RIBONUCLEIC-ACID SYNTHESIS IN ISOLATED DROSOPHILA NUCLEI

We have developed a system for studying transcription in nuclei isolated from Drosophila melanogaster tissue culture cells. These nuclei synthesize RNA at the rate of 75-100 pmol/μg of DNA in 30 min. About 20% of the RNA synthesized is released from the nuclei, and both released and nonreleased RNAs have poly(A) on them. By use of mercury-agarose affinity chromatography and 5'-[γ-S]GTP or 5'-[γ-S]ATP precursors as an assay for initiation, it has been determined that approximately 16% of the radioacivity incorporated into RNA is in in vitro purine initiated RNAs. Alkaline hydrolysis of the initiated RNAs has confirmed that the sulfur is present at the 5' end of newly synthesized RNAs and is serving as an accurate probe for initiation. Some of the in vitro initiated RNAs also have poly(A) added to them. Sucrose gradient sedimentation of in vitro synthesized RNAs shows them to sediment in a broad peak centered at about 20 S, with RNA as large as 50 S detectable. The transcription system has been optimized for NaCl, Mg2+, and Mn2+ concentrations (100, 1.25, and 0.75 mM, respectively), and addition of BSA has been shown to stabilize the RNA once it has been synthesized. By using the inhibitors α-amanitin and rifampicin, we have been able to demonstrate that RNA polymerases la, lb, II, and III are active [in addition to the poly (A) polymerase], with enzymes lb and II accounting for about 80% of the synthesis in these nuclei. This system should prove useful for the study of transcriptional control mechanisms. © 1979, American Chemical Society. All rights reserved.