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Book Summary:
Mobile group II introns use a mobility mechanism termed retrohoming in which the excised intron RNA inserts directly into a double-stranded DNA target site and is then reverse transcribed by the intron-encoded reverse transcriptase. The DNA target site for most group II introns includes â?¼30 nucleotides surrounding the intron insertion site, with a â?¼15 nucleotide region recognized by base pairing to the intron RNA. Alteration of the base-pairing interaction permits retargeting of the intron into virtually any desired DNA target. Here, I present the detailed base pairing requirements of the Ll.LtrB intron with its DNA target, including the minimal requirements for protein recognition. I developed a procedure for using retargeted group II introns for highly efficient chromosomal gene disruption in Escherichia coli and other bacteria. The retargeted introns can also be used to introduce targeted double-strand breaks, which can be repaired by cotransformation with a homologous DNA fragment, enabling the introduction of point mutations into chromosomal genes without the addition of a selectable marker. I investigate the in vivo splicing and mobility characteristics of a number of other group II introns. Those originally identified in Pylaiella littoralis and the Archaen Methanosarcina acetivorans were found to splice in vivo, while an intron identified in E. coli, and two in Novosphingobium aromaticivorans showed both splicing and mobility. I present the DNA target-site requirements of significantly mobile introns from E. coli and N. aromaticivorans, and their potential applications in biotechnology and genetic engineering are discussed. |
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