In vivo production of oligodeoxyribonucleotides of specific sequences: application to antisense DNA.
Retrons, bacterial retroelements found in Gram-negative bacteria, are integrated into the bacterial genome expressing a reverse transcriptase related to eukaryotic reverse transcriptase. The bacterial reverse transcriptases are responsible for the production of multicopy, single-stranded (ms) DNA consisting of a short single-stranded DNA that is attached to an internal guanosine residue of an RNA molecule by a 2',5'-phosphodiester linkage. Reverse transcriptases use an RNA transcript from the retrons, not only as primer, but also as template for msDNA synthesis. By studying the structural requirement, it was found that for msDNA synthesis an internal region of msDNA can be replaced with other sequences. msDNA can thus be used as a vector for in vivo production of an oligodeoxyribonucleotide of a specific sequence. Artificial msDNAs containing a sequence complementary to part of the mRNA for the major outer membrane lipoprotein of Escherichia coli effectively inhibited lipoprotein biosynthesis upon induction of msDNA synthesis. This is the first demonstration of in vivo synthesis of oligodeoxyribonucleotides having antisense function. Since we have previously demonstrated that bacterial retrons are functional in eukaryotes producing msDNA in yeast and in mouse NIH/3T3 fibroblasts, the present system may also be used to produce a specific oligodeoxyribonucleotide inside the cells to regulate eukaryotic gene expression artificially. We also describe a method to produce cDNA to a specific cellular mRNA using the retron system.
Inouye
M
M
Robert Wood Johnson Medical School, Department of Biochemistry, Piscataway, NJ 08854-5635, USA.
Mao
J R
JR
Shimamoto
T
T
Inouye
S
S
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D002352:Carrier Proteins; D016373:DNA, Antisense / Q000096:biosynthesis*; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D029968:Escherichia coli Proteins; D015870:Gene Expression; D008074:Lipoproteins; D051379:Mice; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000096:biosynthesis*
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D016373:DNA, Antisense; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C099932:Lpp protein, E coli; D016376:Oligonucleotides, Antisense