endogenous ade6 locus on chromosome III (gray bar) and a strain carrying an ade6-469 allele at the z7 integration site on chromosome II (black bar). The M26 allele was converted to ade6+, without (A) or with (B) associated crossing over. Open circle, centromere; open box, ade6 allele; open box with +, ade6+ allele. (A) Nonreciprocal recombination (conversion without crossing over) in the original cross. The Ade phenotype (+ or -) of each spore from the test crosses is indicated. Only two of the four spores are shown for each segregation pattern. The other two spores generated from the second meiotic division would be identical to the pair illustrated. The Ade+ tester strain that produces Ade- spore colonies in a cross with the recombinant identifies the donor locus in the original cross. (B) Reciprocal recombination (conversion with associated crossing over) in the original cross, generating a balanced translocation. Both the Ade phenotype and predicted spore viability are indicated. Because the translocation chromosomes must segregate together to produce a balanced genome and a viable spore, no viable Ade- spores are produced.

In crosses in which the M26 hotspot was active it was a recipient of wild-type information in 97–99% of the recombination events (Table 5, lines 1 and 3). This result is consistent with tetrad analysis of allelic recombination (GUTZ 1971 ). However, in crosses in which the M26 hotspot was inactive there was incomplete correlation with hotspot activity. There was parity in the direction of information transfer with M26 in the z15 integration, in which the hotspot was inactive (Table 5, line 12). In other crosses in which the hotspot integrated at ura4 was inactive, M26 was predominantly a recipient in 86–95% of the recombination events (Table 5, lines 5, 8, and 10). Furthermore, the nonhotspot ura4-154::ade6-M375(3.0) allele was also a recipient in 79–88% of recombination events (Table 5, lines 6, 9, and 11). These results suggest that locus-specific rather than allele-specific factors determine the direction of information transfer in ectopic recombination involving the ura4 locus. To further test this idea, we reversed the orientation of the alleles in the ura4 x z7 cross (Table 5, line 7). The ura4 locus remained predominantly a recipient. The ade6 locus showed a similar pattern in which it was predominantly a recipient with either the M26 or M375 allele (Table 5, lines 1–4). The direction of information transfer in ectopic recombination appears to depend on locus-specific factors, which may obscure allele-specific effects.

We confirmed the genetic analysis by comparison of the results with a Southern blot (Figure 3). Each lane represents ClaI-digested DNA from either parental (lanes 1, 20, 21, and 40) or recombinant (lanes 2–10, 12–19, 22–29, and 31–39) isolates from an ade6-M26 x z7-3::ade6-469 cross, probed with pJV8. Since there is no ClaI site in the ade6 gene or in the integration plasmid, a single band represented the ade6 and z7 loci in the parental strains. Above each lane is the designation for reciprocal (R) or nonreciprocal (N) recombinant, based on the segregation patterns in the test crosses (Figure 2). In the nonreciprocal recombinants one or both parental bands were present. One band represents the recombinant ade6⁺ allele, and the other band represents a parental allele, which should segregate at random. In recombinants with one band at the position of a parental allele (lanes 8, 22–25, 32, 33, 36, and 37), this locus could be assigned as the recombinant ade6⁺ allele (the recipient of wild-type information), since it must be present in the recombinant prototroph. In all these lanes the single band comigrated with the parental ade6 band, which was the recipient by the genetic assay in all the isolates represented (data not shown). This confirmation of the results from the test crosses indicates that the complexities of information transfer in ectopic recombination are not due to technical artifacts.

View larger version (62K): In this window In a new window Download PPT slide   Figure 3. Southern blot with meiotic recombinants. DNA from parental (lanes 1, 20, 21, and 40) and recombinant (lanes 2–10, 12–19, 22–29, and 31–39) strains was digested with ClaI prior to electrophoresis and hybridization with pJV8 (Figure 1). Lanes 11 and 30 represent phage lamba DNA digested with HindIII. Above each lane is the designation for reciprocal (R) or nonreciprocal (N) recombinant, according to results from test crosses (Figure 2). In the lanes with DNA from parental strains, the bands representing the ade6 and z7 loci are indicated. In the lanes with DNA from recombinants, the bands corresponding to the translocation chromosomes are indicated. The band assignments in the reciprocal recombinants were made by probing the same blot with probes specific for the S. pombe genomic DNA at each end of the z7 integration (data not shown).

Crossing over and reciprocal translocations:
As another means of comparing the mechanisms of ectopic and allelic recombination, we determined the frequency of crossing over associated with ectopic intragenic recombination. Crossing over between similarly-oriented loci on nonhomologous chromosomes results in a balanced translocation, the only type of rearrangement likely to produce a viable haploid spore. An assessment of the frequency of crossing over is gained from test crosses to strains with ade6⁺ integrations (Figure 2 and Table 5). Recombinants with balanced translocations should produce only Ade⁺ progeny in both test crosses because the translocation chromosomes show "pseudolinkage;" i.e., the translocation chromosomes must segregate together to produce a viable haploid spore (Figure 2; SZANKASI et al. 1986 ). As confirmation, Southern blot analysis of a subset of these recombinants showed a pattern consistent with reciprocal translocations (Figure 3). In each lane with a reciprocal recombinant, as determined by the test crosses, both parental bands were lost and two new recombinant bands appeared. In crosses with such recombinants the calculated frequency of crossing over associated with ectopic intragenic recombination was 34–58% (Table 5), consistent with previous observations in allelic ade6 recombination (GRIMM et al. 1994 ). The proportion of crossovers was similar for M26 and M375. In two crosses, only one or two of ~90 recombinants examined did not segregate Ade^- spores in either test cross (Table 5, lines 1 and 10). The nature of these rare recombinants has not been further studied. Based on the pattern of translocations obtained with the other crosses, simple balanced translocations that create pseudolinkage were not expected from the crosses in lines 1 and 10 (see below).

The production of viable translocations, or lack thereof, was combined with chromosomal position information to infer the orientation of the different integrations relative to the centromere (Figure 1). All of the Ade⁺ recombinant progeny tested from some crosses produced Ade^- spores in one test cross (Table 5, lines 2, 5–7, 11, and 12), inferring that the original crosses did not produce viable balanced translocations. When the two recombining loci in the original cross were on different chromosomes, then the lack of translocations was most likely due to the ade6 alleles being in opposite orientations relative to their respective centromeres. In this case, crossing over would result in a dicentric chromosome and an acentric fragment and spore inviability. Therefore, the ade6 and z15 loci are inferred to be in opposite orientations relative to cenIII and cenI, respectively, and ade6 and z7 are inferred to be in the same orientations relative to cenIII and cenII, respectively. The ura4-117(3.0) and ura4-154(3.0) integrations produced translocations with z15 but not z7, a pattern opposite to the endogenous ade6 gene. Combining this information with the known orientation of the ade6 gene relative to cenIII (GRIMM et al. 1994 ), the inferred orientations of the ade6 gene in the different integrations are shown in Figure 1.

In crosses between the endogenous ade6 locus and the ura4-117(3.0) and ura4-154(3.0) integrations (Table 5, lines 10 and 11), which are on opposite arms of chromosome III and in opposite orientations relative to cenIII, crossing over would result in a dicentric chromosome III and an acentric fragment. A dicentric chromosome III resulting from crossing over between the ade6 locus and the ura4 integrations might result in a viable spore if one centromere were inactivated. However, a dicentric chromosome III would not be expected to cause pseudolinkage in the same way as a heterochromosomal translocation.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have found that the M26 recombination hotspot is active in meiotic ectopic recombination. The efficiency of ectopic recombination varies widely depending on the chromosomal positions of the interacting loci. The chromosomal context effects on recombination hotspot activity and the frequency of crossing over associated with intragenic recombination are similar in ectopic and allelic recombination, suggesting similar mechanisms.

M26 hotspot activity and chromosomal context:
The M26 hotspot was active in ectopic recombination and showed the same chromosomal context dependence as in allelic recombination. Previous studies have suggested that a specific chromatin structure is required for M26 hotspot activity, and this structure is disrupted by some chromosomal insertions (VIRGIN et al. 1995 ; FOX et al. 1997 ). Crosses between strains carrying genomic insertions of different ade6 alleles at the same or different chromosomal sites have allowed a direct comparison of hotspot activity in allelic and ectopic recombination. The M26-specific hotspot was active in allelic recombination in some ura4 integrations [ura4(5.9) and ura4(Spe)] but not others [ura4(3.0) and ura4(Sac)], and was inactive at several random integration sites (Table 4; VIRGIN et al. 1995 ). In ectopic recombination M26 hotspot activity, and inactivity, was observed at the same integration sites as in allelic recombination (Table 4). Hotspot activity was dependent only on the site of the M26 allele, and was not affected by the position of the 469 allele with which M26 recombined. Thus, the chromosomal context effects on hotspot activity observed in allelic recombination affect ectopic recombination in the same manner.

Direction of information transfer in ectopic recombination:
As another test of the mechanism of M26 action in ectopic recombination, we determined the direction of information transfer between the recombining alleles (Table 5). In tetrad analysis of M26 x ade6⁺ crosses, there is disparity in the direction of information transfer, with M26 converted to ade6⁺ in 46 of 52 (88%) recombinant tetrads (GUTZ 1971 ). In contrast, the M375 allele was converted to ade6⁺ in only three of nine recombinant tetrads (33%). These results led GUTZ 1971 to propose a model of M26 action in which M26 is a site of DNA strand breakage and initiation of recombination events. The broken chromosome is repaired using the intact homologous chromosome as a donor of genetic information (GUTZ 1971 ). This model is similar to the double-strand break repair model of recombination (RESNICK 1978 ; SZOSTAK et al. 1983 ), for which there is considerable supporting evidence from studies in S. cerevisiae (reviewed in LICHTEN and GOLDMAN 1995 ). However, thus far DNA strand breaks at the M26 hotpsot have not been reported.

In ectopic recombination the direction of information transfer was not directly correlated with hotspot activity. In all crosses in which the M26 hotspot was active it was predominantly a recipient of wild-type information (Table 5), similar to the results from allelic recombination (GUTZ 1971 ). However, in the same crosses the M375 allele was also predominantly a recipient, indicating that the direction of information transfer was not strictly dependent on hotspot activity. The insertions at the ura4 loci were predominantly recipients in all crosses analyzed, regardless of the locations of the M26, M375, or 469 alleles. These results indicate that factors in addition to, or other than, hotspot activity influence the direction of information transfer in ectopic intragenic recombination. These factors could be related to the integration structures (ura4) or to the specific chromosomal loci (ade6).

Crossing over and chromosomal rearrangements:
The M26 hotspot stimulates both nonreciprocal and reciprocal recombination. Tetrad analysis reveals that intragenic recombination at the ade6 locus is associated almost exclusively with 3:1 tetrads or gene conversion events (GUTZ 1971 ). Crossovers in an 11-cM interval spanning ade6 are associated with 52–65% of ade6 intragenic recombination events, and the M26 hotspot stimulates crossovers to the same extent as intragenic recombination (GRIMM et al. 1994 ). In ectopic recombination between the two copies of an artificially constructed ade6 duplication, M26 stimulates crossing over approximately threefold relative to M375 (SCHUCHERT and KOHLI 1988 ). We found a crossover frequency of 34–58% associated with ade6 ectopic recombination (Table 5), similar to allelic recombination. The proportion of crossovers associated with intragenic recombination was similar for M26 and M375. In the ade6 x z7 cross, in which the M26 hotspot was active, there were 2.7 x 10^-5 translocations per viable spore with M26 in the cross [0.58 x (4.7 x 10^-5 recombinants per viable spore); Table 3 and Table 5]. With M375 in the cross there were 2.2 x 10^-6 translocations per viable spore (Table 2 and Table 5). This 12-fold induction of translocations by M26 is a direct demonstration that a meiotic recombination hotspot can stimulate chromosomal translocations. In humans, clustering of exchange points within specific regions of repetitive DNA associated with chromosomal rearrangements infers that the events are mediated by ectopic recombination and are stimulated by a recombination hotspot (LOPES et al. 1996 ; MATASSI et al. 1997 ).

The high frequency of crossing over associated with ectopic recombination between artificially integrated markers differs from the rare crossing over associated with recombination between naturally repeated sequences. In ectopic recombination between S. pombe tRNA genes, only one reciprocal translocation has been reported as a result of crossing over, and it occurred during mitotic recombination between sup3 and sup9 (SZANKASI et al. 1986 ). Among ~550 meiotic recombinants analyzed in a spore inviability assay, no translocations were identified (SZANKASI et al. 1986 ). Similar differences in crossover frequencies between ectopic recombination with artificial or natural repeats have also been observed in S. cerevisiae (JINKS-ROBERTSON and PETES 1986 ; KUPIEC and PETES 1988A , KUPIEC and PETES 1988B ; LICHTEN et al. 1987 ; GOLDMAN and LICHTEN 1996 ). It seems likely that crossing over in ectopic recombination between natural repeats is suppressed to prevent potentially deleterious chromosomal rearrangements.

The two most obvious factors that could mediate suppression of ectopic recombination between natural repeats are the degree and length of homology. In E. coli and S. cerevisiae, both reciprocal and nonreciprocal ectopic recombination between diverged (homeologous) sequences are reduced relative to nearly identical (homologous) sequences (RAYSSIGUIER et al. 1989 ; BAILIS and ROTHSTEIN 1990 ; HARRIS et al. 1993 ; SELVA et al. 1995 ; DATTA et al. 1996 ; CHAMBERS et al. 1996 ). In mutants defective in mismatch repair the levels of homeologous recombination are increased (RAYSSIGUIER et al. 1989 ; SELVA et al. 1995 ; DATTA et al. 1996 ; CHAMBERS et al. 1996 ). These studies suggest that ectopic recombination between naturally dispersed diverged sequences is suppressed by the recognition of multiple mismatched base pairs in heteroduplex recombination intermediates. Below a certain level of homology, mismatch repair proteins may cause a failure to form mature recombination products.

The effects of length of homology on mitotic recombination have been studied in E. coli (WATT et al. 1985 ; SHEN and HUANG 1986 ), S. cerevisiae (JINKS-ROBERTSON et al. 1993 ), and mammalian cells (RUBNITZ and SUBRAMANI 1984 ; AYARES et al. 1986 ; WALDMAN and LISKAY 1988 ). In each species, there is a range in which levels of recombination are dependent on the length of homology in a linear fashion. The minimum effective processing segment has been estimated as approximately 50 bp in E. coli and 200–250 bp in S. cerevisiae and mammals. Recombination occurs below the minimum effective processing segment but it is very inefficient and the length dependence is nonlinear. In S. cerevisiae both the rate of conversion and the proportion of crossovers decrease with decreasing length of homology (JINKS-ROBERTSON et al. 1993 ). The effects of length of homology on meiotic ectopic recombination have not been reported.

In S. pombe, the tRNA genes differ from the artificially integrated ade6 genes in length of homology. There are three copies of the serine tRNA genes dispersed in the S. pombe genome (MUNZ et al. 1982 ). There are only two divergent nucleotides between the sup3 and sup9 genes over a 180-bp region of homology (99% identity), including a 15-bp intervening sequence (AMSTUTZ et al. 1985 ). The sup12 gene is divergent by six additional nucleotides (~95% identity with sup3 and sup9), all clustered within the intron near the site of the recombining alleles. The reduction from 99% to 95% identical sequences, even with clustering of the divergent nucleotides, does not substantially affect meiotic ectopic recombination, since sup12 x sup3 and sup9 x sup3 recombinations occur at comparable frequencies (AMSTUTZ et al. 1985 ). The ade6 integrations in this study share 3–7 kb of perfect homology, except for the two single-nucleotide auxotrophic markers. These results raise the possibility that below a certain length of homology, crossovers are inhibited, as observed in mitotic ectopic recombination in S. cerevisiae (JINKS-ROBERTSON et al. 1993 ). However, because the sup and artificially dispersed ade6 alleles are different sequences in different chromosomal contexts, it not possible to conclude directly that length of homology is the factor responsible for the differences in reciprocal recombination.

Chromosomal position effects:
Ectopic recombination is markedly reduced relative to allelic recombination in S. pombe. The ectopic recombinant frequencies were in the range of 10^-4 to 10^-6 Ade⁺ per viable spore and were reduced 10–1000-fold relative to the corresponding allelic recombinant frequencies (Table 2 and Table 3). The frequencies of ectopic recombination between the artificially integrated ade6 alleles are comparable to ectopic recombinant frequencies between naturally dispersed homologous tRNA genes (10^-5–10^-7 recombinants per viable spore; MUNZ et al. 1982 ; AMSTUTZ et al. 1985 ). Thus for both natural and artificial repeats there is a marked reduction in ectopic relative to allelic recombination.

The low frequencies of ectopic recombination could be due to effects of chromosomal position and inhibition of the homology search. Alignment of the homologs during meiosis may simultaneously promote allelic recombination and inhibit ectopic recombination. In S. cerevisiae ectopic recombination between closely spaced loci is as frequent as allelic recombination (GOLDMAN and LICHTEN 1996 ). Distantly spaced loci on the same chromosome and heterochromosomal recombination occur at similar frequencies, and are reduced 5–20-fold relative to allelic recombination. These results suggest that ectopic recombination is limited in S. cerevisiae by homolog alignment. In this study there was a nearly 300-fold range of frequencies in heterochromosomal recombination, suggesting a lack of correlation with chromosomal position. When the ectopic recombination efficiencies were calculated according to GOLDMAN and LICHTEN 1996 to account for differences due to chromosomal context and marker effects, the same wide range was found (data not shown). However, since the integration structures were not all identical, it is unclear if this range of ectopic recombination efficiencies is related to interactions between specific chromosomal loci or to differences in the integration structures.

One feature of ectopic recombination that was consistent in S. pombe was the low recombinant frequencies between ade6 and all other loci (Table 2 and Table 3). The ade6 locus is approximately 5 cM from the centromere of chromosome III (MUNZ et al. 1989 ). In S. pombe the centromeres are clustered near the spindle pole body in early meiosis and later become dispersed as they trail the telomeres during meiotic chromosome movement (CHIKASHIGE et al. 1997 ). These results raise the possibility that ectopic recombination between ade6 and other loci is inhibited by sequestration of the centromeres, and that spatial positioning of chromosomes within the nucleus is an important factor governing the frequency of ectopic recombination. Ectopic recombination assays with other centromere-linked loci should be helpful in testing this possibility.

Summary:
This study demonstrates that ectopic recombination and chromosomal rearrangements in S. pombe are subject to control by the M26 recombination hotspot. The similar chromosomal context effects on M26 hotspot activity and the similar frequencies of crossing over suggest overlap in the mechanisms of ectopic and allelic recombination. However, ectopic recombination occurs at a much lower frequency than allelic recombination in meiosis, indicating differential control of ectopic and allelic recombination. The role of different factors, such as homologous chromosome alignment and reduced homology in suppressing ectopic recombination in S. pombe, remain to be determined. The ability to create specific chromosomal rearrangements through ectopic recombination, similar to studies in S. cerevisiae, will be a valuable asset in furthering our understanding of chromosome dynamics.

	ACKNOWLEDGMENTS

We thank GERALD SMITH for yeast strains and helpful advice. We thank GERALD SMITH, SUE AMUNDSEN and MARY FOX for helpful comments on the manuscript. This work was supported by an institutional research grant from the Barbara Ann Karmanos Cancer Institute and the American Cancer Society.

Manuscript received February 13, 1998; Accepted for publication March 19, 1998.

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