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Transcript
SUPPLEMENTARY DATA (Gallardo et al.)
SUPPLEMENTARY METHODS
Growth media, drug treatment and yeast strains.
Yeast strains used in this study are listed in Table SI and plasmids in Table SII. The
yeast cells were grown either in synthetic growth media lacking the nutrients indicated or
in rich media.
Transformations were performed according to the protocol described by
(Schiestl & Gietz, 1989).
Yeast gene disruption cassettes were created by PCR
amplification of the loxP-KAN-loxP construct in plasmid pUG6 and pFA6a and primers
specific for the gene of interest (Longtine et al, 1998). Strains were then selected on the
appropriate selective media and specific disruption was confirmed by PCR analysis of
genomic DNA. For the LMB treatment, cells were exposed to 20 to 100ng/mL of HPLC
purified LMB (LC Labs) for 2h at 30°C.
Plasmid construction
To generate the deletion of the yKu binding stem in the TCL1 RNA (tlc1-
stemyku), two PCR reactions (PCR1 and PCR2) were performed using the appropriate
primers (see Table SIII) as follows: typically, each 25-µl reaction contained 10 mM TrisCl, pH 8.8, 25 mM KCl, 5 mM (NH4)2SO4, 2.5 mM MgCl2, 0.2 mM each dNTP, 1.52.5U Pwo polymerase (Roche), 20 pmol of each primer and 10 ng of template DNA
pADCEN36 (Dandjinou et al, 2004). Amplification was carried out with 1 cycle at 94˚C
for 2 min, 35 cycles at 94˚C for 15 s, 50˚C for 30 sec and 72˚C for 2 min, and one final
1
cycle at 72˚C for 5 min. PCR1: primers SPE-931TLC1FWD and TLC1 329:282 REV;
PCR2 : primers +1502TLC1ECOREV and TLC1 269:323 FWD
PCR1 and PCR2 products were gel-purified with QIAquick gel extraction kits
(Qiagen) and joined during the PCR3 reaction as follows: typically, each 25-µl reaction
contained 10 mM Tris-Cl, pH 8.8, 25 mM KCl, 5 mM (NH4)2SO4, 2.5 mM MgCl2, 0.2
mM each dNTP, 1.5-2.5 U Pwo polymerase (Roche), 20 pmol of each primer and 30 to
120 ng of equimolar amounts of purified PCR1 and PCR2 products. Joining was carried
out with 1 cycle at 94˚C for 2 min, 10 cycles at 94˚C for 15 s, 50˚C for 30 sec and 72˚C
for 2 min, followed by 25 amplification cycles at 94˚C for 15 s, 50˚C for 30 sec and 72˚C
for 2 min and one final cycle at 72˚C for 5 min. 20 pmoles of each external primer (SPE931TLC1FWD and +1502TLC1ECOREV) were added just before the amplification
cycles.
The mutated PCR3 fragments were digested with SpeI and EcoRI, gel purified as
previously and cloned into the SpeI-EcoRI site of pADCEN26 (Dandjinou et al, 2004).
Expected deletion in each construct was confirmed by sequencing. Plasmid pADCEN26
(negative control) was transformed into the yeast diploid strain CSHY76. The diploid
CSHY76 was sporulated, dissected and a tlc1 haploid spore was selected. This haploid
strain was transformed with pADCEN36 (WT TLC1) and pADCEN63 (TLC1 deleted
from the KU stem – tlc1-stemyku). All the mutants can complement the tlc1 rad52
deletion up to 100 generations.
Preparation of the TLC1 fluorescent probes
2
A set of 5 oligonucleotide probes was used to detect the endogenous TLC1 RNA. Each
probe is about 50nt long and contains five modified T (aminoallyl-T), distanced by about
10 nt each (Sequence of the probes are in the Table SIV). The probes were labeled with
FluoroLink Cy3 monofunctionnal reactive dye (Amersham Biosciences, Piscataway,NJ)
with at least two Cy3 incorporation per probes (usually 3 to 4) and used to detect the
endogenous TLC1 RNA. Briefly, 10μg of probes were lyophylized in a speed-vac and
resuspended in 35 μL of 0, 1M sodium carbonate pH 8,8. The Cy3 reactive pack was
resuspended in 30μL of DEPC-treated water. 15μL of resuspended Cy3 was mixed to the
35μL of probes, vortexed, and the reaction was incubated at room temperature for 16-24
hours in the dark. To remove the unincorporated Cy3, the reaction mix was purified using
a Sephadex G25 column. The absorbance in the pellet was estimated by measuring the
OD at 260nm for the probe and the Cy3 concentration at 552nm.
Production of oxalyticase
The oxalyticase was prepared using DH5α cells containing the pUV5 plasmid expressing
this lyticase under the control of the T7 promoter. A single colony was first inoculated in
2ml of LB medium in presence of 100μg/ml of ampicilin (Roche) and incubated at 37°C
overnight. To produce the enzyme, 1ml of the overnight culture was inoculated in 500ml
of LB medium in presence of ampicillin and brought to a OD of 0,5 at 600nm before
protein induction by Isopropyl--thiogalactoside (IPTG) (Sigma). The cells were induced
with a final concentration of 0,4mM IPTG during 5 hours at 37°C. After which, the cells
were harvested by centrifugation for 30 min at 3400rpm and washed once with 25mM
Tris pH 7,4. The cells were resuspended in 10ml of 25mM Tris pH 7,4 containing 2mM
3
of EDTA. An equal volume of 40% sucrose buffer (40% sucrose in 25mM Tris pH 7,4)
was added and the cells were mixed gently for 20 min. The sucrose buffer was removed
following centrifugation at 7500rpm for 10 min in a GSA rotor (Sorvall). All previous
steps were performed at room temperature to ensure better shocking for the cells. The
cells were submitted to a cold shock in 10ml of ice cold 0,5mM MgSO4 and mixed gently
on ice for 20 minutes. Finally, the cells were spun at 10 000rpm for 10 minutes in a SS34
rotor (Sorvall) and the supernatant was separated in 200μl aliquots. The oxalyticase
containing aliquots were lyophylized to a volume of 20μl and stored at -20°C until use.
4
SUPPLEMENTARY TABLES
TABLE SI: Yeast strains used in this study
________________________________________________________________________
Strain
genotype
source
W303
Mat a, ura3-1, leu2-3, his3-11, trp1-1, ade2-1
R. Jansen
BY4742
Mat α, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0
Open Biosystems
BY4743
Mat a/α his3/his3 leu2/leu2 lys2/LYS2
Open Biosystems
MET15/met15Δ0, ura3Δ0/ura3Δ0
CSHY76
tlc1
MATa/ ade2/ade2 ura3/ura3 leu2/leu2 his3/his3 trp1/trp1
tlc1::LEU2/TLC1 rad52::TRP1/RAD52.
C. Greider
Mata, ura3-1, leu2-3, his3-11, trp1-1, ade2-1,
R. Wellinger
TLC1::LEU2, RAD52::TRP1
tgs1
W303 ΔTGS1::KAN
this study
RAP1
Myc13
W303, RAP1-13xMyc KAN
this study
est1
BY4743, EST1/EST1::KAN
Open Biosystems
est2
BY4743, EST2/EST2::KAN
R. Wellinger
est3
BY4743, EST3/EST3::KAN
Open Biosystems
yku70
W303 YKU70::KAN
R. Wellinger
Y464
his, leu, trp, CRM1::KAN (pDC-crm1T539C-LEU2)
M. Rosbash
Y464yku70
Y464 YKU70::HIS3
this study
xpo1-1
W303 XPO1::LEU2 (pKW456-xpo1-1-HIS)
C. Guthrie
xpo1-1
yku70
xpo1-1 YKU70::TRP1
this study
Nsp1ts10A
Mat? Ade2-1, can1-100, leu2-3, lys1-1, ura3-52,
5
P-E Gleizes
NSP1:URA3, nsp1Ts10A-URA3
Nsp1ts10A
yku70
Nsp1ts 10A YKU70::KAN
this study
mex67-5
Mat a, ade2, his3, leu2, trp1, ura3, MEX67::HIS
C. Cole
(pun100-leu2-mex67-5)
mex67-5
yku70
mex67-5 YKU70::TRP1
this study
MS739
Mat α, leu2-3, ura3-52, ade2-101, kar1-1
kap123
BY4742 KAP123::KAN
Open Biosystems
kap120
BY4742 KAP120::KAN
Open Biosystems
msn5
BY4742 MSN5::KAN
Open Biosystems
sxm1
BY4742 SXM1::KAN
Open Biosystems
kap122
BY4742 KAP122::KAN
Open Biosystems
kap114
BY4742 KAP114::KAN
Open Biosystems
tel1
BY4742 TEL1::KAN
Open Biosystems
mre11
BY4742 MRE11::KAN
Open Biosystems
xrs2
BY4742 XRS2::KAN
Open Biosystems
PSY1201
ura3-52 trp1-63 leu2-1 pse1-1
P. Silver
PSY1199
nmd5::HIS3 in PSY1183
P. Silver
Y1171
Mat ade2-1 his3-11,15 ura3-52 leu2-3,112 mtr10::HIS3
E. Hurt
L5850
leu2-3,112 ura3-1 ade2-1 his3-11,15 trp1 srp1-31
G. Fink
P. Silver
________________________________________________________________________
6
TABLE SII: Plasmids used in this study
Plasmids
Description
Source
pADCEN36
TLC1, CEN, URA3
pADCEN63
tlc1ΔstemIIc(Stem Ku) in
pADCEN36
pNop1-GFP
NOP1-GFP in pRS315
URA3
S. Abou Elela.
pRL134
LOC1-6xMyc cloned in
pESC-URA3
R. Long
pVL399
Empty 2µ vector, pADHTerm-ADH, LEU2.
V. Lundbald.
pVL784
EST1 in pVL399
V. Lundbald.
pVL999
EST2 in pVL399
V. Lundbald.
7
R.J Wellinger
this study
TABLE SIII: Sequences of the primers used for cloning
Primer
Sequence
SPE-931TLC1FWD
5’-GGGTACACTAGTAGCCTTTCTAGAGGTTCC-3’
TLC1 329:282 REV
TLC1 269:323 FWD
5’-CGGTTTGATAAAAAACCACAAATTGCGCACACACAAGC-3’
5’-GGTTTTTTATCAAACCGTAAATTCTTAAACACTGCTATTGC-3’
+1502TLC1ECOREV
5’-AACAGAATTCGGGAAGGTAAATACCACC-3’
________________________________________________________________________
Underlined nucleotides indicate either Spe1 and EcoR1 restriction sites.
8
TABLE SIV: Sequences of the oligonucleotide probes against TLC1
Probe
Sequence
TLC1-1
5’-t*gcgcacacacaagcat*ctacactgacaccagcat*actcgaaattctt*tg-3’
TLC1-2
5’-ct*aataaacaatt*agctgtaacatt*tgtgtgtggggt*gtggtgatggt*aggc-3’
TLC1-3
5’-tt*ccagagttaacgat*aagatagacat*aaagtgacagcgct*tagcaccgt*c-3’
TLC1-4
5’-ttacgt*tcttgatctt*gtgtcattgtt*cagttactgat*cgcccgcaaacct*-3’
TLC1-5
5’-tgcat*cgaaggcat*taggagaagt*agctgtgaat*acaacaccaagat*tca-3’
________________________________________________________________________
* aminoallyl modified-T
9
SUPPLEMENTARY FIGURES LEGENDS
Figure S1: The TLC1 RNA is exported from the nucleus independently from the
ribosomal RNA and mRNA export pathways. The nuclear export mutants nsp1ts10a,
specific for rRNA export (Gleizes et al, 2001) and mex67-5, specific for mRNA export
(Segref et al, 1997), were used to define the nuclear export pathway of TLC1 RNA. A)
FISH against the TLC1 RNA in a nsp1ts10a yku70 strain was performed at T= 0h or 2hrs
of shift at restrictive temperature (37ºC). TLC1 RNA keeps its cytoplasmic accumulation
even after 2 hours at 37°C. Scale bar = 1µm. B) FISH against the TLC1 RNA in a
mex67-5 yku70 strain was performed at T= 0h or 2hrs of shift at restrictive temperature
(37ºC). TLC1 RNA keeps its cytoplasmic accumulation even after 3hrs at restrictive
temperature (data not shown). Scale bar = 1µm.
Figure S2:
Control for nucleus leakage during the heterokaryon shuttling assay.
Heterokaryons were created by mating a Mata strain deleted of the TLC1 gene (tlc1) with
a Mat strain carrying the kar1-1 allele, a wild-type TLC1 gene and a galactose-inducible
Loc1p-myc (kar1-1+pRL134). After transient expression of Loc1p-myc, mating and
immunofluorescence, the distribution of Loc1p-myc in the nuclei of the heterokaryons
was determined. In all heterokaryons observed, Loc1p-myc was detected in only one
nucleus. Scale bar = 1µm.
10
SUPPLEMENTARY REFERENCES
Dandjinou AT, Levesque N, Larose S, Lucier J-F, Elela SA, Wellinger RJ (2004) A
Phylogenetically Based Secondary Structure for the Yeast Telomerase RNA. Current
Biology 14(13): 1148-1158
Gleizes P-E, Noaillac-Depeyre J, Leger-Silvestre I, Teulieres F, Dauxois J-Y, Pommet D,
Azum-Gelade M-C, Gas N (2001) Ultrastructural localization of rRNA shows defective
nuclear export of preribosomes in mutants of the Nup82p complex. J Cell Science
155(6): 923-936
Longtine MS, McKenzie A, 3rd, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen
P, Pringle JR (1998) Additional modules for versatile and economical PCR-based gene
deletion and modification in Saccharomyces cerevisiae. Yeast (Chichester, England)
14(10): 953-961
Schiestl RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using
single stranded nucleic acids as a carrier. Curr Genet 16(5-6): 339-346
Segref A, Sharma K, Doye V, Hellwig A, Huber J, Luhrmann R, Hurt E (1997) Mex67p,
a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores.
EMBO J 16(11): 3256-3271
11