Transposon Tn7 gene insertion into an evolutionarily conserved human homolog of Escherichia coli attTn7.
Escherichia coli transposon Tn7 can integrate into its target DNA sequence, attTn7 at the 3' end of glmS, with high specificity and efficiency. Remarkably, the insertional recognition sequence in the E. coli genome displays a high degree of identity with the corresponding region at the 3' end of the corresponding human gene for glutamine-fructose-6-phosphate transaminase (GFPT), located at 2p13. It was therefore of interest to determine whether Tn7 could recognize the corresponding human sequence, and transpose at that site. Strains of E. coli DH5alpha were prepared carrying the tnsA-E genes on one plasmid, and attTn7 or the human equivalent on a second recipient plasmid within the alpha-complementation fragment of the lacZ gene. Each strain was transformed with a donor plasmid carrying a gentamycin resistance gene within the Tn7L and Tn7R cassettes. Restriction mapping and sequence analysis of recipient plasmids isolated from white colonies demonstrated that Tn7 inserted the gentamycin resistance gene both into the E. coli attTn7 sequence, and into its human counterpart. No nonspecific insertion was observed in a control plasmid containing only the lacZ fragment. These results provide a basis to investigate whether TnsA-D proteins can mediate gene insertion into comparably conserved sites in eukaryotic chromosomes.
Cleaver
S H
SH
Department of Microbiology and Immunology, Kimmel Cancer Center, and Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Wickstrom
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E
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004251:DNA Transposable Elements / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D019143:Evolution, Molecular; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); C000731191:component S, glutamate mutase protein, Bacteria