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 7 U H , 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 rearrangerelatively 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 andduplications in subsequent generations because of
breakage-fusion-bridge cycles. MCCLINTOCK (1953, 1954) also found that large deletions of the chro-
mosomal material flanking
Ds
could occur at lowfrequency. More recently, analyses at the molecular
level showed that the maize transposable elements Ac
and Mutator can be associated with deletions of flank-
ing 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 tran-
sposons 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 se-
quences flanking transposons and insertion se-
quences 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 se-
quences 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-recom-
bination 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 rear-
rangements occur relative to transposon excision
differs for different types of mobile element. This paper describes a series of large-scale rear-
rangements induced by the transposable element
Tam3 at the nivea locus of Antirrhinum majus. T h e 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
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 se- quence. 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 chro-
mosome 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 rearrange- ments 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 homo- zygotes 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 pigmenta- tion, o r 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 pigmen- tation 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 nitrocel- lulose (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. T h e agarose containing the required fragments was cut out and frozen for 2 hr at -20°C. T he 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. T h e 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 rear-
rangements 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 hybrid-
izes (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 . T h e 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.
T h e most frequent event found in nivrec: 98 prog-
eny 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 frag- ment 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
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 associ- ated with rather modest sequence rearrangements. However, insertions and deletions of upstream se- quence 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 frag-
ment A, some plants retained one or both DdeI sites
progenitor nivrec:98
T=7=
e'
t!!
2 n i v - : 5 6 0
3 n i v ' : 5 6 2
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
174 C. Martin, S. MacKay and R. Carpenter
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 K p t I 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 intwo 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
Chromosomal Restructuring 175
there was a 0.8-kb deletion in the line niv- :536,
including sequences from Tam3 and the nivea locus.
T h e 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. T h e 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:
T h e third type of deletion found in two lines derived from nivrec: 98 involved much larger regions of DNA. One wasidentified in a heterozygote carrying the stable
niv- : 45 allele. T h e 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 se-
quences. Genomic DNA from the heterozygote was
digested with EcoRI and probed with fragment A.
T h e 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 homo- zygous for the deletion. A heterozygote carrying
niv- : 529 and niv- : 45 was selfed. From 15 progeny
examined at the molecular level five were homozy-
gous for niv- :45 and 10 were heterozygotes. No
homozygotes carrying niv- : 529 were found. An al-
ternative 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. T h e 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. T h e ratio of dark to
palely pigmented plants was 1 :2 in 95 plants exam- ined. 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 homozy-
gotes and heterozygotes indicated that there was no loss of' viability in the heterozygote.
T h e structure of niv- :529 was mapped in more
detail. Digestion of genomic DNA with EcoRI and
BglI, PvuII, BstEII or SmaI and probing with frag-
ment 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 flankedT a d .
T h e 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 se-
quences was obtained in genomic mapping of this
derivative plant. This evidence for somatic and ger- minal 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 homozy- gote 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
176 C. Martin, S. MacKay and R. Carpenter
(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 large3 - 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). T h e 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 chromo- some, or by recombination between Tam3 copies on
tion and loss caused by acentric chromatid formation
Alternatively they might arise from multiple chro- mosome breaks and subsequent religation. Exclusion of particular chromosome fragments during religa- tion would give rise to large deletions. Cloning the
-23 I* tions described adjacent to Ac (DOOSER 1985) and
-94
e
-
-geminel excision529allele 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. n i r - : 5 2 0 c
_ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - _ - _ _ - _ - _ - - - J
&
(iii)
- 8 -c
"
r tt
=ab
t
FIGURE 4.-(i) (A) Southern blots of EcoRI-digested genomic DNA from a heterozygote carrying n i v - : 5 2 9 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 chro- mosome 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
T h e 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 rearrange- ments found in these two plants are not identical. T h e 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 hybri-
dised. Thus, sequences homologous to A were, after rearrangement, contained within new and separate EcoRI fragments (Figure 5 ) .
T h e 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 ho-
mologous 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 frag- ment 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
Chromosomal Restructuring 177
(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 innivrc':98
line it appeared that sequencesin 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
pro- vided molecular and genetic evidence that both Tam3 copies remained functionally intact, and could trans-pose independently. Genomic DNA from niv"':
53
1kb kb A and B were both still flanked by copies of Tam3
I?!!:
-
1 & ? ?--:w.5
The rearrangement of DNA appears to leave the5- - 5
PROBEA PROBE B
b
kb 1 2
13- -Y
m
- I 69.5- 0
1 2 kb homozygotes, digested with EcoRI and probed with
4
..
-
12.5 fragment A, showed a major band at13
kb and alsoa faint band at
9.5
kb (Figure5).
Two progeny plantsexamined randomly in a larger scale screening had
a fainter band of
13
kb and a much stronger one atthe majority of nivnc:
531
plants was exactly3.5
kbsmaller 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 ina
ni+: 98.
A y yt
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 rear- rangement 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 pro-genitor band of
16
kb. Germinal excision of theTam3 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 about50%.
Together, these results suggest that the dispersion rearrangement leaves the two copies of Tam3 intact
and that they retain the ability to excise. The fre-
quency of excision may be somewhat reduced, al-
though the crossing to niv- :44 involved in the deri-
vation of nivnc:
53
1 may have influenced the excisionfrequency by changing the genetic background of
niv"':
531
relative to nivrcc:98.
Mechanisms for generating dispersion rearrange-
ments: 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 se-
quences preexisting on the same molecule or follow-
ing intramolecular transposition (Figure
5)
(Ross,
C. Martin, S. MacKay and R. Carpenter
9.4-
4.3-
2.3-
2.0-
probed with A
b
probed with B
band h O m 0 1 0 0 0 U s b a n d hornolopou.
I O I I O e
d s p s n d l n p O n
vmrlabl. l l I s C O n S l * n l S I 1 0
I e n p l h 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 be-
tween dispersed transposons on the same chromo- some 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 di-
gested 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 duplica- tion 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 ex- tended 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 frag-
ment 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 DNAfrom lines niv'"": 557 and niv"':554 was cut with these enzymes, new bands of the predicted size were ob-
served. 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
Chromosomal Restructuring 179
a
ut
'1
t
lragrnent 2.Skb
b
Z "
e 3 8 8 "
e
9
1
kb cb
-9.4
W" - 6 4
-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, re-
vealed 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 se-
quence 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. T h e dupli- cation 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 RStEIIn o r e l B s l E l l fragment
4 . 6 k b
VI
e - ~ m
m (D
b
.9.4
-6.4
"
-
-4.3
-2.3
-2.0
BstEll
FIGURE 7.-(a) Diagrammatic re- presentation of how inverted dupli- cation of sequences to the right of Tam3 might give rise to novel re- striction 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 frag- ments in D S A cut with BglII and BamHI. niu"':334 carried the pre- dicted novel BstEII fragment, but
niurrr: 337 did not.
as 2 kb further downstream, although the duplication
could be much larger.
T h e 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 hybridized180 C. Martin, S. MacKay and R. Carpenter
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 down-
stream from T a d .
Tam3 copies remain active after inverted dupli- cation 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.
T h e 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 homo- zygote. 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 hybrid-
ized to both fragments A and B. T h e 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
-
6.44.3
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
Chromosomal Restructuring 181
.,%
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, sug- gesting 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 rear- rangement 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. T h e description of these rearrangements raises questions about how they arise and about their significance to the plant. T h e 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: ( l a )
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 ele-
ments, such as those described by DOOSER (1985)
and TAYLOR and WALBOT (1985), might arise by
transposon-recombination, unless transposition oc- casionally 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 rear- rangement. T h e number of putative inversions pro- duced by Tam3 is too low, at present, to draw similar conclusions. However, the chromosome breakage and religation model proposed by COEX, CARPESTER and
182 C. Martin, S. MacKay and R. Carpenter
T A B L E 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 rec :561 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 rec :657 Pale pigmentation concentrated on face with a few dark sites sequence
Chromosomal Restructuring 183
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 occasion-
ally 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 reli-
gation is the mechanism for generating these changes.
T h e 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 ar- gued for small sequence rearrangements or transpo-
son 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 evolu-
tion. 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. T h e 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 se-
quence, 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 expres-
sion 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 ob-
served 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, chro-
mosomal 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 rear-
rangement of DNA associated with transposable ele-
ments may provide a significant contribution to varia- tion for the evolution of higher plant genomes.
We wish to thank ESRICO COES, T I M ROBBISS, ASDREW HLDSOS,
JORCE ALMEIDA, ASDREA PRESCOTT, J E R E M Y 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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