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PubMed1981

A method for isolation of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells.

When cytoplasmic polyadenylated ribonucleic acid [poly(A+)RNA] from HeLa cells was treated with ribonuclease H (RNase H) and oligodeoxythymidylate [oligo(dT)] to remove its 3'-poly(A) tail, an increased binding to poly(A)-agarose was observed. The bound material, which comprised 4-6% of the initial RNA, contained 65-80% of the oligo(uridylic acid) [oligo(U)] sequences generated by RNase T1 digestion. Oligo(U) isolated from the bound fraction was shown to be 83% U and to have a U/G ratio of 33. In contrast, oligo(U) from the unbound material was 77% U and had a U/G ratio of 13, suggesting that it is shorter and less U rich than the oligo(U) in the bound fraction. On sucrose gradients, oligo(U+)RNA consistently sedimented with a larger s value than oligo(U-) RNA. The oligo(U) content of oligo(U+) RNA suggests one oligo(U) tract of 33 nucleotides per RNA molecule of 2000-3000 residues.

Base Sequence↗
PubMed1981

Early steps in the path of nascent ribonucleic acid across the surface of ribonucleic acid polymerase, determined by photoaffinity labeling.

The photoaffinity probes beta-(4-azidophenyl) adenosine 5'-diphosphate (N3PhppA) and beta-(4-azidophenyl) adenylyl-(3'--5')-uridine 5'-diphosphate (N3PhppApU) were used to determine the RNA polymerase subunit contacts made by the 5' ends of three nascent RNA chains. Ternary enzyme-poly[d(A-T)].oligonucleotide complexes were prepared in which the nascent oligonucleotide contained a photoaffinity label at the 5' end and a 32P radiolabel only at the 3' end. The length of the RNA was fixed at two, three, or four nucleotides. Photolysis of the ternary complexes was followed by dissociation, polyacrylamide gel electrophoresis, autoradiography, and scintillation counting. With a dinucleotide probe, the enzyme subunits labeled were beta' (71%) and sigma (21%). Photolysis of the ternary complex containing trinucleotide RNA also resulted in labeling of the beta' (64%) and sigma (35%) subunits. With a tetranucleotide, the beta' subunit was very heavily labeled (88%), and a small amount of labeling of the beta (7%) and sigma (4%) subunits was observed. The alpha subunit was not labeled with any of the probes. These results imply that a conformational change, possibly involving dissociation of the sigma subunit, occurs in the enzyme as the ribonucleotide is elongated from a tri- to a tetranucleotide.

Adenosine Diphosphate↗
PubMed1980

The detection of messenger ribonucleic acid sequences in heterogeneous nuclear ribonucleic acid fractions of the oestrogen-stimulated rat uterus.

cDNA (complementary DNA) complementary to the abundant sequences of mRNA isolated from oestrogen-stimulated uterus was hybridized to polyadenylated and non-polyadenylated uterine hnRNA (heterogeneous nuclear RNA). High-molecular-weight polyadenylated hnRNA, isolated under denaturing conditions, was able to saturate the cDNA, but complementary sequences were low abundance when compared with homologous mRNA. The mRNA sequence content of the polyadenylated hnRNA increased considerably during oestrogen-induced growth of the uterus. Non-polyadenylated hnRNA also contained sequences complementary to the cDNA.

Animals↗