ABSTRACT
Covalent complexes of nucleic acids and proteins are widespread among viruses. Covalent complexes of RNA and proteins are proposed to exist in eukaryotic cells. The goal of this work was to obtain specific antibodies to the covalent linkage unit (CLU) between virus RNA and protein to search cellular RNA-protein complexes. Antibodies were generated by direct immunization of a rabbit with the BSA-coupled EMC virus RNA-VPg complex. By a dot-blot immunoassay and immunofluorescent microscopy it was found that the antibodies specifically recognize both EMC virus RNA-VPg and synthetic CLU-containing compounds. Thus, a fraction of the antibodies was directed to CLU.
Subject(s)
Antibodies/immunology , Encephalomyocarditis virus/chemistry , Oligoribonucleotides/immunology , Tyrosine/immunology , Viral Core Proteins/immunology , Animals , Antibodies/metabolism , CHO Cells/cytology , CHO Cells/immunology , CHO Cells/virology , Cricetinae , Encephalomyocarditis virus/immunology , Fluorescent Antibody Technique , Immunoblotting , Phosphotyrosine/analogs & derivatives , Phosphotyrosine/immunology , RNA, Viral/immunology , RNA, Viral/metabolism , Serum Albumin/immunology , Tyrosine/metabolism , Viral Proteins/immunology , Viral Proteins/metabolismSubject(s)
Cloning, Molecular/methods , Genetic Vectors , Bacteriophage lambda , Lac Operon , Restriction MappingABSTRACT
A modified procedure in two versions (micro, for 10 ml of phage lysate, and macro, for 200-500 ml) is described for preparing lambda phage DNA. The main advantage of the modified method is that it gives a possibility to isolate high-quality DNA from lambda phage lysates in 2-3 hrs. Only standard solutions (TE, NaCl, SDS, MgCl2, EDTA, RNAse A) were used throughout the whole protocol. Incubation with DNAse I and proteinase K was omitted and in microvariant concentration of the phage by PEG 6000 was excluded. Digestion by RNAse A was performed in solution with EDTA and SDS and leads to RNA degradation. The yields of DNA (0.5-2 micrograms per ml of L-broth) are similar to those obtained by other methods. DNA quality is better than in the samples of DNA prepared by other express-methods and practically the same as after CsCl centrifugation. DNA can be used for splitting by restriction enzymes, cloning and gene library construction.