5.10.1 Yeast cell transformation
Lithium acetate treated yeast cells were transformed with plasmid-DNA following the description of Gietz et al. (1992) with minor modifications.
Lithium acetate/TE (pH 7.5): 100 mM Lithium acetate 10 mM Tris-HCl
1 mM EDTA
PEG/Lithium acetate/TE (pH 7.5): 40 % (v/v) PEG 4000 100 mM Lithium acetate 10 mM Tris-HCl
1 mM EDTA
Yeast cells were grown in appropriate liquid media at 25°C, until log-phase (1-2x107 cells/ml) was achieved. Then, the cells were harvested by centrifugation at 4 100 x g for 5 min, washed once in 1 ml of lithium acetate/TE and finally resuspended in an appropriate volume of lithium acetate/TE to obtain a cell density of 1x109 cells/ml. For
one transformation reaction, 50 µl of this cell suspension (5x107 cells) were mixed with 1 µg plasmid-DNA and 50 µg Salmon sperm-carrier DNA, which was boiled for 5 min and cooled down on ice for 2 min before usage. Subsequently, 300 µl of PEG/lithium acetate/TE was added to the sample, which was thoroughly mixed, incubated with agitation at 25°C for 30 min and then heat-shocked at 42°C for 15 min. Afterwards, the cells were pelleted by centrifugation for 60 sec at 21 100 x g, the supernatant was removed and the cells were resuspended in 100 µl of sterile water to plate them onto a selective agar plate. Finally, the plates were incubated at 25°C for two to three days and subsequently, growing single colonies were streaked onto new selective agar plates.
5.10.2 Extraction of chromosomal DNA from yeast cells
Chromosomal / genomic DNA (gDNA) from yeast cells was extracted, as described previously (Rose et al., 1990).
Detergent lysis buffer (pH 8.0): 2 % (v/v) Triton X-100 1 % (w/v) SDS
100 mM NaCl 10 mM Tris-HCl 1 mM EDTA TE-buffer (pH 8.0): 10 mM Tris-HCl
1 mM EDTA
Yeast cells were grown in 10 ml liquid cultures to saturation and collected by centrifugation at 4 100 x g for 5 min. Upon washing once with water, the pellet was resuspended in 100 µl of H2O and 200 µl of detergent lysis buffer, 200 µl of phenol/chloroform/isoamyl alcohol (25:24:1) (P/C/I) and 300 µl of glass beads were added. The cells were disrupted by using twice the FastPrep-24 machine at 5 m/s for 20 sec. Subsequently, 200 µl of TE-buffer was added and the phases were separated by centrifugation for 5 min at 21 100 x g and room temperature (RT).
Then, the upper phase was transferred into a fresh tube and the same amount of P/C/I was added, vigorously mixed and centrifuged again for 5 min at 21 100 x g and RT. This procedure was repeated, until the interphase remained clear. Finally, the chromosomal DNA from the upper phase was precipitate by the addition of 6 µl of 7.5 M ammonium acetate and 1 ml of 100 % ethanol and centrifugation for 10 min at 21 100 x g and 4°C. Afterwards, the precipitate DNA was washed once with 70 %
ethanol, air dried and resuspended in 50-100 µl of deionized water. Storage was performed at -20°C.
5.10.3 Extraction of total RNA from yeast cells
Yeast cells were grown in 50 ml of YPD medium to log-phase (~2x107 cells/ml), shifted for 1 h to their restrictive temperatures and harvested by centrifugation for 5 min at 2 050 x g and 4°C. The total RNA of these pellets was extracted by using the kit “NucleoSpin RNA” (Macherey-Nagel) following the manufacturer’s protocol that includes the DNase I treatment. Finally, the RNA was eluted in 60 µl of nuclease-free water, the concentration was measured with the NanoDrop spectrophotometer and adjusted to 1 µg/µl (if possible). RNA samples were stored at -80°C.
5.10.4 Synthesis of digoxigenin (DIG)-labeled RNA-probes
Digoxigenin (DIG)-labeled RNA-probes were needed for the detection of specific RNAs in fluorescence in situ hybridization experiments (see section 5.11.3) and in non-radioactive Northern blot analyses (see section 5.10.5). The synthesis of these probes was performed, as suggested by the manufacturer of the DIG-labeling mix (Roche Diagnostics, DIG Application Manual for Filter Hybridization, 2008).
The DIG-labeled RNA-probes that were designed in this study for the recognition of ribosomal RNAs detect always their precursor molecules as well, as these molecules arise in a series of cleavage events from the common 35S precursor (see Figure 1B).
Thus, the 20S pre-rRNA probe is complementary to the 5’ITS1 and the 27S pre-rRNA probe to the ITS2, but both recognize also 35S, 33S and 32S pre-rRNAs. The mature rRNA probes detect all precursor molecules.
To produce templates for the synthesis of specific RNA-probes, PCR-products with a T7-RNA-polymerase promoter on the antisense strand were generated by using the primer pairs HK1138+1139 (25S rRNA probe), HK1140+1141 (18S rRNA probe), HK1893+1894 (20S pre-rRNA probe), HK1895+974 (27S pre-rRNA probe) and HK1723+1724 (U2 snRNA probe) in a PCR reaction with gDNA and were purified from agarose gels with the “peqGOLD Gel Extraction Kit” (Peqlab). These purified PCR-products were used as templates in an in vitro transcription assay with the T7-RNA-polymerase and the “DIG RNA labeling mix” (Roche) to generate antisense RNA probes. In such reaction, DIG-11-UTP will be incorporated in average every 20th to 25th nucleotide in the newly synthesized RNA. The following reaction mix (20 µl) was incubated for 2 h at 37°C:
250 ng template (purified PCR product) 1x DIG RNA labeling mix (Roche)
20 U RiboLock RNase Inhibitor (Thermo Scientific) 1x Transcription buffer (Thermo Scientific)
40 U T7-RNA-polymerase (Thermo Scientific)
Afterwards, the produced RNA was precipitated by LiCl-precipitation (for >100 nt).
Hyb-Mix: 50 % (v/v) deionized formamide 5x SSC
1x Denhardt’s 0.1 mg/ml Heparin
Filled up with DEPC-treated water 50x Denhardt’s: 1 % (w/v) Ficoll
1 % (w/v) Polyvinylpyrrolidone 1 % (w/v) BSA
For precipitation, the reaction mix was filled up to 50 µl with RNase-free water and ¼ volume of 4 M LiCl, 100 µg of tRNA and 3 volumes of 100 % ethanol was added and incubated overnight at -20°C. Then, the RNA was pelleted by centrifugation for 60 min at 21 100 x g and 4°C, washed with 70 % ethanol, dried on ice and resus-pended in 25 µl of 1 M TE pH 7.5 + 1 mg/ml heparin. Afterwards, 25 µl of deionized formamide and 50 µl of Hyb-Mix were added and the RNA-probes were stored at -20°C.
5.10.5 RNA-formaldehyde gels and Northern blotting
To analyze the steady state level of ribosomal RNAs in different yeast strains, their total RNA was extracted (see section 5.10.3) separated on denaturing RNA-formaldehyde agarose gels and analyzed by Northern blotting, as basically described previously (Sambrook et al., 1989; Wu et al., 2014). Non-radioactive Northern blotting with DIG-labeled RNA probes was performed according to the suggestions of the manufacturer (Roche Diagnostics, DIG Application Manual for Filter Hybridization, 2008).
Before starting the experiment, all solid materials were incubated with 0.1 M NaOH for 30 min and all solutions were treated with diethyl dicarbonate (DEPC) to inactivate RNases and prevent RNA degradation. For that, 1 ml of DEPC was added to 1 l deionized water, stirred overnight and subsequently autoclaved.
10x MOPS (pH 7.0): 200 mM MOPS
50 mM Sodium acetate 10 mM EDTA
20x SSC (pH 7.0): 3 M NaCl
300 mM Sodium citrate Hybridization buffer (pH 7.2): 0.5 M Na-phosphate pH 7.2
7 % (w/v) SDS 1 mM EDTA
1M Na-Phosphate buffer (pH 7.2): 68.4 ml 1M Na2HPO4
31.6 ml 1M NaH2PO4
5x Maleic acid buffer (pH 7.5): 0.5 M Maleic acid 0.75 M NaCl
10x Blocking stock solution: 10 % (w/v) Blocking reagent (Roche) 1x Maleic acid buffer
1x Blocking buffer: 10x Blocking stock solution diluted in 1x Maleic acid buffer (freshly prepared before use)
Washing buffer: 1x Maleic acid buffer 0.3 % (v/v) Tween 20 Detection buffer: 0.1 M Tris pH 9.5
0.1 M NaCl
RNA Loading Dye: 50 % (v/v) deionized formamide 6 % (v/v) Formaldehyde
1x MOPS
25 ng/ml Ethidium bromide 10% Glycerol (RNase-free)
Bromophenol blue and Xylene cyanol
At the beginning, a 1 % agarose gel containing 2 % formaldehyde was poured under the hood. For that, 1.5 g agarose was dissolved in 127 ml of nuclease-free water, cooled down to 55°C and mixed with pre-warmed 15 ml of 10x MOPS and 8.1 ml of formaldehyde (37 % from Applichem). During polymerization, the RNA samples were prepared. For each strain 1 µg of total RNA was diluted in 5 µl of DEPC-water, mixed with 10 µl of freshly prepared RNA-Loading Dye, denatured for 10 min at 65°C and chilled on ice. The complete samples were loaded into the wells of the formaldehyde-agarose gel, which was subsequently run in 1x MOPS at 80 V for 4-5 h. Afterwards, the quality of the RNA was examined with an UV transilluminator. To remove the formaldehyde before Northern blotting, the gel was washed once in DEPC-water and twice in 20x SSC for 15 min. A dry Northern blot was performed overnight and the following sandwich was set up:
(1) the gel facing down on a glass plate and wrapped with parafilm (2) a dry, positively charged nylon membrane (Amersham Hybond-N+)
with the size of the gel
(3) three dry Whatman papers (0.8 mm) in the same size (4) a stack of paper towels and a 200-500 g weight
The next day, the RNA was cross-linked by placing the membrane on a Whatman paper pre-soaked with 2x SSC and exposing it to UV light for 7 min at 5000 J/cm2 and afterwards, by baking the membrane for 2 h at 80°C. Then, the membrane was stored dry or was pre-hybridized with hybridization buffer in a closed glass tube for 1 h at 68°C. For one detection, 1 µl of each DIG-labeled RNA probe (for preparation see section 5.10.4) was pipetted in 5 µl of hybridization buffer, denatured for 5 min at 55°C and added to the hybridization solution with the membrane. Hybridization was done at 68°C overnight. Afterwards, the following washing steps were performed for 15 min each: in 2x SSC/ 0.1 % SDS at RT, in 1x SSC/ 0.1 % SDS at RT and twice in 0.5x SSC/ 0.1 % SDS at 68°C. For detection, the membrane was washed for 5 min in washing buffer, blocked for 30 min in 1x blocking buffer and incubated for 30-60 min with anti-Digoxigenin-alkaline phosphatase, Fab fragments (diluted 1:10 000 in blocking buffer, from Roche). After washing twice for 15 min in washing buffer, the membrane was equilibrated for 2-5 min in detection buffer and incubated with the chemiluminescent substrate CSPD (diluted 1:100 in detection buffer, from Roche) for 5 min. The membrane was enclosed in an autoclave bag and incubated for 10 min at 37°C to reduce the background. To detect the emitted light, the membrane was exposed to X-ray films (Fuji), which were subsequently developed with an X-ray film processor. For hybridization with an additional DIG-labeled RNA-probe, the procedure was repeated starting at the pre-hybridization step.