The Plant Journal (1997) 11(1), 125-135
Ds excision from extrachromosomal geminivirus vector
DNA is coupled to vector DNA replication in maize
Uwe Wirtz 1,t, Brian Osborne 2 and Barbara Baker 2,3,*
1Department of Plant Pathology,
2Department of Plant Biology, University of California,
Berkeley, CA 94720, USA, and
3plant Gene Expression Center, University of California
and United States Department of Agriculture, Agricultural
Research Service, Albany, CA 94710, USA
Summary
Analysis of transposition products generated after Activator (Ac) excision from the P locus in maize suggest that
Ac excises either during or after replication of the P locus.
The frequency of excision of the non-autonomous Ac
derivative, Dissociation (Ds), from extrachromosomal
replicating and nonreplicating vector DNAs in transfected
black mexican sweet maize protoplasts was compared to
assess directly a role of extrachromosomal vector DNA
replication in Ds excision~ Replicating (rep +) and nonreplicating (rep-} vector DNAs comprised a Ds element
that harbored a geminivirus, wheat dwarf virus (WDV),
origin of replication and WDV genes required for viral DNA
replication (rep + ) or mutant, inactive derivatives of these
genes (rep-). Excision of Ds was detected only in those
cell nuclei co-transfected with the replicating Ds-vector
DNA and a transposase expression vector. Quantitative
reconstruction experiments showed that Ds excised at
least 3 x 10S-fold more frequently from replicating vector
DNA as compared with nonreplicating vector DNA. Therefore, these results provide direct evidence for a coupling
of Ds excision from extrachromosomal vector DNA to
vector DNA replication in maize.
Introduction
The 4.6-kb maize controlling element Activator (Ac) and its
non-autonomous derivative Dissociation (Ds) were first
identified and studied genetically by Barbara McClintock
(McClintock, 1947; McClintock, 1950). Ac and Ds elements
have been cloned, sequenced and molecular features of
this transposon family have been described (for review,
see Fedoroff, 1989). Ds elements are typically deletion
derivatives of Ac which lack sequences encoding a func-
Received29 April 1996;revised6 September1996;accepted17 October
1996.
*For correspondence(fax510 559 5678;
[email protected]).
tPresentaddress:I. G. S. BiotecGmbH,Hauptstrasse8, D-06408Biendorf,
Germany.
tional transposase. Therefore, Ds requires the presence
of Ac-encoded transposase for transposition (Kunze and
Starlinger, 1989). In addition, Ac deletion analysis has
shown that approximately 200 bp of 5' and 3' terminal
sequences are required in cis for transposition (Coupland
et aL, 1989).
However, the mechanism and the regulation of Ac and
Ds transposition are not yet understood in detail. One
central question concerns the role of DNA replication in
Ac and Ds transposition. Genetic and molecular analysis
of the products of Ac transposition events from the P locus
in maize suggest that most Ac transposition events occur
during or shortly after replication of the locus bearing Ac
(Chen et al., 1987, 1992; Greenblatt, 1966, 1968, 1974, 1984;
Greenblatt and Brink, 1962). The maize P gene product is
required for red pigmentation of the kernel pericarp tissue
and the cob glumes (Styles and Ceska, 1977). Ac insertion
at P can prevent red pigmentation of these tissues and
excision of Acfrom Pcan restore red pigmentation (Athma
et aL, 1992; Lechelt et aL, 1989). Two types (I and II) of
twinned red and light-variegated sectors of kernels were
identified after Ac excision from P. In both types, Ac had
excised from the P locus in the red sector and was
retained at P in the corresponding light-variegated sector
(Greenblatt, 1974). In type I twin sectors, the transposed
Ac was found in the red and the light-variegated sector at
identical chromosomal sites. Type II twin sectors harbored
a transposed Ac only in the light-variegated sector (Chen
etaL, 1992). Based on these findings, it was proposed that,
following replication of the region bearing Ac, Ac can
excise from one of the two daughter chromatids. Following
excision, Ac can reinsert into either unreplicated recipient
DNA (type I) or into a replicated recipient site of the
daughter chromatid retaining Ac at the donor site (type II).
This analysis of Ac transposition from the P locus in maize
shows a positive correlation between Ac transposition
and donor DNA replication. However, it remains unclear
whether donor DNA replication is essential for Ac and
Ds excision.
We have assessed the role of extrachromosomal vector
DNA replication in Ds excision. We chose to investigate
the relationship between Ds excision and DNA replication
using a replicon derived from the 2.7-kb genome of the
geminivirus, wheat dwarf virus (Macdowell et aL, 1985).
WDV DNA replication parallels that of plant chromosomal
DNA replication in the following ways: (i) DNA replication
of geminiviruses takes place in the cell nucleus (Horns and
Jeske, 1991; Rushing et al., 1987), (ii) replicating doublestranded DNAs of geminiviruses exist as minichromo125
126
U w e Wirtz et al.
Figure 1. Diagrams of the plasmids pDsW +, pDsW-, pDs, pill/IV and pTps.
(a) pDsW ÷ and pDsW-. Ds sequences are shown as shaded boxes. Terminal
Ds-inverted repeats are indicated as black triangles. A wheat dwarf virusderived replicon (WDV; open box) and ~AN7 plasmid sequences (hatched
box) are inserted into Ds. The position and orientation of WDV ORFs III
and IV are indicated (arrows), and the location of the WDV 'starting' (SIR)
and 'terminating' (TIR) intergenic regions are shown. The remaining plasmid
sequences are indicated by the flanking thin line. Styl (S) restriction enzyme
sites are indicated, pDsW- is identical to pDsW + except that 90 bp between
the Styl sites are deleted in pDsW-.
(b) pDs, pDs is the immediate precursor plasmid of pDsW ~ and pDsW-.
(c) pill/IV, A fragment harboring WDV ORFs III and IV (open box) is inserted
between the strong maize ubiquitin-1 promoter and its first intron (Pr.
Ubi÷i; large filled arrow) and the 3'-untranslated region of the nopaline
synthase gene (3'NOS; filled box).
(d) pTps. A fragment harboring the Ac transposase ORF (shaded box) is
inserted between the Pr. Ubiti (large filled arrow) and 3'NOS (filled box).
The position and orientation of the Ac transposase ORF is indicated (arrow
within the shaded box).
The Pr. Ubi÷i (c, d) and Ac transposase ORF (d) are not drawn to scale.
somes (Pilartz and Jeske, 1992; Saunders et al., 1991;
Stenger et al., 1991), and (iii), except for the participation
of the virus-encoded replication protein(s), DNA replication
of geminiviruses is dependent on the cellular replication
machinery (Elmer et al., 1988; Etessami et al., 1991). In
contrast to plant chromosomal DNA replication, geminivirus DNA replication occurs probably by a rolling circle
mechanism (Laufs et al., 1995; Saunders et al., 1991;
Stenger et al., 1991). WDV encodes four open reading
frames (ORF I-IV). Studies of WDV mutants have shown
that ORFs III and IV are required for WDV DNA replication,
whereas ORFs I and II are dispensable for WDV DNA
replication (Laufs et al., 1990; Matzeit et al., 1991; Schalk
et al., 1989; Woolston et al., 1989). Furthermore, a WDV
derivative in which ORFs III and IV were fused by deletion
of the WDV intron, was shown to replicate (Schalk et al.,
1989). Two intergenic regions exist between the virion
sense ORFs I and II and the complementary sense ORFs III
and IV (Figure la). The 'starting' intergenic region (SIR) of
WDV was shown to contain promoter sequences that are
required for expression of ORFs I-IV (MacDowell et al.,
1985; Revington et al., 1989) and sequences that are essential in cis for WDV DNA replication (Heyraud et aL, 1993;
Kammann et al., 1991; Revington et al., 1989). The replication protein of the geminivirus, tomato golden mosaic
virus, was shown to bind specifically to a region within its
SIR (Fontes et aL, 1992; Lazarowitz et al., 1992). The
replication protein of the geminivirus, tomato yellow leaf
curl virus, has been shown to initiate viral-strand DNA
synthesis by introducing a nick in the plus strand within a
specific sequence of the SIR, identical to all geminiviruses
(Laufs etal., 1995). Furthermore, post-cleavage, the replication protein remains bound to the 5' end of the cleaved
strand. The replication protein has an ATPase activity
required for viral DNA replication (Desbiez et al., 1995).
The 'terminating' intergenic region was also shown to
be required in cis for WDV DNA replication (Kammann
et al., 1991).
Ac transposase-dependent excision of Ac and Ds from
extrachromosomal replicating WDV-derived vector DNA in
transfected black Mexican sweet (BMS) maize, Triticum
monococcum and Oryza sativa protoplasts has been
demonstrated previously (Laufs et al., 1990). Excision of
Dsl from the genome of maize streak virus, a geminivirus
closely related to WDV, was detected upon introduction
of the vector DNA via Agrobacterium infection into Ac
transposase-expressing maize plants (Shen and Hohn,
1992; Shen et al., 1992). In the Ac/Ds/WDV studies (Laufs
et aL, 1990), it was noted that excision of Ac was only
detected from extrachromosomal replication-competent
vector DNA. However, no rigorous conclusion regarding
the coupling of Ac excision to DNA replication could be
drawn from these studies as the recircularized empty donor
product carried the WDV origin of replication. Therefore,
the detection of Ac excision from replicating vector DNA
and not from nonreplicating vector DNA could be attributed
to amplification of the replication-competent circular empty
donor product.
The experiments described here do not suffer from this
ambiguity as the analogous empty donor product does not
bear the WDV origin of replication. We describe experiments comparing Ds excision frequency from replicating
vector DNAs and nonreplicating derivatives in transfected
BMS maize protoplasts. The results provide direct evidence
for a coupling of Ds excision to extrachromosomal geminivirus vector DNA replication.
Results
Plasmids
Constructions of the plasmids used for these studies are
described in detail in the Experimental procedures and
shown in Figure 1. The plasmid pDsW ÷ (Figure la) carries
a mini Ds that harbors a WDV-derived replicon, pDsW- is
a nonreplicating derivative of pDsW ÷. pDsW- is replicationincompetent because the 90-bp Styl fragment at the 5' end
of the WDV ORF III of pDsW ~ (Figure la) is deleted, pDs is
the immediate precursor plasmid of pDsW- and pDsW-
Significance of DNA replication for excision of Ds
127
(Figure lb). The plasmid pill/IV is a WDV ORF III and ORF
IV expression vector (Figure lc). The plasmid pTps is an
Ac transposase expression vector (Figure ld).
pDsVi/+ replicates in BMS maize cells
Replication of pDsW + and pDsW- in BMS maize cell nuclei
was assessed in transfected protoplasts derived from BMS
maize suspension cells. We used the methylation state of
the sequence GATC in transfected pDsW + and pDsWplasmids to determine the extent of DNA replication.
pDsW + and pDsW- DNA used for the transfection experiments were propagated in Escherichia coil dam + cells.
Consequently, adenine residues within the sequence GATC
were methylated. We used two different approaches to
assess the methylation state of GATC sequences in transfected pDsW + and pDsW- plasmids.
In the first approach, we used the restriction enzyme
Mbol. Mbol only digests DNA containing unmethylated
adenine residues within its recognition sequence, GATC
(Heinzel et al., 1991; McWhinney and Leffak, 1989). Therefore, transfected pDsW ÷ and pDsW- plasmid DNA is initially
resistant to Mbol digestion, but following two rounds of
semi-conservative replication in maize cells, the plasmids
become sensitive to Mbol digestion. Total DNA was isolated
from nuclei-enriched fractions at 2, 4 and 8 days after
transfection with pDsW ÷ and pDsW- and was digested
with Mbol and Scal. Scal was used to linearize any Mbolundigested vector DNA. Southern blot hybridization analysis using a 638-bp fragment of pDsW + or pDsW- as
a probe showed that Mbol digestion released a 638-bp
fragment that was detected in increasing amounts at 2, 4
and 8 days in pDsW ÷ transfected protoplasts (Figure 2a and
b). This fragment was not detected in pDsW- transfected
protoplasts (Figure 2b). This result shows that pDsW ÷
DNA becomes Mbol-sensitive in transfected protoplasts,
whereas pDsW- does not. We conclude that pDsW ÷ replicates and pDsW- does not.
Using a second approach to assess replication of pDsW +
and pDsW- in BMS maize, we tested the methylation state
of pDsW + and pDsW- using the restriction enzyme Dpnl
(Figure 2c and d). Dpnl only digests DNA containing methylated adenine residues within its recognition sequence
GATC and consequently only digests the unreplicated,
methylated pDsW ÷ and pDsW- DNA transfected into maize
protoplasts (McWhinney and Leffak, 1989; Peden et al.,
1980). DNA which replicates only once in the maize cells
should become resistant to Dpnl digestion due to the
incorporation of unmethylated adenine residues into one
of the DNA strands. We examined DNA isolated from
pDsW + and pDsW-transfected protoplasts for the presence
of Dpnl-resistant forms of the two vector DNAs. Total DNA
was isolated from protoplasts transfected with pDsW ÷ and
pDsW- and digested with Dpnl and Scal followed by PCR
Figure 2. Analysis of pDsW+ and pDsW- replicationin transfected BMS
maizeprotoplssts.
(a) Diagramof the locationof the single Scal (S) restrictionsite and two
(of the 40 total) Mbol (M) restrictionsitesthat generatea 638-bpfragment
within pDsW+ and pDsW-. The 638-bpfragmentof pDsW+ or pDsW-was
used as a hybridizationprobe(filled bar).
(b) Southern blot hybridizationof DNA isolated from nuclei-enriched
fractions 2, 4 and 8 daysaftertransfectionwith pDsW+ (7.5kb) and pDsW(7.4 kb). IsolatedDNA was digestedwith Mbol and Scal and hybridized
with the 638-bpfragmentindicatedin Figure2a.
(c) Diagramof the locationof the single Scal (S) restrictionsite and four
(of the 40 total) Dpnl (D) restriction sites within pDsW+ and pDsW-. The
location and orientationof the primers 5 and 6 (arrows) are also shown
(primers and plasmidDNA are not drawnto scale).
(d) Ethidiumbromide-stainedgel of PCRproductsamplifiedusingtemplate
DNAisolatedfrom BMS maizeprotoplasts2, 4 and 8 daysaftertransfection
with the indicatedplasmids. DNA was digestedwith Scal and Dpnl and
primers 5 and 6 were used in the PCRamplification.
amplification using primers 5 and 6 of a region of pDsW +
and pDsW- containing four Dpnl sites (Figure 2c and d).
Replicated DNA is expected to yield a 563-bp, Dpnl-resistant
DNA fragment, whereas unreplicated DNA should yield
five Dpnl-digested DNA fragments. These Dpnl DNA fragments are less that 100 bp and thus are not dectected by
this analysis. We detected a 563-bp, Dpnl-resistant, PCR
product in increasing amounts at 2, 4 and 8 days posttransfection in pDsW+-transfected protoplasts but not in
pDsW--transfected protoplasts (Figure 2c and d). These
results show that Dpnl-resistant pDsW + DNA accumulated
over time.
128
Uwe Wirtz et al.
Based on these analyses, we conclude that pDsW~
replicates in transfected BMS maize protoplasts and pDsWdoes not.
Ds excises from pDsW ~ in BMS maize cells
To assess excision of Ds from pDsW+ and pDsW-, BMS
maize protoplasts were co-transfected with pDsW+ and
pTps or pDsW- and pTps. Aliquots of the same protoplast
preparation used to evaluate replication of pDsW~ and
pDsW- were used for this experiment. Excision of Ds from
pDsW + and pDsW- was analyzed by PCR amplification of
DNAs isolated from nuclei-enriched fractions at 2, 4 and 8
days after transfection. PCR amplification using primers 1
and 2 will produce a 311-bp product only if Ds excision
has occurred (Figure 3a). The 311-bp empty donor product
was detected in pDsW+/pTps-transfected protoplasts 2, 4
and 8 days after transfection but not in pDsW-/pTpstransfected protoplasts (Figure 3b). These results were
repeated in five independent transfection experiments.
The results from one of these experiments are shown in
Figure 3b. In control experiments, it was shown that Ds
does not excise from pDsW- when pTps is omitted from
the transfection (data not shown). These results show that,
as expected, Ds excision is dependent on Ac transposase
and further suggest that Ds excises more efficiently from a
replicating substrate than from a nonreplicating substrate.
The possibility that the WDV ORF Ill/IV product(s),
encoded by pDsW ÷, played a direct role in Ds excision
was examined in complementation experiments using the
plasmid constructions pDs and pill/IV (Figure 3a and b).
pill/IV carries the WDV ORFs III and IV and was shown to
complement replication of pDsW- in trans (data not shown).
It was further demonstrated that Ds excised from pDsWin the presence of pill/IV and pTps by PCR amplification of
the 311-bp excision product from template DNA isolated
from
two
independent transfection experiments
(Figure 3b). However, Ds did not excise in protoplasts
transfected with pDs (a plasmid lacking WDV sequences
required in cis for replication) in the presence of pill/IV and
pTps. No 311-bp excision product was detected in two
independent experiments (Figure 3b). PCR produced a
1667-bp product indicating that Ds remained in place in
pDs (Figure 3a and b). These results show that efficient Ds
excision from the vector DNA is dependent, in cis and in
trans, on the WDV replication apparatus.
We further examined whether potential WDV ORF AStylIll/IV gene product(s) from pDsW- inhibited excision of Ds
from pDsW-. We assessed whether Ds excision from
pDsW ~ was reduced or inhibited in the presence of pDsW-.
No difference in the amount of the 311-bp excision product
was detected when DNA isolated from pDsW+/pTps - or
pDsW+/pDsW-/pTps-transfected protoplasts was used as
template in the quantitative PCR amplification (data not
Figure 3. PCR analysis of De excision from pDsW ~, pDsW- and pDs
transfected into BMS maize protoplasts.
(a) Diagram of the location and orientation of the primers 1 and 2 (arrows)
within pDsW ~, pDsW- and pDs (primers and plasmid DNA are not drawn
to scale). The BstEII (B) restriction enzyme site is indicated, A 311-bp PCR
product is expected upon excision of Ds from pDsW +, pDsW and pDs
using primers 1 and 2. A 1667-bp PCR product is generated from pDs with
primers 1 and 2. The large PCR products expected to be generated from
pDsW ~ and pDsW (3.6 and 3.5 kb) with primers 1 and 2 were not detected
under the conditions used in these experiments.
(b) Ethidium bromide-stained gel of PCR products amplified using template
DNA isolated from BMS maize protoplasts 2, 4 and 8 days after transfection
with the indicated plasmids. Plasmids were co-transfected with pTps.
Primers 1 and 2 were used in the PCR amplification. Controls are lane 1:
primers alone; lane 2: primers and untransformed BMS maize DNA.
(c) Ethidium bromide-stained gel of PCR products amplified using template
DNA isolated from BMS maize protoplasts 2, 4 and 8 days after transfection
with the indicated plasmids (CH3 : unmethylated and BstEIl-linearized input
plasmid DNA; CH3": methylated and BstEIl-linearized input plasmid DNA.
Plasmids were co-transfected with pTps, Primers 1 and 2 were used in the
PCR amplification. Control is lane 1: primers alone.
shown). This result was confirmed in a second independent
transfection experiment. Therefore, potential expression
of WDV ORF ~Styl-III/IV gene product(s) does not inhibit
the excision of Ds from the vector DNA.
Significance of DNA replication for excision of Ds
Furthermore, we wished to determine whether loss of
bacterial methylation of the vector DNA alone converts the
vector into a Ds excision substrate. We assessed whether
Ds is able to excise from nonreplicating unmethylated
vector DNA. We isolated pDsW ÷ and pDsW- plasmid DNA
from dam/dcm E. coli JM110 cells (Yanisch-Perron et al.,
1985) and verified the absence of plasmid DNA methylation
by restriction enzyme analysis (data not shown; see
Experimental procedures). BMS maize protoplasts were
co-transfected with pDsW + and pTps or pDsW- and pTps.
Protoplasts were also transfected with dam+/dcm ÷ pDsW +
and pDsW- vector DNA as controls, pDsW + and pDsWwere transfected as BstEIl-linearized plasmid DNAs
(Figure 3a). Excision of Ds from pDsW + and pDsW- was
analyzed as described above by PCR amplification. The 311bp empty donor product was not detected in protoplasts 2,
4 and 8 days after co-transfection with the unmethylated
or methylated pDsW- and pTps input DNAs (Figure 3c).
However, the 311-bp empty donor product was detected
after co-transfection with the unmethylated or methylated
pDsW+/pTps input DNAs (Figure 3c). We conclude that Ds
excision is dependent on vector DNA replication and not
on vector DNA demethylation, because Ds did not excise
from the nonreplicating unmethylated substrate.
Taken together, these results suggest the coupling of Ds
excision to extrachromosomal vector DNA replication.
Quantification of Ds excision from pDsWQuantitative measurements comparing the frequency of
Ds excision from pDsW ÷ and pDsW- were performed on a
dilution series of DNAs isolated from 8-day-old pDsW÷/
pTps- and pDsW-/pTps-transfected protoplasts. These
DNAs were diluted in 10-fold steps to a final 109-fold
dilution. The 311-bp excision product was detected in the
undiluted and 10-1 to 10-3 diluted pDsW+/pTps DNAs
following PCR amplification with primers 1 and 2 (Figure 4a
and b). The 311-bp excision product was not detected in
any of the DNA samples from pDsW-/pTps-transfected
protoplasts (Figure 4b). This result was verified by PCR
analysis of DNA isolated from a second independent transfection experiment. Both experiments suggest that Ds
excises at least 103-fold more frequently from replicating
pDsW + than from nonreplicating pDsW-.
A 211-bp PCR excision product was detected following
a second round of amplification using primers 3 and 4
(Figure 4a and c). The substrates were the PCR reactions
performed with primers 1 and 2 of the undiluted to 108-fold
diluted aliquots of pDsW+/pTps DNA. No 211-bp excision
product was detected following reamplification of any
pDsW-/pTps aliquot (Figure 4c). The same result was
obtained using DNA from a second independent transfection experiment. Therefore, these experiments suggest that
129
Figure 4. QuantitativePCRanalysisof Dsexcisionfrom pDsW÷ and pDsW8 days post-transfection.
(a) Diagram of the location and orientation of the primers 1, 2, 3 and 4
(arrows)within pDsW+, pDsW-and pDs (primers and plasmid DNA are not
drawn to scale). A 311-bp PCR product is expected upon excision of Ds
from pDsW÷ and pDsW- using primers 1 and 2, and a 211-bp PCR Ds
excision product is expectedwhen primers 3 and 4 are used.
(b) Ethidiumbromide-stainedgel of PCRproductsamplified usingtemplate
DNA isolatedfrom pDsW+/pTpsand pDsW-/pTpsco-transfectedBMS maize
protoplasts 8 days after transfection. The template DNA was undiluted
(1 x) or diluted by 10-fold steps to a final 10-e-folddilution as indicated,
and the primers 1 and 2 were used in the PCR amplification. Control is
lane 1: primers 1 and 2 alone.
(c) Ethidium bromide-stained gel of excision fragments produced by
reamplification of PCR products shown in Figure 4b using primers 3 and
4. Control is lane 1: primers 3 and 4 alone.
replication enhances Ds excision from these vector DNAs
at least 10e-fold under these conditions.
Further quantification of Ds excision frequency from
pDsW+
The 108-fold difference in Bs excision frequency observed
between replicating and nonreplicating vector DNAs could
be affected by three features of our system.
First, the amount of empty donor product detected by
PCR in transfected protoplasts is dependent on the relative
amount of pDsW + and pDsW- DNA. A difference in the
relative amount of pDsW ÷ and pDsW- DNA would most
likely be attributed to the replication of pDsW ÷. For
130
Uwe Wirtz et al.
example, if a higher concentration of pDsW + than pDsWwere present in transfected protoplasts, then the amount
of Ds excision from pDsW ÷ would be overestimated by a
factor equal to the difference in the concentrations of
pDsW ÷ and pDsW-. To determine the relative concentration
of pDsW + and pDsW-, PCR was performed using primers
5 and 6 (Figure 5a) on a dilution series of DNAs isolated
from 8-day-old pDsW ÷- and pDsW--transfected protoplasts.
These DNAs were diluted in 10-fold steps to a final 109fold dilution. Primers 5 and 6 amplify a 563-bp PCR product
from vector DNA harboring Ds (Figure 5a). pTps was
omitted from these experiments to avoid possible depletion
of the pDsW ÷ template by excision of Ds. Tenfold more
DNA from pDsW--transfected protoplasts was required to
give the same amount of PCR product as DNA from pDsW ~transfected protoplasts (Figure 5b).
We further showed that Ds excision from pDsW ÷ did not
significantly deplete the pDsW ÷ template required for
amplification of the 563-bp PCR product. PCR was performed using primers 5 and 6 on a dilution series identical
to the aformentioned dilution series of DNA isolated from
pDsW+/pTps - and pDsW-/pTps-transfected protoplasts. A
10-fold difference in the relative concentrations of pDsW ÷
and pDsW- was also observed when pTps was included in
the transfection (data not shown).
The results of Southern blot hybridization analysis of
DNA isolated from pDsW÷/pTps - and pDsW-/pTps-transfected protoplasts confirmed that 10-fold more pDsW ÷
DNA was present than pDsW- DNA (data not shown).
Therefore, the 108-fold increase in Ds excision frequency
from pDsW ÷ compared with pDsW- was adjusted to 107
due to the 10-fold difference in the relative concentrations
of pDsW ~ and pDsW in the nuclei of the transfected
protoplasts 8 days after transfection.
Secondly, the 107 factor was adjusted further, because
WDV DNA replication is dependent on the host cell replication machinery. Three per cent of the transfected protoplasts were in a state of cell division 8 days posttransfection (see Experimental procedures). Therefore, only
3% of the cells could have contributed to the measured
10-fold increase of pDsW ~ concentration. In order to calculate the difference in frequency of Ds excision between
pDsW +- and pDsW--transfected cells, it is necessary to
consider only those cells that are competent to support
WDV replication. Otherwise, the observed fold-difference
would be directly proportional to the percentage of dividing
cells, which would yield an overestimate. Thus, we divided
the 107 factor by 30 which results in a minimum 3×105fold increase in Ds excision frequency from pDsW- compared with pDsW-.
The third factor that might affect our measurement of
relative Ds excision frequency is the possibility that pDsW T
replicated after Ds excision and thus contributed to an
overestimation of its relative concentration compared with
Figure S. QuantitativePCR analysis of pDsW" and pDsW- present in
transfectedcell nucleiof BMS protoplasts8 dayspost-transfection.
(a) Diagram of the locationand orientationof primers 5 and 6 (arrows)
within pDsW~or pDsW-(primersand plasmidDNAare not drawnto scale).
The sizeof the resultingPCRproductis 563 bp.
(b) Ethidiumbromide-stainedgel of PCRproductsamplifiedusingprimers
5 and 6 and the samedilutionseriesof templateDNAas usedin Figure3c.
Control is lane 1: primers 5 and 6 alone.
pDsW- in transfected protoplasts. We demonstrated, however, that pDsW + does not replicate in BMS cells following
Ds excision. This was shown by creating the circular
pDsW ÷ empty donor product in vitro (see Experimental
procedures), introducing it into maize protoplasts, and
analyzing the extracted DNA by the Mbol- and Dpnldigestion/vector DNA replication assays described above.
The empty donor product does not replicate (data not
shown). As expected, the empty donor product also does
not replicate in the presence of pDsW" (data not shown)
since this product does not contain a WDV origin of
replication.
To determine whether Ds excised at low frequency from
pDsW-, we determined the sensitivity of our PCR excision
assay (data not shown). We made a dilution series of known
amounts of the aforementioned empty donor plasmid DNA
and mixed the empty donor plasmid DNA with same
amounts of genomic BMS maize DNA to resemble the
experiments described above. We performed two rounds
of PCR using the same conditions as described for the
experiment shown in Figure 4. We were able to detect the
211-bp PCR excision fragment by adding 20 fg of plasmid
DNA, but could not detect PCR products with 2 fg or less
of plasmid DNA. Twenty femtogram of a 7 kb plasmid
corresponds to approximately 3000 plasmid molecules.
Therefore, our assays can detect Ds excision from at least
3000 plasmid molecules. We conclude that there is a 3x 10sfold increase in Ds excision frequency from replicating,
extrachromosomal pDsW ~ DNA compared with a nonreplicating pDsW-.
Significance of DNA replication for excision of Ds
Discussion
We have investigated whether Ds excision is coupled to
vector DNA replication in maize by examining the influence
of vector DNA replication on the frequency of Ds excision.
Our approach was to compare the frequency of Ds excision
from extrachromosomal replicating and nonreplicating
vector DNAs in transfected BMS maize protoplasts. Ds was
found to excise only from replicating vector DNA and not
from the nonreplicating derivative.
We have shown that Ds excises at least 3× 10S-fold more
frequently from replicating vector DNA as compared with
the nonreplicating derivative. The relative excision frequency is derived from three findings. First, we demonstrated that there is at least a 108-fold excess of empty
donor product generated in protoplasts transfected with
the replicating Ds-vector DNA over the amount generated
with the nonreplicating derivative. Secondly, we found a
10-fold excess of replicating Ds-vector DNA over nonreplicating Ds-vector DNA and therefore we corrected the
relative Ds excision frequency from 108 to 107. Third, only
3% of the transfected protoplasts were in a state of cell
division and therefore could facilitate WDV DNA replication.
Therefore, we adjusted the Ds excision frequency from 107
to 3×105 . We reasoned that in order to calculate accurately
the difference in frequency of Ds excision between pDsW +and pDsW--transfected cells we should consider only those
cells competent to support WDV replication. The calculated
difference in frequency between the two samples is most
likely underestimated due to the low percentage of replicating cells in these experiments. Furthermore, we demonstrated that the empty donor product does not replicate
and thus does not affect the measurement of Ds excision
frequency.
To distinguish the coupling of Ds excision to DNA replication from a coupling of excision to the replication protein(s)
expressed from the replicating vector DNA, we have
demonstrated that Ds excision is not directly coupled to
the presence of the WDV replication protein(s). We showed
that the Ds-vector DNA, which does not harbor sequences
required in cis for vector DNA replication, did not replicate
in the presence of the WDV replication protein(s) and Ds
did not excise. Thus, the WDV replication protein(s) are
only indirectly involved in excision by facilitating WDV
vector DNA replication.
To distinguish the coupling of Dsexcision to DNA replication from a coupling to the loss of the bacterial vector DNA
methylation pattern, we showed that Ds does not excise
from unmethylated pDsW-.
The results of our experiments show for the first time
that Ds excision, and presumably Ac excision, from extrachromosomal vector DNA in maize is directly coupled to
the replication of the vector DNA bearing the transposable
element. Furthermore, our results show that the coupling
131
of Ds/Ac excision to DNA replication is not restricted to
the P locus in maize.
Possible role of DNA replication in Ds excision at the
molecular level
The role of DNA replication in Ds excision from vector
DNA in maize remains to be determined at the molecular
level. The coupling of Ds excision to WDV vector DNA
replication may reflect shared biochemical and/or conformational requirements of these processes. However,
the molecular basis for the coupling of excision to DNA
replication has been determined for the bacterial transposable element IS10 (Tnl0) in dam + E. coli cells (Roberts
et al., 1985). IS10 has two dam sites, one within the
transposase promoter and one within the inner terminus
where transposase presumably binds. The absence of
methylation at both of these sites completely accounts for
increased transposition in dam-E, coli cells. Transposition
of IS10 is also increased when it is hemi-methylated. These
results suggest that in dam + E. coli IS10 will transpose
preferentially when it is hemi-methylated. Hemi-methylated copies of the transposon exist transiently after DNA
replication, thus coupling IS10 transposition to donor DNA
replication. However, Tnl0 also excises from nonreplicating X DNA and reinserts into the E. coli genome via
transposition (Bender and Kleckner, 1986). We have shown
that Ds excises at least 3x105-fold more frequently from
replicating vector DNA as compared with nonreplicating
vector DNA. Our result, however, does not rule out inefficient excision of Ds from nonreplicating vector DNA.
The methylation status of the Ac and Ds sequences that
bind Actransposase might also be important in Ac and Ds
excision. Actransposase, purified from a baculovirus/insect
cell expression system, binds in vitro to 5' and 3' Ac
subterminal regions (Kunze and Starlinger, 1989). The
binding sites contain multiple 5'-AAACGG-3' DNA
sequences and the transposase also binds synthetic concatemers of this motif. One of the two cytosine-hemimethylated forms of this motif has a higher affinity for
transposase than the unmethylated form. The holo-methylated motif and the other hemi-methylated form do not
bind at all. Ac and Ds excision may be coupled to DNA
replication due to increased binding of Ac transposase to
one of the two hemi-methylated forms of Ac and Ds
analogous to the transposition of the aforementioned IS10.
However, even if methylation plays a role in Ds and Ac
excision in vivo, additional features associated with the
replication fork may be essential in Ac and Ds excision
(reviewed in Kornberg and Baker, 1992). In fact, our results
indicate that absence of methylation of the cytosine
residues in the transposase binding motif on both DNA
strands is not sufficient to allow Ds excision from nonreplicating vector DNA. However, although our Ds-vector
132
Uwe Wirtz et al.
DNA was isolated from bacteria that do not methylate the
cytosine residue in the sequence 5'-AAACGG-3', we did not
investigate whether the transfected DNA became rapidly
methylated at those cytosine residues.
Since the role of DNA replication in Ds excision has not
been determined at the molecular level, it remains to be
determined whether our findings can be extended to the
excision of Ds in other systems. Nicotiana plumbaginifolia
plants harboring only Ds were regenerated after transfection of protoplasts with pUC-Ds plasmid and a transposase
expression vector. This finding suggests that Dstransposed
from extrachromosomal pUC DNA vector into the genome
(Houba-H~rin et al., 1994). The pUC-Ds vector does not
carry a known plant origin of replication. However, this
result does not necessarily mean that Ds excision occurs
independently from DNA replication in these transfected
protoplasts. The Ds-bearing plasmid might replicate as
extrachromosomal DNA in the transfected protoplasts,
Alternatively, the molecular requirement(s) for Ds excision
that are provided in our system by vector DNA replication
might be provided by another mechanism in transfected
Nicotiana plumbaginifolia protoplasts.
Implications for experiments employing Ds excision from
vector DNA
Our results imply that replicating vector DNA should be
used to study features of Ds (Ac) excision from vector DNA
in maize and presumably other monocots. Furthermore,
Dstransposition from extrachromosomal replicating vector
DNA into plant genomes can be exploited for the creation
of transgenic plants harboring single copy genes (Lebel
et al., 1995; Sugimoto et al., 1994).
Experimental procedures
Plasmid constructions
Plasmid construction was performed as described previously (Ausubel et al., 1988; Maniatis et al., 1982).
pDs. Our mini Ds was generated by PCR using Ac (Laufs et al.,
1990) as template DNA and the Ac oligonucleotides 7 (5'-TGGCCATATTGCAGTCATCCCG-3') and 8 (5'-GTGCTACATTAAACTATGTGTG-3') as primers. The mini Ds consists of 254 bp and 320 bp
of the Ac 5- and 3- termini, respectively. Ds termini are separated
by an Xhol linker d(pCCTCGAGG) and Ds was cloned via its Xhol
site into the Sail site of the 885-bp plasmid ~AN7 (Lutz et al.,
1987) making the plasmid pDs7. The orientation of Ds is such that
the 5' Ds terminus is at the 5- end of the supF gene of ~AN7. Ds
was sequenced to verify an unaltered sequence of the Ac termini
(data not shown) using a sequencing kit (United States Biochemical
Corporation).
The remainder of plasmids pDs, pDsW÷ and pDsW- are identical
(flanking thin line in Figure la and b). This portion of the vector
is called Ds-donor DNA. Details of the construction are available
upon request.
Circular in vitro-generated empty donor product. The Dsdonor DNA of pDs was released from pDs by Sstl digestion and
the fragment was recircularized with T4DNA ligase. Monomeric
Ds-donor DNA circles were purified by gel electrophoresis and
used for the transfection experiments.
pDsW +. The plasmid pWDVA4X6 (Laufs et aL, 1990) carries a
1839-bp derivative of the 2749-bp WDV genome. ORFs I and II of
WDV are replaced by a Xhol linker in this derivative and the WDV
replicon is cloned via its Sstl restriction site in pUC18. The 0.641and 1.198-kb Sstt-Xhol WDV fragments of pWDV&4X6 (Laufs et al.,
1990) were ligated to Sall-tinearized ~AN7 to create the plantbacteria shuttle vector pW +. The Bglll site of the 1.872-kb SmalBglll fragment of pW + harboring the WDV replicon was filled in
using Ktenow polymerase and the fragment was inserted into the
Xmnl site of pDs creating the 7.5-kb plasmid pDsW~.
pDsW-. The 90-bp Styl fragment at the 5' end of WDV ORF III
(Laufs et al., 1990) was removed from pW + and the remaining
fragment was filled in using Klenow polymerase. This fragment
was recircularized and resulted in the Styl-resistant plasmid pW(Laufs et al., 1990). The Bglll site of the t.786-kb SmaI-Bglll
fragment of pW- harboring the AStyl WDV sequence was filled in
using Klenow polymerase. This fragment was inserted into the
Xmnl site of pDs creating the 7.4-kb plasmid pDsW-.
pTps. The 0.265-kb PstI-Hindlll fragment of pCaMVNEO (Fromm
et al., 1986) harbors the 3'-untranslated region of the nopaline
synthase gene (3'NOS). The 3'NOS fragment was treated with
T4DNA polymerase and inserted into Sail-digested and Klenow
polymerase-treated pUC19 making the plasmid p3'N. The Asp718
site of p3'N was filled in using Klenow polymerase and the
1.989-kb Pstl fragment harboring the maize ubiquitin-1 promoter
and its first intron (Pr. Ubi+i; Christensen et al., 1992) was treated
with T4DNA polymerase. The two fragments were ligated and
resulted in the plasmid pPrU3'N. The 3.282-kb Banll fragment of
Ac, encoding Ac transposase, was treated with T4DNA polymerase, the BamHI linker d(pCGGATCCG) was ligated to the ends
of the fragment and the BamHI-digested fragment was inserted
into the BamnHIsite of pPrU3'N creating the 8.3-kb plasmid pTps.
pill/IV. The Bglll and the Ncol sites of the 1.36-kb BgllI-Ncol
fragment from pW + (see pDsW÷), harboring WDV ORFs III and
IV, were filled in using Klenow polymerase. The BamHI linker
d(pCGGATCCG) was Iigated to the ends of the fragment and the
BamHI-digested fragment was inserted into the BamHI site of
pPrU3'N creating the 6.3-kb plasmid pill/IV.
Preparation of plasmid DNA for protoplast transfection
pDs, pDsWL pDsW-, pTps and pill/IV were cloned in dam+/dcm +
E. coil DH5a cells (Grant et al., 1990). pDsW÷ and pDsW- DNA
were also cloned in dam/dcm-E, coliJM110 cells (Yanisch-Perron
et al., 1985) to produce unmethylated DNA (an EcoK site is not
present within pDsW~ or pDsW-). The absence of dam/dcm
methylation within the JM110-cloned plasmid DNA was verified
by restriction enzyme digestion (data not shown), pDsW* and
pDsW- DNA from E. coil JM110 cells was digested with Mbol but
not with Dpnl, showing that the plasmid DNA was unmethylated
at its damnsites. As expected, pDsW÷ and pDsW- isolated from
dam + DH5(z cells was resistant to Mbol digestion but sensitive to
Significance of DNA replication for excision of Ds
Dpnl digestion. Furthermore, we demonstrated that DNA isolated
from JM110 cells was fully digested with the dcm, methylationsensitive restriction enzyme, ScrFI, showing that it was unmethylated at its dcm sites, pDsW + and pDsW- isolated from dcm+ DH5~
was partially digested with ScrFI showing that not all dcm sites
were methylated. Plasmid DNA from both strains was completely
digested with the dcm, methylation-insensitive restriction
enzyme, BstNl.
Plasmid DNAwas purified by CsCI/ethidium bromide equilibrium
centrifugation (Ausubel eta/., 1988).
Maize cell suspension culture and protoplast transfection
A BMS maize cell suspension was propagated and protopiasts
were prepared as described previously (Fromm et al., 1987).
Protoplasts (6×106) in 1.5 ml protoplast growth medium (Fromm
et al., 1987) were transfected with 150 p,g of each plasmid DNA
dissolved in 150 I~1 TE buffer (20 mM Tris-HCI, pH 7.4; 10 mM
EDTA, pH 8.0). Transfection was mediated bythe addition of 1.5 ml
of a polyethylene glycol solution (25 % polyethylene glycol,
molecular weight 6000; 0.1 M Ca(NO3)2; 0.25 M mannitol; pH 9.0).
After 30 minutes of incubation at 22°C, 37.5 ml of a 0.275 M
Ca(NO3)2 solution (pH 6.0) was added. The protoplasts were
collected by centrifugation at 200 g for 10 min at 22°C and
carefully resuspended in 30 ml of protoplast growth medium.
Intact protoplasts were counted in a hemocytometer. Seventy per
cent of the protoplasts (4.2×106) were found to be intact. The
transfected protoplasts were cultured as 10 ml aliquots (1.4× 106)
in 100 × 25 mm petri dishes in the dark at 26°C. Seventy per cent
of the protoplasts had resynthesized cell walls as judged by the
altered shape of the protoplasts 2 days after transfection. At this
time point, 0.05% of the cells were in a state of cell division. Eight
days after protoplast transfection, 3% of the cells were in a state
of cell division.
Cell nuclei preparation and DNA isolation
A cell nuclei-enriched fraction from transfected protoplasts was
prepared as described by Saxena et al. (1985). Protoplasts and
protoplast-derived cells were harvested 2, 4 and 8 days after
transfection and incubated in 10 ml of protoplast enzyme solution
(Fromm etal., 1987). The protoplasts were collected by centrifugation (see above) and resuspended in 5 ml of NIB solution (10 mM
2-[N-morpholino] ethane sulfonic acid; 0.2 M sucrose; 5 mM
13-mercaptoethanol; 2.5 mM EDTA; 0.1 mM spermin, 10 mM NaCI;
10 mM KCI; 0.02% Triton X-100; pH 5.2) on ice. The protoplasts
were incubated for 3 min on ice and passed three times through
a 26-gauge needle. The material was filtered through two layers
of miracloth and the nuclei were collected by centrifugation at
500 g for 9 min at 4°C. The pellet was resuspended in 5 ml of
NIB-T (TIB without Triton X-100) on ice. The centrifugation was
repeated and the nuclei-enriched fraction finally resuspended in
500 I11 of NIB-T.
Phenosafranine-stained nuclei were inspected in a hemocytometer (Saxena et al., 1985). The nuclei preparation efficiency was
70% (1×106) with respect to the starting number of protoplasts
(1.4× 106) and was identical for preparations made from 2-, 4- and
8-day-old transfected protoplasts.
Total DNA was isolated from the nuclei-enriched fraction by
adding 300 pl of DNA extraction buffer (0.1 M NaCI; 0.1 M TrisHCI, pH 8.0; 0.1 M EDTA, pH 8.0; 0.5 % SDS; 250 I~g proteinase K/
300 I~1 DNA extraction buffer) to 1× 106 nuclei. DNA was purified
by extracting once with phenol, once with 1:1 phenol:chloroform
133
and twice with chloroform. The DNA was ethanol-precipitated,
washed once with 70% ethanol and finally dissolved in 100 I~1TE
buffer. The DNA concentration was determined by comparing
aliquots of this DNA with aliquots of known amounts of DNA on
a gel. DNA yields of 0.5 pg I~1-1were recovered from the nucleienriched fractions isolated 2, 4 and 8 days after transfection.
DNA analysis by Southern blot hybridization
Vector DNA replication assay by Mbol digestion (Figure 2a
and b). Total nuclear DNA (5 ~tg) was digested with Mbol and
Scal. The digested DNA was size-fractionated by electrophoresis
through a 1% agarose gel and blotted to BA83 (Schleicher &
Schuell) nitrocellulose filter (Southern, 1975). The 638-bp Dpnl
fragment of pDsW + was labeled with (c(-32p)dCTP using a random
oligonucleotide priming kit (Amersham), and was used as a probe.
Dpnl has the same cleavage site as Mbol, but digests plasmid DNA
propagated in dam+ E. cofiwhich Mbol does not. Hybridization was
performed for two days at 42°C (Hughes et al., 1978). The filter
was washed four times in SDS/SSC buffer (0.1% SDS; 15 mM
NaCI; 1.5 mM Na-citrate) at 50°C and exposed to XAR-5 film
emulsion (Kodak).
DNA analysis by the polymerase chain reaction
Vector DNA replication assay by Dpnl digestion (Figure 2c
and d). Total nuclear DNA (1 pg) was digested with Dpnl and
Scal. The digested DNA was subjected to 14 PCR cycles of 1 min
at 94°C, 2 min at 54°C and 3 min at 72°C using two units of
Thermus aquaticus polymerase (Promega), 0.1 ~g of primers 5
(5'-CTCTTCATAGCCTTATGCAGTTG-3' and 6 (5'-CGCCAGCAACGCAAGCTTCTAG-3') in a volume of 100 pl containing PCR buffer
(50 mM Tris-HCI, pH 9.0; 50 mM NaCI; 2.5 mM MgCI2) and
200 pM each of dATP, dCTP, dGTP and TTP. PCR amplification was
performed in the DNA Thermal Cycler 480 from Perkin-Elmer. A
30 pl sample of the products was fractionated on a 2% agarose
gel containing 0.1 ~g m1-1 ethidium bromide after completion of
the PCR.
Ds excision assay (Figures 3 and 4). For the first amplification,
1 ~g of total nuclear DNA was linearized with Scal or BstEIl. The
linearized DNA was subjected to 35 PCR cycles (see above) using
0.1 I~g of the primers t (5'-GCATATGCAGCAGCTATATGTG-3') and
2 (5'-AGCGGTTCCATCCTCTAGAGGA-3'). For PCR reamplification,
2 ~1 of the PCR products from the first amplification were used as
template DNA and subjected to 35 cycles (see above) using 1 ~g
of the primers 3 (5'-CCTGCCI-rCATACGCTATFTAT-3') and 4 (5'TTTACCAACAGTACCG GAATG C-3').
pDsW+ and pDsW- quantification assay (Figure 5). Aliquots
of 1 ~g of total nuclear DNA and dilutions thereof were linearized
with Scal. The linearized DNA was subjected to 35 PCR cycles
using 0.1 Ilg of the primers 5 and 6 (sequence of primers 5 and
6: see above).
Acknowledgments
We are grateful to Dr Bruno Gronenborn for several helpful
discussions and critical reading of the manuscript, This work was
supported by the Rockefeller Foundation,
134
U w e Wirtz et al.
References
Athma, P., Grotewald, E. and Peterson, T. (1992) Insertional
mutagenesis of the maize P gene by intragenic transposition of
Ac. Genetics, 131, 199-209.
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman,
J.G., Smith, J.A. and Struhl, K. (1988) Current Protocols in
Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E.,
Moore, D. D., Seidman, J. G., Smith J. A. and Struhl, K., eds).
New York: Greene and Wiley Interscience.
Bender, J. and Kleckner, N. (1986) Genetic evidence that Tnl0
transposes by a nonreplicative mechanism. Cell, 45, 801-815.
Chen, J., Greenblatt, I.M. and Dellaporta, S.L. (1987) Transposition
of Ac from the P locus of maize into unreplicated chromosomal
sites. Genetics, 117, 109-t16.
Chen, J., Greenblatt, I.M. and Dellaporta, S.L. (1992) Molecular
analysis of Ac transposition and DNA replication. Genetics, 130,
665-676.
Christensen, A.H., Sharrock, R.A. and Quail, P.H. (1992) Maize
polyubiquitin genes: structure, thermal perturbation of
expression and transcript splicing, and promoter activity
following transfer to protoplasts by electroporation. Plant Mo/.
BioL 18, 675-689.
Coupland, G., Plum, C., Chatterjee, S., Post, A. and Starlinger, P.
(1989) Sequences near the termini are required for transposition
of the maize transposon Ac in transgenic tobacco plants. Proc.
Nat/Acad. ScL USA, 86, 9385-9388.
Desbiez, C., David, C., Mettouchi, A., Laufs, J. and Gronenborn,
B. (1995) Rep protein of tomato yellow leaf curl geminivirus has
an ATPase activity required for viral DNA replication. Proc. Nat/
Acad. Sci. USA, 92, 5640-5644.
Elmer, J.S., Brand, L., Sunter, G., Gardiner, W.E., Bisaro, D.M. and
Rogers, S.G. (1988) Genetic analysis of the tomato golden
mosaic virus t1. The product of the ALl coding sequence is
required for replication. Nuc/. Acids Res. 16, 7043-7060.
Etessami, P., Saunders, K., Watts, J. and Stanley, J. (1991)
Mutational analysis of complementary-sense genes of African
cassava mosaic virus DNA A. J. Gen. Virol. 72, 1005-1012.
Fedoroff, N. (1989) Maize transposable elements. In Mobile DNA
(Berg, D.E. and Howe, M.M., eds). Washington, DC: American
Society for Microbiology, pp. 375-411.
Fontes, E.P.B., Luckow, V.A. and Hanley-Bowdoin, L. (1992) A
geminivirus replication protein is a sequence-specific DNA
binding protein. Plant Ceil, 4, 597-608.
Fromm, M.E., Taylor, L.P. and Walbot, V. (1986) Stable
transformation of maize after gene transfer by electroporation.
Nature, 319, 791-793.
Fromm, M., Callis, J., Taylor, L.P. and Walbot, V. (1987)
Electroporation of DNA and RNA into plant protoplasts. Methods
Enzymo/. 153, 351-366.
Grant, S.G.N., Jessee, J., Bloom, F.R. and Hanahan, D. (1990)
Differential plasmid rescue from transgenic mouse DNAs into
Escherichia coil methylation-restriction mutants. Proc. Natl
Acad. Sci. USA, 87, 4645-4649.
Greenblatt, I.M. (1966) Transposition and replication of modulator
in maize. Genetics, 53, 361-369.
Greenblatt, I.M. (1968) The mechanism of modulator transposition
in maize. Genetics, 58, 585-597.
Greenblatt, I.M. (1974) Movement of modulator in maize: a test
of an hypothesis. Genetics, 77, 671-678.
Greenblatt, I.M. (1984)A chromosome replication pattern deduced
from pericarp phenotypes resulting from movements of the
transposable element Modulator in maize. Genetics, 108, 471485.
Greenblatt, I.M. and Brink, R.A. (1962) Twin mutations in medium
variegated pericarp maize. Genetics, 47, 489-501.
Heinzel, S.S., Krysan, P.J., Tran, C.T. and Calos, M.P. (1991)
Autonomous DNA replication in human cells is affected by the
size and the source of the DNA. Mol. Cell BioL 11, 2263-2272.
Heyraud, F., Matzeit, V., Kammann, M., Schaefer, S., Schell, J. and
Gronenborn, B. (1993) Identification of the initiation sequence
for viral-strand DNA synthesis of Wheat Dwarf Virus. EMBO J.
12, 4445-4452.
Horns, T. and Jeske, H. (1991) Localization of abutilon mosaic
virus (AbMV) DNA within leaf tissue by in situ hybridization.
Virology, 181,580-588.
Houba-Herin, N, Domin, M. and Pedron, J. (1994) Transposition
of a Ds element from a plasmid into the plant genome in
Nicotiana p/umbaginifo/ia protoplast-derived cells. Plant J. 6,
55-66.
Hughes, S.H., Shank, RR., Spector, D.H., Kung, H.-J., Bishop, J.M.,
Varmus, H.E., Vogt, P.K. and Breitman, M.L. (1978) Proviruses
of avian sarcoma virus are terminally redundant, co-extensive
with unintegrated linear DNA and integrated at many sites. Cell,
15, 1397-1410.
Kammann, M., Schatk, H.-J., Matzeit, V., Schaefer, S., Schell, J.
and Gronenborn, B. (1991) DNA replication of wheat dwarf
virus, a geminivirus, requires two cis-acting signals. Virology,
184, 786-790.
Kornberg, A. and Baker, T.A. (1992) DNA Replication (Kornberg,
A. and Baker, T. A., eds). New York: W.H. Freeman and Company.
Kunze, R. and Starlinger, P. (1989) The putative transposase of
transposable element Ac from Zea mays L. interacts with
subterminal sequences of Ac. EMBO J. 8, 3177-3185.
Laufs, J., Wirtz, U., Kammann, M., Matzeit, V., Schaefer, S., Schell,
J., Czernilofsky, A.P., Baker, B. and Gronenborn, B. (1990)
Wheat dwarf virus Ac/Ds vectors: expression and excision of
transposable elements introduced into various cereals by a viral
replicon. Proc. Natl Acad. Sci. USA, 87, 7752-7756.
Laufs, J., Traut, W., Heyraud, E, Matzeit, V., Rogers, S.G., Schell,
J. and Gronenborn, B. (1995) In vitro cleavage and joining at
the viral origin of replication by the replication initiator protein
of tomato yellow leaf curl virus. Proc. Natl Acad. Sci. USA, 92,
3879-3883.
Lazarowitz, S.G., Wu, L.C., Rogers, S.G. and Elmer, J.S. (1992)
Sequence-specific interaction with the viral ALl protein
identifies a geminivirus DNA replication origin. Plant Cell, 4,
799-809.
Lebel, E.G., Masson, J. Bogucki, A. and Paszkowski, J. (1995)
Transposable elements as plant transformation vectors for long
stretches of foreign DNA. Theor. Appl. Genet. 91,899-906.
Lechelt, C., Petarson, T., Laird, A., Chen, J., Dellaporta, S.L.,
Dennis, E., Peacock, W.J. and Starlinger, P. (1989) Isolation and
molecular analysis of the maize P locus. Mol. Gen. Genet. 219,
225-234.
Lutz, C.T., Hollifield, W.C., Seed, B., Davie, J.M. and Huang, H.V.
(1987) Syrinx 2 A: an improved X phage vector designed for
screening DNA libraries by recombination in vivo. Proc. Natl
Acad. ScL USA, 84, 4379-4383.
McClintock, B. (1947) Cytogenetic studies of maize and
Neurospora. Carnegie Institute Washington Year Book, 46,
146-152.
McClintock, B. (1950) Mutable loci in maize. Carnegie Institute
Washington Year Book, 49, 157-167.
MacDowell, S.W., MacDonald, H., Hamilton, W.D.O., Coutts, R.H.A.
and Buck, K.W. (1985) The nucteotide sequence of cloned wheat
dwarf virus DNA. EMBO J. 4, 2173-2180.
Significance o f DNA replication for excision o f Ds
Man/at/s, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular
Cloning A Laboratory Manual Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press.
Matzeit, V., Schaefer, S., Kammann, M., Schalk, H.-J., Schell, J.
and Gronenborn, B. (1991) Wheat dwarf virus vectors replicate
and express foreign genes in cells of monocotyledonous plants.
Plant Cell, 3, 247-258.
McWhinney, C. and Leffak, M, (1989) Autonomous replication of
a DNA fragment containing the chromosomal replication origin
of the human c-myc gene. Nucl. Acids Res. 18, 1233-1242.
Peden, K.W.C., Pipas, J.M., Pearson-White, S. and Nathans, D.
(1980) Isolation of mutants of an animal virus in bacteria.
Science, 209, 1392-1396.
Pilartz, M. and Jeske, H. (1992) Abutilon mosaic gem/n/virus
double-stranded DNA is packed into m/n/chromosomes.
Virology, 189, 800-802.
Revington, G.N., Sunter, G. and Bisaro, D.M. (1989) DNA
sequences essential for replication of the B genome component
of tomato golden mosaic virus. Plant Cell, 1,985-992.
Roberts, D., Hoopes, B.C., McClure, W.R. and Kleckner, N. (1985)
IS10 transposition is regulated by DNA adenine methylation.
Cell, 43, 117-130.
Rushing, A.E., Sunter, G., Gardiner, W.E., Dute, R.R. and Bisaro,
D.M. (1987) Ultrastructural aspects of tomato golden mosaic
virus infection in tobacco. Phytopathology, 77, 1231-1236.
Saunders, K., Lucy, A. and Stanley, J. (1991) DNA forms of the
gem/n/virus African cassava mosaic virus consistent with a
rolling circle mechanism of replication. Nucl. Acids Res. 19,
2325-2330.
Saxena, P.K.,Fowke, L.C. and King, J. (1985) An efficient procedure
135
for isolation of nuclei from plant protoplasts. Protoplasma, 128,
184-189.
Schalk, H.-J., Matzeit, V., Schiller, B., Schell, J. and Gronenborn,
B. (1989) Wheat dwarf virus, a gem/n/virus of graminaceous
plants needs splicing for replication. EMBO J. 8, 359-384.
Shen, W.-H. and Hohn, B. (1992) Excision of a transposable element
from a viral vector introduced into maize plants by agroinfection.
Plant J. 2, 35-42.
Shen, Wo-H., Das, S. and Hohn, B. (1992) Mechanism of Dsl
excision from the genome of maize streak virus. Mol. Gen.
Genet. 233, 388-394.
Southern, E.M. (1975) Detection of specific sequences among
DNA fragments separated by gel electrophoresis. J. Mol. Biol.
98, 503-517.
Stenger, D.C., Revington, G.N., Stevenson, M.C. and Bisaro, D.M.
(1991) Replicational release of gem/n/virus genomes from
tandemly repeated copies: evidence for rolling-circle replication
of a plant viral DNA. Proc. Natl Acad. ScL USA, 88, 8029-8033.
Styles, E.D. and Ceska, O. (1977) The genetic control of flavonoid
synthesis in maize. Can. J. Genet. Cytol. 19, 289-302.
Sugimoto, K., Otsuki, Y., Saji, S. and Hirochika, H. (1994)
Transposition of the maize Ds element from a viral vector to
the rice genome. Plant J. 5, 863-871.
Woolston, C.J., Reynolds, H.V., Stacey, N.J. and Mullineaux, P.M.
(1989) Replication of wheat dwarf virus DNA in protoplasts and
analysis of coat protein mutants in protoplasts and plants. Nucl.
Acids Res. 17, 6029-6041.
Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved
phage cloning vectors and host strains: nucleotide sequence of
the M13mp18 and pUC19 vectors. Gene, 33, 103-119.