Abstract
The yeast retroviruslike element Ty3 inserts at the transcription initiation sites of genes transcribed by RNA polymerase III (Pol III). An in vitro integration assay was developed with the use of Ty3 viruslike particles and a modified SUP2 tyrosine transfer RNA (tRNA(Tyr)) gene target. Integration was position-specific and required Ty3 integrase, Pol III transcription factor (TF) IIIB-, TFIIIC-, and Pol III-containing fractions showed that TFIIIB and TFIIIC, together, were sufficient for position-specific Ty3 integration, but not for transcription. This report demonstrates that in vitro integration of a retroelement can be targeted by cellular proteins.
Highlights
Title Requirement of RNA polymerase III transcription factors for in vitro position-specific integration of a retroviruslike element
Powered by the California Digital Library University of California to 4 weeks in medium supplemented with geneticin G418 [(Gibco-BRL, Gaithersburg, MD), at 400 ptg/ml for 1 week; 200 pg/ml, 2 to 3 weeks] and screened for human IKB-cx by immunoblotting
Virol. 67, 288 (1993)]; the mutants were cloned into PMT2T as 1 550-bp Eco RI fragments encoding fulllength IKB-oL. 11
Summary
Title Requirement of RNA polymerase III transcription factors for in vitro position-specific integration of a retroviruslike element. In vitro integration reactions were performed with the standard conditions as described [16] or with variations in order to ( 284 779 ) 1 2 3 4 5 6 7 8 9 10 identify essential reaction components 278-279 PCR with 2 ng of DNA; lower paneslu,g2g8e4s-t2e8d5that integration in vitro depended. Scription complex, as had been observed in for Ty3 integration immediately upstream mutations at the initiation site was competvivo [12] To test this directly, we intro- of the tRNA gene was formed when the itive as a target for integration in vivo [12]. Duced a mutation into the tDNA promoter. target was wild-type, but not when it was
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