Blocked and unblocked 5'termini in vesicular stomatitis virus product RNA in vitro: their possible role in mRNA biosynthesis.
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RNA, Messenger↗
Biomedical subjects
R J Colonno
Publications and source records attributed to R J Colonno.
Synthesis of complementary RNA containing polyadenylic acid by Sendai virions in vitro.
Sendai virus synthesized, in vitro, [32P]AMP- and [3H]AMP-labeled RNA that ranged in size from 3 to 25S with major peaks at 7S and 13S. Both labeled products were predominantly single-stranded RNA and were complementary in base sequence to 50S virion RNA. Passage of the 3 to 25S in vitro RNA transcripts through a polyuridylic acid-cellulose column revealed that only the larger (predominantly 18S) RNA transcripts contained polyadenylic acid[poly(A)] segments capable of binding to the column. After treatment with a combination of RNase A and T1, the majority of the in vitro poly(A) sedimented at 6S although the product ranged in size from 3 to 9S. Proof that the RNase-resistant material was indeed poly(A) was obtained by nearest-neighbor analysis when 95% of the radioactivity was recovered in AMP.
Base Sequence↗
Isolation of a transcriptive complex from Newcastle disease virions.
An active transcriptive complex was isolated from purified virions of Newcastle disease virus. After disruption with Triton X-100 and high salt, soluble and particulate fractions were separated by density gradient centrifugation. The transcriptive complex, recovered at a density of 1.275 g/cm3, appeared as a nucleocapsid structure by electron microscopy. When analyzed by polyacryl-amide gel electrophoresis, the nucleocapsids consisted of the nucleocapsid protein, a minor protein of 53,000 molecular weight, and the large L protein. Nucleocapsids possessed less than 1% of the hemagglutinating and neuraminidase activities originally associated with virions. The active complex synthesized predominantly 11 to 20S RNA in vitro and approximately one-fourth of the RNA molecules contained polyadenylic acid segments. In the presence of S-adenosyl-L-methionine, the RNA molecules were capped and methylated at the 5' termini. The transcriptive complex was also capable of methylating exogenous Escherichia coli RNA in the absence of viral RNA synthesis.
Capsid↗
Methylation of messenger RNA of Newcastle disease virus in vitro by a virion-associated enzyme.
Purified Newcastle disease virus contains an enzyme that incorporates the methyl group from S-adenosyl-L-methionine into RNA synthesized in vitro by the virion-associated RNA polymerase (RNA nucleotidyltransferase). Incorporation of radioactivity from S-adenosyl-L-[methyl-3H]methionine was totally dependent upon RNA synthesis. The methylation reaction was completely inhibited by S-adenosyl-L-homocysteine, suggesting the transfer of only the methyl group of S-adenosyl-methionine to RNA products. Velocity sedimentation and hybridization of the in vitro product RNA indicated that both [3H]methyl and [32P]GMP labels resided in single-stranded 18S RNA molecules which were virus specific. Approximately 1 to 2 methyl groups were incorporated per RNA molecule. DEAE-cellulose chromatography of product RNA after alkaline hydrolysis suggested that the 5' terminus was the site of methylation.
Chromatography, DEAE-Cellulose↗