Neurospora Crassa Endonuclease Research Articles

Nucleotide 'maps' of digests of deoxyribonucleic acid.

Various digests of (32)P-labelled DNA were examined by two-dimensional ionophoresis on cellulose acetate and DEAE-cellulose paper. The products from digestion with pancreatic deoxyribonuclease and Neurospora crassa endonuclease were qualitatively closely similar, but very complex, and were used to investigate the mapping behaviour of nucleotides in various ionophoretic systems. Ionophoresis on DEAE-cellulose paper in triethylamine carbonate, pH 9.7, followed by ionophoresis in the second dimension at pH1.9 gave high resolution of nucleotides in very complex mixtures and permitted the fractionation of larger quantities than is possible on cellulose acetate. High resolution of nucleotides in compact spots was obtained with two-dimensional ionophoresis on cellulose acetate and AE-cellulose paper, a system that is a useful supplement to those based on DEAE-cellulose paper.

Isolation of Escherichia coli transfer RNA-gene hybrids

Escherichia coli transfer RNA-DNA hybrids have been isolated from a hybridization complex prepared by hybridization of homologous transfer RNA and DNA in solution. The RNA-DNA hybrid was released from reannealed and unhybridized DNA by Neurospora crassa endonuclease and purified by methylated albumin kieselguhr chromatography, cesium sulfate density-gradient centrifugation and Sephadex G100 chromatography. The purified transfer RNA-gene hybrid had a molecular weight of 50,000 to 60,000, estimated by gel filtration, a density in cesium sulfate of 1.500 g/cm 3, a T m of 81 °C in 0.3 m-saline and a ratio of DNA to RNA of 1.0.

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

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.

Isolation of Neurospora crassa endonuclease specific for single-stranded DNA.

A method for the preparation from Neurospora crassa conidia of an endonuclease specific for single-stranded DNA is described. The enzyme was recovered from sonicates of conidia in high yield (51%) and was remarkably stable.