Rapid rescue of cellular transcriptional activator elements by amplification of a single copy selection gene.
Cellular transcriptional activator sequences from a Syrian hamster cell line (baby hamster kidney (BHK] were rescued by a double selection procedure. An enhancer-deficient SV40 promoter was linked to the neomycin resistance (NEO) gene and transfected into BHK cells. Genomic DNA fragments of G418-resistant cell clones containing multiple copies of integrated plasmid DNAs were used for a second transfection of BHK cells, resulting in the genomic integration of a single copy plasmid which expresses the NEO gene efficiently. For rapid cloning of the integrated promoter and adjacent cellular DNA sequences, these cell clones were fused to COS-1 cells, thereby providing SV40 large T antigen and the monkey cell permissive factor necessary for SV40 replication. Resulting from this fusion, the integrated plasmid and adjacent sequences were amplified to about 1000 extrachromosomal copies giving rise to an abundant pool of promoter elements which thus can be cloned into a plasmid very easily for further investigations. Promoter analyses of three clones in the chloramphenicol acetyltransferase transient expression assay demonstrated that the recombination with cellular DNA enables the initially defective SV40 promoter to express at wild type levels.
Garbrecht
S
S
Institute of Biochemistry, Munich, Federal Republic of Germany.
Kruczek
I
I
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004742:Enhancer Elements, Genetic; D005784:Gene Amplification; D005786:Gene Expression Regulation; D008647:Mesocricetus; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection
D015345:Oligonucleotide Probes; D014157:Transcription Factors; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase