Recovery of recombinant bacterial plasmids from E. coli transformed with DNA from microinjected mouse cells.
We have previously described the isolation of thymidine kinase positive (TK+), human beta-globin gene-containing colonies following co-microinjection of mouse TK- L cells with two recombinant pBR322 plasmids, one containing the TK gene of herpes simplex virus type I (plasmid pXl), and the second containing a human genomic DNA fragment within which is the human beta-globin gene (plasmid pRKl). DNA isolated from one such clone was used in bacterial transformation experiments with a selection for tetracycline-resistant colonies (that is, for cells containing pRKl). A total of forty-two tetracycline-resistant colonies were isolated, thirty of which contained circular pRK1 molecules identical to those originally injected. The remaining twelve colonies contained unique plasmids that were grouped into five different classes of recombinant molecules. All five of these unique recombinant classes appear to contain a common deletion endpoint occurring at a specific region of the pBR322 segment of pRKl. Four of the unique recombinant classes appear to have arisen from the deletion of a segment of a pRKl trimer or dimer molecule, while the fifth class appears to have resulted from recombination between pRKl and pXl followed by a deletion event within this recombinant. It is uncertain whether these deletions are occurring within the eukaryotic cell or upon subsequent transformation of the bacterial cell. If the latter, then the passage of the plasmid DNA through the eukaryotic cell alters a specific site of the pBR322 DNA in such a way that deletions can occur at a high frequency in this region when the plasmid DNA is introduced back into a bacterial cell. Thus, we have established a prokaryote-eukaryote-prokaryote DNA transfer and recovery system which should be useful in studies on DNA replication and the regulation of gene expression in higher eukaryotes.
D000818:Animals; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005914:Globins / Q000235:genetics; D006801:Humans; D007739:L Cells / Q000378:metabolism; D051379:Mice; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D013937:Thymidine Kinase / Q000172:deficiency; D014169:Transformation, Bacterial
D004274:DNA, Recombinant; D005914:Globins; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes