Protocol 1: Yeast splicing extracts preparation
Extract preparation for in vitro pre-mRNA splicing. YPD cultures of S. cerevisiae mutant strain 3.2 A1D [MATa, prp2-1, ade2, his3, lys2-801, ura, first described in 19991] were grown at 26°C until they reached late logarithmic phase (A600 of 3-5). 2 mls of the protease inhibitor phenylmethyl sulfonyl fluoride [0.5M PMSF] per liter of culture were added and the culture was allowed to grow for another 30 min.
Cells were collected by centrifugation at room temperature for 10 min at 7000 (3800 x g) in a Sorvall GS3 rotor. The wet weight of the cells (30g) were measured and this value was used for the rest of the extract preparation procedure. Pellets were re-suspended in 150mls of TE Buffer [50mM Tris-HCl (pH 8.0), 0.2mM EDTA (pH 8.0)] at 50mls per 10g of cells.
Cells were collected by centrifugation at room temperature for 6 min at 6000 (2800 x g) in a GS3 rotor. Pellets were then re-suspended in 90mls of SB30 buffer [1M Sorbitol, 50mM Tris-HCl (pH 7.8), 10mM MgCl2, 30mM DTT] at 30 mls per 10g of cells. Cells were collected by centrifugation at room temperature for 6 min at 6000 rpm (3900 x g) in a Sorvall GSA rotor. Pellets were next re-suspended in 60mls of SB3 buffer [1M Sorbitol, 50mM Tris-HCl (pH 7.8), 10mM MgCl2, 3mM DTT] at 20mls per 10g of cells. 10ul of suspended cells were added to 1ml of 10% SDS and the A800 was measured.
The suspended cells were treated with the supernatant from the addition of 9 mg of zymolyase (from ICN, TM-100T)in 600ul of zymolyase buffer [20mM KPO4 (pH 7.8), 5% glucose] at 3 mg and 200ul per 10g of cells, respectively. The cells were shaken at 30°C for 75 min at 120 rpm and then the A800 was re-measured by adding 10ul of the zymolyase-treated cell suspension plus 1ml of 10% SDS. Before proceeding, the A800 of the suspension must decrease by >80% from the initial absorbance reading.
Zymolyase (from Seikagaku) treated cells, called spheroblasts, were collected by centrifugation at room temperature for 5 min at 3000 rpm in a GSA rotor. The pellets were gently re-suspended in 90mls of SB3 containing 0.5mM PMSF at 30mls per 10g of cells. The spheroblasts were collected at room temperature for 5 min at 3000 rpm in a GSA rotor. Spheroblast pellets were chilled on ice for 30 min.
Pellets were re-suspended in 30mls of buffer A containing protease inhibitors [10mM HEPES (pH 7.8), 1.5mM MgCl2, 10mM KCl, 0.5mM DTT, 1uM leupeptin, 1uM pepstatin, 1um benzamdine, 1uM PMSF] at 10mls per 10g of cells. Re-suspended pellets were poured into a dounce glass homogenizer (type B) and lysed by 14 hard strokes [7 up and 7 down], with 2 min between each pair of strokes to allow heat to dissipate.
The homogenate volume was measured and transferred to a beaker and 1/10 volume of KCl (0.2M) was added dropwise. The solution was stirred on ice for 30 min and then centrifuged at 4°C for 30 min at 17,000 rpm (22,500 x g) in a Sorvall SS-34 rotor. The supernatant was collected and centrifuged at 4°C for 60 min at 37,000 rpm (140,000 x g) in a Beckman 60Ti rotor. The clear middle layer was collected by a pasteur pipette. The top (lipid-rich) and bottom (cellular debris-rich) layers were discarded. The extract was frozen with liquid N2 and stored at -80°C.
The next day the frozen extract was thawed under tap water. The extract was placed in a Spectra/Por 1 dialysis tubing (6000-8000 MWCO) and dialyzed against 1L of buffer D [20mM HEPES (pH 7.8), 20% glycerol, 50mM KCl, 0.2mM EDTA (pH 8), 0.5mM DTT, 0.5mM PMSF] at 4°C, for 3 hrs. The tubing was inverted every 20 min and the buffer was changed after 1 and 2 hrs. The dialyzed mixture was centrifuged at 4°C for 30 min at 17,000 rpm (22,500 x g) in a Sorvall SS-34 rotor. The supernatant was carefully aliquoted into 1.5ml microcentrifuge tubes. The dialyzed extract was frozen in liquid N2 and stored at -80°C