Derepression of a mouse alpha-fetoprotein expression vector in COS-1 cells by amplification of specific cis-acting sequences of the AFP promoter.
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.
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
J P
JP
Rabek
P R
PR
Hoyt
D E
DE
Zhang
M G
MG
Izban
J
J
Papaconstantinou
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D005784:Gene Amplification; D000882:Haplorhini; D051379:Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012709:Serum Albumin / Q000235:genetics; D000509:alpha-Fetoproteins / Q000096:biosynthesis / Q000235:genetics*
D012709:Serum Albumin; D000509:alpha-Fetoproteins