ABSTRACT
Hybridization of labeled low molecular weight (LMW) nuclear RNA's to pre-mRNA from rabbit non-matured erythroid bone marrow cells or globin mRNA from reticulocytes revealed three RNA species having approximately 90, 100 and 160 nucleotides which are were specifically hybridized with purified cytoplasmic globin messenger RNA, while one (100 nucleotides) was also hybridized with rabbit 18S rRNA. The identity of these rabbit RNAs to LMW RNAs described for other animal species, as well as their possible hybridization sites and function are discussed.
Subject(s)
Bone Marrow/analysis , Globins/genetics , Nucleic Acid Hybridization , Nucleic Acid Precursors/analysis , RNA, Messenger/analysis , RNA/analysis , Animals , Base Sequence , Bone Marrow Cells , Chromatography, Agarose , Electrophoresis, Polyacrylamide Gel , Erythroblasts/analysis , In Vitro Techniques , Nucleic Acid Conformation , RNA Precursors , RNA Splicing , RNA, Small Nuclear , RabbitsABSTRACT
Inverse transcriptase of bird myeloblastosis virus is a unique instrument for artificial synthesis of structural genes of viruses, plants, animals. Methods for the virus production in preparative amounts are developed due to selection of the corresponding line of chickens, conditions of their maintenance, diet infection methods and myeloblastosis diagnostics. Main demands to the inverse transcriptase preparations (their high activity, absence of nuclease impurities, high concentration of the enzyme preparation solutions and their stability in storage) are ensured by zonal centrifugation purification of the virus in a sucrose density gradient, described methods of inverse transcriptase isolation and purification as well as conditions of its storage.
Subject(s)
Avian Leukosis Virus/enzymology , Avian Myeloblastosis Virus/enzymology , RNA-Directed DNA Polymerase/isolation & purification , Animals , Avian Leukosis/diagnosis , Avian Sarcoma Viruses/enzymology , Chickens , Chromatography, DEAE-Cellulose , Escherichia coli/enzymology , Leukemia, Experimental/microbiology , Mice , Microbiological Techniques , Phosphorylation , RNA-Directed DNA Polymerase/metabolism , Rauscher Virus/enzymologyABSTRACT
cDNA synthesized on rabbit bone marrow erythroid cells pre-mRNA was cloned in bacterial plasmids. Cold phenol extracted pre-mRNA was a several times more effective template in the reaction of reverse transcription without oligo(dT) 10-primer than hot phenol extracted pre-mRNA. There was no yield increase of DNA-product on hot phenol extracted pre-mRNA in the reaction of reverse transcription with the oligo(dT)10-primer addition. The "hot phenol" poly (A)+-pre-mRNA was used to obtain the representative, full-sized cDNA. The double-stranded form of this cDNA, obtained with the help of DNA-polymerase I, was inserted into the PstI-site of pBR322 plasmid. About 25% E. coli JC5183 clones, transformed with this hybrid plasmid, were found to contain the globin sequences.
