Open AccessEnzymic Purification of a Hybrid between Ribosomal Ribonucleic Acid and Deoxyribonucleic Acid
Author(s) -
Spadari Silvio,
Sgaramella Vittorio,
Mazza Giorgio,
Falaschi Arturo
Publication year - 1971
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1971.tb01318.x
Subject(s) - dna , rna , chromatography , size exclusion chromatography , nitrocellulose , chemistry , bacillus subtilis , hydrolysis , ribosomal rna , sepharose , biochemistry , biology , bacteria , enzyme , membrane , gene , genetics
A procedure for the isolation of a pure RNA : DNA hybrid is described. The isolation was accomplished through the following steps:1 32 P‐labeled ribosomal RNA of Bacillus subtilis was hybridized with 3 H‐labeled DNA of the same organism; 2 the excess of unhybridized DNA was partially hydrolyzed with exonuclease I from Escherichia coli ; 3 the mixture was purified by gel‐filtration on a Sepharose 4B column, thus eliminating the products of hydrolysis and a considerable fraction of residual unhybridized DNA; 4 filtration through a nitrocellulose column freed the mixture of another portion of residual denatured DNA without loss of hybrid; 5 two successive CsCl gradients allowed the isolation of material having a DNA/RNA ratio very close to 1, to be compared to an original value of 250 at the beginning of the purification.The overall yield, in different runs, was of approximately 5%. The isolated hybrid does not adsorb to nitrocellulose filters, as expected for its double‐stranded structure. The densities of the pure hybrid in CsCl and Cs 2 SO 4 are, respectively, of 1.788 and 1.480 g/cm 3 . The molecular weight of the DNA moiety of the isolated hybrid is of approximately 50000. The procedure is highly reproducible and can be applied in principle to any hybrid for which a pure RNA is available.