Increase of specificity of RNase from Bacillus amyloliquefaciens (barnase) by substitution of Glu for Ser57 using site-directed mutagenesis.

Bacterial ribonucleases from Bacillus amyloliquefaciens and Bacillus intermedius show the specificity towards the nature of a nucleoside at the O3' end of the phosphodiester bond to be split in the preference order G > A >> U > C in the cleavage reactions of polynucleotides. It follows from the X-ray data that the substrate guanosine base is bound at the active site of these RNases in the same manner as for high-specificity guanylic RNases. We supposed that the difference in specificity for the two types of RNases is due to the additional hydrogen bond between the protein and a purine base in the case of bacterial guanyl-preferring RNases in contrast to the high-specificity guanylic RNases.

[The isolation of highly purified bacillary ribonucleases from recombinant strains of Escherichia coli].

Active and inactive highly purified extracellular alkaline ribonucleases (barnase) from recombinant Escherichia coli have been prepared according to the industrial technology of Bacillus intermedius ribonuclease purification. Electrophoretic parameters and ultraviolet spectra characterize these preparations as homogeneous proteins. Immunochemical test system of identification of inactive RNAse for its purification has been worked out.

[The cloning and expression of the RNAse structural gene of Bacillus intermedius].

The structure gene of extracellular alkaline ribonuclease Bacillus intermedius (binase) has been cloned in E. coli cells in composition of pMT 316 plasmid carrying the inhibitor gene (barstar of barnase--binase structure homologue. The possibility to use such vector has been proved during the barstar action on binase catalytic activity.

[Isolation of mRNA from protoplasts of spore-forming bacteria and study of its translation products].

Total RNA was isolated from the protoplasts of Bacillus intermedius 7P which actively produced alkaline exocellular RNAse. A fraction of polyadenylated RNA was isolated from this RNA using chromatography on poly(U) Sepharose. The total RNA and poly(A) +RNA were translated in Xenopus laevis oocytes.

[Isolation and study of polyadenylated RNA from Bacillus intermedius].

A poly(A)+RNA fraction was isolated from the overall RNA of Bacillus intermedius using chromatography on poly(U) Sepharose and was shown to be electrophoretically heterogeneous. The presence of a polyadenylate segment was confirmed by hybridization with polyuridine. The biological activity of the poly(A)+RNA was proved by the translation in Xenopus laevis oocytes.