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 intronencoded
protein (IEP). My dissertation focuses on two mobile group II introns:
Lactococcus lactis Ll.LtrB and Escherichia coli EcI5, which belong to structural
subclasses IIA and CL/IIB1, respectively. Previous studies showed that the Ll.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 Ll.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 sitespecific
Tx1 integrations by simple screening. To facilitate screening for embryos with
targeted integrations in protein-encoding genes, I constructed an intron carrying a GFPRAM
(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. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/6654 |
| Date | 23 October 2009 |
| Creators | Zhuang, Fanglei |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Format | electronic |
| Rights | Copyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works. |
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