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Vol. 11, No. 21,
pp. 2910-2924,
November 1, 1997
1 Departments of Molecular Genetics, Biochemistry, and Medical Biochemistry, The Ohio State University, Columbus, Ohio 43210-1292 USA; 2 New York State Department of Health, Albany, New York 12201-0509 USA; 3 Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455-0312 USA
The Lactococcus lactis group II intron Ll.ltrB is similar to mobile yeast mtDNA group II introns, which encode reverse transcriptase, RNA maturase, and DNA endonuclease activities for site-specific DNA insertion. Here, we show that the Lactococcal intron can be expressed and spliced efficiently in Escherichia coli. The intron-encoded protein LtrA has reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II intron-encoded protein. As for the yeast mtDNA introns, the DNA endonuclease activity of the Lactococcal intron is associated with RNP particles containing both the intron-encoded protein and the excised intron RNA. Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cleaves the antisense strand. The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA in E. coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA. Furthermore, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component. Expression in E. coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.
[Key Words: Key Words: Gene therapy; intron mobility; Lactococcus lactis; retrotransposition; ribozyme]
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