1. The reaction of site-specific cleavage of tRNA at a 7-methylguanine residue, including subsequent treatment with sodium borohydride and aniline [Wintermeyer, W. and Zachau, H. G. (1975) FEBS Lett. 58,306-3091, was shown to work only within a certain range of tRNA concentrations (higher than 30 pM). The Escherichia coli 16s rRNA, which contained a unique m7G (position 527), could not be split by this method when taken at any concentration.2. It was found that the presence of statistically methylated carrier RNA in the reaction mixture at the borohydride stage significantly stimulates site-specific fragmentation of 16s rRNA and 32P-labeled tRNAs.3. Direct sequencing proved that 16s rRNA and tRNAs are cleaved by this procedure successfully at the m7G residue.4. The E. coli 16s rRNA was preparatively cleaved by the described procedure into two fragments. The 5'-terminal fragment (1 -526) and the 3'-terminal fragment (528 -1542) were isolated in the pure form and their secondary structure investigated by the circular dichroism method. The results of this study showed that the secondary and tertiary structures of the 5'-terminal one-third of the 16s rRNA are at least as ordered as those of intact 16s rRNA or its 3'4erminal two-thirds.The site-specific fragmentation of a polynucleotide chain is widely used for the study of the structure and functions of RNA and ribonucleoprotein complexes. Apart from the enzymatic approaches El-41, the chemical methods of splitting native RNAs at modified bases hold promise. Previously Zachau and co-workers described the procedure for splitting the yeast tRNAPhe at some minor nucleotides [5-71. The procedure of tRNA cleavage at a 7-methylguanine residue was based on reducing this base with sodium borohydride and subsequently splitting the polynucleotide chain at the reduced base with aniline.However, we have not been successful in our attempts to make direct use of this method for splitting the Escherichiu coli 16s rRNA at the unique 7-methylguanine residue or the 3'-terminally labeled yeast [32P]tRNAPhe. A closer study of the reaction has shown that the addition of the methylated RNA-carrier (methyl-RNA) at the borohydride reduction stage considerably enhances the efficiency of the reaction and makes it possible to cleave site-specifically 16s rRNA or tRNA with a 50-70% yield. Using this method we could cleave relatively large amounts of E. coli 16s rRNA at the unique residue of 7-methylguanine at position 527. A 526-nucleotide-long 5'-terminal fragment and a 1 01 5-nucleotidelong 3'-terminal fragment have been isolated and their secondary structure investigated by the circular dichroism (CD) method. MATERIALS AND METHODSThe 16s rRNA was isolated by phenol extraction from the 30s ribosomal subunits of Escherichia coli (strain MRE600).Abbreviations. Methyl-RNA, carrier RNA statistically methylated with dimethylsulfate; CD, circular dichroism.The phenylalanine and valine tRNA from yeast were kindly provided by Dr T.V. Venkstern. The RNase H preparation from E. coli was furnished by...

No abstract

No abstract

No abstract

The study of enzymatic and protective properties of recombinant IgA1 protease in active and mutant form showed that active form of IgA1 protease exhibited species – and type-specificity for mouse and human immunoglobulins. Mutant form, which did not exhibit enzymatic activity, had protective properties against meningococcal infection, induced by meningococcus serogroup A, B and C protecting the mice from lethal infection by living virulent culture of heterologous serogroups of meningococcus. Obtained results make it possible to consider IgA1 protease as a perspective preparation at the stages of development of polyvalent vaccine for protection the people from meningococcal infection of various etiology