A study on premature segregation of unreplicated chromosomes 2

Chapter 2 Materials and Methods
2.1 Materials
Table 1. Reagents used in this study.

Description
Source
Laboratory Chemicals
BDH Laboratory Supplies (UK)
Sigma Chemical Company (USA)

Media Components
Difco Laboratories (USA)

DNA modifying and restriction enzymes
New England Biolabs (USA)
Amersham (UK)

Glusulase
DuPont NEN Research Products (USA)

Lyticase
Sigma-Aldrich (USA)

Alpha factor
Biotechnology Centre (Singapore)

Nocodazole
Sigma-Aldrich (USA)

MG132 proteasome inhibitor

Sigma-Aldrich (USA)




Table 2. Antibodies used for immunofluorescence and protein analyses.

Antibodies
Source
Mouse monoclonal anti-HA
Roche Diagnostics

Mouse monoclonal anti-cmyc
Santa Cruz Biotechnology Inc

Rabbit polyclonal anti-HA
Santa Cruz Biotechnology Inc

Rabbit polyclonal anti-cmyc
Santa Cruz Biotechnology Inc

Rabbit polyclonal anti-G6PDH
Sigma-Aldrich

Rat monoclonal anti-tubulin YOL 1\34
Serotec

Rabbit polyclonal anti-Clb2
Kim Nasmyth (Institute of Molecular
Pathology, Vienna)

Rabbit polyclonal anti-Cdc5
Santa Cruz Biotechnology Inc

Mouse monoclonal anti-Cdc6
Abcam

Rabbit polyclonal anti-Cdc28
Kim Nasmyth (Institute of Molecular
Pathology, Vienna)



Table 3. List of S. cerevisiae strains used in this study.

Strain
Genotype
Source
US1363
Wild type bar- (unmarked). MATa, ade2-1, trp1-1, can1-
100, leu2-3, 112, his3-11, 15, ura3, GAL. Psi+, bar1:hisG.
It is unmarked bar- version of US841.

This
study
US1688
MATa, his3, cdc34-1, leu2, trp1, bar1D:hisG-URA3-hisG.
cdc34-1 bar- (Ura-)

This

study
US3335
Mat a, ade2-1 trp1-1 can1-100, leu2-3,112, his3-11, 15
GAL, psi +, SCC1 myc18:: TRP1
Halo test shown this strain is bar+

This
study
US3538
PDS1-HA3 in WT US 1363 Mat a, PDS1-HA3-URA3,
trp1,his3,leu2

This
study
US4122
Wild type with endogenously tagged Cin8
MATa, CIN8-HA
3
-HIS3 ade2-1, trp1-1, can1-100, leu2-3,
ura3, bar1 GAL. Psi+.

This
study
US4262
MATa, ade2-1, can1-100, leu2-3,112, his3-11,15, GAL,
cdc6::hisG, trp1-1, ura3::URA3 GAL-ubiR-CDC6.
SCC1myc18:TRP, cdc23-1

This
study

US4275
MATa, ade2-1, can1-100, leu2-3,112, his3-11,15, GAL,
cdc6::hisG, trp1-1, ura3::URA3 GAL-ubiR-CDC6.

Simonett
a Piatti
US4344
MATa, ade2-1, can1-100, leu2-3,112, his3-11,15, GAL,
cdc6::hisG, trp1-1, ura3::URA3 GAL-ubiR-CDC6.
SCC1myc18:TRP

This
study
US4364
MATa, ade2-1, can1-100, leu2-3,112, his3-11,15, GAL,
cdc6::hisG, trp1-1, ura3::URA3 GAL-ubiR-CDC6.
PDS1HA:URA3

This
study
US4366
cdc6∆GalCDC6 CIN8-HA
Mat a, GalCDC6:URA3, CIN8-HA:HIS3 bar+ (halo test)

This
study

US4423
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6

hisGURA3hisG looped out on 5-FOA, Ura-

This
study
US4677
Wild type bar- (unmarked). MATa, ade2-1, trp1-1, can1-
100, leu2-3, 112, his3-11, 15, ura3, GAL. Psi+, bar1:hisG.
It is unmarked bar- version of US841.
KIP1-HA transplaced into US 1363 WT

This
study
US5239
cdc34-1 carrying endogenously-tagged CIN8-HA (HIS-
marked) MATa trp- leu- ura- HIS+ bar- ts at 36˚C.

This
study
US5582
cdc7 deletion (Gal CDC7-3HA integrated into CDC7 locus
with Kanamycin marker)
One step PCR using pUS2112 as template
Mat a, ade2-1 trp1-1 can1-100, leu2-3,112, his3-11, 15
GAL, psi +, SCC1 myc18:: TRP1, checked by southern.
Halo test shown this strain is bar+

This
study
US5585
cdc45 deletion (Gal CDC45-3HA integrated into CDC45

locus with Kanamycin marker)
One step PCR using pUS2112 as template
Mat a, ade2-1 trp1-1 can1-100, leu2-3,112, his3-11, 15
GAL, psi +, SCC1 myc18:: TRP1, checked by southern.
Halo test shown this strain is bar+

This
study
US5677
cdc34-1 HA3-CDH1.
MAT a, bar-, cdc34-1 HA3-CDH1-TRP1, leu2, ura3, his3.

This
study
US5722
cdc34-1 sic1∆:URA3 HA3-CDH1
MAT a bar-, cdc34-1 sic1∆:URA3, HA3-CDH1-TRP1, leu2,
his3.

This
study
US5690
cdc34-1 cdc6∆ MET-CDC6 sic1∆
Mat a, cdc34-1 cdc6∆:HIS3 MET-CDC6:TRP1
sic1∆:URA3

This
study
US6005
cdc34-1 mutant with 3X SPC42-GFP-TRP1.

MATa, cdc34-1, 3X SPC42-GFP-TRP1, ura3 leu2, his3.

This
study
US6342
cdc34-1 KIP1-HA3
Mat a, bar-, cdc34-1, KIP1-HA3-HIS3, leu2, trp1, ura3. ts
at 37 degree.

This
study
US6343
cdc6∆ KIP1-HA3
MATa, ade2-1, can1-100, leu2-3,112, GAL, cdc6::hisG,
trp1-1, ura3::URA3 GAL-ubiR-CDC6, KIP1-HA3-HIS3.

This
study

US6527
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6
hisGURA3hisG looped out on 5-FOA, so it is Ura-, SCC1-
cmyc12-KAN

This
study
US6537
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6

hisGURA3hisG looped out on 5-FOA, SCC1-cmyc12-KAN,
pGAL-HA3-PDS1 CEN URA3.

This
study
US6936
Mat a, cdc34-1 cdc6∆:HIS3 MET-CDC6:TRP1
cdh1∆:KAN, SPC72-GFP:LEU2, KIP1HA:Hygromycin

This
study
US6938
Mat a, cdc34-1 cdc6∆:HIS3 MET-CDC6:TRP1
cdh1∆:KAN, SPC72-GFP:LEU2, ASE1HA:Hygromycin

This
study
US7007
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6
hisGURA3hisG looped out on 5-FOA
so it is Ura- Dicentric plasmid URA3 +

This
study
US7008
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6
hisGURA3hisG looped out on 5-FOA so it is Ura-, Ycplac
33 (5.6 kb)- a URA3 selectable CEN4 plasmid (#1955).

This
study
US7009
Wild type bar- (unmarked).
MATa, ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15,
ura3, GAL. Psi+, bar1:hisG. It is unmarked bar- version of
US841. ASE1-HA in pRS303 at its own locus

This
study
US7010
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6
hisGURA3hisG looped out on 5-FOA, so it is Ura-
ASE1-HA in pRS303 at its own locus

This
study
US7011
cdc34-1 bar- (Ura-)
ASE1-HA in pRS303 at its own locus, 3' end tagged with
HA; digest with EcoRV for integration at the endogenous
locus and select for HIS

This
study
US7012
cdc34-1 cdc6∆ MET-CDC6
Mat a, cdc34-1 cdc6∆:HIS3 MET-CDC6:TRP1 SPC72-

GFP:LEU2

This
study
US7013
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6
hisGURA3hisG looped out on 5-FOA GALndSIC1:URA3

This
study
US7014
cdc34-1 cdc6∆ MET-CDC6
This
Mat a, cdc34-1 cdc6∆:HIS3 MET-CDC6:TRP1

study
US7015
Mat a, cdc34-1 CDH1∆:KanMX

This
study
US7016
cdc34-1 cdc6∆ MET-CDC6 CDH1∆:KanMX
Mat a, cdc34-1 cdc6∆:HIS3 MET-CDC6:TRP1 SPC72-
GFP:LEU2

This
study
US7018

cdc34-1 bar- (Ura-)
HA-tagged CIN8 non-degradable version in pUS20 (Ura3
Integrative vector)

This
study
US7019
cdc34-1 bar- (Ura-)
ASE1 non-degradable version

This
study
US7020
cdc34-1 CDH1∆:KanMX
HA-tagged CIN8 non-degradable version in pUS20 (Ura3
Integrative vector)


This
study
US7021
cdc34-1 CDH1∆:KanMX
ASE1 non-degradable version

This
study
US7022
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6
hisGURA3hisG looped out on 5-FOA, so it is Ura-

GAL-HA-HCT1 on Kan/CEN plasmid, CIN8HA3: HIS

This
study
US7023
MATa, cdc34-1, 3X SPC42-GFP-TRP1, ura3 leu2, his3.
Non-degradable version of CIN8 under GAL1-promoter in
URA3, CEN plasmid (pUS18)

This
study
US7024
cdc34-1 cdc6∆ MET-CDC6 CDH1∆:KanMX
Non-degradable version of Cin8 under GAL1-promoter in
URA3, CEN plasmid (pUS18)

This
study
US7025
MATa, cdc34-1, 3X SPC42-GFP-TRP1, ura3 leu2, his3.
GAL-KIP1 nd-HA no degradable version in 2 micron URA
plasmid.

This
study
US7026
cdc34-1 cdc6∆ MET-CDC6 CDH1∆:KanMX
GAL-KIP1 nd-HA no degradable version in 2 micron URA
plasmid.
This

study
US7027
MATa, cdc34-1, 3X SPC42-GFP-TRP1, ura3 leu2, his3.
GAL-ASE1-cmyc on HIS3, 2µ plasmid.
This
study
US7028
cdc34-1 cdc6∆ MET-CDC6 CDH1∆:KanMX
GAL-ASE1-cmyc on HIS3

This
study
US7029
cdc34-1 cdc6∆ MET-CDC6
Mat a, cdc34-1 cdc6∆:HIS3 MET-CDC6:TRP1
This
study
SIC1 (p40) c-myc tag under its own promoter in pUS15
(LEU2)

US7031
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, MET-CDC6
trp1-1::TRP1 hisGURA3hisG looped out on 5-FOA
so it is Ura-, cin8∆:KanmX

This
study
US7032
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,

ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6
hisGURA3hisG looped out on 5-FOA
ase1∆:KanmX, so it is Ura-

This
study
US7033
Mat a, cdc34-1 cdc6∆:HIS3 MET-CDC6:TRP1
cdh1∆:KAN, CIN8-HA on URA3, CEN plasmid.

This
study
US7034
cdc34-1 cdc6∆ MET-CDC6 sic1∆
Mat a, cdc34-1 cdc6∆:HIS3 MET-CDC6:TRP1
sic1∆:URA3 erg6∆::KANMX plasmid.

This
study
US7036
cdc34-1 bar- (Ura-)
GAL-ASE1-cmyc on HIS3, 2u plasmid

This
study
US7037
cdc34-1 bar- (Ura-)
Gal-Cin8-myc on URA3 CEN plasmid.

This

study
US7038
Mat a, cdc34-1cdh1∆:KAN, GAL-ASE1-cmyc on HIS3, 2u
plasmid

This
study
US7039
Mat a, cdc34-1 cdh1∆:KAN,
Gal-CIN8-myc on URA3 CEN plasmid.

This
study
US7040
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG,
trp1-1::TRP1 MET-CDC6
hisGURA3hisG looped out on 5-FOA
so it is Ura-, GAL-ASE1-cmyc on HIS3, 2u plasmid

This
study
US7041
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6
hisGURA3hisG looped out on 5-FOA
so it is Ura-, Gal-CIN8-myc on URA3 CEN plasmid.

This
study

US7042
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6
hisGURA3hisG looped out on 5-FOA
so it is Ura-, cdh1∆:KAN
GAL-ASE1-cmyc on HIS3, 2u plasmid
This
study
US7043
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG,
This
study
trp1-1::TRP1 MET-CDC6 hisGURA3hisG looped out on 5-
FOA, so it is Ura-, cdh1∆:KAN
Gal-CIN8-myc on URA3 CEN plasmid.

US7044
cdc34-1 bar- (Ura-)
GAL-cmyc6-CDC5 on YIPlac 211 (3.79 kb) - a URA3
selectable integrative vector
This
study
US7045
cdc34-1 bar- (Ura-)
GAL-cmyc6-CDC5 (N209A) on URA3, integrative plasmid.

This
study
US7046

Mat a, cdc34-1cdh1∆:KAN,
GAL-cmyc6-CDC5 on YIPlac 211 (3.79 kb) - a URA3
selectable integrative vector.

This
study
US7047
Mat a, cdc34-1cdh1∆:KAN,
GAL-cmyc6-CDC5 (N209A) on URA3, integrative plasmid.

This
study
US7048
Mat a, cdc34-1 cdc6∆:HIS3 MET-CDC6:TRP1
lys1∆:KANMX.

This
study
US7049
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6
hisGURA3hisG looped out on 5-FOA
so it is Ura-, YIPlac211-GAL-cmyc6-CDC5 -URA3

This
study
US7050
MAT a ade2-1, cant1-100, leu2-3,112, his3-11,15,
ura3,GAL,cdc6::hisGURA3hisG, trp1-1::TRP1 MET-CDC6
hisGURA3hisG looped out on 5-FOA so it is Ura-,

cdh1∆:KAN YIPlac211-GAL-cmyc6-CDC5 -URA3

This
study





Name
Description
Source
pUS18
Ycplac 33 (5.6 kb)- a URA3 selectable CEN4 plasmid
(#1955).

US Lab
pUS639
CIN8-HA on URA3, CEN plasmid.

US Lab
pUS654
SIC1 (p40) c-myc tag under its own promoter in pUS15
(LEU2, 2µ)

US Lab
pUS785
SIC1 (p40) c-myc triple tag in pUS20 intergrative vector
(URA3, CEN).

US Lab
pUS875
GAL-ASE1-cmyc on HIS3, 2µ plasmid.

US Lab
pUS2700
Dicentric minichromosome pT431 (from Tomo tanaka's
lab) centromere 1 is normal and centromere 2 is under the
control of Gal-promoter, Tet-operater sequence is
inserted inbetween the two centromere. ARS is
surrounded by recombinase site, upon expression of
recombinase (under Met promoter), the ARS will be
looped out. This plasmid is marked by Ura and the
selection is by ampicillin-resistence.

Tomo
Tanaka
pUS2170
ASE1-HA in pRS303 at its own locus
3' end tagged with HA; digest with EcoRV for
integration at the endogenous locus and select for HIS

US Lab
pUS2199
HA-tagged CIN8 non-degradable version in pUS20 (Ura3
Integrative vector)

US Lab
pUS2201
ASE1 non-degradable version

US Lab
pUS1383
GAL-HA-HCT1 on Kan/CEN plasmid

US Lab
pUS2433
YIPlac211-GAL-cmyc6-CDC5 -URA3

US Lab
pUS2216
Non-degradable version of CIN8 under GAL1-promoter
in URA3, CEN plasmid (pUS18)

US Lab
Table 4. List of plasmids used in this study.

pUS2259
Gal-CIN8-myc on URA3 CEN plasmid.

US Lab
pUS2560
erg6∆::KANMX plasmid.

US Lab
pUS2581
GAL-KIP1 nd-HA no degradable version in 2 micron
URA plasmid.

US Lab

pUS2433
GAL-cmyc
6
-CDC5 on YIPlac 211 (3.79 kb) - a URA3
selectable integrative vector.

US Lab
pUS2718
GAL-cmyc
6
-CDC5 (N209A) on URA3, integrative
plasmid.

US Lab





Primer
Description
Source
OUS2814
5’GGATTTGATGATACCAGTAT3’
to be used with OUS2815 to check 3' tagging of Ase1 by
plasmid pUS2170

US Lab
OUS2815
5’ATTGATGAAGAAGCTGATGA3’

to be used OUS2814 to check 3' tagging of Ase1 by plasmid
pUS2170

US Lab
OUS1545
5’GGA AAA ATG AGC AAG TTT CGA AAT TGA ATG
GAT TCT CCT TTA CAG ATA TTC GGA TCC CCG
GGT TAA TTA A3’
Sequences not underlined belong to top strand of Ase1, just
before STOP codon. The underlined sequences correspond
to F2 sequences from Yeast 14: 953-961 (1998). To be used
with OUS 1546 to generate a 2325 bp PCR product
(template pUS 2098, pFA6a-13Myc-KANMX6) for one step
tagging of ASE1-cmyc
13
at the endogenous locus.

US Lab
OUS1546
5’AAA ATA AAT AAG AGA TCA GAG GGT TAA ATG
AAT GCG CTT TTA AGA AAT GAA TTC GAG CTC
GTT TAA AC3’
Sequences not underlined belong to bottom strand of Ase1,
just after but not including STOP codon. The underlined
sequences correspond to R1 sequences from Yeast 14: 953-
961 (1998). To be used with OUS 1545 to generate a 2325
bp PCR product (template pUS 2098, pFA6a-13Myc-
KANMX6) for one step tagging of ASE1-cmyc
13
at the

endogenous locus.

US Lab
OUS1490
5' GCG GGA TCC GAA TGG ATT CTC CTT TAC AG3’
Forward oligo at 3' end of Ase1 gene for making a
disruption

US Lab
OUS1491
5' GCG GAA TTC CTG GTA TCT AAG GGA ACG G3’
REVERSE oligo at 3' end of Ase1 gene for making a
disruption

US Lab
Table 5. List of the main oligonucleotides used in this study.

OUS1165
5’CCAGTGAAAATGTGGACAATGAGGGCTCGAGAA
AAATGTTAAAGATTGAATTTCATCTCCGGTTCTGCT
GCTAGT 3'
Forward oligo to tag the endogenous copy of cin8 with HA/
myc by one step PCR 50 bp before the stop codon
UNDERLINED SEQUENCES ARE THOSE OF THE HA
PLASMID

US Lab
OUS1166
5’TACTTTGTTTTTATTAACCACTAGTTTGAATAT3’
ATATTCGACTGAAAGGCAATATCAA

CGTCGACCTCGAGGCCAGAAGACTAAGAGG 3'
UNDERLINED SEQUENCES ARE THOSE OF THE HA
PLASMID

US Lab
OUS1183
Forward oligo to tag the endogenous kip1 by one step PCR:
50 BP JUST BEFORE THE STOP CODON
5'AGAAGAAACTGAAAATAATGACATACTGCAAAA
TAAAAAACTTCATCAATTTCATCTCCGGTTCTGCTG
CTAGT 3'
UNDERLINED SEQUENCES ARE THOSE OF THE HA
PLASMID

US Lab
OUS1184
Reverse oligo to tag the endogenous kip1 by one step PCR :
50 BP JUST AFTER THE STOP CODON
5'ACACTCAATGCATATAGTGATACAAATATTTTAC
AATGGCTATATCCCCCGTCGACCTCGAGGCCAGAA
GACTAAGAGG 3'
UNDERLINED SEQUENCES ARE THOSE OF THE HA
PLASMID

US Lab
OUS2107
5’GTCACCGGAAGGAGAGCCCTGT3’
PCR with ous2107 and ous2108 to obtain a 2.8kb fragment
in wild type CDH1 strain. Designed to test for cdh1
deletion. Primers anneal to 597bp upstream of START and

420bp downstream of STOP.

US Lab
OUS2108
5’TCGCCCATCCTGACGCCTGTAA3’
PCR with ous2107 and ous2108 to obtain a 2.8kb fragment
in wild type CDH1 strain. Designed to test for cdh1
deletion. Primers anneal to 597bp upstream of START and
420bp downstream of STOP.

US Lab
OUS1540
5’TAG AGG TAT ATT CTC TGG AAG AAC AAT TTT
GG AAT ATT CAG AAC AGA ATT CGA GCT CGT
TTA AAC3’
Underlined sequences are identical to F4 primer sequences
in Table 1 (yeast 14: 953-961, 1998). The other sequences
are identical to cdc6 sequences (-80 to -40 bp).
To be used with OUS OUS 1542 and template pUS 2112
(pFA4a- KanMX6- PGAL1-3HA) to generate a ~ 2kb PCR
product for one step tagging of GAL-HA
3
-CDC6 in WT
strain.

US Lab
OUS1542
5’ATT TCT TCT GAT ACG CTT AGT TGG AGT TAT
TGG TAT AGC TGA CAT GCA CTG AGC AGC GTA
ATC TG3’

Underlined sequences are identical to R3 primer sequences
in Table 1 (yeast 14: 953-961, 1998). The other sequences
are identical to cdc6 sequences (40 bp downstream of
START, including the START codon).
To be used with OUS OUS 1540 and template pUS 2112
(pFA4a- KanMX6- PGAL1-3HA) to generate a ~ 2kb PCR
product for one step tagging of GAL-HA
3
-CDC6 in WT
strain.

US Lab
OUS2295
Forward oligo to confirm cdc6 disruption
5' GGCTTGCGATTTGTTGTGTTTTGA 3'
200bp before start, using with ous 2296 to confirm cdc6
disruption.

US Lab
OUS2296
Reverse oligo to confirm cdc6 disruption
5' ATTACGTTTTATTGTGTCTT3'
200bp after stop, using with ous 2295 to confirm cdc6
disruption.

US Lab
OUS1972
Forward oligo for confirm sic1 disruption.
5' TCCAGAGGGACTAGGTA 3'
used with ous 1973

US Lab
OUS1973
Reverse oligo for confirm sic1 disruption.
5' ATATAATCGTTCCAGAAACT 3'
used with ous 1972

US Lab


2.2 Methods
2.2.1 Escherichia coli strains and culture conditions
For cloning purposes and plasmid DNA amplification, E. coli DH5α or XL1 Blue
cells (Stratagene) were used as bacterial hosts. The E. coli cells were cultured in
either 2X TY liquid medium (1.6% bacto- tryptone, 1% bacto- yeast extract, 0.5%
NaCl) or on 2X TY plates containing 2% bacto agar at 37
o
C. 100µg/ml Ampicillin
(Sigma) was added to the medium or plates for selection of cells carrying recombinant
plasmids.

2.2.2 Yeast strains and culture conditions
All Saccharomyces cerevisiae strains were congenic to the background W303 and
listed in 3. Mutant strains from other genetic background were made isogenic by
backcrossing three times to the parental wild-type strain (W303 background).
Yeast cells were grown in YEP medium (1.1% yeast extract, 2.2% peptone and
50mg/l adenine) supplemented with 2% raffinose/ 0.5% glucose, 2% glucose or 2%
galactose/2% raffinose depending on the objectives of the experiments. Since
deletion of CDC6 is lethal, cdc6Δ cells were kept alive with a methionine-repressible
MET-CDC6 or galactose-inducible GAL-CDC6 construct. cdc6Δ MET-CDC6 cells

were grown in methionine free synthetic media (0.67% yeast nitrogen base from
Difco, amino acid drop out mix) with 2% raffinose/ 0.5% glucose, 2% glucose or 2%
galactose/2% raffinose. Whereas, methionine was added to media (at a final
concentration of 200 µg/ml) in experiments requiring repression of the MET3
promoter (e.g. shut of CDC6 transcription). Yeast cells were grown in a water bath at
24
o
C with constant shaking for good aeration at 200rpm. Temperature sensitive
mutants were cultured at the permissive temperature of 24
o
C, but filtered and
transferred to various restrictive temperatures (33˚C or 35˚C or 36˚C) depending on
strains. Cells treated with proteasome inhibitor MG132 carried an ERG6 disruption to
facilitate permeability of the inhibitor.
For sporulation, diploid strains were patched onto sporulation plates containing 0.22%
yeast extract, 2% potassium acetate and 2% glucose or 2% galactose and 2% raffinose.
Recombinant strains were obtained by tetrad analysis.

2.2.3 Cell cycle synchronization
For synchronization at G1, S. cerevisiae cells were grown to exponential phase at
24˚C, diluted to OD
600
of 0.2 and α-factor was added at a final concentration of 5
µg/ml (for BAR1) cells and 1 µg/ml (for bar1Δ) cells.
For synchronization at the pre-nuclear division stage, nocodazole (US Biologicals)
was added at a final concentration of 15 µg/ml. The cells were incubated with the
drug for about 3-3.5 hours till more than 90% of the cells had arrested with an
undivided nucleus and no detectable spindle.

2.2.4 Yeast transformation

Yeast transformation was performed by the lithium acetate method. Yeast cultures
were grown overnight. Cells were collected and spun down at 3500 rpm for 2 minutes.
Subsequently, the pellets were washed once with Li-TE buffer (0.1M lithium acetate,
10mM Tris-HCl pH 7.5, 1mM EDTA) and then resuspended in 2ml of Li-TE buffer.
The cells were incubated on a roller at room temperature for 1 hr. For each
transformation, 10µl of 1mg/ml salmon sperm DNA, linearized DNA of interest or
plasmid DNA, 100µl of cells in Li-TE buffer and 140µl of 70% PEG 6000 were
mixed and incubated at 24
o
C for 3 hrs. This was followed by a heat shock (15mins) at
42
o
C. Finally, cells were spun down at low speed (1.2K for 1 min), resuspended in
50µl sterile H
2
0 and spread onto the appropriate selective plates.

2.2.5 Isolation of plasmid DNA from yeast
S. cerevisiae cultures were grown overnight in 5 ml YEPD medium and cells were
harvested by centrifugation. The cells were then resuspended in 0.3 ml of lysis buffer
(2% Triton X-100, 1% SDS, 0.1 M NaCl, 10 mM Tris pH8.0 and 1 mM EDTA); 0.3
ml of phenol:chloroform (1:1) and 0.2 ml acid-washed glass beads were subsequently
added to the cell suspension before vortexing vigorously for 6 min on a IKA Vibrax
shaker. After centrifugation, the supernatant was transferred to a fresh tube and
extracted twice with phenol:chloroform:isoamylalcohol (25:24:1) before precipitating
the plasmid with ethanol. The plasmid DNA was pelleted by centrifugation for 10
min, washed with 70% ethanol, dried and dissolved in 40 µl TE. For amplification of
the DNA, 5 µl of the plasmid DNA was used to transform into 20 µl of E. coli XL1-
Blue cells.

2.2.6 Yeast chromosomal DNA extraction
Yeast cells were harvested from 5 ml cultures, washed with ddH
2
0 and resuspended in
0.2 ml of spheroplasting mix containing 80% SCE (1M Sorbitol, 0.1 M sodium
citrate, 0.06 M EDTA, pH 7.0), 10% lyticase and 10% β-mercaptoethanol. The
mixture was incubated at 37
o
C with occasional shaking for 1 hr or more when
required (cells were checked under microscope for complete digestion of cell). After
verifying that spheroplasting was complete, 0.2ml SDS solution (2% SDS, 0.1M Tris-
HCl, 0.05M EDTA) was added to the mixture and the mixture was heated at 65
o
C for
5mins to allow lysis. This was followed by addition of 0.2ml 5M KOAc and the
mixture incubated on ice for 20mins. The mixture was centrifuged at 13000rpm for
5mins. The supernatant was transferred to a fresh tube containing 0.2ml 5M NH
4
OAc
and 1ml isopropanol and the DNA precipitated in dry ice for 5mins. The tubes were
mixed by inverting and chromosomal DNA was pelleted by centrifugation for a few
seconds at low speed centrifugation, washed once in 70% ethanol, and dissolved in
50µl TE or ddH
2
0 after removing all traces of isopropanol. To confirm proper
integration by PCR methods, 1 µl of the chromosomal DNA was diluted in 5 µl with
distilled water.

2.2.7 Southern blot analysis
Chromosomal DNA was digested at 37

o
C (> 12 hrs) in a total volume of 100µl. The
restriction enzyme digest was set up as follows: 10µl DNA, 10µl 10X restriction
buffer, 1µl EcoR1, 1µl 10mg/ml RNAse and 78µl sterile water. The digested DNA
was precipitated by addition of 10µl 3M sodium acetate (pH 5.2), 1µl 0.5M EDTA
(pH 8) and 200µl cold ethanol in dry ice for a minimum of 10mins. The samples were
centrifuged at 13000rpm for 10mins. The supernatant was discarded and the pellets
rinsed in 80% ethanol. The DNA pellet was dried and dissolved in 15µl 1X gel
loading buffer (6X gel loading buffer: 0.25% bromophenol blue, 0.25% xylene cyanol
FF, 15% Ficoll [Type 400; Pharmacia] in water). The restriction enzyme digested
yeast chromosomal DNA samples were size fractionated on a 1% agarose gel in 1X
TBE (Tris-borate/ EDTA; 0.09M Tris-borate, 0.002M EDTA) running buffer. A few
µl of ethidium bromide (10mg/ml) was added to the gel to stain the DNA fragments.
The DNA fragments were imaged using a UVIdoc gel documentation system (Uvitec,
Cambridge). Subsequently, the gel was denatured with 0.5N NaOH, 1.5M NaCl for 1
hr and neutralized with 1M TrisHCl (pH 7.4), 1.5M NaCl for 1 hr. The DNA was
then transferred onto nylon membrane (Amersham Hybond N, GE Healthcare) using
20X SSC as the transfer buffer as described by Sambrook et al 1989 (Sambrook et al.
1989). After the 18 h transfer, the membrane was baked for 2 h at 80˚C and this was
followed by a UV cross-linking step in order to immobilize DNA onto the membrane.
The membrane was then prehybridized in a Hybaid rotary oven in a 20 ml pre-
hybridization solution for a minimum of 1 hr and hybridized overnight at 65
o
C with
purified radioactive α-
32
P dATP labeled probe (prepared using random priming kit
from Roche Boehringer Mannheim and radioactive label from NEN) in hybridization
solution. The membrane was washed in 2X SSC containing 0.1% SDS at 65˚C. This
was followed by several washes in 0.1X SSC containing 0.1% SDS at 55˚C until the

background count was negligible. Finally, the membrane was exposed to X-ray film
at -80˚C.

2.2.8 Immunofluorescent staining (IF)
Indirect immunofluorescence analysis was performed with synchronized yeast cells.
Yeast samples were immediately fixed with 3.7% formaldehyde, collected by
centrifugation and, resuspended in 1ml KPF buffer (0.1M KH
2
PO
4
pH6.4, 3.7%
formaldehyde). After 1-2 hours at room temperature or overnight fixation at 4˚C, the
cells were harvested and washed 3 times with 0.1 M KH2PO4 pH 6.4 to remove all
traces of formaldehyde. Subsequently the cells were washed once in 1ml sorbitol
phosphor-citrate buffer (1.2 M sorbitol, 0.1M K2HPO4 pH5.9) and stored in the same
buffer at 4˚C. The yeast cells were suspended in 0.2ml 1.2 M sorbitol phosphor-
citrate buffer and spheroplasted by treatment with 20 µl glusulase and 5 µl 10mg/ml
lyticase in the sorbitol phosphor-citrate buffer at 37
o
C for 15-90 mins. Following
complete spheroplasting, cells were washed once in sorbitol-citrate solution and re-
suspended in 20µl of the same buffer. 5µl of cells were immobilized for in situ
immunofluorescence by transferring them onto a multi-well slide previously coated
with 0.1% polylysine (Sigma). After aspirating excess cells, the slide was immersed
into methanol at -20
o
C for 6 mins, followed by immersion in acetone at -20
o
C for 30
secs. Next, the slide was air-dried and the cells were first preincubated with BSA-

PBS (PBS containing 1 mg/ml BSA) solution for 2 min. Subsequently, the cells were
incubated with primary antibody diluted in BSA-PBS either overnight at 4
o
C or for 2
h at 30˚C. Cells were then washed 3 times with PBS-BSA (1% BSA, 0.04M K
2
HPO
4
,
0.01M-KH
2
PO
4
, 0.15M-NaCl, 0.1% NaN
3
) and incubated with appropriate secondary
antibodies for 2 hrs at 30
o
C. All incubations with antibodies were carried out in dark
moist chambers. Cells were again washed three times in BSA-PBS before mounting
in antifade solution Vectashield (Vector Laboratories) containing DAPI (4’,6
diamidino-2-phenylindole ) for visualizing DNA. Cover slips were added to protect
the samples and the cover slips sealed in place with nail polish. For visualization of
tubulin, rat monoclonal anti-tubulin YOL1/34 was the primary antibody (Serotec) and
Alexa Fluor 594 goat anti- rat IgG (Invitrogen, Molecular Probes) the secondary
antibody. All cells were observed and photographed using the Leica DMRX
Microscope connected to a Hamamatsu charge-coupled device camera driven by the
Metamorph software (Universal Imaging Corporation).

2.2.9 Microscopy

Cells collected at various time points were frozen immediately in dry ice without
fixation and stored until further use. Cells were later thawed and mounted on slides
with Vectashield containing DAPI (Molecular Probes). The images were captured
using a Zeiss AxioImager upright motorized microscope with Plan Apochromat 100X
objective equipped with EXFO 120W metal halide illuminator and attached to a
Photometrics CoolSNAP HQ2 high sensitivity monochrome camera driven by the
Metamorph software (Universal Imaging Corporation) or Zeiss Axiovert 200M
Microscope connected to a Photometrics COOLSNAP HQ digital camera driven by
Metamorph software.
For time-lapse imaging, cells were placed between a cover slip and a thin slab of 2%
agarose containing 2% glucose in low immunofluorescence yeast nitrogen base with
complete drop-out medium supplemented with adenine. Microscopy was performed
in an enclosed chamber maintained at 25˚C or 35˚C with a Zeiss Axio inverted
microscope equipped with a Plan-Apochromat 100X objective and a Yokogawa
CSU22 spinning disk confocal system with dual line argon krypton ion laser
(wavelength 488 nm / 568 nm). Stack images were taken at 30 s intervals (7 planes
spaced 0.5 µm apart) with a Cascade:512B camera (Roper Scientific, Trenton, NJ),
acquired and analyzed with Metamorph software (Universal Imaging Downington,
PA). Spindle lengths were measured as pole-to-pole distances using the Caliper
function in Metamorph software.

2.2.10 Flow cytometry analysis (FACS)
Cell samples were collected from liquid cultures for analyzing the distribution and
content of DNA by fixing cells in 1ml 70% ethanol and incubated at room
temperature for 1 hr or overnight at 4
o
C. After fixation, cells were washed with 0.2M
Tris-HCl pH7.5, 20mM EDTA, and resuspended in 100µl 0.2M Tris-HCl pH7.5,
20mM EDTA, 0.1% RNase A (1mg/ml), before incubating at 37
o

C for 4 hrs. The
samples were washed 1X in PBS and incubated in 0.1ml PI solution (50mg/ml
propidium iodide in PBS) at 4
o
C overnight. The samples were diluted by addition of
900µl PBS and samples were sonicated for 5 secs. The DNA content was determined
using the FAC Scan flow cytometer (Becton Dickinson Immunocytometry Systems,
USA). The data was analyzed using WinMDI program.

2.2.11 Preparation of cell extracts for Protein analysis
2.2.11.1 Protein extraction using Tri-Chloroacetic Acid (TCA)
Cells harvested at various time-points were resuspended in 1 ml ice-cold ddH
2
0. Next,
the samples are normalized to absorbance of 2.0 at OD600 (1 ml final volume). To
each normalized sample, 150µl YEX lysis buffer (1.85 M NaOH, 7.5% β-
mercaptoethanol) was added. After 10 mins’ incubation on ice, 150µl 50% TCA was
subsequently added and the mixture again incubated on ice for 10mins. The
precipitate was collected by centrifugation at 4
o
C for 10mins at 13000rpm and
resuspended in 50µl of 1X gel loading buffer and 10µl 1M Tris-HCl (pH 8.0). The
samples were boiled for 5mins, and 8µl were loaded onto SDS-PAGE gels for
Western blot analysis.

2.2.11.2 Protein extraction using acid-washed glass beads
For immunoprecipitation, protein was extracted using acid washed glass beads. Cells
were spun down and washed once with Stop Mix buffer (0.9%NaCl, 1mM NaN3,
10mM EDTA, and 50mM NaF). Cell pellets were immediately frozen by liquid
nitrogen, and stored in -20

o
C for later use. Cell pellets were later thawed on ice. 0.2
ml of ice cold lysis buffer with protease inhibitors (1% Triton X-100, 1% sodium
deoxycholate, 0.1% SDS, 50mM Tris- HCl pH 7.2, 1mM PMSF, 20µg/ml leupeptin,
40µg/ml aprotinin, 0.1mM Na-orthovanadate, 15mM p-nitrophenylphosphate) and
150-200µl of acid-washed glass beads (Biospec) were added. The cells were lysed by
vigorous vortexing at 4
o
C (IKA-Vibrax shaker). After centrifugation at 4
o
C for 15
mins at 13000 rpm, the supernatant was transferred to a fresh Eppendorf tube, quick
frozen in liquid nitrogen and stored at -80
o
C for later use. Protein concentration was
determined by using the Bradford Protein Assay (Bio-Rad) according to
manufacturer’s instructions.

2.2.12 Western blot analysis
Protein samples were resolved by sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) using 10% acrylamide:bis-acrylamide (29:1) gels. The
proteins resolved on SDS-PAGE were then transferred onto Hybond-C nitrocellulose
membrane (Amersham) using the Biorad Wet Transfer system in transfer buffer (48
mM Tris, 39 mM glycine, 10% methanol). The membrane was then incubated 3X 10
min in blocking buffer (PBS containing 0.1% Tween-20 and 3% skimmed milk) at
room temperature. It was then incubated overnight at 4˚C or 2 h at room temperature
with the primary antibody in PBS-Tween containing 3% skimmed milk. It was then
washed with three changes of PBS-Tween for 10 mins and subsequently incubated in
PBS containing 3% skimmed milk and the horseradish peroxidase-conjugated
secondary antibody (Amersham; 1:5000 or 1:10000 dilution) for 1 h at room

temperature. The membrane was then washed with three changes of PBS-Tween for
10 mins and subsequently in PBS for 5 mins. Antibody-antigen complexes were
visualized using the Enhanced Chemiluminescence (ECL) system (Santa Cruz
biotechnology).

2.2.13 Immunoprecipitation
Collected samples were lysed using the glass bead method as described above; the
lysis buffer was modified: NP40 buffer (20mM Tris HCl pH 8, 150mM NaCl, 10%
glycerol (Fresh), 1% Nonidet P-40 or NP-40, 2mM EDTA, 1mM DTT) supplemented
with protease inhibitors (1mM PMSF, 20µg/ml leupeptin, 40µg/ml aprotinin, 0.1mM
Na-orthovanadate, 15mM p-nitrophenylphosphate). Next, 1mg of cell lysate was
transferred to an Eppendorf tube containing 40µl of antibody-conjugated beads (Santa
Cruz). The final volume was adjusted to 1ml with lysis buffer containing protease
inhibitors so that the cell lysate can interact with the antibody-conjugated beads
completely. This was followed by 3-4 hrs of incubation in a roller at 4
o
C. The beads
were then washed 4-6 times with either RIPA buffer (1% Triton X-100, 1% Na-
deoxycholate, 0.1%SDS, 150 mM NaCl and 50 mM Tris-HCl pH 7.2) or low salt
buffer (50 mM Tris-HCl pH7.5 and 50 mM NaCl). After removing all the remaining
supernatant, 5µl of 5× gel loading buffer was added to the beads. The samples were
heat inactivated by boiling for 5mins and loaded for SDS- PAGE electrophoresis and
subsequent Western blot analysis.

2.2.14 PCR-based strategy for fluorescent protein and epitope
tagging of yeast genes
Long template PCR system (Roche) was used to amplify the cassettes for fluorescent
protein and epitope tagging following the manufacturer’s instructions. These
amplified cassettes are 5’-(gene specific sequence)- GGTGACGGTGCTGGTTTA-3’
F5 and 5’-(gene specific sequence)- TCGATGAATTCGAGCTCG-3’ R3 for

fluorescent protein tagging; and 5’-(gene specific sequence)-
ATCGATGAATTCGAGCTCG-3’ and 5’-(gene specific sequence)-
CGTACGCTGCAGGTCGAC-3’ for epitope protein tagging. The forward primer
consisted of the 60 3’ nucleotides of the gene to be tagged (excluding the stop codon)
fused to F5 or S2; the reverse primer consisted of the reverse complement of the 60
nucleotides 3’ of the stop codon fused to R3 or S3. After PCR purification (Qiagen
PCR kit), all purified PCR product was transformed into yeast cells plated on
selective drop-out plates. Tagging of the targeted gene was confirmed by Southern
blot and colony PCR to confirm integration of gene at its own locus (Janke et al. 2004;
Sheff and Thorn 2004).

2.2.15 Pulse-chase assay
For investigating the stability of proteins, G1-sunchronized cells were released into
galactose medium at the appropriate temperatures for 30-60 mins to induce GAL1-
driven expression. For monitoring of the fate of the pulse, cells were then transferred
to medium containing 2% glucose and cycloheximide (1 mg/ml) to repress
transcription and translation respectively. Proteins were extracted from whole-cell
lysates and results were analyzed by Western blotting.

2.2.16 Sample preparation for SILAC mass spectrometry
Samples for mass spectrometry were prepared as described in (de Godoy et al. 2008;
Gruhler and Kratchmarova 2008). To prepare samples for SILAC (Ong et al. 2002),
the yeast strains must be defective in endogenous lysine or arginine production, thus
facilitating the incorporation of exogenous amino acids. Therefore, we only deleted
LYS1 as the strain we used is not viable in both ARG4 and LYS1 deletion. Control
yeast strain was grown overnight in liquid labeling medium (6.7% Yeast nitrogen
base and 30mg/L [
13
C
6

/
15
N
2
] H-lysine, Cambridge Isotope Laboratories, and other
normal amino acid mix), while the other strain was grown in non-labeling medium
(6.7% Yeast nitrogen base supplemented with complete normal amino acid mix
including 30mg/L unlabeled lysine). Both cultures were supplemented with the
desired carbon source. Samples were collected and frozen in liquid nitrogen
immediately. The samples were thawed on ice, and the total proteins were extracted
using the glass bead method (refer to Section 2.2.11.2). Prior to boiling, the beads
were resuspended in sample buffer (NuPAGE LDS Sample Buffer, Invitrogen).
Finally, the protein samples were sent to our collaborator (Dr Gunraratne from Walter
Blackstock’ Lab) for subsequent 1D gel electrophoresis, in-gel trypsin digestion and
mass spectrometric analysis.