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METHODS
Plasmid construction
To generate the expression plasmids pHL2574, pHL2575, and pHL2578 we
amplified the hermes transposase from pGal-hermes-native (pHL2569, N. Craig) and
with primers HL1357, HL1389 added an upstream XhoI and a downstream XmaI site.
The fragment was ligated into the XhoI and XmaI sites in the polylinker of the vectors
Rep3X, Rep41X, and Rep81X (FORSBURG 1993).
To construct plasmids pHL2623, pHL2624, and pHL2625 a PCR product
encoding the transposase with the T317A mutation was generated using primers HL1477
and HL1478. The resulting product was cut with EcoRI, which cuts within primer
HL1478, and BglII, which cuts immediately downstream of primer HL1477. The
resulting fragment was cloned into the EcoRI and BglII sites of pGal-hermes-native
(pHL2569, N. Craig). These plasmids were digested with SalI and BglII and inserted into
vectors pHL2574, pHL2575, and pHL2578 at the same sites to form vectors pHL2623,
pHL2624, and pHL2625, which contain the T317A mutation in the transposase.
The donor plasmid pHL2577 was built by fusion PCR. Hermes right, kanMX6,
and hermes left were amplified separately using as template pHL2572 for the hermes
TIRs and pHL2205 for kanMX6. The three components were combined by fusion PCR
to yield the full-length fragment. Restriction sites EagI and XhoI were added via outside
primers HL1364 and HL1392. Terminal A’s were added to the 2887 bp fragment and it
was inserted into pCR2.1 (invitrogen) making pHL2576. The new construct was digested
with EagI and XhoI and the fragment containing hermes right, kanMX6, and hermes left
was cloned into pHL390 using EagI and XhoI of the polylinker.
To construct pHL2641, PCR primers HL1503 and HL1504 were used to produce
an 850 bp PCR product containing the p15A origin of replication (ori) from pACYC184
(ROSE 1988). The ori was inserted into the NdeI and XmaI sites of pHL2576. The new
element containing the ori was excised with EagI and XhoI and cloned into the same sites
of pHL2577, generating vector pHL2641.
To generate plasmid pHL2651, the BglII , PmeI fragment of NatR from pCR2.1Nat (SATO et al. 2005) (pHL2621) was inserted into the same sites in the kanMX6 gene
in pHL2577.
Media
Edinburgh minimal liquid (EMM) with 2 g/L of dropout mix (all amino acids in
equal weights plus adenine in 2.5 time more weight) was used when selecting for
auxotrophs {Moreno, 1991 #1429}. 10uM thiamine was added to repress the expression
of the nmt1 promoter when necessary. 5-Fluoroorotic acid (5-FOA) was added at 1
mg/mL for counter-selection of URA3 (BOEKE et al. 1987). The complete media (YES)
included 30 g/L dextrose, 5 g/L yeast extract, and 2 g/L of complete dropout mix, a
combination of all amino acids, uracil, and adenine. To select for insertions, G418 at a
concentration of 500 µg/mL was added along with 5-FOA to YES plate media. To select
for insertions of hermes-nat, 100 µg/mL of nourseothricin (WERNER BioAgents) was
added to YES. Yeast extract media (YE) was used to screen for ade- colonies and is
identical to YES but lacking the complete dropout mix.
Transposition assay
Cells were initially grown as patches on EMM-ura-leu+B1 for 2 days. Plates were
replica printed to EMM-ura-leu-B1 and grown for 2 days to induce transcription of
hermes transposase. Plates were then replica printed to EMM-leu+FOA+B1 and grown
for 2 days to remove the donor plasmid. Finally, plates were replica printed to
YES+FOA+G418 and grown for 40 hours to obtain patches of cells containing insertions
of the transposon.
Mapping insertion sites
Following the transposition assay, plasmids were removed from the cells by
expanding single colonies on YES plates. Inverse PCR was performed by digesting 2 µg
of prepared genomic DNA (see DNA blotting) overnight with 40 units of EcoRI (NEB).
The resulting digestion was phenol extracted, and diluted to 1 ng/µl. The dilution was
religated with 1 U/µl T4 DNA Ligase at 18 °C overnight. The resulting material was
ethanol precipitated and 100 ng of circularized DNA was amplified using primers
pHL1430 and pHL1431. The PCR products were inserted into pCR2.1 (Invitrogen) and
sequenced. The target site duplication was verified using a primer in hermes left
(HL1893) and one in the flanking genomic DNA as identified by the inverse PCR. The
sequence composition of the target site duplications was displayed as a sequence logo
using weblogo.berkeley.edu.
DNA blotting
Fifty mL cultures were grown to a final OD of 8.0 at 600 nm and pelleted. Cells
were resuspended in solution Sp1 (1.2 M sorbitol, 50 mM citric acid monohydrate, and
50 mM Na2HPO4*7H2O, 40 mM EDTA, pH 5.6) containing 15 mg Zymolyase.
Spheroblasted cells were resuspended in 7.5 mL 5X TE + 1% SDS and incubated for one
hour to lyse cells. Following centrifugation and resuspension, 2.5 mL of 5M KOAc was
added and incubated for 30 minutes. Cellular debris was removed via centrifugation and
the nucleic acids were precipitated by adding 2.5 volumes of cold 2-propanol. RNase A
digestion was performed for one hour at a concentration of 100 µg/mL. Overnight
digestion with Proteinase K (50 µg/mL) was followed by three phenol, chloroform
isoamyl alcohol extractions. Finally, DNA was precipitated in 0.1 volume of 5M NaCl
and 2.5 volumes of ethanol, and then resuspended in 40 uL of 1x TE. DNA blotting was
performed by digesting two µg of prepared genomic DNA overnight with 40 units of
EcoRI. Following electrophoresis and transfer, the membrane was hybridized using a
radiolabeled fragment of kan sequence.
Immunoblot Analysis of Hermes Transposase
A whole cell protein extract was obtained using a standard bead-beater protocol.
Cells were grown to a final OD of 1.0 and 40 OD units of cells were pelleted. The cells
were washed first with water and then with extraction buffer consisting of 15 mM KCl,
10 mM HEPES-KOH (pH 7.8), and 5 mM dithiothreitol. The cells were then resuspended
in 0.2 ml of extraction buffer plus 5.0 mM dithiothreitol, 2.0 mM phenylmethylsulfonyl
fluoride and a mixture of protease inhibitors including a protease inhibitor cocktail (1
tablet in 50 ml, Roche), as well as 0.7 g/ml pepstatin, 0.5 g/ml leupeptin and 1 g/ml
aproteinin. 100 l of acid-washed glass beads were added, and the samples were put into
the mini-beadbeater 8 (Biospec products Inc., Bartlesville, OK) for 3 cycles of 30
seconds to break the cells. Twelve µg of protein was then subjected to electrophoresis on
a 10-20% Tris-Glycine (Invitrogen) gel and transferred to a membrane. The membrane
was probed overnight at room temperature with an anti-transposase antibody added at a
concentration of 1:1000. The antibody was polyclonal and was raised in rabbit (X. Li,
and N. Craig, unpublished).
Table S1: Plasmids*
Plasmid
pHL390
pHL423
Rep3X
Markers
amp,
URA3
amp,
LEU2
pHL2205
amp,
LEU2
amp,
LEU2
amp,
LEU2
amp
pHL2569
amp
pHL2572
amp,
LEU2
Tet, cat
Rep41X
Rep81X
pACYC184
pHL2574
pHL2575
pHL2576
pHL2578
pHL2621
amp,
LEU2
amp,
LEU2
Amp, kan
amp,
LEU2
amp, kan
pHL2623
amp,
LEU2
pHL2624
amp,
LEU2
pHL2625
amp,
LEU2
pHL2577
Amp,
Description
pSP2 lacking hermes TIR’s
Rep3 nmt1 promoter, control
plasmid lacking hermes
transposase
nmt1 promoter, high expression
nmt1 promoter, medium
expression
nmt1 promoter, low expression
pFA6a-kanMX6
hermes transposase driven by
GAL promoter of S. cerevisiae.
hermes left/right flanking LEU2
of S. cerevisiae.
cloning vector with p15A origin
of replication
Rep3X nmt1 promoter expressing
wt hermes transposase
Rep41X nmt1 promoter
expressing wt hermes transposase
hermes TIR’s flanking kanMX6
in cloning plasmid pCR2.1
rep81X nmt1 promoter
expressing wt hermes transposase
nat resistance gene in cloning
plasmid pCR2.1
Rep3X nmt1 promoter expressing
mutant hermes transposase
(T317A)
Rep41X nmt1 promoter
expressing mutant hermes
transposase (T317A)
Rep81X nmt1 promoter
expressing mutant hermes
transposase (T317A)
hermes TIR’s flanking kanMX6
Ref.
(COTTAREL et
al. 1993)
(MAUNDRELL
1993)
(FORSBURG
1993)
(FORSBURG
1993)
(FORSBURG
1993)
(BAHLER et al.
1998)
N. Craig
N. Craig
(ROSE 1988)
this study
this study
This study
this study
(SATO et al.
2005)
this study
this study
this study
this study
pHL2641
pHL2651
URA3
Amp,
URA3
Amp,
URA3
hermes TIR’s flanking kanMX6
with bacterial origin of
replication p15A
hermes TIR’s flanking nat with
bacterial origin of replication
p15A
*The genes URA3 and LEU2 came from S. cerevisiae.
this study
this study
Table S2. Oligonucleotides
Oligonucl
eotide
HL1364
HL1376
Sequence (5’ to 3’)
AGTAACAAAGGAACCTAGAGGG
HL1430
TTAATTAACCCGGGGATCCGCCG
AAGT
CGGTGATGCTGTCGCCG
CGGCGACAGCATCACCGACTTCG
GCGGATCCCCGGGTTAATTAA
CCGAGAATTAAAAACTGTTTCAG
GGGAATTCGAGCTCGTTTAAAC
GTTTAAACGAGCTCGAATTCCCCT
GAAACAGTTTTTAATTCTCGG
TTAACAATAACGGGCGGCCGCAG
AGAACAACAACAAGTGGC
AACAGGAAAGACCTCCTCGAGCA
TATGCAGAAAATGGACAATTTGG
AAGTG
CACATTACACTTCCCGGGTTATAT
ATCTAATTTACAAAAATTTTTG
GCCTCGACATCATCTGCCC
HL1431
CTCTAGCGGTGATCTTAACATC
HL1477
GTCGGCCTTTTTCGGCCGTCTCTG
HL1478
GTGGAATTCCACCGTGCAGGGCA
CTCAC
HL1503
GCATCGTACAGGTACATATGGAA
GTGCTTCATGTGGCAGG
ACCACAGTGCAGCTCCCGGGGGC
TGACTTCAGGTGCTACATT
ACCCGAGTGTCGATGAATCAATG
AA
HL1379
HL1367
HL1368
HL1392
HL1389
HL1357
HL1504
HL1893
REFERENCES
Use
5’ Primer to amplify Hermes
right in the fusion PCR product
3’ Primer to amplify Hermes
right in the fusion PCR product
5’ Primer to amplify KanMX-6
for the fusion PCR
3’ Primer to amplify pFA6KanMX-6 the fusion PCR
5’ Primer to amplify Hermes left
in the fusion PCR product
3’ Primer to amplify Hermes left
in the fusion PCR product
5’ Primer to PCR Hermes
Transposase
3’ Primer to PCR Hermes
Transposase
5’ Primer in pFA6-KanMX-6 for
inverse PCR
3’ Primer in Hermes Right for
inverse PCR
5’ Primer to amplify BglII /
EcoRI fragment containing
T317A
3’ Primer to amplify BglII /
EcoRI fragment containing
T317A
5’ Primer to amplify p15A origin
of replication
3’ Primer to amplify p15A origin
of replication
In hermes-left to PCR amplify
junction with genomic DNA
BAHLER, J., J. Q. WU, M. S. LONGTINE, N. G. SHAH, A. MCKENZIE et al., 1998
Heterologous modules for efficient and versatile PCR-based gene targeting in
Schizosaccharomyces pombe. Yeast 14: 943-951.
BOEKE, J. D., J. TRUEHEART, G. NATSOULIS and G. R. FINK, 1987 5-Fluoro-orotic acid as
a selective agent in yeast molecular genetics. Meth Enzymol 154: 164-175.
COTTAREL, G., D. BEACH and U. DEUSCHLE, 1993 Two new multi-purpose multicopy
Schizosaccharomyces pombe shuttle vectors, pSP1 and pSP2. Curr Genet 23:
547-548.
FORSBURG, S. L., 1993 Comparison of Schizosaccharomyces-Pombe Expression Systems.
Nucleic Acids Research 21: 2955-2956.
MAUNDRELL, K., 1993 Thiamine-repressible expression vectors pREP and pRIP for
fission yeast. Gene 123: 127-130.
ROSE, R. E., 1988 The Nucleotide-Sequence of Pacyc184. Nucleic Acids Research 16:
355-355.
SATO, M., S. DHUT and T. TODA, 2005 New drug-resistant cassettes for gene disruption
and epitope tagging in Schizosaccharomyces pombe. Yeast 22: 583-591.