A study on premature segregation of unreplicated chromosomes 2

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 Sant

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Chapter 2 Materials and Methods

2.1 Materials

Table 1 Reagents used in this study

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)

Alpha factor Biotechnology Centre (Singapore)

MG132 proteasome inhibitor Sigma-Aldrich (USA)

Table 2 Antibodies used for immunofluorescence and protein analyses

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)

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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

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

Simonett

a Piatti

SCC1myc18:TRP

This study

PDS1HA:URA3

This study

US4366 cdc6∆GalCDC6 CIN8-HA

Mat a, GalCDC6:URA3, CIN8-HA:HIS3 bar+ (halo test)

This study

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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

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

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

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hisGURA3hisG looped out on 5-FOA, so it is Ura-,

SCC1-cmyc12-KAN

This study

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

cdh1∆:KAN, SPC72-GFP:LEU2, ASE1HA:Hygromycin study This

hisGURA3hisG looped out on 5-FOA

so it is Ura- Dicentric plasmid URA3 +

This study

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

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

hisGURA3hisG looped out on 5-FOA GALndSIC1:URA3

This study

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

study

US7016 cdc34-1 cdc6∆ MET-CDC6 CDH1∆:KanMX

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 study This

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 study This

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

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

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

GAL-ASE1-cmyc on HIS3

This study

US7029 cdc34-1 cdc6∆ MET-CDC6

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

This study

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SIC1 (p40) c-myc tag under its own promoter in pUS15

ase1∆:KanmX, so it is Ura-

This study

cdh1∆:KAN, CIN8-HA on URA3, CEN plasmid study This

US7034 cdc34-1 cdc6∆ MET-CDC6 sic1∆

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 study This

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

US7039 Mat a, cdc34-1 cdh1∆:KAN,

Gal-CIN8-myc on URA3 CEN plasmid

This study

ura3,GAL,cdc6::hisGURA3hisG,

trp1-1::TRP1 MET-CDC6

so it is Ura-, GAL-ASE1-cmyc on HIS3, 2u plasmid

This study

so it is Ura-, Gal-CIN8-myc on URA3 CEN plasmid

This study

ura3,GAL,cdc6::hisGURA3hisG, study This

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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 study This

US7046 Mat a, cdc34-1cdh1∆:KAN,

GAL-cmyc6-CDC5 on YIPlac 211 (3.79 kb) - a URA3

This study

US7047 Mat a, cdc34-1cdh1∆:KAN,

GAL-cmyc6-CDC5 (N209A) on URA3, integrative plasmid

This study

so it is Ura-, YIPlac211-GAL-cmyc6-CDC5 -URA3

This study

hisGURA3hisG looped out on 5-FOA so it is Ura-,

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

This study

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Name Description Source

pUS18 Ycplac 33 (5.6 kb)- a URA3 selectable CEN4 plasmid

pUS654 SIC1 (p40) c-myc tag under its own promoter in pUS15

pUS785 SIC1 (p40) c-myc triple tag in pUS20 intergrative vector

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

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

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pUS2259 Gal-CIN8-myc on URA3 CEN 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

US Lab

pUS2718 GAL-cmyc 6 -CDC5 (N209A) on URA3, integrative

plasmid

US Lab

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Primer Description Source

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

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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

OUS1183 Forward oligo to tag the endogenous kip1 by one step PCR:

50 BP JUST BEFORE THE STOP CODON

OUS1184 Reverse oligo to tag the endogenous kip1 by one step PCR :

50 BP JUST AFTER THE STOP CODON

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

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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

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OUS1972 Forward oligo for confirm sic1 disruption

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 37oC 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

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