Streptolysin O-permeabilized cell system for studying trans-acting activities of exogenous nuclear proteins.
Efficient transfer of exogenous proteins into culture animal cells can be achieved by Streptolysin O (SLO) permeabilization of plasma membranes. We used this method to establish an in vitro transcription system for early response genes. The promoters of many early response genes contain an essential DNA motif known as the Serum Response Element (SRE). Recent data has shown that this DNA sequence is recognized by Serum Response Factor (SRF) and its associated proteins. Our initial experiments showed that HeLa nuclear extracts induced the transcription of the c-fos gene in serum-starved murine fibroblasts which were permeabilized by either physical method (glass beads) or cytolytic pore-forming protein (SLO). Plasma membrane permeabilization presumably permits passive diffusion of macromolecules into target cells and we showed that a truncated SRF expressed in bacteria was translocated into the nucleus within 30 minutes after SLO permeabilization. HeLa crude extracts were fractionated in order to identify the active nuclear factors. SRF was purified by binding to Wheat Germ Agglutinin (WGA)-agarose but the active factors remained in the WGA-unbound fractions. Our results demonstrate that this permeabilized cell in vitro transcription system is simple, efficient and can be used to test crude nuclear fractions as well as purified proteins expressed in bacteria; it will be an useful tool for the reproduction of transcriptional regulation on chromatin templates in vitro as well as the investigation of the biochemical functions of specific transcription factors or signal transduction effectors.
Liu
S H
SH
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, China.
Chu
J C
JC
Ng
S Y
SY
D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D015152:Blotting, Northern; D002463:Cell Membrane Permeability; D004247:DNA; D016762:Genes, fos; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D013301:Streptolysins / Q000494:pharmacology*; D015534:Trans-Activators / Q000494:pharmacology*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D009687:Nuclear Proteins; D013301:Streptolysins; D015534:Trans-Activators; C025964:streptolysin O; D004247:DNA