New procedure for the direct analysis of in vitro reverse transcription of Rous sarcoma virus RNA.

Based on the observation that in vitro transcription of Rous sarcoma virus (RSV) RNA by avian myeloblastosis virus DNA polymerase renders the RNA PROGRESSIVELY MORE SENSITIVE TO Escherichia coli RNase H digestion, a new procedure for the in situ analysis of this process has been developed. In vitro transcription products of 32P-labeled RSV RNA are first treated with RNase H, the resistant fraction is then digested to completion with RNase T1, and the oligonucleotides are analyzed by a fingerprint technique. By using the established order of these oligonucleotides along the RNA molecule, a comparison of the yields of each oligonucleotide, before and after transcription, allows qualitative and quantitative in situ analyses of the transcription process.

Existence and possible roles of transcriptional barriers in T7 DNA early region as shown by electron microscopy.

Transcription of T7 DNA in vitro by Escherichia coli RNA polymerase in the absence or presence of termination factor rho has been studied extensively using electron microscopy. It is demonstrated that sequences located between the early genes behave as transient transcriptional barriers in the absence of rho and are recognised both as rho-dependent terminators and as RNaseIII cleavage sites.

Stimultaneous purification of Escherichia coli termination factor rho, RNAase III and RNAase H.

This communication describes a method for the stimultaneous purification of Escherichia coli termination factor rho, RNAase III and RNAase H, which is rapid, reproducible and high in yield. Depending on how cells are grown 0.5 to 1 mg of RNAase III, 1 to 2 mg of RNAase H and 1 to 2 mg of rho are obtained from 100 g wet cells.