Ac Insertion Site Affects the Frequency of Transposon-Induced Homologous Recombination at the Maize p1 Locus

Corresponding author: Thomas Peterson, 2206 Molecular Biology Bldg., Iowa State University, Ames, IA 50011., thomasp{at}iastate.edu (E-mail)

Communicating editor: V. SUNDARESAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The maize p1 gene regulates the production of a red pigment in the kernel pericarp, cob, and other maize floral tissues. Insertions of the transposable element Ac can induce recombination between two highly homologous 5.2-kb direct repeat sequences that flank the p1 gene-coding region. Here, we tested the effects of the Ac insertion site and orientation on the induction of recombination at the p1 locus. A collection of unique p1 gene alleles was used, which carry Ac insertions at different sites in and near the p1 locus, outside of the direct repeats, within the direct repeat sequences, and between the direct repeats, in both orientations. Recombination was scored by the numbers of colorless pericarp sectors (somatic frequency) and heritable mutations (germinal frequency). In both the somatic and germinal tests, the frequency of homologous recombination is significantly higher when Ac is inserted between the direct repeats than when Ac is inserted either within or outside the repeats. In contrast, Ac orientation had no significant effect on recombination frequency. We discuss these results in terms of the possible mechanisms of transposon-induced recombination.

IT has been widely reported that transposable elements can induce genome rearrangements, such as deletions, duplications, and inversions, in both eukaryotes and prokaryotes (reviewed in SAEDLER and GIERL 1996 ). In Escherichia coli, transposons Tn3 and Tn7 can increase the frequency of homologous recombination in nearby regions (KONDO et al. 1989 ; HAGEMANN and CRAIG 1993 ). Also, IS10 intermolecular transposition stimulates homologous recombination between the donor and acceptor molecules at the transposition site (EICHENBAUM and LIVNEH 1995 ). In Saccharomyces cerevisiae, homologous recombination between Ty elements produces deletions, inversions, and translocations (reviewed in BOEKE 1989 ). In Drosophila, transposition of the P element leads to increased recombination frequencies in both the male germline (HIRAIZUMI 1971 ; KIDWELL and KIDWELL 1976 ) and somatic cells (SVED et al. 1990 ) and to the generation of a variety of chromosome rearrangements (SVOBODA et al. 1995 ; PRESTON et al. 1996 ). Additionally, P-element excision promotes efficient gene conversion and enables efficient site-specific gene replacement (ENGELS et al. 1990 ; GLOOR et al. 1991 ; LANKENAU et al. 1996 ; PRESTON and ENGELS 1996 ).

In plants, initial reports indicated that maize Ac or Ds transposable element insertions either decreased (MCCLINTOCK 1953 ) or had no effect (FRADKIN and BRINK 1956 ) on meiotic recombination in the region flanking the element. At the maize bz1 locus, the insertion of a Ds element reduced the intragenic recombination frequency about fourfold (DOONER and KERMICLE 1986 ). Also, a Mu1 insertion in bz1 did not stimulate intragenic meiotic recombination (DOONER and RALSTON 1990 ). In a study of meiotic recombination at the maize a1 locus, insertion of an inactive Mu1 element reduced recombination rates in a nearby 377-bp interval by 50% (XU et al. 1995 ). In this latter case, the Mu1 insertion was inactive and, in the hemizygous condition, may have interrupted pairing or branch migration of the Holliday junction (BISWAS et al. 1998 ). More recently, DOONER and MARTINEZ-FEREZ 1997 reported that an Ac insertion could destabilize a tandem duplication in the maize bz locus, but the same insertion did not stimulate meiotic recombination or homology-dependent repair.

In contrast, several reports have demonstrated a marked ability of transposons to stimulate premeiotic homologous recombination. Transposon-induced recombination has been associated with insertion of transposable elements at the maize p1 and knotted loci (ATHMA and PETERSON 1991 ; LOWE et al. 1992 ). The p1 gene encodes a Myb-homologous transcriptional regulator of flavonoid pigmentation in kernel pericarp and cob (STYLES and CESKA 1977 , STYLES and CESKA 1989 ; GROTEWOLD et al. 1994 ). The p1 coding region is flanked by two 5.2-kb direct repeat sequences (LECHELT et al. 1989 ), and recombination between the two repeats can be easily detected as a loss of p1 gene function giving rise to colorless sectors on maize kernels (ATHMA and PETERSON 1991 ). In a previous study, it was shown that insertion of the maize transposable element Ac in the p1 gene increases the frequency of homologous recombination 100-fold (ATHMA and PETERSON 1991 ). Similarly, the Mu1 transposon is reported to increase recombination 100–2000- fold at the Kn1-O tandem direct duplication locus in maize (LOWE et al. 1992 ). In the absence of a transposon insertion, the homologous recombination rates are very low at both the p1 and knotted loci. Further studies indicated that a Mu element insertion in the Kn1-O locus stimulates meiotic recombination and gene conversion frequencies (MATHERN and HAKE 1997 ). In transgenic tobacco, the Ac element was reported to induce somatic recombination between homologous ectopic sequences (SHALEV and LEVY 1997 ). Moreover, in a transgenic Arabidopsis system, an active Ds transposon can enhance intramolecular homologous recombination >1000-fold over the spontaneous recombination frequency (XIAO and PETERSON 2000 ). Transposon excision is thought to generate transient DNA double-strand breaks (DSBs) that may initiate recombination (SZOSTAK et al. 1983 ). Indirect evidence in plants suggests that a DSB is most often repaired by nonhomologous end-joining (NHEJ, also termed illegitimate recombination; COEN et al. 1989 ; MORTON and HOOYKAAS 1995 ; SCOTT et al. 1996 ; RINEHART et al. 1997 ). However, our previous results (ATHMA and PETERSON 1991 ; XIAO and PETERSON 2000 ) show that homologous recombination can occur as an alternative to the NHEJ pathway when homologous repeat sequences flank the transposon. To study the mechanism of transposon-induced recombination in plants, we tested the effects of transposon position and orientation on the frequency of recombination observed at the maize p1 locus. The results indicate that position of the transposon within the p1 locus, but not its orientation, strongly affects the recombination frequency.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Terminology, maize stocks, and analysis of mutants:
The maize p1 gene encodes a transcription factor that regulates phlobaphene pigmentation of the pericarp, cob, and other floral organs (STYLES and CESKA 1972 , STYLES and CESKA 1989 ; GROTEWOLD et al. 1994 ). The pericarp is the outer layer of the mature kernel; it is derived from the ovary wall and therefore is of maternal origin. Maize p1 alleles are conventionally identified by a suffix that indicates their expression in pericarp and cob: P1-rr specifies red pericarp and red cob, P1-wr specifies white (colorless) pericarp and red cob, P1-ww specifies white (colorless) pericarp and cob, P1-vv specifies variegated pericarp and cob, and P1-ovov specifies orange-variegated pericarp and cob. This study utilized six p1 alleles, each containing Ac insertions at different sites in or near the p1 locus (Fig 1); all the alleles were derived directly or indirectly from P1-vv (EMERSON 1917 ) via intragenic transposition of Ac. The alleles P1-ovov-Val (F. A. VALENTINE, unpublished results), P1-rr-11:666, P1-ovov-1114, and P1-ovov-12:1-1 were derived from P1-vv, whereas the P1-9D36A and P1-9D47B alleles were derived from P1-ovov-1114 (PETERSON 1990 ; ATHMA et al. 1992 ). Genetic crosses and mutant screens were performed as described previously (ATHMA and PETERSON 1991 ).

View larger version (70K): In this window In a new window Download PPT slide   Figure 1. Ear phenotypes of p1 alleles used in this study. From left to right: P1-rr:11:666; P1-9D47B; P1-ovov-Val; P1-9D36A; P1-ovov-1114; P1-ovov-12:1-1. Each allele is in heterozygous condition with allele P1-wr, which specifies colorless pericarp and red cob. Cell clones with losses of p1 function are visible as colorless pericarp sectors; numbers of these sectors are given in Table 1.

Frequency of colorless sectors:
Colorless sectors were scored on ears from plants in which the P1::Ac insertion allele was heterozygous with a P1-wr allele. The colorless sector frequency was calculated from the percentage of kernels with visible colorless sectors, regardless of the size of the sector. Sector size reflects the developmental stage at which a loss-of-function mutation occurs; the later in development a mutation occurs, the smaller the resulting sector. The average colorless sector frequency of each allele was determined by counting the number of kernels with colorless sectors divided by the estimated total number of kernels. For each allele, sectors were scored on 32 randomly picked ears, except for the P1-ovov-12:1-1 allele for which 16 ears were used. The number of kernels per ear was estimated as the product of the number of kernel rows times the mean number of kernels per row. P values of the t-tests were generated by Microsoft Excel 97.

DNA isolation, Southern blot hybridizations, and PCR:
The approximate site of Ac insertion in each allele was determined by genomic Southern hybridization. Genomic leaf DNA was isolated from seedling leaves of individual plants as previously described (COCCIOLONE and CONE 1993 ), digested with restriction enzymes SalI or EcoRI, electrophoresed through 0.75% agarose gels, and transferred to nylon membranes (Micron Separations, Westborough, MA; SAMBROOK et al. 1989 ). Blots were hybridized with random-prime-labeled (Pharmacia, Piscataway, NJ) p1 locus DNA fragments 15 and 8B (Fig 2). Together, these probes detect a 26-kb contiguous region encompassing the p1 gene and its flanking sequences. The exact Ac insertion sites were then determined by PCR amplification, using primers in the Ac sequence and a second primer homologous to p1 gene sequences. Genomic DNA templates were amplified by PCR for 35 cycles as follows: denaturation at 94° for 30 sec; annealing at 59° for 30 sec; and elongation for 1 min at 72°. PCR products were cloned into pT7Blue T-vector (Novagen) and sequenced at the Iowa State University DNA sequencing and synthesis facility.

View larger version (16K): In this window In a new window Download PPT slide   Figure 2. (Top) Ac insertion sites in different maize p1 gene alleles. (Bottom) Schematic diagram showing Ac-induced homologous recombination. () 5.2-kb direct repeats; () p1 gene transcription start site; () Ac insertion; () Ac transcription direction; () fragment 15; () fragment 8B; () exons; () 5' leader sequence and 3' untranslated region; I1 and I2, intron 1 and intron 2; S, SalI site; S*, methylated SalI site; 1, P1-rr:11:666; 2, P1-9D47B; 3, P1-ovov-Val; 4, P1-9D36A; 5, P1-ovov-1114; 6, P1-ovov-12:1-1.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Ac insertion sites of different p1 alleles:
To assess the effect of the Ac insertion site on the frequency of homologous recombination, we used a collection of six p1 alleles that carry Ac elements inserted at various sites in and near the p1 gene. The position of Ac insertion in each allele was approximated by Southern blot hybridization and then precisely determined by PCR amplification and sequencing of Ac/p1 junction fragments. Ac insertion site positions are depicted in Fig 2. The P1-rr-11:666 allele contains Ac inserted outside the p1 locus 5.2-kbp direct repeat, at position 6757 bp 5' of the P1-rr transcription start site. The P1-9D47B allele has Ac inserted within the 5.2-kbp P1-rr direct repeat, at position 5024 bp 5' of the P1-rr transcription start site. Four of the alleles contain Ac inserted at various sites between the two 5.2-kbp direct repeats: at 49 bp 5' of the transcription start site (P1-ovov-Val); in intron 1 at 473 bp downstream of the transcription start site (P1-9D36A); and at two sites within the large second intron (P1-ovov-1114 and P1-ovov-12:1-1), at positions 4338 and 4490 bp, respectively, downstream of the transcription start site (ATHMA et al. 1992 ). Ac insertions within the p1 gene introns exhibit an orientation-dependent effect on p1 expression; when Ac is inserted in the same transcriptional orientation as p1, transcripts that initiate from the p1 gene promoter terminate within the Ac element. Hence, in the P1-vv allele, p1 gene expression is blocked by Ac except in the cell clones in which Ac has excised. The resulting kernel pigmentation phenotype consists of colorless pericarp with red sectors (LECHELT et al. 1989 ). In contrast, when Ac is inserted in the opposite transcriptional orientation relative to the p1 gene, Ac does not markedly interfere with p1 transcription and the Ac insertion is apparently spliced out of the p1 transcript (PETERSON 1990 ). This orientation-dependent effect has been documented in the case of Ds insertions in the maize waxy locus (WESSLER et al. 1987 ). Whereas the P1-ovov-Val allele gives orange-variegated pericarp, even though Ac is inserted in the same transcriptional orientation as the p1 gene, in this case, Ac is located 49 bp upstream of the p1 transcription start site and hence would not be expected to exhibit the orientation-dependent phenotypic effect observed with Ac/Ds insertions in transcribed regions.

Comparison of colorless sector frequency of different p1 alleles:
Each of the six p1 alleles studied here specifies red or orange-variegated pericarp pigmentation (Fig 1); when these alleles are heterozygous with a colorless pericarp allele (either P1-ww or P1-wr), loss-of-function mutations produce easily visible colorless pericarp sectors (Fig 1). A colorless pericarp sector can result from any somatic mutation that eliminates p1 gene function. Because Ac frequently transposes to linked sites (GREENBLATT 1984 ; DOWE et al. 1990 ; PETERSON 1990 ; GROTEWOLD et al. 1991 ; ATHMA et al. 1992 ; MORENO et al. 1992 ), some sectors may result from intragenic transpositions to other sites in the p1 locus essential for P function. In a previous study, it was found that the great majority of colorless sectors (49 out of 52 analyzed) arising from the P1-ovov-1114 allele were p1 deletions caused by recombination between the long 5.2-kb direct repeats flanking the P1-rr gene (ATHMA and PETERSON 1991 ). Thus, the number of colorless pericarp sectors provides an indication of the frequency of homologous recombination between the p1 locus flanking repeats. In the absence of Ac at the p1 locus (P1-rr-4026) the colorless sector frequency is 0.031% (ATHMA and PETERSON 1991 ). This frequency is increased from 50- to 150-fold in the Ac insertion alleles characterized here (Table 1). Additionally, we characterized an allele (P1-9D27B) containing a Ds insertion generated by an internal deletion in the Ac element in the P1-ovov-1114 allele; hence, this Ds element is at the same site as the Ac element in the P1-ovov-1114 allele. In the absence of Ac, this allele exhibited two colorless sectors in 2008 kernels (0.1%); in the presence of an Ac in trans, this allele produced 65 colorless sectors among 2025 kernels (3.2%). These results confirm the finding of XIAO and PETERSON 2000 that Ac transposase is essential for induction of recombination by a Ds element.

To determine the possible effects of Ac position and orientation on recombination frequency, we used a t-test for significant differences in the colorless sector frequencies between alleles. The results (Table 2) indicate that the six alleles fall into three statistical groups: Group 1 comprises the alleles P1-ovov-Val, P1-ovov-1114, and P1-ovov-12:1-1, with colorless sector frequencies ranging from 3.6 to 5.0%. Group 2 comprises the P1-rr-11:666 allele (colorless sector frequency 2.4%). Finally, Group 3 comprises alleles P1-9D47B and P1-9D36A, with 1.6 and 1.8% colorless sector frequency, respectively. However, if the alleles are grouped on the basis of their insertion location, the average colorless sector frequency is higher for Ac elements inserted between the two direct repeats (group A, 3.5%) than for Ac elements inserted outside and within the repeats (group B, 2.0%). A t-test analysis shows that the frequencies of colorless sectors of these two groups are significantly different (P value = 2.48 x 10^-5). Overall, these results show that Ac insertions between the two direct repeats induce a higher frequency of recombination than Ac insertions outside or within the repeats.

Developmental timing of recombination:
While the numbers of sectors indicate frequency of recombination, sector size indicates the developmental time at which mutation occurs. As shown in Table 3, colorless sectors ranged in size from <¹/₄ kernel to sectors covering more than six kernels. In all six alleles, smaller sectors were much more numerous than larger sectors; this most likely reflects the fact that more cells are available for mutation at later stages of development. We also considered the possibility that sector size could affect the frequency measurements of different alleles, because the area available for mutation is slightly altered by the fact that once an area has mutated it is no longer available for mutation (ANDERSON and EYSTER 1928 ). We found that larger sectors (>1 kernel) were observed only in the P1-9D36A, P1-ovov-Val, P1-ovov-1114, and P1-ovov-12:1-1 alleles. Thus, the potential complication of sector size actually did not affect the conclusion that alleles with Ac inserted between the two direct repeats have more colorless sectors than alleles with Ac inserted outside or within the direct repeat.

Comparison of germinal mutations of different alleles:
As noted above, the colorless pericarp sectors scored in the somatic assay can result from mutations other than recombination of the flanking repeats. To assess the types of mutations generated, we isolated a number of heritable mutants derived from each insertion allele and analyzed these mutants by Southern blot hybridization (Fig 3). The results allowed us to compare the frequencies of germinally transmitted recombination events produced by different alleles (Table 4). From the P1-rr-11:666 allele, 5 P1-ww mutants were recovered, but none were derived from homologous recombination. From the P1-9D47B allele, 7 P1-ww mutants were obtained, and only 1 resulted from recombination. In contrast, from the four alleles (P1-ovov-Val, P1-9D36A, P1-ovov-1114, and P1-ovov-12:1-1) with Ac insertions between the two 5.2-kb direct repeats, 85 heritable P1-ww mutants were obtained, and 61 of these were deletions generated by recombination of the flanking repeats. The other 24 P1-ww mutants were generated by intragenic Ac transposition or other transposition-related rearrangements (data not shown). These results suggest that the somatic assay is a reasonable indicator of recombination frequency for the alleles that carry Ac insertions between the repeats, but that it may overestimate the true recombination frequency for alleles that carry Ac inserted within or outside the direct repeats.

View larger version (78K): In this window In a new window Download PPT slide   Figure 3. Representative Southern blot analysis of new p1 mutations. Genomic DNA samples from individual plants were digested with SalI and hybridized with p1-specific probe P15, a genomic fragment from the P1-rr allele (Fig 2; LECHELT et al. 1989 ). Lane 1, P1-rr gives bands of 3.4, 3.0, and 1.2 kb; the 1.2-kb band is a doublet arising from the duplicated sequences flanking the P1-rr gene. Lane 2, P1-ww-1112 is deleted for the 3.4-kb band and one copy of the 1.2-kb fragment; this mutation is the archetype of the class arising via recombination of the p1-flanking duplications (ATHMA and PETERSON 1991 ). Lanes 3–5, p1 mutations derived from the P1-rr-11:666 allele; lanes 6–15, p1 mutations derived from the P1-9D36A allele. The banding patterns in lanes 6, 13, and 15 identify these as mutations arising by recombination. The banding patterns in the other lanes do not match those of P1-ww-1112 (lane 2), and thus these mutations have other types of lesions in the p1 locus.

Effect of Ac orientation:
The six alleles characterized here contain Ac insertions in one of two orientations with respect to the p1 gene: the VV group (P1-rr-11:666 and P1-ovov-Val) in which Ac is inserted in the same transcriptional orientation as the p1 gene and the OVOV group (P1-9D47B, P1-9D36A, P1-ovov-1114, and P1-ovov-12:1-1) with Ac inserted in the opposite orientation. The average colorless sector frequency among both the VV and OVOV group alleles is 3.0%. More importantly, comparisons among the alleles with Ac insertions between the direct repeat sequences show no significant differences in somatic recombination frequency between P1-ovov-Val and P1-ovov-1114, nor between P1-ovov-Val and P1-ovov-12:1-1 (Table 2). There is a significant difference in somatic recombination frequency between P1-ovov-Val and P1-9D36A, but it is unclear whether this is related to the orientation of the Ac insertion in P1-9D36A (see DISCUSSION). Additionally, comparison of germinal mutation frequencies also shows no significant effect of Ac orientation on the frequency of homologous recombination (Table 4).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The aim of this study is to test the influence of the position and orientation of Ac elements inserted at the maize p1 locus on the induction of homologous recombination in nearby sequences. The results indicate that Ac insertions in the 8.2-kbp interval between the 5.2-kbp direct repeats can significantly increase recombination frequencies when compared to insertion within or outside of the direct repeats. These results were initially observed in a somatic assay and were even more pronounced when germinal mutations were examined. Here, we discuss the implications of these results for proposed mechanisms of DSB repair and transposon-induced recombination in plants.

Homologous recombination initiated by a double-strand break:
In yeast, it is well documented that DNA DSBs promote mitotic recombination (ORR-WEAVER et al. 1981 ; OSMAN et al. 1996 ). In plants, the frequency of intrachromosomal homologous recombination can be enhanced by X-ray, gamma-ray, and UV irradiation (TOVAR and LICHTENSTEIN 1992 ; LEBEL et al. 1993 ; PUCHTA et al. 1995 ) and by chemical agents that induce DSBs, such as methylmethanesulfonate and mitomycin C (PUCHTA et al. 1995 ). Moreover, homologous recombination in plants is enhanced by in vivo induction of DNA double-strand breaks by a site-specific endonuclease (PUCHTA et al. 1993 ; CHIURAZZI et al. 1996 ). In Drosophila, the P element can undergo precise excision in a homology-dependent process requiring P trans- posase (ENGELS et al. 1990 ). In Caenorhabditis elegans, the frequency of precise reversion following excision of the Tc1 element is much higher when heterozygous with a wild-type homologous chromosome (PLASTERK 1991 ). Both of these examples indicate that DSBs generated by transposon excision can be repaired using the homologous locus as a template. The precise mechanism of Ac/Ds transposition is unknown, but genetic evidence indicates that it occurs via a "cut and paste" mechanism (GREENBLATT and BRINK 1962 ; CHEN et al. 1992 ). Together, these studies are consistent with the hypothesis that transposon-induced homologous recombination is initiated by a DNA double-strand break generated by transposon excision.

Mechanism of transposon-induced recombination in plants: Ac insertion site and recombination frequency:
A substrate with two direct repeats separated by an internal sequence was used to study DSB-induced intrachromosomal recombination in S. cerevisiae. The recombination frequency was >10 times higher when the DSB was induced in the internal sequence than at a site outside the repeats. In both cases, >90% of the recombinants were of the deletion type, in which the internal sequence was deleted but one direct repeat was retained (PRADO and AGUILERA 1995 ). This recombination product is similar to what we observed at the maize p1 locus. Similar results were obtained in the yeast Schizosaccharomyces pombe, where 99.8% recombinants were of the deletion type when the DSB was generated between the direct repeats, whereas if the DSBs were generated within one repeat, the deletions were reduced to 77% (OSMAN et al. 1996 ). These deletions were proposed to occur via the single-strand annealing (SSA) pathway (LIN et al. 1984 ). The SSA model proposes that the free 5' ends produced by a DSB are degraded by a 5' to 3' exonuclease, exposing regions of homology on both sides of the break. The 3' single strands anneal at complementary regions, and the protruding nonhomologous ends are removed by a flap endonuclease (Fig 4, steps c and d). According to this model, the reduction in deletion frequency observed when a DSB is induced within the repeat sequences reflects the reduced chance that a free 3' single-stranded DNA end generated by 5' to 3' exonucleolytic digestion will find its complementary counterpart, due to the nonsymmetrical position of the DSB.

View larger version (9K): In this window In a new window Download PPT slide   Figure 4. Models for transposon-induced homologous recombination. Red lines represent the inserted transposon; green lines, homologous direct repeats; thick black lines, sequences internal to the direct repeats; thin black lines, other genomic sequences. (a) Transposase binds to the terminal and subterminal regions of transposon causing the association of the transposon ends and generating a DNA double-strand break. (b) Exonuclease degrades from 5' to 3' at the DSB site and generates 3' single-strand DNA. The transient DSB is repaired by one of three pathways: single-strand annealing (SSA; c and d); annealing-dependent synthesis (ADS; e–g); one-sided invasion (OSI; h–k). (c) The 5' to 3' degradation reaches the homologous region and two 3' single-strand homologous sequences pair with each other. (d) Heterologous DNA is digested by nuc- lease and gaps are ligated. (e) Both strands of DSB are degraded by exonucleases that process the 5' end faster than the 3' end to form a 3' overhang. (f and g) Once 3' end degradation reaches homologous regions, complementary strands can anneal with each other to prime DNA synthesis and fill the remaining gaps. Green dotted lines indicate newly synthesized strands. (i–k) The 3' end invades the homologous repeat copy. The heterologous DNA at the 3' end is removed by nuclease. (j) D-loop is nicked at the red arrow, and DNA synthesis follows to generate a Holliday structure. (k) Resolution of the Holliday junction results in deletion of the p1 gene and retention of one copy of the direct repeat sequence.

In the case of the maize p1 locus, the interval between the two flanking repeats is 8.2 kbp. According to the SSA model, induction of a DSB at a site that is more centrally located within this interval would be expected to produce a higher frequency of recombination of the homologous flanking repeats compared to sites that are closer to one side of the interval, assuming that exonucleolytic degradation proceeds in both directions from the DSB at approximately equal rates. However, the recombination frequency of P1-ovov-Val is not significantly different from that of P1-ovov-12:1-1 and P1-ovov-1114 (Table 2), even though the locations of the Ac insertion alleles are very different: the Ac insertion site of allele P1-ovov-Val is 979 bp downstream of the end of the 5' direct repeat of the p1 gene, while the Ac insertion sites of alleles P1-ovov-1114 and P1-ovov-12:1-1 are 5.4 kb downstream of the end of the 5' direct repeat of the p1 gene. Recombination in allele P1-ovov-Val via the SSA model would necessitate degradation of >12 kbp on the downstream side of the DSB to expose a complementary sequence for pairing with the upstream repeat.

A variation of the SSA model is termed synthesis-dependent strand annealing (SDSA) (FORMOSA and ALBERTS 1986 ). In this model, the free 3' ends resulting from a DSB invade a homologous duplex and act as primers for DNA synthesis. The newly synthesized DNA strands anneal with each other, followed by further DNA synthesis to fill remaining gaps. On the basis of the SDSA model, we propose a model of annealing-dependent synthesis (ADS). Following formation of a DSB, both broken ends are degraded by exonucleases that process the 5' end more rapidly than the 3' end, resulting in 3' overhanging strands on both sides of the break (Fig 4E). Once the 3' end degradation reaches a region of homology (in the p1 locus within the flanking 5.2-kbp direct repeats), the complementary strands can anneal with each other, and the 3' ends prime DNA synthesis to fill remaining gaps (Fig 4F and Fig G). This ADS model can account for the observed generation of the p1 mutants containing a single copy of the 5.2-kbp direct repeat sequence.

An alternative pathway for the repair of DSBs has been termed one-sided invasion (OSI; BELMAAZA and CHARTRAND 1994 ); the OSI mechanism appears to be a major pathway in plant somatic recombination (PUCHTA et al. 1996 ; PUCHTA 1998 ). To explain the formation of p1 gene deletions, the OSI model proposes that a free 3' end generated by DSB would invade the opposite duplex. Complementary regions within the homologous repeat sequences would pair, and the nonhomologous 3' end sequences would be removed by a flap endonuclease. Resolution of the resulting Holliday junction would result in deletion of the interval region and one repeat (Fig 4, h–k). A potential complication in the case of the p1 locus is that the invading 3' ends may have several kilobase pairs of heterologous sequence arising from the interval between the direct repeats, and these heterologous sequences could affect the homology search and pairing with the downstream repeat sequences.

The behavior of an allele that contains Ac inserted within the direct repeat sequences provides a test of the predictions of the ADS and OSI models. Formation of a DSB by excision of Ac from the upstream direct repeat sequence would generate a free end homologous to the downstream direct repeat, and this should be the most efficient substrate for the OSI model, whereas the same free end would be a less efficient substrate for the ADS model: Exonucleolytic degradation in both directions from the DSB would likely result in deletion of the remaining 1 kbp portion of the upstream 5' flanking repeat before the degradation of the downstream strand had proceeded the 12 kbp required to expose the complementary region in the downstream repeat sequence. The observed low frequency of deletions generated by the P1-9D47B allele is consistent with the expectations of the ADS model, but not the OSI model.

It should be noted that our ability to detect recombination events is based on a screen for loss of p1 expression; hence, we would not have detected gene conversion events that restore p1 function, if they did occur. Molecular analysis indicates that Ds elements may arise via double-strand gap repair following Ac transposition (RUBIN and LEVY 1997 ; YAN et al. 1999 ); however, such repair synthesis appears to be rare compared to the frequency of simple end-joining following Ac excision, regardless of whether homologous sequences are present in cis or in trans (DOONER and MARTINEZ-FEREZ 1997 ). Relevant to this study, DOONER and MARTINEZ-FEREZ 1997 observed no cases of conversion of the Ac insertion site by the adjacent duplication in the bz(Dp26)-m1 allele, which is structurally similar to P1-9D47B (i.e., a cis duplication with Ac inserted in one repeat copy). This suggests that gene conversion events that restore p1 function, if they occur, are probably too infrequent to account for the low frequency of recombination observed in the P1-9D47B allele.

How do transposons stimulate recombination?
In addition to the importance of a DSB, transposon-induced recombination may involve the recruitment of host factors by transposase. Our data from transgenic Arabidopsis show that active transposons can greatly increase intrachromosomal homologous recombination (>1000-fold higher than control; XIAO and PETERSON 2000 ). This frequency is 10–100 times higher than that observed for direct induction of a DSB (CHIURAZZI et al. 1996 ). One possible explanation for this high level of enhancement is that the efficiency of generating DSBs is different in these two experiments. Another explanation is that Ac transposase might recruit host factors that promote homologous recombination. There is some evidence that host plant factors are involved in transposition: First, host proteins that bind subterminal regions of Ac elements were found in both tobacco and maize (BECKER and KUNZE 1996 ; LEVY et al. 1996 ). Second, a recessive mutation found in Arabidopsis can increase the frequency of Ac transposition (JARVIS et al. 1997 ). Possibly, the Ac transposition process may recruit host factors required for a nonhomologous end-joining and/or homologous recombination. Following transposition, a DSB generated by transposon excision may be repaired by alternative pathways depending on the sequences or chromatin structure near the break (SCOTT et al. 1996 ). Further experiments will be required to elucidate the relative roles of DSBs, host factors, and chromatin structure in transposon-induced recombination.

The observation that Ac induces recombination between directly duplicated sequences contrasts with a recent report that Ac does not stimulate homologous meiotic recombination in the maize bz1 gene (DOONER and MARTINEZ-FEREZ 1997 ). To reconcile these apparently conflicting results, it is important to note that the transposon-induced recombination events we observed in maize (this report) and Arabidopsis (XIAO and PETERSON 2000 ) are premeiotic. That is, these events occurred during sporophytic development to generate clonal sectors of genetically distinct cells. If these somatic sectors of mutant cells are included in the lineage that gives rise to the gametophytes, the premeiotic mutations can be transmitted to the next generation. In contrast, the events studied by DOONER and MARTINEZ-FEREZ 1997 appeared to occur at or near meiosis. The lack of effect of Ac excision on meiotic recombination may be due to temporal or spatial differences in the occurrence of Ac transposition relative to meiotic recombination. For example, Ac transposition is known to occur predominantly during or shortly after DNA replication (GREENBLATT and BRINK 1962 ; CHEN et al. 1992 ), whereas meiotic recombination occurs after pairing of homologous chromosomes. Alternatively, Ac transposase may be spatially excluded from chromosomal regions that are undergoing meiotic recombination.

Anomalous mutation frequency of the P1-9D36A allele:
Although the Ac insertion position in the P1-9D36A allele is between the two direct repeats, its colorless sector frequency is second lowest among all alleles. Nevertheless, the germinal recombination frequency of this allele is similar to that of other alleles with Ac inserted between the repeats. Thus, the P1-9D36A allele is suppressed in frequency of somatic mutations, yet it has a normal germinal mutation frequency. This mutation bias is opposite to that effected by a dominant mutation that suppresses germinal reversion of an allele of the maize waxy gene with a Ds insertion (EISSES et al. 1997 ). The observed low somatic mutation rate of the P1-9D36A allele could result from suppression of the activity of the Ac transposon. It has been demonstrated that the Ac element activity can vary depending on the Ac copy number and location (MCCLINTOCK 1964 ; HEINLEIN 1995 ; for review see FEDOROFF and CHANDLER 1994 ). Each of the alleles studied here was crossed to a r-sc:m-3 Ds tester stock; in this test, multiple copies of Ac elements delay the occurrence of Ds excision, resulting in small colored revertant sectors in the kernel aleurone (KERMICLE 1980 ; SCHWARTZ 1984 ). Ac activity of the P1-9D36A allele in the aleurone appeared normal; thus, suppression of Ac activity in pericarp cells, if it occurred, would have had to occur without affecting Ac activity in aleurone cells. Ac activity has also been shown to be inversely correlated with Ac methylation (SCHWARTZ and DENNIS 1986 ; CHOMET et al. 1987 ; SCHWARTZ 1989 ; BRUTNELL and DELLAPORTA 1994 ). However, Southern hybridizations show that the BamHI site at the Ac 5' end is unmethylated in the P1-9D36A allele (not shown); moreover, the Ac insertion in P1-9D36A is located in a region of the p1 gene that is relatively free of DNA methylation (DAS and MESSING 1994 ; CHOPRA et al. 1998 ). Therefore, the low frequency of colorless sectors in the P1-9D36A allele cannot be attributed to increased methylation of the Ac element, nor to the proximity of a hypermethylated region in the p1 locus. The basis for the anomalous somatic sector frequency of P1-9D36A remains to be determined.

	ACKNOWLEDGMENTS

We thank Yu-Feng Wang for assistance with statistical tests and Lyudmila Sidorenko for assistance in PCR amplification. This work was supported by a grant from the Iowa Corn Promotion Board, journal paper no. J-18886 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. 3297, and supported by the Hatch Act and State of Iowa funds.

Manuscript received May 8, 2000; Accepted for publication August 24, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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