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Nucleic Acids Research, 1990, Vol. 18, No. 22 6677-6682
© 1990

MOLECULAR BIOLOGY

Derepression of a mouse {alpha}-fetoprotein expression vector in COS-1 cells by amplification of specific cis-acting sequences of the AFP promoter

J.P. Rabek, P.R. Hoyt+, D-E. Zhang, M.G. Izban§ and J. Papaconstantinou*

Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch Galveston, TX 77550, USA

* To whom correspondence should be addressed

Received March 1, 1990. Revised September 24, 1990. Accepted September 24, 1990.

The existence of trans-acting regulatory factors has been demonstrated by in vivo competition with cis-acting sequences from both viral and eukaryotic genomes. Plasmids containing a functional SV40 origin of replication when transfected into permissive SV40 T-antigen producing COS-1 cells will amplify to high copy numbers (5,000 to 10,000) without inflicting toxic effects upon the host cell. This amplification vector (pSVori) has been used to amplify cis-acting regulatory elements which can act as competitors for positive and negative trans-acting factors in vivo. Using this amplification system we conducted experiments to determine whether amplification of {alpha}-fetoprotein (AFP) and albumin cis-acting promoter sequences could activate a corresponding co-transfected AFP-promoter-CAT or Alb-promoter-CAT expression vector in COS-1 cells. We used pMoMLV(–1009)AFPcat, or p(–308)Albcat-MoMLV as reporter genes and pSVori to amplify specific promoter sequences of the AFP or albumin promoter. Our experiments indicated that amplification of a region from – 53 to – 202 of the AFP promoter resulted in the activation of the pMoMLV(–1009)AFPcat and p( – 308)Albcat-MoMLV expression vectors in COS-1 cells. Surprisingly, amplification of the albumin promoter sequences failed to activate either the pMoMLV(–1009)AFPcat or p(–308)Albcat-MoMLV plasmids.

+Present addresses: Oak Ridge National Laboratory, Biology Division, PO Box Y, Oak Ridge, TN 37830

§Present addresses: University of Cincinnati, College of Medicine, ML522, 231 Bethesda Ave, Cincinnati, OH 45267, USA


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