Purification of Neurospora crassa endonuclease-resistant DNA-RNA hybrid.

Abstract

Denatured calf thymus DNA has been transcribed in vitro with RNA polymerase of Micrococcus lysodeikticus. The DNA–RNA hybrid which formed in this system was found to be highly resistant to a purified Neurospora crassa endonuclease, while the unhybridized DNA and RNA were rapidly degraded. DNA–RNA hybrid with a purity of 62–75% was isolated in one step from the digest in 69–86% yield by chromatography on methylated albumin – kieselguhr. Further purification was obtained by density gradient centrifugation in Cs2SO4 solution. The purified hybrid had a buoyant density of 1.49 g cm−3 in Cs2SO4, intermediate between that of mouse tRNA marker (1.63 g cm−3) and calf thymus DNA (1.43 g cm−3). During sucrose density gradient centrifugation, the hybrid sedimented slightly faster than mouse tRNA marker. Evidence for the double-stranded nature of the hybrid was found by thermal denaturation of the complex. The RNA of the hybrid became increasingly sensitive to ribonuclease as the hybrid was heated at successively higher temperatures.