2.  Materials and Methods

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Strains between horizontal lines are isogenic

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The Cse4 peptide used in this study was synthesized by Sigma Genosys. It consists of the N-terminal amino acids 112-134 with the following sequence (putative acetylation sites are marked in red):

1 mg/ml peptide was solubilized in dH₂O and stored at -80°C.

The primers used in this study were synthesized by Metabion and applied in a dilution of 10 pmol/μl. Sequences derived from the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/) were used to design optimal primers with the assistance from the Oligonucleotide Properties Calculator (http://www.basic.nwu.edu/biotools/oligocalc.html).

The cultivation of bacteria with plasmids was carried out in LB-media or on LB-plates at 37°C unless indicated otherwise. For the bacterial expression of 3xHA-Cse4, nutrient rich TY-median was used. For the maintenance of plasmids the appropriate antibiotics were added to the media (ampicillin, kanamycin, chloramphenicol).

Yeast strains were grown on YPD full media or on YM minimal media at 30°C or 23°C (temperature sensitive mutants). Minimal media were supplemented as appropriate.

Chemically competent bacterial strains were obtained from Gibco BRL (DH5α) and Invitrogen (TOP10, BL21 (DE3) Star). The transformation was carried out as suggested by the manufacturer.

For the preparation of competent yeast cells and the transformation of DNA into yeast we used a method described from (Klebe, et al., 1983). Competent yeast cells were used immediately or were stored after the addition of DMSO to 5.5% at

-80°C.

The isolation of plasmids from bacteria was performed with the Mini- or Midi-Kit from QIAGEN according to the manufacturers instruction.

Genomic yeast DNA was isolated as described from (Hoffman and Winston, 1987). For PCR (polymerase chain reaction) mediated analysis of gene knock-outs in yeast, a single yeast colony was heated for one minute in a microwave. The PCR mix was added subsequently and the DNA amplified in a thermocycler.

The epitope tagged HA-CSE4 plasmid was constructed by inserting three HA-tags into the XbaI-site of CSE4 analogous to (Stoler, et al., 1995). For the insertion of the N- and C-terminal CSE4 fragments into the pACT2 and pBTM117c vector the amino acids 11-139 and the amino acids 137-229 were PCR- amplified with CSE4-primers containing specific restriction sites and subcloned into pCR®BluntII-TOPO (Invitrogen). For insertion into pACT2, the N-terminus was excised with PstI/SpeI and the C-terminus with SacI/PstI. The overlapping ends were filled in with T4 DNA-polymerase and the fragments were cloned into SmaI-linearized pACT2. To obtain the N-/C-terminal CSE4 in the LexA-vector, the fragments were excised with AccI/NotI and inserted into AccI/NotI-linearized pBTM117c.

To generate yeast strains with a specific genotype, the appropriate strains were crossed, sporulated and the resulting tetrads were dissected. For this purpose, small amounts of the yeast strains to be crossed were mixed in dH₂O and were allowed to mate for ≥ 8 h. To select for diploid cells, the cells were then transferred onto supplemented YM and grown at 30°C. After two days, diploids were restreaked onto YPD before they were transferred to sporulation plates and incubated at 30°C for 3-4 days. The cell wall of the sporulated strain was digested with zymolyase solution for 10 minutes at room temperature; the reaction was stopped by the addition of 160 μl dH₂O. The spores from the asci were separated under the microscope (Zeiss Axioskop FS) with a micromanipulator (Narishige) and analyzed for their genotype.

For specific gene knock-outs, we took advantage of homologous recombination. In most cases the kanMX-system was used, which lead after successful homologous recombination to geneticin (G418) resistance in S. cerevisiae.

The basic principle of the system is to amplify the kanMX-gene from pF6a-kanMX4 (pAE478) with homologous sequences at the ends from the gene to be knocked out via PCR. The PCR product is then transformed into competent yeast cells, which are plated onto G418 plates (YPD + 200 mg/l G418). After 2-3 days at 23°C/30°C, the grown colonies are analyzed with PCR.

Standard conditions:

PCR for subsequent cloning: repeat step 2-4 for 20-25 cycles
Control PCR: repeat step 2-4 for 25-30 cycles

Sequencing of DNA templates was performed with the ABI PRISM^TM Dye Terminator Cycle Sequencing Ready Reaction Kit. For this purpose, 0.5-1 μl DNA was mixed with 4 μl Terminator Ready Reaction Mix, 1 μl Primer and dH₂O and amplified in the thermocycler.

PCR conditions for 25 cycles were:

After amplification, the DNA was precipitated with Na-acetate/EtOH, dried and analyzed in the sequencing department of the MPIMG.

The Two-hybrid system can be used to detect protein-protein interactions in yeast. The method is based on the GAL4 gene from Saccharomyces cerevisiae, whose gene product activates several genes from the galactose synthesis pathway. For their activation, Gal4p binds with its DNA-binding domain (DNA-BD) to sequences upstream of the genes (UAS).

Additionally, Gal4p consists of an activation domain (AD), which binds via proteins to the DNA-binding domain and leads to transcription of the reporter genes (lacZ, HIS3). If the two domains remain separated from each other, the transcription from the reporter genes is blocked.

For our experiments we used the pACT2-vector with the activation domain and pBTM117c with the DNA-BD. pBTM117c contains instead of the Gal4 DNA-BD LexA, a bacterial protein that normally binds to lexA promotors. The advantage of LexA based two-hybrid is that less false positive interactions occur and that weak interactions are effectively amplified due to multiple LexA operators. The proteins to be tested were cloned into the vectors and both plasmids were [page 35↓]cotransformed into the two-hybrid strain L40c. Protein-protein interactions were monitored with the β-galactosidase filter assay and the expression of HIS3.

Upon activation of the reporter gene lacZ, the enzyme β-galactosidase is expressed in the cells. In this assay, the enzyme uses the colourless X-Gal (5-Brom-4-chlor-3-indolyl-β-D-galactopyranoside) as a substrate instead of galactose, and cleaves it into the dark blue 5-brom-4-chlor-indigo. For this purpose the yeast cells were transferred onto a nitrocellulose filter and broken in liquid nitrogen. The filter was incubated with 2,5 ml buffer Z and 50 μl X-gal for up to 2 h at 30°C. During this time course, a positive interaction between the proteins should lead to a blue coloration.

The reporter gene expression was also tested with the HIS3 reporter assay. The two-hybrid strain with the appropriate plasmids was streaked onto media lacking histidine. L40c itself is auxotroph for histidine, but due to the interaction of the proteins to be tested, HIS3 gene expression is activated, and the strain can grow in the absence of histidine.

To perform FACS analysis, yeast cells were cultured in an appropriate volume (~3 ml) until they reached OD₆₀₀=0.05-0.1. After brief centrifugation and washing, cells were fixed in 70% EtOH at 4°C over night. Cells were then incubated for 4 h in 20x TE + 1 μg/ml RNase A, washed twice in PBS and subsequently stained over night in PBS + 100 μg/ml propidium-iodide (Sigma). After the suspension was 10x diluted, cell were separated from each other by sonication (3x 5 sec, 60%) and maintained in the dark. FACS analysis was performed with a flow cytometer (FACSCalibur) at the Deutsche Rheumaforschungszentrum in Berlin.

Protein extract preparation from bacteria

After reaching the desired optical density (OD), the bacterial culture was centrifuged for 10 minutes at 5000 rpm and the pellet resuspended in column buffer. The cells were broken with ultrasound (4 x 1 minute, 60 %) or with the french press. To obtain the protein extract, the broken cells were centrifuged for 30 minutes at 20.000 rpm and the supernatant was frozen at -80°C.

Protein extract preparation from yeast

Yeast cell cultures were grown to an OD₆₀₀= 0,8-1 and harvested by centrifugation for 20 minutes at 5000 rpm at 4°C. After washing the cells with dH₂O, the cell pellet was resuspended in the appropriate buffer.

Native whole cell extracts from yeast cultures <200 ml were prepared in 2x buffer L by glass bead lysis as described by (Moazed and Johnson, 1996), except that the concentration of NaCl was adjusted to 200 mM. For denatured protein extracts, the cells were lysed in Bead Buffer, boiled for 10 minutes at 95°C and diluted in IP dilution buffer for subsequent immunoprecipitation.

Larger cell cultures were resuspended in 2x buffer L or bead buffer, respectively, and the cells were broken using a french press. After 1 h centrifugation at 40.000 rpm, protein extracts were analyzed or frozen at -80°C for further use.

Proteins were analyzed according to their molecular weight with 8/10/12% SDS-PAGE gels. The transfer from the gel to a nitrocellulose membrane was performed either at 0,8 A/cm² for 1 h with a Semi-Dry Blot from BIO-RAD, or at 110 V for 45 minutes with a wet blot from BIO-RAD. The efficiency of the transfer was visualized with Poinceau S dye. Subsequently, the membrane was blocked for 1 h with 5% fat free milk/TBST and incubated with the primary antibody in 5% fat free milk/TBST at 4°C overnight. After washing the membrane four times with TBST, the secondary HRP-conjugated antibody was added for 30 minutes. The membrane was washed with TBST for six times and proteins were then detected with ECL-solution from Amersham.

To detect proteins directly in SDS-PAGE gels, the gel was stained for 1 h with Coomassie Brilliant Blue R250. Subsequently, the gel was destained with 25% methanol/10% acetic acid, such that the staining of the proteins was maintained.

If small protein concentrations (~0,1 ng/mm²) were to be detected, we used the Silver Stain Kit from Bio-Rad. The staining of the proteins in an SDS-PAGE gel was performed according to the manufacturer’s instructions. Protein concentrations in solutions were determined with the Bradford Assay (Bio-Rad Protein Assay Kit).

To concentrate proteins in a smaller volume, Centricon-columns (amicon) with an exclusion matrix of 10 kD were used. After one hour centrifugation at 5000 g, protein solutions were concentrated from 2 ml to approximately 50-200 μl. If [page 38↓]necessary, an additional buffer change was performed with three washing steps in 2 ml buffer.

Immunoprecipitation experiments were performed to isolate proteins and protein complexes. If the immunoprecipitation was carried out under denaturing conditions, the protein extract was boiled before use for 10 minutes at 95°C. The appropriate antibody was added to the whole cell protein extract and incubated for one hour with shaking at 4°C. Subsequently, protein G sepharose beads (Pharmacia) were added and the lysate-antibody-protein G mix was incubated over night.

Immunoprecipitates from native extracts were washed four times with 1x buffer L and resuspended in SDS sample buffer; precipitates from denatured extracts were washed with Urea wash buffer, IP buffer and detergent free wash buffer before resuspension in SDS sample buffer. After boiling for 10 min at 70°C and centrifugation, the immunoprecipitates were analyzed by immunoblotting.

For bacterial expression of large amounts of Cse4, N-terminal His-tagged Cse4 was generated by inserting CSE4 into the XhoI/BamHI-site of pET15b and transforming the resulting plasmid (pAE994) into BL21(DE3) Star cells. The expression of His-Cse4 in 2 l LB-culture was induced by adding 1 mM IPTG to the medium at OD₆₀₀=0.8 and subsequent growth for additional 2h at 37°C. The cells were harvested and proteins extracted in Lysis Buffer by sonication (3x 20 sec, 40-50%). After centrifugation, the supernatant was added to 2 ml 50% Ni-NTA matrix and incubated with rotation for 1 h at 4°C. Proteins that did not bind the to matrix were washed off with 15 ml Wash Buffer.

The bound His-Cse4 was eluted by adding 8x 500 μl Elution Buffer with 250 mM imidazole, 1x with 350 mM imidazole and 1x with 1 M imidazole to the matrix. The samples were dialyzed against water, concentrated and further analyzed for His-Cse4 by immunoblotting.

To investigate the acetylation of proteins, we took advantage of an in vitro acetylation assay. In a total volume of 25 μl we mixed 2 μg recombinant histone H4 and/or 2 μg His-Cse4 together with the enzyme (200 μg rSAS-I or 500 μg recombinant PCAF), 5xHAT-Buffer and 0,5 μl [¹⁴C] acetyl-CoA (50-62 mCi/mmol Amersham). After one hour incubation at 30°C, the mix was run on a 15 % SDS-PAGE gel. The gel was dried in a gel dryer (BioRad) for 1 hour at 80°C, and the acetylated proteins were detected after over night exposure with a phosphoimager.