Poly(Adenosine Diphosphate Ribose) in Wheat

The wheat seed used was stored at 4 f 1 "C until required and before use was washed with approx. 2 litres of distilled water per lOOg of seed. The seeds were germinated in the dark on moist filter papers a t 20"C+ 1°C. The extraction of chromatin from the ungerminated seeds was based on that of Simon & Becker (1976). Approx. 25g of dry seed was washed, then homogenized in approx. lOOml of extraction buffer 1 [0.05~-Tris/HCI (pH 8.1)/15m~-p-mercaptoethanol/0.5 % (v/v) Triton X-1001 for IOmin in a Sorval homogenizer. The homogenate was filtered through two layers of muslin, the retained material was washed with 20ml of extraction buffer 1 and then stirred for lOmin at 4"+1"C. All subsequent steps were carried out a t this temperature. The filtered homogenate was centrifuged a t 4000g for 5min, the precipitate was discarded and a small volume of ~M-(NH,),SO, was added, sufficient t o make the supernatant 0 . 0 5 ~ with respect t o (NH4)$304. The supernatant was recentrifuged at lOOOOg for IOmin. The resulting supernatant was discarded. The pellet was resuspended in 50ml of extraction buffer 2 [0.05 M-Tris/HCI (pH8. I ) / ] 5 r n ~ p mercaptoethanol/0.05~-(NH,),S0~/0.5 % (v/v) Triton X-1001 by using a Teflon pestle; the resulting homogenate was centrifuged at lOOOOg for IOmin, and the pellet made up in 50ml of extraction buffer 3 [0.01 M-Tris/HCI (pH 8.1)/15 mM-D-mercaptoethanol/ 2m~-MgC1,]. This was recentrifuged at IOOOOg for lOmin and the final pellet made up in a small volume of incubation buffer [0.1 M-Tris/HCI (pH 8.2)/0.06~-KC1/0.01 M-