Download Materials and methods (Supplement)

Materials and methods (Supplement)
Construction of a expression plasmid
The coding region of the Tol2 transposase gene Tol2TP (AB032244, 1782 bp) was amplified
by PCR with primers Tol2TP-F (5’– cg ggatcc ACCAT GGAGG AAGTA TGTGA TTCA -3’;
lower cases including a BamHI linker sequence) and Tol2TP-R (5’– gc tctaga ctcgag CTACT
CAAAG TTGTA AAACC TCA –3’; lower cases including an XbaI and XhoI linker sequence).
The amplified fragment was digested with BamHI and XbaI, and subcloned into the BamHI and
XbaI sites of pCS107X, in which the multicloning site of pCS107 was replaced with a new one
(5’- ggatcc atcgat ttcgaa gaattc agatct at gcggccgc at aggcct accggt acgcgt ctcgag tctaga -3’;
made by Asuka Takahashi and M. T.) to construct pCS107X-Tol2TP. Similarly, the Tol2TP
coding region was inserted into the BamHI and XhoI sites of pGEX-6P-1 to make
pGEX-6P-Tol2TP. Plasmid constructs were verified by DNA sequencing using an ABI PRISM
3100 Genetic Analyzer.
Escherichia coli BL21(DE3) cells carrying the plasmid pT-Trx or
pT-GroE for the expression of thioredoxin or chaperon protein GroE, respectively [10] were
transformed with pGEX-6P-Tol2TP for producing GST-Tol2TP fusion protein.
Expression and purification of GST-Tol2TP
E. coli cells carrying pGEX-6P-Tol2TP and pT-Trx or pT-GroE were cultured overnight at
37 ˚C in the LB medium containing 100 g/ml ampicillin and 30 g/ml chloramphenicol,
transferred to pre-warmed LB medium/ampicillin/ chloramphenicol at 1:100 dilution, and
incubated at 18˚C with shaking at 250 rpm. When OD600 of bacterial culture had reached
0.4-0.6, the expression of GST-Tol2TP was induced with 0.1 mM
isopropyl--d-thiogalactopyranoside (IPTG) for 4 h. The cells were collected by
centrifugation and frozen in liquid nitrogen until use. All subsequent steps were performed at
0-4˚C. Frozen cells were resuspended in pre-cooled sonication buffer (150 mM NaC1, 50 mM
Tris-HCl, pH 8.0, 1 mM EDTA, and 1 mM DTT) containing 0.5mM PMSF, and lysed by
sonication. The lysate was supplemented with TritonX-100 and PMSF to make final
concentrations of 1% and 1 mM, respectively, and shaken gently for 30 min and centrifuged at
18,000 x g for 45 min. The supernatant was loaded onto a glutathione-Sepharose 4B column
(GE Healthcare Life Science) pre-equilibrated with sonication buffer containing 1%
TritonX-100. After washing with sonication buffer, GST-Tol2TP was eluted with sonication
buffer containing 10 mM reduced glutathione.
Cleavage and purification of rTol2TP from GST-Tol2TP
The eluate containing GST-Tol2TP was added with PreScission protease (8 U for 5–10 mg
of fusion protein; GE Healthcare Life Science) and dialyzed with PreScission cleavage buffer
(150 mM NaCl, 50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 1 mM DTT, 0.01% TritonX-100) at 4
˚C for 12–16 hr to cleave the Tol2TP portion (rTol2TP) from GST-Tol2TP. The dialysate was
loaded on a glutathione-Sepharose 4B column to obtain rTol2TP as a flow-through fraction.
The flow-through fraction was dialyzed with storage buffer (20 mM HEPES, pH 7.4, 500 mM
NaCl), concentrated with VIVASPIN (Sartorius), added with glycerol to make a final
concentration of 10%, and stored at –80 ˚C until use. The protein concentration of rTol2TP
was determined by SDS-polyacrylamide gel electrophoresis (PAGE) and an image analyzer
(Odyssey; LI-COR) after staining with Coomassie Brilliant Blue R250 (CBB) using bovine
serum albumin (BSA) as a standard. Western blot was performed as described [16] with rabbit
anti-GST IgG antibody (Sigma) and anti-rabbit IgG (H+L) antibody conjugated with IRDye800
(Rockland).
Reference (Supplement)
[16] Shibata, M., Itoh, M., Hikasa, H., Taira, S. and Taira, M. (2005) Role of Crescent in
convergent extension movements by modulating Wnt signaling in early Xenopus
embryogenesis. Mech. Dev. 122, 1322-1339.
[17] Kaufman, P.D. and Rio, D.C. (1992) P element transposition in vitro proceeds by a
cut-and-paste mechanism and uses GTP as a cofactor. Cell 69, 27-39.
[18] Zhou, L., Mitra, R., Atkinson, P.W., Hickman, A.B., Dyda, F. and Craig, N.L. (2004)
Transposition of hAT elements links transposable elements and V(D)J recombination.
Nature 432, 995-1001.
[19] Rommens, C.M., van Haaren, M.J., Nijkamp, H.J. and Hille, J. (1993) Differential repair
of excision gaps generated by transposable elements of the 'Ac family'. Bioessays 15,
507-12.
[20] Kawakami, K., Koga, A., Hori, H. and Shima, A. (1998) Excision of the tol2 transposable
element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio. Gene 225, 17-22.
[21] Kawakami, K. and Shima, A. (1999) Identification of the Tol2 transposase of the medaka
fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish
Danio rerio. Gene 240, 239-44.
[22] Ma, Y. et al. (2004) A biochemically defined system for mammalian nonhomologous
DNA end joining. Mol Cell 16, 701-13.
Supplement figures
Purification of rTol2TP
Pilot experiments to determine the optimal culture conditions were performed as follows.
To suppress the formation of insoluble aggregates, GST–Tol2TP was coexpressed with
thioredoxin (Trx) or GroE in E. coli, and the E. coli cells were cultured at 18 °C. As shown in
Supplement Fig. 1, IPTG induced a 100-kDa protein, which is consistent with the calculated
molecular mass of GST–Tol2TP (100,017 Da), in the presence of GroE but not Trx.
This band
was confirmed as GST–Tol2TP on a western blot probed with anti-GST antibody (see Fig. 1B).
To make a large preparation of rTol2TP, E. coli carrying pGEX–6P–Tol2TP and pT–GroE
were grown in a 2 L culture, and induced with IPTG. SDS–PAGE of the cell lysate showed
that a certain amount of GST–Tol2TP was present in the supernatant, although a larger amount
of GST–Tol2TP occurred in the pellet (Supplement Fig. 1, lanes 3, 4). The GST–Tol2TP in
the supernatant was purified with a glutathione–Sepharose 4B column.
A model of Tol2TP action
The Tol2 element is flanked by an 8-bp direct repeat unit, TCAAGAAC, resulting from
target site duplications during its integration. After the excision of the Tol2 element, this
direct repeat is cancelled to restore the original sequence, possibly via a ‘cut-and-paste’
mechanism (Fig. 2D, type I) [17] (Supplement Fig. 2A). However, additional short sequences
were present in the footprint sequences of the plasmid DNA after excision. These additional
sequences can be categorized as two types. Type II has short direct repeats, as exemplified by
clone 2b, as well as by clones 9 and 10c of a previous report [8], whereas type III has a
complete or partial sequence complementary to the 8-bp direct repeat unit (GTTCTTGA, GTT,
or GA), together with the short direct repeat, as exemplified by clones 5c, 4a, and 2c, as well as
by clones 2, 4, 6, 7, 10a, and 10b of a previous report [8].
Hermes transposase excises the element via double-stranded breaks and forms hairpin
structures at the ends of the DNA. This activity requires the DDE catalytic triad in the
three-dimensional structure of the transposase [18]. Because the DDE catalytic triad is also
conserved in Tol2TP (see Fig. 1A), it is likely that the footprint sequences generated by Tol2TP
are created through hairpin formation in broken DNA. If this is the case, the type II and type
III footprints of rTol2TP can be explained with the model previously proposed by Coen [19]
(Fig. 2D, Supplement Fig. 2D).
It should be noted that the proportions of type I, II, and III footprint sequences seem to vary
greatly between zebrafish [20,21] and Xenopus [[8] and this work]. This implies that Tol2TP
may only excise the Tol2 element from DNA, and that the resultant double-stranded breaks of
the plasmid DNA are joined by cellular repair enzymes, which could differ in activity between
zebrafish and Xenopus embryos.
Figure legend (Supplement)
Supplement Fig. 1. Purification of GST-Tol2TP
Cell cultures with (+) or without (-) IPTG (0.1 mM)-induction and glutathione affinity column
purification steps as indicated were analyzed by SDS-PAGE. Dot, GST-Tol2TP band; f.t.,
flow-through fraction; thick arrow, GST-Tol2TP.
Supplement Fig. 2. Models for the transposition of the Tol2 element
(A) Precise excision of the Tol2 element results in a single direct repeat unit. (B) the “Coen”
model modified by Roomens et al [19].
According to this model, DNA double stranded breaks
with 1 bp staggered cuts are generated and formed hairpin structures at the position adjacent to
the transposable element. These hairpin structures are resolved by nicks and both open ends of
genomic DNA are ligated to create various inversions or direct repeats which depend on the
position of nicks. White and black triangles, direct repeat unit and its complementary strand,
respectively. (C) A model for the formation of footprint type III. Arrow, positions of nicks
for opening hairpin structures; italic, complement sequence. Nonhomologous end joining
(NHEJ) of DBS is likely to be executed by a repair enzyme complex containing
DNA-dependent protein kinase, Artemis endonuclease, and XRCC4:DNA ligase IV [22]. This
model is supported by the observation that, 7 bases, TCAAGAA (left) or CAAGAAC (right),
but not 8 bases of the direct repeat unit were connected to the complementary sequence as
shown in clones 5c and 4a (Fig. 2D)