RESUMO
Two-stage synthesis of double-stranded DNA was performed using purified rat transferrin mRNA as a template, reverse transcriptase and DNA polymerase I. Double-stranded transcripts of transferrin mRNA were cloned as recombinant plasmid derivatives of pBR322. The insert length in these plasmids varied from 150 to 1500 bp. Clones carrying transferrin mRNA sequences were identified using colony hybridization and Southern blot hybridization with 32P-cDNA probe. Nick-translated DNAs from transformed clones hybridized with a single component of rat liver polysomal RNA that corresponded to transferrin mRNA in its molecular weight (0.86 MD). In hybridization selection cell-free translation test cloned plasmid DNAs hybridized specifically with rat liver poly(A) +RNA that programmed the cell-free synthesis of a polypeptide identical to pretransferrin in antigenic properties and molecular weight.
Assuntos
Clonagem Molecular , DNA/biossíntese , RNA Mensageiro/genética , DNA Polimerase Dirigida por RNA/genética , Transferrina/genética , Animais , Sequência de Bases , DNA/genética , Enzimas de Restrição do DNA/genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/genética , Escherichia coli/genética , Hibridização de Ácido Nucleico , Plasmídeos , Biossíntese de Proteínas , Ratos , Recombinação GenéticaRESUMO
Highly purified transferrin mRNA was isolated from rat liver using indirect immunoprecipitation of polysomes with antibodies to rat transferrin and poly(U)-sepharose chromatography. Isolated transferrin mRNA was apparently homogeneous in sedimentation and electrophoretic experiments. Its sedimentation coefficient is 20S and molecular weight 925 000 (chain length 2800 nucleotides). The purified mRNA programmed the synthesis of electrophoretically homogeneous precursor of transferrin. The cell-free translation of transferrin mRNA was highly sensitive to the inhibition by cap analogues (pm7G) that seems being indicative of the capped structure of its 5' end. Hybridization of transferrin mRNA with [3H]poly(U) revealed the discrete length distribution of poly(A) sequences in mRNA (96, 45 and 26 mononucleotides). The proportion of double-stranded (nuclease SI-resistant) regions in transferrin mRNA is as high as 50-60%. Transferrin cDNA was synthesized via the reverse transcription of transferrin mRNA with oligo(dT) primer. This cDNA hydridized with mRNA template and with total polysomal RNA at C0t1/2 values 1.5 X 10(-3) and 3.6 X 10(0) mol nucleotides X 1(-1) Xs, respectively. Hence, the purified mRNA preparation is 2500-fold enriched with transferrin-coding sequences in comparison to the total polysomal RNA from rat liver.
Assuntos
Fígado/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Transferrina/genética , Animais , Sequência de Bases , DNA/metabolismo , Cinética , Peso Molecular , Hibridização de Ácido Nucleico , Polirribossomos/metabolismo , RNA Mensageiro/isolamento & purificação , RatosRESUMO
Detailed study of the obligate-thermophilic aerobic spore-forming bacterium, indentified earlier as Bacillus megaterium, allowed to describe it as a new species--Bacillus thermocatenulatus nov. sp. This organism is characterized by a high content of GC in DNA (69 mole percent).
