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Copyright 8 1988 by the Genetics Society of America

Large-Scale Chromosomal Restructuring Is Induced by the Transposable Element Tam3 at the nivea Locus of Antirrhinum majus

Cathie Martin, Steve MacKay and Rosemary Carpenter Department of Genetics, John Innes Institute, Norwich NR4 7UH, England

Manuscript received August 24, 1987 Accepted January 15, 1988

ABSTRACT The transposable element, Tam?, gives rise to large-scale (greater than 1 kb) chromosomal

rearrangements at a low frequency, when it is inserted at the nivea locus of Antirrhinum majw. Although some deletions may result from imprecise excision of Tam?, rearrangements involving deletion, dispersion and inverted duplication of flanking sequences, where Tam3 remains in situ, have also been identified. These rearrangements have been mapped at the molecular level, and the behavior of Tam? following rearrangement has been observed. It is clear that Tam? has enormous potential to restructure chromosomes through successive rounds of large-scale rearrangements. The mechanisms by which such rearrangements might arise are discussed.

T HE ability of transposable elements to rearrange relatively large regions of chromosomes was

one of their first properties to be described (MCCLIN- TOCK 1946). Chromosomal breaks induced by the transposable element Ds gave rise to deletions and duplications in subsequent generations because of breakage-fusion-bridge cycles. MCCLINTOCK (1953, 1954) also found that large deletions of the chromosomal material flanking Ds could occur at low frequency. More recently, analyses at the molecular level showed that the maize transposable elements Ac and Mutator can be associated with deletions of flanking sequences (TAYLOR and WALBOT 1985; DOONER 1985). These deletions appear similar to the events described cytogenetically by MCCLINTOCK, although their size is presumably much smaller.

Similar deletions are associated with bacterial transposons and insertion sequences (OHTSUBO and OHT- SUBO 1977; REIF and SAEDLER 1975, 1977; Ross, SWAN and KLECKNER 1979; CORNELIS and SAEDLER 1980) [reviewed by KLECKNER (1983) and by HEFFRON (1983)l. In bacteria, deletions and inversions of sequences flanking transposons and insertion sequences are thought to arise from intramolecular recombination and intramolecular transposition (SHAPIRO 1979; COHEN and SHAPIRO 1980; IIDA, MEYER and ARBER 1983; HEFFRON 1983; KLECKNER 1983).

Similar types of DNA rearrangements have been reported for FB elements in Drosophila (BINGHAM 1981; COLLINS and RUBIN 1984). Deletion of sequences flanking an FB element, duplication and inversion of flanking sequences and reciprocal trans- location can occur at low frequency. Large-scale rearrangements of DNA such as inversions, deletions

Genetics 119 171-184 (May, 1988).

and duplications have also been reported in Drosophila associated with retrovirus-like transposons (GOLD- BERG et al. 1983, DAVIS, SHEN and JUDD 1987), hybrid- dysgenesis (reviewed by ENGELS 1983), male-recombination chromosomes (GREEN and SHEPHERD 1979) and unstable X chromosomes (LIM 198 1; LIM et al. 1983). Chromosomal rearrangements of this type appear to center on particular hot spots, which are sites of transposable element insertion.

Rearrangement of large regions of DNA therefore appears to be a feature of transposable element activity although the frequency at which such rearrangements occur relative to transposon excision differs for different types of mobile element.

This paper describes a series of large-scale rearrangements induced by the transposable element Tam3 at the nivea locus of Antirrhinum majus. The nivea locus (chalcone synthase) is involved in antho- cyanin biosynthesis in the flowers. When Tam3 is inserted at the locus in line nivearecu"etrr:98 (nivrec:98), gene expression is reduced and the flowers are palely pigmented. The phenotype is unstable, however, and when Tam? excises somatically, gene expression is increased to give darker red sites or sectors (HARRISON and CARPENTER 1979; SOMMER et al. 1985). Excision in the cells that give rise to the gametes yields full red revertant progeny. Although reversion to full red is the most common event resulting from Tam3 activity at the nivea locus, novel phenotypes may also be generated, suggesting that Tam3 may occasionally give rise to other DNA rearrangements (CARPENTER, MARTIN and COEN 1987).

By examining mutants of niurec: 98 that show mod- ified phenotypes, and also by random screening, we have identified and mapped a number of relatively

172 C. Martin, S. MacKay and R. Carpenter

large DNA rearrangements induced by T a d at the nivea locus. Included in these rearrangements are deletions of various types, dispersal of sequences along the chromosome and a novel type of large insertion resulting from inverted duplication of sequence. T a d frequently remains at the locus and continues to excise, suggesting that it may be able to cause successive changes leading to complex chromosome restructuring. As T a d exists in multiple copies (MARTIN et al. 1985) it therefore has the potential to reorganize a large proportion of the genome.

MATERIALS AND METHODS

Production of Antirrhinum lines: The Antirrhinum line that gave all the rearrangements described was niv"':98 (HARRISON and CARPENTER 1979; SOMMER et al. 1985). This line carries a Tam? insertion 63 bp upstream of the start of chalcone synthase (nivea) gene transcription (SOMMER et al. 1985; SOMMER and SAEDLER 1986). Mutants carrying DNA rearrangements induced by T a d in the germline were identified in three ways.

1. Some rearrangements which altered the phenotype of the flowers by modifying nivea gene expression were identified in the progeny of crosses between niv'"":98 and either of two stable nivea null alleles, niv- : 44 and niv- : 45 (CARPENTER, MARTIN and COEN 1987). All the rearrangements were germinal, and had therefore occurred in the parent. All were found to be associated with the nivrec:98 allele, the other allele being unaffected. Against the acyanic null alleles from niv- :44 and niv- :45, rearrangements that blocked or reduced chalcone synthase (nivea) gene expression could be easily selected. Heterozygous plants carrying one rearranged allele were selfed and the homozygotes were selected in the progeny, except where the rearrangement was inviable in the homozygous form. In such cases the rearranged allele was maintained as a heterozygote.

2. A few rearrangements were selected phenotypically in progeny from self-fertilized nivreC:98. Such plants were generally identified as showing a reduction of pigmentation, or many acyanic sites on the flowers. In such cases the plants were selfed and progeny homozygous for the rearrangements were selected by examining restriction enzyme fragments on Southern blots.

3. Some rearrangements were discovered by random screening of DNA from niv"':98 plants. Plants showing unusual restriction fragments were selfed and homozygotes carrying rearrangements were identified on Southern blots.

Lines carrying rearrangements were given stock numbers and a name designed to convey the phenotype of the plant: niv- for an albino, niv for a plant with pale stable pigmentation and niv"" for a plant with somatically unstable pigmentation.

Mapping of DNA rearrangements generated by T a d : Genomic DNA was extracted as described by MARTIN et al. (1985), and digested with restriction enzymes, separated by agarose gel electrophoresis and transferred to nitrocellulose (SOUTHERN 1975; WAHL, STERN and STARK 1979). To map the DNA rearrangements, several DNA fragments were prepared from two clones of the wild-type nivea locus, pAm3 and pAml (SOMMER et al. 1985; SOMMER and SAEDLER 1986), and a clone of T a d , pAm8 (SOMMER et al. 1985), kindly provided by H. SOMMER.

DNA fragments were prepared by agarose gel electro-

phoresis of the plasmid cut with appropriate restriction enzymes. The agarose containing the required fragments was cut out and frozen for 2 hr at -20°C. The DNA was then removed by squeezing the liquid from the frozen gel through a syringe. Preparations were extracted once with phenol :chloroform (1 : 1) and once with chloroform alone. The aqueous phase was precipitated with 2 vol absolute ethanol and 0.1 vol 3 M sodium acetate pH 5.5. DNA fragments were redissolved in 10 mM Tris pH 8.0, 1 mM EDTA and radioactively labeled with "P-dCTP by nick translation and hybridized to the nitrocellulose filters (MAN- IATIS, FRITSCH and SAMBROOK 1982). The filters were washed twice after hybridization in 0.1 X SSC (0.15 M NaCI, 0.015 M sodium citrate pH 7.0) 0.5% sodium dodecyl sulfate at 65°C for 1 hr.

RESULTS AND DISCUSSION

Fine structure of the nivrec: 98 progenitor allele and analysis of full red revertants

T o develop a rapid method for screening rearrangements of DNA at the nivea locus it was necessary to produce a series of DNA fragments that could be used to probe Southern blots of genomic DNA.

T a d is inserted in the promoter of the nivea gene in nivre":98, within a 158 bp KpnI fragment (SOMMER et al. 1985) (Figure 1). EcoRI does not cut within T a d , so that when genomic DNA from niurec: 98 cut with EcoRI is probed with a clone of the wild- type nivea locus (pAm3) a fragment of 9.2 kb hybridizes (Figure 1). This consists of 5.7 kb of nivea flanking sequences plus the 3.5-kb T a d insertion. Digestion of pAm3 with EcoRI and KpnI gives EcoRIIKpnI fragments of 3.5 kb (A) and 2.0 kb (B) which can be used as probes for the sequences flanking Tam3 to the left and right, respectively. From the sequence of the chalcone synthase gene (SOMMER and SAEDLER 1986) the KpnI site to the right of Tam3 lies only 20 bp from the point of insertion. Cutting genomic DNA with EcoRI and KpnI and probing Southern blots with fragment B showed whether the sequence close to T a d on the right remained intact. The sequence also revealed a DdeI site lying 1 bp to the left of T a d . The 8-bp direct duplication formed on T a d integration also contains a DdeI site so that T a d is flanked by two DdeI sites. Probing genomic DNA cut with DdeI with fragment A showed whether one or both of these DdeI sites remained intact.

The most frequent event found in nivrec: 98 progeny is reversion to full red expression which is dominant to the nivre':98 phenotype. Digestion of DNA from 50 full red revertant plants with EcoRI and probing with the wild-type sequences from pAm3 showed that each revertant carried at least one fragment of 5.7 kb. Since this is the size of the wild-type fragment, reversion to full red in each case was accompanied by excision of T a d .

Excision of transposable elements in higher plants is rarely precise. Small sequence rearrangements

Chromosomal Restructuring 173

a

Chalcone synthase coding sequence

m 4 b-

Fragment C

Fragment A Fragment B

b

1 kb 1 I

FIGURE I.-Restriction maps of the niuea (chalcone synthase) locus in A. majw (a), and the Tam3 insertion present in niu":98 (b) (from SovueR et al. 1989; SOVVER and SAEDLER 1986). Fragments A, B and C, shown in (a), were used as radioactive probes in genomic mapping experiments. For clarity only the DdeI site(s) next to the point of Tam3 insertion are shown.

involving 1 to 30 bp appear to be a common feature of excision (SCHWARZ-SOMMER et al. 1985; SAEDLER and NEVERS 1985). T o determine the extent to which reversion to full red phenotype involved sequence rearrangements, DNA from 20 full red revertants was digested with EcoRI and KpnI or with DdeI and probed with fragment B or fragment A respectively. All 20 plants retained the right hand KpnI site, and in addition, at least one of the DdeI sites. This suggested that excision of Tam3 is normally associated with rather modest sequence rearrangements. However, insertions and deletions of upstream sequence of less than 50 bp would not necessarily have been detected by these methods.

Deletions arising from niv"': 98 Deletions accompanying excision of T a d : After

simple excision, the most common rearrangement arising from nivrec:98 was deletion. Deletions were of various types but the most common involved concomitant loss of T a d . When genomic DNA from several lines was digested with EcoRI and probed with fragments A or B, a band smaller than the wild- type band of 5.7 kb was observed. Three examples are shown in Figure 2. In these alleles it seemed likely that Tam3 had been excised, taking with it some flanking DNA. When genomic DNA from these plants was digested with DdeI and probed with fragment A, some plants retained one or both DdeI sites

progenitor nivrec:98

T=7=

e' t!!

2 n iv- :560

3 n iv ' :562

FIGURE 2.-Restriction maps of mutants arising from imprecise excision of T a d : 1) deletion of about 290 bp of niuea sequences lying mainly to the left of Tam3 (line niu:364); 2) deletion of about 900 bp of niuea sequences to the left of the Tam3 insertion site (niu- :560); 3) deletion of about 800 bp of niuea sequences flanking both sides of Tam3 (niu- : 962). The dotted line indicates sequences that have not been unambiguously identified as present or lost. The size of the EcoRI fragments for each niu allele is shown in comparison with the wild-type (wt) 9.7-kb fragment.


PROBE A a B A B A B A B A B (II 0 0 m m

kb

34

8 4

4.3

"

-4.3

2.3

2.0

0 -4.3

-2a -2.3

b

I h b

FIGURE 3.-(a) Southern blots of niu": 98 and niv- : 536 genomic DNA digested with a range of restriction enzymes and probed with fragments A or B to illustrate the mapping procedure employed to determine the extent and approximate position of the alteration in the mutant. (b) Restriction map of the deletion in niu-:536 compared with that of its niu":98 progenitor.

(niv- : 560) while others had lost them (niv:564; niv- :562). Similarly, when genomic DNA was cut with EcoRI and KpnI and probed with fragment B some plants had lost the right hand KptI site (niv- :560; niv- :562) while others retained it (niv:564) (Figure 2). To date we have identified deletions of this type involving up to 1.4 kb of DNA. The simplest explanation of these events is that they are derived by imprecise excision of T a d . Such imprecision could be accommodated by existing models for transposition (SAEDLER and NEVERS 1985; COEN, CARPENTER and MARTIN 1986) although the distances involved are rather large to be accounted for by transient aberrations in DNA synthesis and repair as suggested in the model proposed by SAEDLER and NEVERS. Alternatively, the Tam3 transposase could occasionally recognize alternative sequences in the flanking DNA at which to initiate excision.

Deletions including part of Tam3 and flanking se-

quences: A second type of deletion was identified in two plants which were albinos, suggesting loss of chalcone synthase gene activity. When genomic DNA was digested with EcoRI and probed with pAm3, a band of 8.4 kb hybridized in line niv- : 536 and one of 7.8 kb in line niv- :563 (Figure 3). The sizes of these fragments suggested that some or all of Tam3 remained at the locus. The extent of the deletions in these lines was mapped by digesting genomic DNA with EcoRI and several other enzymes that cut in the flanking sequences or in Tam3 itself. The mapping of line niv- : 536 is shown in Figure 3. Probing with fragment A showed that the left hand KfmI site of the niveu locus and the left hand BglI sites, the PvuII site and the BstEII site of Tam3 were identical to those in the nivrec:98 progenitor, but the SmaI site of Tam3 was missing. Probing with fragment B showed that the right-hand KfmI site of the niveu locus and the right-hand BglI sites of Tam3 were missing, so


there was a 0.8-kb deletion in the line niv- :536, including sequences from Tam3 and the nivea locus. The line niv- : 563 had a very similar map, showing loss of SmaI and the right hand BgEI sites of Tam3 and the right hand KpnI site of the nivea locus. The deletion in this case appeared a little larger (1.4 kb). In both lines an AvaII site 127 bp downstream of the start of transcription was missing, indicating that the flowers of these plants were albino because part of the nivea coding sequence had been deleted.

Determination of the exact extent of the deletions in these alleles awaits cloning and sequencing. How- ever, it is clear that in both cases an event involving loss of some of Tam3 and some of the nivea flanking sequences has occurred. An abortive excision attempt at one end of Tam3 followed by exonuclease digestion of the nicked DNA strands before religation (SAEDLER and NEVERS 1985) or imprecise religation of hairpin loops formed at the end of Tam3 and in the flanking sequence (COEN, CARPENTER and MARTIN 1986) might generate these deletions.

Deletions adjacent to Tam3: The third type of deletion found in two lines derived from nivrec: 98 involved much larger regions of DNA. One was identified in a heterozygote carrying the stable niv- : 45 allele. The flowers of this heterozygote plant were completely unpigmented, indicating that there had been inactivation of the niuea gene in the nivrec: 98 allele, possibly involving deletion of coding sequences. Genomic DNA from the heterozygote was digested with EcoRI and probed with fragment A. The normal bands from the niv- :45 allele were seen together with a new band of about 17 kb (allele 529), but no band of 9.2 kb. When the same digest was probed with fragment B only the bands from the niv- :45 allele were seen. This suggested that the allele niv- : 529 had lost all the sequences to the right of T a d , at least as far as the first EcoRI site. T o confirm that a deletion of the nivea coding sequences was involved, we attempted to derive a line homozygous for the deletion. A heterozygote carrying niv- : 529 and niv- : 45 was selfed. From 15 progeny examined at the molecular level five were homozygous for niv- :45 and 10 were heterozygotes. No homozygotes carrying niv- : 529 were found. An alternative strategy to produce a homozygote was then adopted. A heterozygote carrying niv- :529 and niv- : 45 was crossed to a nivea line, niv : 532 (niv : 532 gives flowers of a paler intensity when heterozygous with a niv-allele, than when homozygous). A palely pigmented plant from the progeny of this cross, which was shown to be carrying niv- : 529 by Southern blotting, was then selfed. The expected phenotypic segregation of progeny was 1 darkly pigmented (niv: 532lniv: 532): 2 palely pigmented (niv: 5.321 niv:529): 1 albino (niv- :529/niv- :529). In fact, all the progeny were pigmented. The ratio of dark to

palely pigmented plants was 1 :2 in 95 plants examined. Thus, it would appear that the niv- : 529 allele is inviable in the homozygous form. This implies that the deletion involved in this derivative is large, since loss of nivea gene expression itself is not lethal (see niv- :45; niv- : 560; niv-562). The deletion probably involves loss of essential gene function further along the chromosome. The 1 : 2 segregation of homozygotes and heterozygotes indicated that there was no loss of' viability in the heterozygote.

The structure of niv- :529 was mapped in more detail. Digestion of genomic DNA with EcoRI and BglI, PvuII, BstEII or SmaI and probing with fragment A, showed that the upstream structure of the nivea locus in allele 529 was identical to that of niurer: 98. A copy of Tam3 remained in position adjacent to the upstream sequences. There was no evidence for sequences homologous to fragment B associated with the 529 allele although homologous sequences from the 532 allele were always seen (Figure 4). Instead a new piece of DNA flanked T a d .

The exact point of the deletion could not be mapped by Southern blotting. The SmaI site of Tam3 remained intact and it seemed likely that the end of the deletion lay immediately adjacent to T a d .

DNA from plants heterozygous for niv- : 529 and niv:532, digested with EcoRI showed a faint band of 13.5 kb that hybridized to fragment A as well as the major band at 17 kb. Since this band was 3.5 kb smaller than the major band it seemed likely that this resulted from somatic excision of Tam3 from niv- :529. In one plant, the 17-kb band was entirely replaced by the 13.5-kb band (Figure 4). This would appear to be a germinal excision event. No evidence for Tam3 remaining adjacent to the upstream sequences was obtained in genomic mapping of this derivative plant. This evidence for somatic and germinal excision showed that the Tam3 copy remaining in niv- :529 was still capable of transposition and confirmed that the deletion of downstream sequences occurred immediately adjacent to T a d , because the element was still functionally active.

A second deletion of' this type was found involving loss of sequence upstream of T a d . Again, we have not so far been able to establish this allele (nivreC: 561) in a homozygous form, suggesting that the homozygote is inviable and the deletion involved is relatively large. The deletion must involve at least 3.7 kb because no sequences homologous to fragment A were detected. Genomic mapping showed that a copy of Tam3 remains adjacent to the downstream nivea sequences. The phenotype of this allele (in a heterozygote with a niv- allele) is unstable (Table l ) , showing that the Tam3 copy is still active and that the deletion occurs immediately adjacent to the end of T a d .


(i) Deletions of this type appear very similar to those occurring adjacent to Ds in maize (MCCLISTOCK 1953,

A) B) =, q; 1954). Many of the maize deletions involved large 3 - g$!

2mnbm= ZN pz regions of DNA (several map units) and they were

r n m r m 8? f &' often inviable in the homozygous form. At the mo-

kb 7 *c" lecular level the deletions generated by Tam3 in the adjacent flanking sequences appear similar to dele-

Mutator (TAYLOR and WALBOT 1985). The mechanism by which such deletions might arise is unclear. Large deletions could arise by recombination between Tam3 copies in the same orientation on the same chromosome, or by recombination between Tam3 copies on

tion and loss caused by acentric chromatid formation

Alternatively they might arise from multiple chromosome breaks and subsequent religation. Exclusion of particular chromosome fragments during religation would give rise to large deletions. Cloning the

-23 I* tions described adjacent to Ac (DOOSER 1985) and -94 e - -geminel excision

529allele 0

532 attele - - -84 %?i?%- 41111)1

PROBED WITH different chromosomes, giving reciprocal transloca- FRAGMENT A

(ill (NEVERS, SHEPHERD and SAEDLER 1986) (Figure 4).

a$ 3

**P 5 5 PIOO."bIO, n i r ' ~ : e e new sequences flanking Tam3 in the niv- : 529 and

I niv"': 561 alleles and analysis of their immediate progenitors should establish whether recombination or some other mechanism was involved in generating these deletions.

5 t.

.11.1. nir- :520 c _ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - _ - _ _ - _ - _ - - - J

&

(iii)

- 8 c- " r t

t

=ab t

FIGURE 4.-(i) (A) Southern blots of EcoRI-digested genomic DNA from a heterozygote carrying niv- :529 and niv:532. Probe A shows homology to niv- : 529 and niv: 532 while probe B shows homology only to niv: 532. (B) Southern blot of genomic DNA cut with EcoRI and probed with fragment A, showing the niv"':98 progenitor compared to a heterozygote carrying niv- : 529 and niv:532. Genomic DNA from a single plant found amongst the progeny of the 5291532 heterozygote is also shown. This plant carries a band of reduced size corresponding to a change in the 529 allele, probably resulting from germinal excision of T a d . A faint band of the same size corresponding to somatic excision can be seen in the parental 5291532 heterozygote. (ii) Restriction map of the deletion found in niv-:5?9. A = AvaII, B = BgII, Bs = BstEII, E = EcoRI, K = KpnI, P = PvuII, S = SmaI. (iii) Diagrammatic represention of (a) how recombination between Tam3 copies (boxed) in the same orientation on the same chromosome might give rise to a deletion of the niv- :.i29 ~ y p e and (h) how recombination between Tam3 copies on non-homologous chromosomes could give rise to an acentric chromosomal fragment which would cause deletion of sequences flanking Tam3 in the progeny.

Rearrangements involving dispersion of sequences flanking Tam3 along the chromosome

The second type of rearrangement found at the niveu locus in plants derived from niv"":98 involved reassortment of the sequences flanking Tam3 without their loss. This type of rearrangement was found in two plants (nivrcc: 53 1 and nivrer: 566). The rearrangements found in these two plants are not identical. The rearrangements were identified when genomic DNA digested with EcoRI and probed with fragment A showed a new EcoRI fragment ( i e . , not 9.2 kb). When the same filter was stripped and probed with fragment B a second novel EcoRI fragment hybridised. Thus, sequences homologous to A were, after rearrangement, contained within new and separate EcoRI fragments (Figure 5 ) .

The rearrangement carried by the line nivrec:531 was examined in more detail. Analysis of plants segregating for the nivre': 531 rearrangement and a nia- : 44 allele in the Fp, showed that, in eight plants examined, the sequences of the rearrangement homologous to A and those homologous to B always segregated together despite being spatially separated. This showed that the two EcoRI fragments must still be linked. Restriction digests with enzymes that cut infrequently failed to identify a single genomic fragment that carried both sequences. I t therefore seems likely that the two halves of the niaea locus are now separated by at least 20 kb of DNA.

Genomic DNA from niore':531 was digested with EcoKI and the enzymes BgII, PauII, BstEII and SmuI,


(I) a and probed with fragments A and B. When the 3 * ;> homologous restriction fragments were compared * a 2 5 v)

B r r h with those in nivrc':98 line it appeared that sequences

in the same orientation as in the progenitor.

copies of Tam3 intact. In addition to the evidence for this from restriction site mapping, large scale screening of plants homozygous for niv'"": 531 provided molecular and genetic evidence that both Tam3 copies remained functionally intact, and could transpose independently. Genomic DNA from niv"': 53 1

kb kb A and B were both still flanked by copies of Tam3

I?!!: - 1 & ? ? --:w.5 The rearrangement of DNA appears to leave the

5- -5

PROBEA PROBE B b

kb 1 2 13- -Y m - I 6

9.5- 0

1 2 kb homozygotes, digested with EcoRI and probed with 4 .. - 12.5 fragment A, showed a major band at 13 kb and also

a faint band at 9.5 kb (Figure 5). Two progeny plants examined randomly in a larger scale screening had a fainter band of 13 kb and a much stronger one at

the majority of nivnc: 531 plants was exactly 3.5 kb smaller than the major one, and was probably a band produced by somatic excision of the Tam3 copy flanking A. In the two plants where the 9.5-kb band was much stronger, the excision appears to have

p 84 tis occurred germinally in one allele. The other allele in v these plants retained its copy of Tam3 next to A. In *A B a similar way, a number of other plants were found

(li) 9.5 kb. The faint band showing homology to A in a ni+: 98

. A y y

t

E PK - " - - - - - - - - -

€ E r -" - - - - - - - - - - - - - - -

Jlh niw '=: 531

(111)

t 5 D c

t

FIGURE 5.-(i) (a) Southern blots revealing the change in size of EcoRI fragments carrying sequences homologous to fragments A and B in the allele niurec:531. The heterozygotes with niv-44 show that the EcoRI fragments containing A and B sequences differ in size in the 531 allele. (b) Molecular evidence that Tam3 can still excise from its positions next to A or B in niu":531 despite the rearrangement. Homozygotes carrying the 531 rearrangement showing a faint band caused by somatic excision of Tam3 ( l ) , were compared to progeny showing one allele in each case with the parental structure and one allele of the size predicted for a germinal excision of Tam3 (2). Tam3 appeared to excise from its position next to A independently of excision of the copy next to B. (ii) Restriction map of the rearrangement in niu":531. Although linked, the relative orientation of sequences A and B with respect to each other on the chromosome has not yet been established. A = AuuII, B = BglI, Bs = BstEII, E = EcoRI, K = KpnI, P = PvuII, S = SmaI. (iii) Diagrammatic representation of how recombination between Tam3 copies (boxed) in opposite orientation on the same chromosome might generate an inversion of the intervening sequences.

with two sizes of EcoRI fragment when probed with B. The new band was 3.5 kb smaller than the progenitor band of 16 kb. Germinal excision of the Tam3 copy flanking A had no phenotypic effect. However, excision of the copy flanking B could be scored phenotypically because it gave rise to a stable pale pigmentation of the flower compared to the unstable recurrem type when Tam3 was present. Five plants from 119 examined showed this phenotype, giving an estimated germinal excision frequency of about 2%. The germinal excision frequency for the progenitor niv":98 allele was about 50%.

Together, these results suggest that the dispersion rearrangement leaves the two copies of Tam3 intact and that they retain the ability to excise. The frequency of excision may be somewhat reduced, although the crossing to niv- :44 involved in the derivation of nivnc: 53 1 may have influenced the excision frequency by changing the genetic background of niv"': 531 relative to nivrcc: 98.

Mechanisms for generating dispersion rearrangements: The simplest explanation for the formation of this type of rearrangement in a single step is that it arises from an inversion of sequences between two Tam3 copies. One mechanism by which this could arise is by recombination between Tam3 copies in opposite orientation on the same chromosome, as occurs with bacterial transposons and insertion sequences preexisting on the same molecule or following intramolecular transposition (Figure 5) (Ross, SWAN and KLECKNER 1979; CORNELIS and SAEDLER

C. Martin, S. MacKay and R. Carpenter

9.4-

4.3-

2.3-

2.0-

probed with A

b

probed with B

band hOm01000Us b a n d hornolopou. I O I I O e

dspsndlnp On vmrlabl. l lIs C O n S l * n l S I 1 0

Ienplh 01 duDIIC.llOn

4.Ohb

FIGURE 6.-(a) Southern blot analysis of genomic DNA from lines homozygous for niu"':554 and niu":557 compared to wild type (Niu+ : 7) and the progenitor (niu"': 98). DNA was digested with EcoRI and probed with fragment A. The filter was subse- quently stripped and reprobed with fragment B. (se: somatic excision band.) (b) Diagrammatic representation of how an inverted duplication of flanking sequence might give rise to the banding patterns observed in niv"': 554 and niu": 557.

1980; ISING and BLOCK 1981; WEINERT, SCHAUS and GRINDLEY 1983; KLECKNER 1983; HEFFRON 1983; IIDA, MEYER and ARBER 1983). Recombination between dispersed transposons on the same chromosome has been suggested to give rise to chromosomal restructuring in unstable X chromosomes in Droso- phila (LIM 1979, 1981) and in yeast (ROEDER and FINK 1983). Alternatively, chromosome breakage at the ends of two transposable elements and religation of the intervening chromosomal fragment in reverse

orientation (ENGELS, 1983) could give rise to this type of rearrangement.

Insertions involving inverted duplications The third type of large rearrangement observed

in plants derived from niv"': 98 involved insertion adjacent to T a d . Several examples were found in the progeny from a single phenotypically mutant inflorescence (CARPENTER, MARTIN and COEN 1987). The plants carrying insertions had almost colourless flowers with a pale pigmentation on the cheeks of the corolla lobes. Occasional sites of pale or full red pigmentation were observed in some lines (Table 1).

When genomic DNA from these plants was digested with EcoRI and probed with fragment B, a band of 4.0 kb hybridized (Fig. 6). When the same filter was reprobed with fragment A, a second band hybridized. In one line the band that hybridized with A was 7.5 kb (niv"':557) and, in another, 9.2 kb (niv": 554).

The simplest model to account for these results was that an insertion involving an inverted duplication of the sequences flanking Tam3 to the right (B) had occurred. As large inverted duplications had not been previously reported at the molecular level we undertook detailed mapping of the lines to establish the identity of the changes. If the duplications extended at least as far downstream as the first EcoRI site, but were each of different length, then a constant band twice the size of the distance from the point of Tam3 insertion to the nearest downstream EcoRI site (4.0 kb) would be observed after probing with fragment B (Fig. 6). A band of size determined by the nearest duplicated EcoRI site would be observed after probing with fragment A. This band would vary in different lines if the length of the inserted duplication was different in each. If an inverted duplication of the sequences homologous to B had occurred, there should also have been duplication of BumHI and BglII sites to create new fragments homologous to B of 3.6 kb and 2.9 kb, respectively (Fig. 7). When DNA from lines niv'"": 557 and niv"':554 was cut with these enzymes, new bands of the predicted size were observed. BstEII cuts the niveu locus about 2.3 kb downstream of the Tam3 insertion site, beyond the first EcoRI site. If the inverted duplication extended as far as this site, a novel band of 4.6 kb homologous to fragment B would be predicted. In BstEII digests of DNA from lines niv"': 557 and niv'": 554, only niv"': 554 showed a novel band of this size, suggesting that the inverted duplication in niv"":557 ran from the point of Tam3 insertion and ended between 2.0 and 2.3 kb downstream between the first EcoRI site and the BstEII sites. The duplication in line niv"': 554 appeared to be larger.

The size of the duplications was confirmed by examination of the EcoRI fragments homologous to


a

ut '1 t

lragrnent 2.Skb

b

Z " e 3 8 8 " e 9 1 kb cb

-9.4

W" -64

-4.3

-b

-2.3

-2.0

8g111 Barn HI

4.4

-6.4

-43

-2.3

-2.0

A. Restriction digests of lines niv"': 557 and nivrec: 554 with EcoRI and BgZI, PvuII, and BstEII, as described previously, and hybridization with fragment A, revealed a copy of Tam3 remaining adjacent to A in niv"': 557 and niv"':554. Therefore, in line niv"':557 an EcoRI fragment homologous to A would consist of 3.7 kb upstream from Tam3 plus 3.5 kb of Tam3 plus about 300 bp of duplicated downstream sequence before the first EcoRI site. Together, these sequences would give the observed band of 7 .5 kb EcoRI.

In the line niv"":554 the EcoRI fragment that hybridizes to fragment A is 9.2 kb long. The duplication in this plant must therefore run past the second EcoRI site to the right of T a d , and at least as far

t BStEll RStEII

norelBslEll fragment

4.6kb

VI

e - ~ m m (D

b

.9.4

-6.4

" - -4.3

-2.3

-2.0

BstEll

FIGURE 7.-(a) Diagrammatic representation of how inverted duplication of sequences to the right of Tam3 might give rise to novel restriction fragments in genomic DNA cut with BgIII, BamHI and BsfEII and probed with fragment B. (b) Southern blots showing that lines niu"':Xi4 and nivrrc:337 carry the predicted novel restriction fragments in DSA cut with BglII and BamHI. niu"':334 carried the predicted novel BstEII fragment, but niurrr: 337 did not.

as 2 kb further downstream, although the duplication could be much larger.

The structure proposed for line nivrec:557 was tested using a probe made from the 0.56-kb EcoRI fragment (Fig. 1: fragment C ) that lies downstream to B. When genomic DNA from the progenitor nivrpc:98 line was digested with EcoRI, only the 0.56 kb EcoRI fragment hybridized. When genomic DNA from niv"':557 was digested with EcoRI and probed with fragment C the 0.56-kb fragment hybridized but also a band of 7.5 kb in niv"":557 (Fig. 8). When the filter was reprobed with fragment A the 7.5-kb band that hybridized was exactly the same size as that which hybridized to C . This confirmed that there had been a duplication of sequences to the right of


Tam3 and a reorganization of its position. Insertions involving inverted duplication of flanking sequence are the simplest explanation of these results.

Exact sizing of the duplication of niv"":554 was not possible because clones for sequences further downstream from the nivea locus were not available. Digestions of genomic DNA with BglII suggested that there was no other BglII site between the one in the inverted duplication and the sequences homologous to A. Together, mapping data suggest that this line carries one inverted duplication of about 4.3 kb extending 1.8 kb beyond the second EcoRI site downstream from T a d .

Tam3 copies remain active after inverted duplication of flanking sequence: Restriction mapping of genomic Southern blots of DNA from nivre":557 and nivre':554 revealed that Tam3 copies remained in place after inverted duplication. Lines nivRC: 554, niv"':557 and niv"":98 were grown at 15" to promote somatic excision (CARPENTER, MARTIN and COEN 1987). Genomic DNA from these plants, restricted with EcoRI and probed with fragment A, showed a somatic excision band, 3.5 kb smaller, for all lines (Fig. 9) . Comparison of the intensity of hybridisation to the somatic excision bands of nivre":554 and ni- v"": 557 to that of nivre":98 indicated that the ability of Tarn3 to excise was apparently unaffected by the inverted duplications.

Plants homozygous for germinal excision of Tam3 were also derived from niv"": 557 and niv"": 554. These plants were similar to those of their unstable progenitors, with very palely pigmented corolla lobes, but the flowers had no, or very few, red sites (Fig. 10). Genomic DNA from these plants showed that Tam3 had left the nivea locus to give an EcoRI fragment of 5.7 kb homologous to fragment A in the derivative of niv"":554 and one of 4.0 kb in the derivative of niv"": 557.

The primary excision event of Tam3 therefore appears to give rise to cells which show the same phenotype as their unstable progenitors because of the presence of the inverted duplication. However, the progenitor lines niv":557 and nivrec: 554 also show a number of more darkly pigmented sites on the flowers (Fig. 10). These must arise from other DNA rearrangements, possibly involving loss of the inverted duplications.

Two plants with darkly pigmented flowers (near full-red) were derived from selfing a nivnc: 557 homozygote. Genomic DNA from these plants digested with EcoRI showed that they had one allele with a restored wild-type fragment of 5.7 kb which hybridized to both fragments A and B. The other allele retained the inverted duplication plus T a d . This suggested that a secondary excision can occur which removes both Tam3 and the inverted duplication, although genomic mapping was not sensitive enough

probed with A pmbedwithc

9.4 - 6.4

4.3

2.3

2.0

FIGURE 8.-Southern blot of genomic DSA from lines niv"': 554 niu": 557 and niv"':98 cut with EcoRI and probed with fragments A and C . Following inverted duplications of sequences flanking Tam3 to the right, line niu":557 carries an EcoRI fragment of 7.5 kb containing sequences homologous to both A and C. The inverted duplication in line n i P : 5 5 7 is larger and so A and C sequences are not carried on the same EcoRI restriction fragment.

to reveal if the duplication had been completely removed.

Reexamination of EcoRI-digested genomic DNA from nivre":557 plants grown at 15" revealed a very faint band of 5.7 kb that hybridised to both fragments A and B (Fig. 9) . This suggests that there may be a secondary excision event of Tam3 which involves loss of both Tam3 and the inverted duplication it created. This leads to restoration of gene expression to give darkly pigmented sites, or darkly pigmented plants if the event occurs germinally. This event appears to be associated with Tam3 activity because plants in which Tam3 has been excised, leaving behind the


.,% PROBE A PROBE B

FICCRE 9.--Southern blot analysis of genomic D S A from ni- v":554, niv'":557 and niv'":98 grown at 15" to promote Tam3 excision. D S A was cut with EcoRI and probed with fragment A or B. The somatic excision band in each line is clearly seen (se). The intensity of hybridisation of fragment A to the somatic excision band in niv":98 and niv":554 is approximately equivalent, suggesting that the inverted duplication in niv"':554 does not mark- edly reduce the ability of Tam3 to excise. The somatic excision band in niv"':557 is also strong, suggesting that Tam3 excision remains high in this rearrangement. In addition, D S A from niv"':557 shows a faint band of 5.7 kb which corresponds to the size of the wild-type fragment: this band is also seen when the filter is hybridised to fragment B. This secondary somatic rearrangement may indicate that the inverted duplication in niv":554 is relatively unstable and is lost along with Tam3 at an appreciable frequency.

inverted duplication, show no (or very infrequent) darkly pigmented somatic sites (Fig. 10).

CONCLUSIONS

Several large-scale rearrangements of DNA have been generated as a result of Tam3 activity at the nivea locus of A. majus. These have been characterized at the molecular level and fall into three categories- deletions, dispersions and insertions-although this list may not be exhaustive. The description of these rearrangements raises questions about how they arise and about their significance to the plant. The simplest explanation of their production is that all changes from the very small to the very large arise by the same mechanism, possibly as the products of aberrant transposition attempts, or, as has been demonstrated for bacterial transposons and insertion sequences, from recombination between transposon copies. In some. examples of eucaryotic transposon-associated rearrangements, recombination has been assumed to be the mechanism giving rise to large-scale changes (ROEDER and FISK 1983; COLLISS and RUBIS 1984; DAVIS, SHES and JLDD 1987). However, when consid- ering transposons that move by excision, it is possible that the transposition process itself is involved in

FI( . I .KL 10,"Phenotypic effect of Tam3 excision from alleles carrying inverted duplications of the niven coding sequences: ( la) niv":554: (Ib) line homozygous for excision of Tam3 from n i P : 5 5 4 which shows loss of dark sites and a more intense pigmented flush on the face of the corolla lobes. (2a) niv"':557: (2b) line homozygous for excision of Tam3 from niv"':557 which shows loss of dark sites.

producing large-scale rearrangements. Indeed, there have been reports of rearrangements in Drosophila that cannot easily be explained by recombination between transposons (ESGELS 1983; COLLISS and RUBIS 1984). I t is difficult to see how large inverted duplications could arise through recombination or how smaller deletions adjacent to transposable elements, such as those described by DOOSER (1985) and TAYLOR and WALBOT (1985), might arise by transposon-recombination, unless transposition occasionally involves replication. Bacterial transposons such as TnlO can transpose replicatively and excise by separate mechanisms (FOSTER et al. 1981). ESGELS (1983) has argued against recombination being the mechanism for generating inversions and deletions in Drosophila on the basis of the frequency at which P elements flank each end of a chromosomal rearrangement. The number of putative inversions produced by Tam3 is too low, at present, to draw similar conclusions. However, the chromosome breakage and religation model proposed by COEX, CARPESTER and MARTIN (1986) may explain adjacent deletions and


TABLE 1 Effect o f rearrangements induced by Tam3 on the expression

of the nivea locus. STOCK No. PHENOTYPE MOLECULAR CHANGE

Wild type N i v * : 7 Full red

Tam3 insertion niv :98 Pale pigmentation with full red sites

T

DELETIONS ACCOMPANYING TAM3 EXCISION

Loss of 250bp of promoter niv:564 Pale pigmentation concentrated on face of lobes

Loss of OOObp of promoter and coding sequence

niv-:560 Albino

DELETION

Loss of 5' promoter region niv :561 rec

Pale pigmentation concentrated on face of lobes with pale and dark sites

DISPERSION OF FLANKING SEQUENCE

Displacement of 5' promoter region

Medium pigmentation with dark and pale sites

niv:565 Displacement of promoter and subsequent Tam3 excision

Palely pigmented

INVERTED DUPLICATION

niv :657 rec

Pale pigmentation concentrated on face with a few dark sites sequence

Inverted duplication of coding


inverted duplications that occur on a small scale. In addition, if chromosome breakage occurs during abortive excision attempts at more than one site on the chromosome and is followed by religation of chromosome fragments, sequences might occasionally be inverted or deleted. This hypothesis is attrac- tive because it provides a mechanism for producing both small- and large-scale rearrangements. We hope to be able to identify particular copies of Tam? involved in each dispersion and adjacent deletion by cloning them, sequencing their ends, and looking for minor sequence differences. In this way we may be able to establish if chromosome breakage and religation is the mechanism for generating these changes.

The more important conclusion arising from this analysis of the rearrangements associated with Tam? is that Tam? has an enormous potential to restructure the genome. The ability of transposable elements to modify gene expression has been convincingly argued for small sequence rearrangements or transposon footprints (SCHWARZ-SOMMER et al. 1985). How- ever, it is also clear that transposable elements can reorganize much larger pieces of DNA within a chromosome. This must be of importance in evolution. Deleterious effects arise from deletions adjacent to Tam? that are inviable in the homozygous form. Other rearrangements may modify gene expression by virtue of deletion or insertion, or possibly by changing the position of a gene in a chromosome. The rearrangements arising at the niuea locus are good examples of how a transposon may generate phenotypic variation (Table 1). One of the novel aspects of these mutations is that they comprise a series of alterations in the relationship between the chalcone synthase gene promoter and coding sequence, so transposons may alter the regulation of a gene to produce new variation. Although many of the changes in gene expression observed in this case arise as a result of the specific insertion site of Tam?, large-scale rearrangements may also modify expression of neighboring genes. Furthermore, so long as the transposable element remains at a particular site, sequential rounds of rearrangement may be possible. Secondary rearrangements have indeed been observed in the progeny of lines niure':331, niure':557 and allele niu- : 329 (our unpublished results).

Although large DNA rearrangements have been recognized as a major consequence of the activity of bacterial transposons and T y elements in yeast, chromosomal rearrangements have appeared to be less significant for those eukaryotic transposable elements that transpose by excision, because their frequency is much lower than that of excision. However, the frequency of germinal events involving change of more than 1 kb of DNA, associated with Tam3 (at least 5% of progeny), suggests that large-scale rearrangement of DNA associated with transposable ele-

ments may provide a significant contribution to variation for the evolution of higher plant genomes.

We wish to thank ESRICO COES, TIM ROBBISS, ASDREW HLDSOS, JORCE ALMEIDA, ASDREA PRESCOTT, JEREMY BARTLETT and M A R K BUTTSER for helpful and stimulating discussions, and CLARE LISTER and DAVID HOP\VOOD for reading and discussion of the manuscript. We are particularly grateful to H. SOMMER at the Max Planck Institute in Cologne for generous gifts of nzuea clones, and extensive sequence data prior to publication. We also thank PETER SCOTT and ASDRLW D.AVIS for the photography, and ASSE WILLIAMS for typing the manuscript.

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59 1-597.

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