||Ohio State Univ., Columbus.;Environmental Protection Agency, Cincinnati, OH. Risk Reduction Engineering Lab.;Department of Energy, Washington, DC.;National Inst. on Aging, Bethesda, MD.
Polyadenylated (poly(A)+) RNA molecules have been isolated from Methanococcus vannielii. Approximately 16% of the label in RNA isolated from cultures allowed to incorporate (3)Huridine for 3 min at 37C was poly(A)+ RNA. Electrophoretic separation of poly(A)+ RNA molecules showed a heterogeneous population with mobilities indicative of sizes ranging from 900 to 3,000 bases in length. Polyadenylate (poly(A)) tracts were isolated by digestion with RNase A and RNase T1 after 3' end labeling of the poly(A)+ RNA with RNA ligase. The radioactively labeled poly(A) oligonucleotides were shown by electrophoresis through DNA sequencing gels to average 10 bases in length, with major components of 5, 9, 10, 11, and 12 bases. Poly(A)+ RNA molecules from M. vannielii were labeled at their 5' termini with T4 polynucleotide kinase after dephosphorylation with calf intestine alkaline phosphatase. Pretreatment of the RNA molecules with tobacco acid pyrophosphatase did not increase the amount of phosphate incorporated into poly(A)+ RNA molecules by polynucleotide kinase, indicating that the poly(A)+ molecules did not have modified bases (caps) at their 5' termini. (Copyright (c) 1985, American Society for Microbiology.)