[page 28↓]

2  Materials and Methods

2.1 Materials

2.1.1  E. coli strains

TOP10

F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araΔ139Δ(ara‑leu)7697 galU galK rpsL (StrR) endA1 nupG(chemically or electro-competent; Invitrogen)

DH5α

F-φ80dlacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17(rk -, mk +) phoA supE44λ- thi-1 gyrA96 relA1 (chemically competent; Gibco)

2.1.2 Yeast strains

Table 2.1: Yeast strains used in this study.

Strain

Genotype

Source*

AEY1

MATαade2-1 ura3-1 his3-11,15 leu2-3,112 trp1-1 can1-100 (=W303-1B)

 

AEY2

MAT a ade2-1 ura3-1 his3-11,15 leu2-3,112 trp1-1 can1-100 (=W303-1A)

 

AEY5

MATαHMR SS ΔI

 

AEY24

MAT a orc2-1 rho°

J. Rine

AEY71

MATαHMR-EΔ300-256 (ΔABF)

A. Brand

AEY80

MAT a nat1-5::LEU2

R. Sternglanz

AEY81

MATαHMR-EΔ331-324 (ΔRAP)

A. Brand

AEY84

MATαHMR-EΔ352-358 (ΔACS)

A. Brand

AEY1017

MATα TEL VII-L::URA3

J. Berman

AEY1224

MAT a SUM1-1

D. Shore

AEY1227

MATαnat1-5::LEU2

 

AEY1273

MATαHMR SS ΔI nat1Δ ::LEU2

 

AEY1275

MATαHMR SS ΔI 5xGal4-RAP-ABF

 

AEY1276

MATαHMR SS ΔI 5xGal4-RAP-ABF nat1Δ::LEU2

 

AEY2144

MATαHMR-EΔ331-324 (ΔRAP) nat1Δ::LEU2

 

AEY2146

MATαHMR-EΔ352-358 (ΔACS) nat1Δ::LEU2

 

AEY2148

MATαHMR-EΔ300-256 (ΔABF) nat1Δ::LEU2

 

AEY2371

MATα TEL VII-L::URA3 nat1Δ::LEU2

 

AEY2947

AEY1276 sir1Δ ::kanMX

 

AEY3008

MATαsum1Δ::URA3 nat1Δ::LEU2

 

AEY3068

MATa ORC1-HA-URA3

 

AEY3070

MATa nat1-5::LEU2 ORC1-HA-URA3

 

AEY3134

MAT a ADE2 lys2Δnat1Δ::LEU2

 

AEY3161

MAT a orc2-1 nat1Δ::LEU2 pRS316-ORC2

 


[page 29↓]

Table 2.1 (continued)

Strain

Genotype

Source*

   

AEY2864

MATαade2-1 ura3-1 his3-11,15 leu2-3,112 trp1-1 can1-100 (=W303-1B) HMR SS abf1 - ΔI orc1Δ::HIS5-GFP pAE405

 

AEY2866

MATαade2-1 ura3-1 his3-11,15 leu2-3,112 trp1-1 can1-100 (=W303-1B) HMR SS ΔI orc1Δ::HIS5-GFP pAE405

 

AEY2867

MAT a ade2-1 ura3-1 his3-11,15 leu2-3,112 trp1-1 can1-100 (=W303-1A) HMR SS ΔI orc1Δ::HIS5-GFP pAE405

 

AEY2877

AEY2866 orc1Δ1-10::LEU2 withoutpAE405

 

AEY2879

AEY2866 orc1Δ1-51::LEU2 withoutpAE405

 

AEY2880

AEY2866 orc1Δ1-100::LEU2 withoutpAE405

 

AEY2883

AEY2864 orc1Δ1-10::LEU2 withoutpAE405

 

AEY2887

AEY2867 orc1Δ1-10::LEU2 withoutpAE405

 

AEY2888

AEY2867 orc1Δ1-51::LEU2 withoutpAE405

 

AEY2889

AEY2867 orc1Δ1-100::LEU2 withoutpAE405

 

AEY2903

AEY2866 orc1-A2V::LEU2 without pAE405

 

AEY2904

AEY2864 orc1Δ1-51::LEU2 withoutpAE405

 

AEY2905

AEY2864 orc1Δ1-100::LEU2 withoutpAE405

 

AEY2907

AEY2866 orc1Δ1-28::LEU2 withoutpAE405

 

AEY2908

AEY2864 orc1Δ1-28::LEU2 withoutpAE405

 

AEY2910

AEY2864 orc1Δ29-51::LEU2 withoutpAE405

 

AEY2911

AEY2867 orc1Δ29-51::LEU2 withoutpAE405

 

AEY2912

AEY2867 nat1Δ ::kanMX

 

AEY2913

AEY2867 orc1-A2V::LEU2 without pAE405

 

AEY2916

AEY2866 nat1Δ ::kanMX

 

AEY2937

AEY2867 orc1Δ1-28::LEU2 withoutpAE405

 

AEY3000

AEY2867 sir1Δ::kanMX

 

AEY3002

AEY2888 sir1Δ::kanMX (transformant #1)

 

AEY3003

AEY2888 sir1Δ::kanMX (transformant #2)

 

AEY3031

AEY2887 TEL VII-L::URA3

 

AEY3032

AEY2888 TEL VII-L::URA3

 

AEY3034

AEY2889 TEL VII-L::URA3

 

AEY3036

AEY2911 TEL VII-L::URA3

 

AEY3038

AEY2913 TEL VII-L::URA3

 

AEY3040

AEY2937 TEL VII-L::URA3

 

AEY3102

AEY2867 orc1-A2P::LEU2 without pAE405

 

AEY3103

AEY2866 orc1-A2P::LEU2 without pAE405

 

AEY3105

AEY3102 TEL VII-L::URA3

 

AEY3144

AEY2867 sir3-A2T::TRP1

 

AEY3145

AEY2866 sir3-A2T::TRP1

 

AEY3147

AEY3102 sir3-A2T::TRP1

 

AEY3148

AEY2913 sir3-A2T::TRP1

 

AEY3149

AEY3103 sir3-A2T::TRP1

 

AEY3151

AEY2903 sir3-A2T::TRP1

 

AEY743

MAT a ade2Δ::hisG ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 orc1Δ::TRP1 HIS3::HMR-URA3 P -ADE2-E pSPB162 (pURA3 ORC1)

S. Bell

AEY2333

AEY743 orc1Δ1-51::LEU2 without pSPB162

 

AEY2335

AEY743 orc1Δ1-100::LEU2 without pSPB162

 

AEY2587

AEY743 orc1Δ1-10::LEU2 without pSPB162

 

AEY2589

AEY743 orc1Δ1-28::LEU2 without pSPB162

 

AEY2721

AEY743 orc1-A2V::LEU2 without pSPB162

 


[page 30↓]

Table 2.1 (continued)

Strain

Genotype

Source*

   

AEY2760

AEY743 orc1Δ29-51::LEU2 without pSPB162

 

AEY3101

AEY743 orc1-A2P::LEU2 without pSPB162

 

AEY3109

AEY743 nat1Δ::LEU2

 

AEY1558

MAT a leu2 trp1 ura3-52 prc1-407 pep4-3 prb1-112

E.W. Jones

AEY2719

AEY1558 ORC1(1-250)-TAP::URA3

 

AEY2758

AEY1558 nat1Δ::kanMX orc1(1-250)-TAP::URA3

 

AEY3107

AEY1558 orc1-A2P(1-250)-TAP::URA3

 

AEY3110

AEY1558 orc1-A2V(1-250)-TAP::URA3

 

AEY3171

AEY1558 SIR3(1-235)-TAP::URA3

 

AEY3173

AEY1558 nat1Δ::kanMX SIR3(1-235)-TAP::URA3

 

AEY160

MATα his3Δ200 leu2Δ1 ura3-167 trp1Δ633 met15 Δ1 RDN::Ty1::MET15

J. Boeke

AEY2786

AEY160 nat1Δ::kanMX

 

AH109

MAT a ade2-101 trp1-901 his3200 leu2-3 met -

MATCHMAKER Two Hybrid strain with reporter genes ADE2, HIS3, lacZ, MEL1

Clontech

AEY3028

AH109 pAE951 pAE952

 

AEY3099

AH109 pAE966 pAE952

 

* Unless indicated otherwise, strains were constructed during the course of this study or were from the laboratory strain collection. Groups of strains between horizontal lines are isogenic.

2.1.3 Growth conditions and media

E.coli strains used for plasmid amplification were cultured according to standard procedures (Sambrook et al. 1989) at 37°C in Luria Bertani (LB) medium supplemented with either 100 μg/ml ampicillin or 50 μg/ml kanamycin. S. cerevisiae strains were cultured according to standard procedures (Guthrie and Fink 2002) either in complete (YPD) or minimal (YM) medium supplemented as appropriate with 20 μg/ml adenine, uracil, tryptophan, histidine and methionine or 30 μg/ml leucine and lysine. Strains were grown at 30°C, unless otherwise noted.

Media

LB

10 g/l caseinpeptone, 5 g/l yeast extract, 5 g/l NaCl

 

SOC

2 g/l tryptone, 500 mg/l yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose

 
 

YPD

10 g/l yeast extract, 20 g/l peptone, 2 g/l glucose

 

YM

6.7 g/l yeast nitrogen base w/o amino acids, 2 g/l glucose

 

CA

YM medium with 40 g/l casamino acids

 

5-FOA

14 g/l yeast nitrogen base w/o amino acids, 4 g/l glucose, 2 g/l 5-FOA, 40 mg/l uracil

 

Lead medium

0.3% peptone, 0.5% yeast extract, 4% glucose, 0.02% (w/v) ammonium acetate, 0.1% Pb(NO3)2

 

Sporulation medium

19 g/l KAc, 0.675 mM ZnAc

(For plates, 20 g/l agar was added to liquid media.)


[page 31↓]

2.1.4  Plasmid constructions

Plasmids used in this study are listed in Table 2.2. Cloning strategies and selection markers for bacteria and yeast are added in brackets. Cloning details are described in chapter 2.2.2.

Table 2.2: Plasmids used in this study.

Plasmid

Description / Construction / Markers*

Source**

YEp24

2μ-based genomic library (Amp, URA)

(Carlson and Botstein 1982)

pAE100

pRS316 ADH1P - GAL4(1-147)-SIR1 (CEN; Amp; URA)

J. Rine

pAE108

pRH98-1 GPDPGAL4(1-147)-ORC2 (CEN; Amp, URA)

J. Rine

pAE109

pRH98-1 GPDPGAL4(1-147)-ORC5

J. Rine

pAE303

YCp50 NAT1 (CEN; Amp, URA)

J. Rine

pAE405

pRS316 ORC1 (BamHI-XhoI ORC1-fragment of pAE246)

 

pAE408

pTT64 GAL4(1-147)-ORC1(5-267) (CEN; Amp, HIS)

R. Sternglanz

pAE516

pRH98-1 GPDPGAL4(1-147)-ORC6

(ORC6-ORF BglII-SalI PCR fragment cloned into BamHI-SalI cut pRH98‑1)

 

pAE580

pRS316 SIR3-GFP

D. Shore

pAE595

pRH98-1 GPDPGAL4(1-147)-ORC3

(ORC3-ORFamplified from pAE338; cloned as BamHI-SalI fragment into BamHI-SalI cut pRH98‑1)

 

pAE597

pRH98-1 GPDPGAL4(1-147)-ORC4

(ORC4-ORF amplified from pAE349; cloned as BamHI-SalI fragment into BamHI-SalI cut pRH98‑1)

 

pAE866

pRH98-3 ORC1

(ORC1-ORF amplified from pAE 246; cloned as BamHI-SalI fragment into BamHI-SalI cut pRH98‑3) (2μ; Amp, URA)

 

pAE877

pRS306 ORC1(1-250)-TAP

ORC1(1-250)-TAP amplified by PCR sewing; cloned as BamHI-SalI fragment into BamHI-SalI cut pRS306 (integrating; Amp, URA)

 

pAE951

pGADT7 ORC1 (2μ; Amp; LEU)

B. Stillman

pAE952

pGBKT7 SIR1(346-678) (2μ; Kan, TRP)

B. Stillman

pAE953

pGADT7

Clontech

pAE964

YEplac112 SSB1

(SSB1 as BamHI-PstI fragment from pAE963; cloned into BamHI‑PstI cut YEplac112) (2μ; Amp, TRP)

 

pAE966

pGADT7 ORC1(52-235)

ORC1(52-235) amplified from pAE246; subcloned into pCR-Blunt II-TOPO; cut out as EcoRI fragment and cloned into EcoRI cut pAE953

 

pAE989

pRS306 orc1-A2P(1-250)-TAP

orc1-A2P(1-250) as BamHI-HindIII fragment from pAE971; cloned into BamHI-HindIII cut pAE877

 

pAE990

pRS306 orc1-A2V(1-250)-TAP

orc1-A2V(1-250) as BamHI-HindIII fragment from pAE881; cloned into BamHI-HindIII cut pAE877

 

pAE1001

YIplac204 sir3-A2T(1-504)

sir3-A2T as KpnI-HindIII fragment from pAE997; cloned into KpnI‑HindIII cut YIplac204 (integrating, Amp, TRP)

 

pAE1007

pRS306 SIR3(1-235)-TAP

sir3(1-235)TAP amplified by PRC sewing; cloned as BamHI-SalI fragment into BamHI-SalI cut pRS306

 

* Amp = ampicillinR, Kan = kanamycinR, URA = URA3, HIS = HIS3, LEU = LEU2, TRP = TRP1
** Unless indicated otherwise, plasmids were constructed during the course of this study or were taken from the laboratory plasmid collection.


[page 32↓]

2.1.5 Oligonucleotides

PCR primers were designed using sequence data of the Saccharomyces Genome Database (http://www.yeastgenome.org/). For cloning of PCR fragments, restriction sites were inserted into primer ends. All oligonucleotides used in this study were synthesized by metabion GmbH.

2.1.6 Buffers

Tris-glycine buffer

25 mM Tris, 192 mM glycine, 0.1% SDS

Blot buffer

25 mM Tris, 192 mM glycine, 10% methanol

TBS-T

20 mM Tris pH 7.5, 500 mM NaCl, 0.05% Tween20

SDS sample buffer

50 mM Tris pH 6.8, 100 mM dithiothreitol; 2% SDS, 0.1% bromphenol blue, 10% glycerol

Zymolyase buffer

1 M sorbitol, 0.1 M NaCitrate; 60 mM EDTA pH 8.0; 5 mg/ml zymolyase (Seikagaku corp., Tokyo)

Zymolyase solution

1.2 M sorbitol, 0.1 M KPO4 pH 7.5, 400 μg/ml zymolyase

Buffer A

20 mM Tris pH 8.0, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 pill Complete (Roche) (protease inhibitor cocktail) ad 50 ml buffer

IPP150

10mM Tris-Cl pH 8.0, 150mM NaCl

TEV cleavage buffer

10mM Tris-Cl pH 8.0, 150mM NaCl, 0.5 mM EDTA, 1 mM DTT

IPP150 Calmodulin binding buffer

10 mM β -mercaptoethanol, 10 mM Tris-Cl pH 8.0,
150 mM NaCl, 1 mM MgAcetate, 1 mM imidazole, 2 mM CaCl
2

IPP150 Calmodulin elution buffer

10 mM β -mercaptoethanol, 10 mM Tris-Cl pH 8.0, 150 mM NaCl, 1 mM MgAcetate, 1 mM imidazole, 2 mM EGTA

2.2 Methods

2.2.1 Yeast strain construction

Strains used in this study were generated either by direct deletion or by chromosomal integration of the gene of interest. Alternatively, strains were derived from crosses between strains from the laboratory stock.

Crossing, sporulation and the dissection of asci

For crosses, some cell material of the 2 parental strains grown over night was smeared together in a drop of water. After 8 h of incubation at 30°C (23°C for ts strains) on a YPD plate, the smear was streaked out on selective medium to isolate diploids.

To induce sporulation, the diploids were plated on sporulation plates and incubated at 30°C for 2-3 d or at 23°C for 3-4 d. For dissection, a loopful of asci was incubated in 10 μl zymolyase buffer for 6-10 min at RT. The reaction was stopped by adding 250 μl H2O. Ascospores were subsequently dissected using a micromanipulator (Narishige) connected to [page 33↓]a Zeiss Axioscope FS microscope. Plates were incubated at 30°C or 23°C for 2-5 d. Marker segregation was followed by standard genetic techniques (Guthrie and Fink, 2002).

The suppression of the nat1∆ SUM1-1 synthetic lethality by orc11-235 was determined as follows: Strain JRY7176 (Rusche and Rine 2001) was transformed with an URA3-SIR2 plasmid in order to give the strain mating ability and to create diploids with AEY3134. The URA3-SIR2 plasmid was then lost from the diploid by counter-selection on 5-FOA containing media. The diploid was sporulated, tetrads were dissected and segregants were analyzed for their genotype. Segregants that were Trp+ and Leu+, genotypically were orc1∆::TRP1 and also LEU2::orc11 235, because orc1∆ alone is lethal. To select segregants among these with nat1∆::LEU2, the fact was exploited that SIR2 and NAT1 are neighboring genes within the yeast genome, making recombination between them highly unlikely. Thus, His- segregants from the cross by interference were also nat1::LEU2. Ten such segregants were chosen, proteins extracted and submitted to SDS-PAGE and Western blotting with α-myc antibody to determine their SUM1-1 status. Several segregants were identified that showed a strong signal, and they were presumed to have the genotype orc1::TRP1 LEU2::orc11-235 nat1::LEU2 7myc-SUM1-1.

Gene disruption

Endogenous ORC1 was disrupted in a diploid strain carrying pAE405 and the two HMR alleles HMR SS ΔI and HMR SS abf1 - ΔI (AEY2729)using the PCR-mediated knockout technique. In brief, the complete open reading frame of ORC1 plus 200 bp of upstream sequence was replaced by a fragment containing SpHIS5-GFP amplified from pAE913. Haploid orc1Δstrains were obtained by sporulation and tetrad dissection. NAT1 and SIR1 were disrupted by replacing them with the kanMX cassette using the PCR knockout strategy according to the guidelines for EUROFAN (Wach et al. 1994).

For both deletion protocols, integrants were selected for by standard genetic techniques and the correct integration was verified by PCR.

Chromosomal integrations

For orc1‑A2V, orc1‑A2P and the orc1 N-terminal deletion strains, mutant orc1 alleles were created by site-directed mutagenesis and cloned into an integrative plasmid (pAE785). These constructs were KpnI-linearized and introduced into the LEU2 locus of AEY2866, AEY2867 and AEY743, followed by elimination of pAE405 on 5-FOA medium. Endogenous ORC1 was HA-tagged in strains AEY3068 and AEY3070 by duplicative integration using XbaI-linearized pSB991(pRS306-ORC1-HA/C; S.Bell). sir3-A2T strains were constructed by integrative transformation of PstI-linearized pAE1001, which carries a KpnI/HindIII fragment of sir3-A2T from pLP189 (Stone et al. 2000). Chromosomal integrations of the TAP-tagged versions of ORC1 and SIR3 into the URA3 locus of AEY1558 and AEY2706 were achieved by transforming the strains with the NcoI-linearized plasmids pAE877, pAE989, pAE990 and pAE1007. Integrants were selected using standard genetic techniques and were verified by Western blotting. Telomeric URA3 was inserted into the appropriate strains by transforming SalI/EcoRI-linearized pVII-L URA3-TEL (Gottschling et al. 1990).


[page 34↓]

2.2.2  Molecular cloning techniques

Standard molecular cloning techniques were performed according to (Sambrook et al. 1989). Chemicals, kits and enzymes were purchased by NEB, Invitrogen, Qiagen, Roche, Bio-Rad, Promega and Stratagene, and were applied according to the guidelines.

Transformation of DNA in E. coli and S. cerevisiae

DNA was transformed into competent E. coli cells (TOP10 or DH5α) according to the protocol of the manufacturers. Competent yeast cells were created and transformed as described by (Klebe et al. 1983) and (Ito et al. 1983).

Preparation of genomic and plasmid DNA

Plasmid DNA was extracted from E. coli by the alkaline lysis procedure (Sambrook et al. 1989), and further purification using the Qiagen plasmid kits. Plasmids were isolated from yeast strains according to the protocol of Jaques Paysan: 1.5 ml yeast culture grown to saturation were pelleted and resuspended in 200 μl zymolyase solution. After 2 hours of incubation at 37°C, plasmids were isolated by alkaline lysis with the Qiagen plasmid kit starting with 400 μl of buffer 2. Genomic DNA from yeast was prepared as described in (Hoffman and Winston 1987).

PCR reactions

As a standard PCR protocol, reactions were carried out in 50 μl volume containing 2.5 U Taq-Polymerase (Promega) or 0.5 U VENT Polymerase (NEB), 5 μl of the respective 10x polymerase buffer, 30 pmol of each of the two primers and 0.2 mM of each dNTP. Mg2+ions and template DNA was added in variable concentrations (by default 1.5 mM Mg2+ and 100 pg DNA). Standard amplification reaction: 5’ 95°C, 23 – 30 cycles [30’’ 95°C, 30’’ annealing temperature (according to the primers), elongation time (according to the fragment length) at 72°C], 5’ 72°C. As benchmark, 1’ was given for the elongation of 1 kb sequence.

ORC1(1-250)-TAP (and likewise orc1-A2P-TAP, orc1-A2V-TAP and sir3(1-235)-TAP)fusions were created by PCR sewing in two steps: Fragments of the TAP-tag and the respective fusion protein, which were overlapping at the projected fusion site were amplified separately. In a second PCR, the two overlapping fragments were joined using the outer primers of the first reaction. The obtained fragments contained restriction sites at their ends (added by the primer) and were directly digested and cloned into the integrative vector pRS306. In other cases, PRC fragments with primer based restriction sites at their 5’ and 3’ ends were at first subcloned into the pCR-Blunt II TOPO vector (Invitrogen) and then excised and inserted into their ultimate plasmid. N-terminal deletion alleles of ORC1 were also created by PRC sewing. Thereby, the deletion region was excised from the ORC1-ORF by amplification and subsequent joining of the surrounding sequences. The fused PCR fragment contained the deletion and primer based restriction sites at the 5’ and 3’ ends, and was cloned into an integrative plasmid (pAE785).

Site-directed mutagenesis

In order to create orc1-A2P and orc1-A2V alleles, point mutations were introduced into the second codon of ORC1 using the Quick Change® site-directed mutagenesis strategy (Stratagene). In brief, the ORC1 encoding plasmid pAE246 was amplified with complementing [page 35↓]primer pairs, whose sequences included the point mutation. Newly synthesized plasmids were selected for by DpnI digestion, which is specific to the methylated parental templates, and were verified by sequencing. For subsequent genomic integration of the mutant alleles, NdeI-NcoI fragments of the mutagenized plasmids were exchanged for the NdeI-NcoI fragment of an integrative plasmid containing orc1 Δ 1-100 (pAE787).

Sequencing of DNA

Sequencing PCR reactions were performed according to the ABI PRISM® Big DyeTM Terminator Cycle Sequencing protocol. The reaction mix contained 1 μl BD Terminator mix, 1-8 μl template DNA, 2 mM primer, ad 10 μl H2O. The cycling profile was: 1’ 96°C, 35 cycles [20’’ 96°C, 10’’ annealing temperature, 4’ 60°C]. The reaction was then precipitated and submitted for sequencing to the service group of the institute.

2.2.3 Silencing assays

Mating assays

Mating assays were performed using AEY264 (MAT a his4) and AEY265 (MATαhis4) as mating-type tester strains. For qualitative mating assays (patch-mating), strains were grown on plates over night and replica-plated with a lawn of the respective tester strain on YM medium, which was selective for diploids. After 2-4 d of incubation, the yield of diploids indicated the mating efficiency of the strain. Quantitative mating assays were performed as described (Ehrenhofer-Murray et al. 1997).

MET15 colony color silencing assays

Silencing of the MET15 reporter gene integrated at the rDNA locus was monitored on lead containing plates (Smith et al. 1999). On this medium, strains that silence the reporter gene become darkly pigmented, whereas strains expressing the gene are white. Photographs were taken after 5 d using a Leica stereoscopic microscope equipped with a Sony DXC-9100p CCD color video camera.

URA3 silencing assays

Silencing of the TEL-VIIL::URA3 (Gottschling et al. 1990) gene was measured by the ability of strains to grow on plates containing 5-fluoroorotic acid (5-FOA), which is counter-selective for URA3 expressing cells (Guthrie and Fink 2002). Test strains were scraped from fresh plates, resuspended in 0.5 ml sterile water and diluted to an OD600 of 0.3. 6-fold serial dilutions thereof were spotted with a cell spotter on plates containing 5-FOA and incubated for 2-3 d at 30°C. As a control for cell viability, the serial dilutions were also spotted onto supplemented minimal medium.

HMR::ADE2 silencing

Silencing of the ADE2 gene inserted at the HMR locus was measured by the ability of strains to grow on medium lacking adenine. For this, serial dilutions of the strains were applied as described for the URA3 silencing assays.


[page 36↓]

α -factor response assays

The α-factor response of MAT a HML α strains was measured by spreading them on YPD plates containing 40 μg/ml α-factor and segregating 100 individual cells per strain using a micromanipulator. After 17 h of incubation at 23°C, cells were scored according to their response to α-factor. Schmoo: Individual cells that formed a mating projection and remained arrested. Schmoo cluster: Individual cells that formed multiple mating projections and eventually divided at least once. Colony: Cells that formed colonies of round cells and thus did not respond to α-factor.

2.2.4 Two-hybrid assay

The yeast two-hybrid assay was carried out using the MATCHMAKER system (Clontech). A pGBKT7 plasmid encoding Sir1(346-678) (Triolo and Sternglanz 1996) was used as bait. Orc1(1-235) and Orc1(52-235) were cloned into pGADT7 as prey. The Sir1(346-678) and Orc1(1-235) plasmids, as well as the AH109 tester strain, are courtesy of B. Stillman. Two-hybrid interactions were tested in strains cotransformed with bait and prey by plating them in serial dilutions on YM medium lacking adenine and histidine, respectively, followed by 2-3 d of incubation at 30°C. The dilution protocol is described with the URA3 silencing assays.

2.2.5 Immunofluorescence on yeast cells

Cells carrying the sequence of a Sir3-GFP fusion protein under the control of the natural SIR3 promoter on a CEN-based plasmid (pAE580) were grown to logarithmic phase in liquid selective medium. 1 ml of cell culture was spun down, washed once with distilled water, and then resuspended in 500 µl of water. DNA was stained by adding 1 μl of Hoechst (1µg/ml). Images were captured with a fluorescence microscope (Axioplan 2, Zeiss) using the FITC filter for GFP.

2.2.6 Biochemical techniques

Yeast protein extract preparation

For Western blotting, crude extracts were prepared according to a protocol from Sigrid Schaper. Strains were grown in selective liquid medium to midlog phase (OD600 = 0.5-1). For each probe, 1.5 ODs of cells were harvested and centrifuged for 2’ at 6500 rpm on a table-top centrifuge. The pellet was resuspended in 30 μl of buffer A (modified from TAP protocol). After the addition of 70 μl of SDS sample buffer and acid washed glass beads, cells were broken by vortexing at full speed for 1’, 5’ boiling at 95-100°C, cooling down at RT, and again vortexing for 1’. 15-20 µl of these probes were applied on SDS gels, alternatively, they were stored at –80°C.

For TAP and IEF experiments, protein extracts were prepared according to the TAP protocol (Rigaut et al. 1999). 2 l of cell culture grown in YPD medium at 30°C to an OD600 of 1.5‑2 were spun at 5000 rpm for 20’ at 4°C, washed with cold water, spun again, resuspended in 50 ml in a Falcon tube and spun at 3000 rpm for 15’ at 4°C. The pellet was frozen in –80°C (without shock freezing in liquid nitrogen). For protein extract preparation, the pellet was resuspended at RT in one volume of buffer A, and then kept at 4°C. Cells were broken in 3 French press [page 37↓]passages. Then, 0.2 mM KCl was added and the suspension was ultracentrifuged at 21,000 rpm for 30’ in a Sw40Ti or 70 Ti rotor (Beckman). The supernatant was centrifuged at 34,000 rpm for 2 h at 4°C. The protein concentration of the obtained extract was determined with the method of Bradford (Bradford 1976), and aliquots were frozen in 17% glycerol in liquid nitrogen and kept at –80°C.

SDS page and Immunoblot

Proteins were separated by SDS-PAGE in Tris-glycine buffer according to standard methods (Sambrook et al. 1989). They were then transferred to nitrocellulose by blotting with the BIO‑RAD Tank Transfer System according to the manufacturers guidelines. Mostly, the blot occurred for 2 h at 70V in blot buffer. The nitrocellulose membrane (Pharmacia) was subsequently blocked for 1 h at RT in 5% milk/ TBS-T. After an overnight incubation at 4°C with the primary antibody in 5% milk/ TBS-T, the blot was washed twice for 10’ each with TBS-T. Next, the blot was incubated with the appropriate secondary antibody in 5% milk/ TBS-T for 1 h at RT. After washing 3 times for 10’ with TBS-T, the SuperSignal West Pico Chemiluminescent Substrate (Pierce) was used for immunochemical detection.

Antibodies used were PAP (Peroxidase anti-peroxidase) (Sigma P2026), α‑HA (Sigma), α‑myc (Invitrogen), α‑Tub27 (Babco), and α‑Orc1 (Santa Cruz Biotechnology).

Isoelectric focusing

Proteins were separated by IEF-PAGE using precast ready gels (pH 3-10) from BIO‑RAD. Gels were run according to the suppliers instructions for 1h at 100V, 2 h at 250V, and 30’ at 500V using the Pharmacia Power Supply EPS 3500 XL. For immunoblotting, gels were equilibrated in blot buffer for 2 h, and then immuno-blotted like SDS gels. The theoretical pI was calculated using (http://us.expasy.org/tools/pi_tool.html).

Tandem affinity purification (TAP)

TAP-tagged proteins were purified according to the TAP protocol of the Séraphin laboratory (http://www‑db.embl‑heidelberg.de/jss/servlet/de.embl.bk.wwwTools.GroupLeftEMBL/ExternalInfo/seraphin/TAP.html), except for omitting of NP40. To prepare a sample for subsequent MALDI‑TOF analysis, protein extract of a 4 l cell culture was applied. The TAP tag consists of a calmodulin binding peptide (CBP) and Staphylococcus aureus protein A, separated by a tobacco etch virus (TEV) cleavage site. Therefore, the purification occurred in three steps. Firstly, 250 μl of IgG agarose beads (Sigma A2909), washed beforehand with 15 ml of IPP150 buffer, were added to 10 ml of protein extract together with 100 μl of 1 M Tris-Cl pH 8.0 and incubated under rotation for 2 h at 4°C in a Poly-Prep® chromatography column (BIO‑RAD). Then, the solution was removed through the column and the remaining beads were washed with 30 ml IPP150 and 10 ml TEV cleavage buffer. Next, 100 units of TEV protease (Invitrogen) were added in 1 ml TEV cleavage buffer, and the protein was eluated from the beads during 2 h of rotation at 16°C. The eluate (1 ml) was recovered from the column under addition of 200 μl TEV cleavage buffer, and supplied with 3 ml calmodulin binding buffer and 3 μl 1 M CaCl2. The mix was added to the second affinity column with 250 μl of a calmodulin beads suspension, washed beforehand with 7.5 ml of IPP150 calomdulin binding buffer, and rotated for 1 h at 4°C. After removal of the solution, the beads were washed with 30 ml of IPP 150 calomdulin binding buffer and eluted with 1 ml of IPP150 calmodulin elution buffer containing EGTA. For precipitation, 5 volumes of ice-cold acetone were added and the sample was incubated at –20°C for 20’. After 30’ centrifugation at full speed in a table-top centrifuge at [page 38↓]4°C, the supernatant was removed and the protein pellet was resuspended in 20 μl SDS sample buffer for application on a SDS gel.

In-gel digestion and peptide mass fingerprinting

TAP-purified Orc1 was separated from a 10% acrylamide gel and visualized by Coomassie G‑250 staining. The protein band was excised and divided into two probes. The probes were cleaved in situ as described previously (Shevchenko et al. 1996) using either AspN (37°C) or GluC (25°C) protease (both Roche, Mannheim) at a final concentration of 11.7 ng/µl or 25 ng/µl, respectively. The reduction and carbamidomethylation step was omitted.

The digest supernatant (0.5 µl) was applied on a fast-evaporation nitrocellulose/ α‑cyano‑4‑hydroxycinnamic acid layer (Vorm et al. 1994) and analyzed by MALDI-TOF mass spectrometry using a Bruker Reflex mass spectrometer (Bruker Daltonics, Bremen) in the reflector mode equipped with pulsed‑ion extraction and a nitrogen laser (337nm). For selected peptides, the amino acid sequence was determined by analysis of fragment ions generated by post-source decay (Chaurand et al. 1999) using the FASTTM method (Bruker).


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