Group II intron mobility and its gene targeting applications in prokaryotes and eukaryotes.

摘要

Mobile group II introns are retroelements that insert site-specifically into DNA target sites by a process called retrohoming. Retrohoming is mediated by a ribonucleoprotein particle (RNP) that contains both the intron RNA and the intron-encoded protein (IEP). My dissertation focuses on two mobile group II introns: Lactococcus lactis L1.LtrB and Escherichia coli EcI5, which belong to structural subclasses IIA and CL/IIB1, respectively. Previous studies showed that the L1.LtrB IEP, denoted LtrA protein, is pole localized in E. coli. First, I found that active LtrA protein is associated with E. coli membrane fractions, suggesting that LtrA pole localization might reflect association with a membrane receptor. Second, I found that EcI5 is highly active in retrohoming in E. coli and obtained a comprehensive view of its DNA target site recognition by selection experiments. I found that EcI5 recognizes DNA target sequences by using both the IEP and base pairing of the intron RNA, with the IEP having different target specificity than for other mobile group II introns. A computer algorithm based on the empirically determined DNA recognition rules enabled retargeting of EcI5 to integrate at ten different sites in the chromosomal lacZ gene at frequencies up to 98% without selection. Finally, I developed methods for gene targeting in the frog Xenopus laevis by using L1.LtrB RNPs for site-specific DNA modification in isolated sperm nuclei, followed by in vitro fertilization to generate genetically modified animals. The site-specific integrations were efficient enough to detect in fifty sperm nuclei for a multiple copy target site, the Tx1 transposon, and several hundred sperm nuclei for protein-encoding genes. Based on these results, I obtained transgenic tadpoles with site-specific Tx1 integrations by simple screening. To facilitate screening for embryos with targeted integrations in protein-encoding genes, I constructed an intron carrying a GFP-RAM (Retrotransposition-Activated Marker). By using this GFP-RAM with introns containing randomized sequences that base pair with the target DNA, I obtained tadpoles with intron integrations at different genomic locations, including protein-encoding genes. The methods for using group II introns for targeted sperm DNA modification in X. laevis may be applicable to other animals.