Selection of DNA clones with enhancer sequences.
A method is described for selection of DNA clones that contain enhancer sequences that activate gene expression. An Escherichia coli-rodent cell shuttle vector, pPyE0, was used that contains polyoma viral DNA without the polyoma enhancer region. Replication of pPyE0 DNA in mouse cells is markedly reduced due to deletion of the polyoma enhancer region. Insertion of mouse genomic DNA fragments that contain putative enhancer sequences into pPyE0 adjacent to the polyoma origin of replication restored, to varying extents, the ability of the recombinant plasmid DNA to replicate in mouse cells. Recombinant plasmids that replicate well in mouse cells, therefore, are amplified selectively. Transfection of mouse neuroblastoma or fibroblast cells that constitutively synthesize polyoma large tumor antigen with a library of mouse genomic DNA fragments inserted in pPyE0 yielded many recombinant plasmids. DNA inserts from each of the 16 clones that were examined stimulated the expression of an enhancerless chloramphenicol acetyltransferase reporter gene. The DNA inserts from 4 clones that were studied resulted in 4- to 13-fold increases in chloramphenicol acetyltransferase mRNA in transfected mouse cells. Nucleotide sequence analysis led to the identification of 5 genomic DNA clones that were obtained by selection. All of the homologies found were to regions of DNA that are thought to be involved in the regulation of gene expression.
Asoh
S
S
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Lee-Kwon
W
W
Mouradian
M M
MM
Nirenberg
M
M
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004261:DNA Replication / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D014162:Transfection; D014407:Tumor Cells, Cultured
D000952:Antigens, Polyomavirus Transforming; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase