Eukaryotic promoters drive gene expression in Escherichia coli

T K Antonucci,P Wen,W J Rutter

doi:10.1016/s0021-9258(19)84621-5

T K Antonucci, P Wen + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(19)84621-5

Copy DOI

Abstract

Eight eukaryotic promoters have been tested for their activity in vivo in Escherichia coli. The rat beta-actin, rat amylase, rat chymotrypsin B, mouse metallothionein I, rat insulin I, human insulin, Rous sarcoma virus long terminal repeat (RSV LTR) and hepatitis B viral precore promoter activities were measured by using the bacterial chloramphenicol acetyltransferase coding sequences as the reporter function and by primer extension RNA analysis. All eight promoter-chloramphenicol acetyltransferase constructs produce chloramphenicol acetyltransferase activity with the following relative strengths: RSV LTR greater than rat beta-actin greater than rat insulin I greater than rat amylase greater than hepatitis B virus precore greater than human insulin greater than rat chymotrypsin B greater than mouse metallothionein I. A primer extension analysis indicates that transcription from the RSV LTR, rat insulin I, and rat beta-actin promoters initiates at the sites expected for eukaryotic rather than prokaryotic promoters. Thus the site of initiation is determined by the DNA sequence rather than by the RNA polymerase.

Highlights

Actin, rat amylase, rat chymotrypsin B, mouse metallothionein I, rat insulin I, human insulin, Rous sarcoma virus long terminal repeat (RSV LTR) andhepatitis B viral precore promoter activities were measured by using the bacterial chloramphenicol acetyltransferase coding sequences as the reporter function and by sin B, rat insulin I, human insulin, anmdouse metallothionein I promoters are all compared to the powerful E. coli trp/lac (TAC) fusion promoter
Bacterial Strains-The plasmids pTAC-chloramphenicol acetyltransferase (CAT) and pRSV-CAwTere expressed in E. coli strain JMlO1(1)while all of the plasmids carrying mammalian or viral promoters were expressed in strain HBlOl [1]
A primer extension analysisindicates that theShine-Dalgarno ribosome bindingsite [9] andthe coding setranscription from the RSV LTR,rat insulin I, and rat &actin promoters initiatesat the sites expected for eukaryotic rather than prokaryotic promoters

Summary

Introduction

Actin, rat amylase, rat chymotrypsin B, mouse metallothionein I, rat insulin I, human insulin, Rous sarcoma virus long terminal repeat (RSV LTR) andhepatitis B viral precore promoter activities were measured by using the bacterial chloramphenicol acetyltransferase coding sequences as the reporter function and by sin B, rat insulin I, human insulin, anmdouse metallothionein I promoters are all compared to the powerful E. coli trp/lac (TAC) fusion promoter. Bacterial Strains-The plasmids pTAC-CAT and pRSV-CAwTere expressed in E. coli strain JMlO1(1)while all of the plasmids carrying mammalian or viral promoters were expressed in strain HBlOl [1]. The extract erally believed that eukaryotic promoters do not function in expressing plasmid prINS-CAT (100 ng of protein) was incubated for prokaryotes [1]and vice versa.

Results

Conclusion