Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.
American Association for Cancer Research

Site Tools

  • AAAS
  • Subscribe
  • Feedback

Site Search

Search Advanced

Science 21 July 2000:
Vol. 289. no. 5478, pp. 452 - 457
DOI: 10.1126/science.289.5478.452
Prev | Table of Contents | Next

Reports

Group II Introns Designed to Insert into Therapeutically Relevant DNA Target Sites in Human Cells

Huatao Guo,1 Michael Karberg,1 Meredith Long,2 J. P. Jones III,2 Bruce Sullenger,2 Alan M. Lambowitz1*

Mobile group II intron RNAs insert directly into DNA target sites and are then reverse-transcribed into genomic DNA by the associated intron-encoded protein. Target site recognition involves modifiable base-pairing interactions between the intron RNA and a >14-nucleotide region of the DNA target site, as well as fixed interactions between the protein and flanking regions. Here, we developed a highly efficient Escherichia coli genetic assay to determine detailed target site recognition rules for the Lactococcus lactis group II intron Ll.LtrB and to select introns that insert into desired target sites. Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retargeted to insert efficiently into virtually any target DNA and that the retargeted introns retain activity in human cells. This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy.

1 Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas, Austin, TX 78712, USA.
2 Center for Genetic and Cellular Therapies, Department of Surgery, Duke University Medical Center, Box 2601, Durham, NC 27710, USA.
*   To whom correspondence should be addressed. E-mail: lambowitz{at}mail.utexas.edu

Read the Full Text


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
The S.ma.I2 class C group II intron inserts at integron attC sites.
C. Quiroga, P. H. Roy, and D. Centron (2008)
Microbiology 154, 1341-1353
   Abstract »    Full Text »    PDF »
Gene Targeting in Gram-Negative Bacteria by Use of a Mobile Group II Intron ("Targetron") Expressed from a Broad-Host-Range Vector.
J. Yao and A. M. Lambowitz (2007)
Appl. Envir. Microbiol. 73, 2735-2743
   Abstract »    Full Text »    PDF »
Dispersion of the RmInt1 group II intron in the Sinorhizobium meliloti genome upon acquisition by conjugative transfer.
R. Nisa-Martinez, J. I. Jimenez-Zurdo, F. Martinez-Abarca, E. Munoz-Adelantado, and N. Toro (2007)
Nucleic Acids Res. 35, 214-222
   Abstract »    Full Text »    PDF »
Restriction for gene insertion within the Lactococcus lactis Ll.LtrB group II intron.
I. Plante and B. Cousineau (2006)
RNA 12, 1980-1992
   Abstract »    Full Text »    PDF »
Multicopy Integration of Heterologous Genes, Using the Lactococcal Group II Intron Targeted to Bacterial Insertion Sequences.
H. Rawsthorne, K. N. Turner, and D. A. Mills (2006)
Appl. Envir. Microbiol. 72, 6088-6093
   Abstract »    Full Text »    PDF »
Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing.
J. Yao, J. Zhong, Y. Fang, E. Geisinger, R. P. Novick, and A. M. Lambowitz (2006)
RNA 12, 1271-1281
   Abstract »    Full Text »    PDF »
Profile of Alan M. Lambowitz.
N. Zagorski (2006)
PNAS 103, 1669-1671
   Full Text »    PDF »
Inaugural Article: A bacterial group II intron-encoded reverse transcriptase localizes to cellular poles.
J. Zhao and A. M. Lambowitz (2005)
PNAS 102, 16133-16140
   Abstract »    Full Text »    PDF »
Construction of an Alpha Toxin Gene Knockout Mutant of Clostridium perfringens Type A by Use of a Mobile Group II Intron.
Y. Chen, B. A. McClane, D. J. Fisher, J. I. Rood, and P. Gupta (2005)
Appl. Envir. Microbiol. 71, 7542-7547
   Abstract »    Full Text »    PDF »
Recruitment of host functions suggests a repair pathway for late steps in group II intron retrohoming.
D. Smith, J. Zhong, M. Matsuura, A. M. Lambowitz, and M. Belfort (2005)
Genes & Dev. 19, 2477-2487
   Abstract »    Full Text »    PDF »
Gene targeting using randomly inserted group II introns (targetrons) recovered from an Escherichia coli gene disruption library.
J. Yao, J. Zhong, and A. M. Lambowitz (2005)
Nucleic Acids Res. 33, 3351-3362
   Abstract »    Full Text »    PDF »
Quantitative Analysis of Group II Intron Expression and Splicing in Lactococcus lactis.
Y. Chen, J. R. Klein, L. L. McKay, and G. M. Dunny (2005)
Appl. Envir. Microbiol. 71, 2576-2586
   Abstract »    Full Text »    PDF »
High-affinity binding site for a group II intron-encoded reverse transcriptase/maturase within a stem-loop structure in the intron RNA.
K. WATANABE and A. M. LAMBOWITZ (2004)
RNA 10, 1433-1443
   Abstract »    Full Text »    PDF »
In vivo selection reveals combinatorial controls that define a critical exon in the spinal muscular atrophy genes.
N. N. SINGH, E. J. ANDROPHY, and R. N. SINGH (2004)
RNA 10, 1291-1305
   Abstract »    Full Text »    PDF »
Conserved Target for Group II Intron Insertion in Relaxase Genes of Conjugative Elements of Gram-Positive Bacteria.
J. H. Staddon, E. M. Bryan, D. A. Manias, and G. M. Dunny (2004)
J. Bacteriol. 186, 2393-2401
   Abstract »    Full Text »    PDF »
A Conjugation-Based System for Genetic Analysis of Group II Intron Splicing in Lactococcus lactis.
J. R. Klein, Y. Chen, D. A. Manias, J. Zhuo, L. Zhou, C. L. Peebles, and G. M. Dunny (2004)
J. Bacteriol. 186, 1991-1998
   Abstract »    Full Text »    PDF »
Intracellular expression of engineered RNase P ribozymes effectively blocks gene expression and replication of human cytomegalovirus.
K. KIM, S. UMAMOTO, P. TRANG, R. HAI, and F. LIU (2004)
RNA 10, 438-447
   Abstract »    Full Text »    PDF »
Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker.
J. Zhong, M. Karberg, and A. M. Lambowitz (2003)
Nucleic Acids Res. 31, 1656-1664
   Abstract »    Full Text »    PDF »
Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection.
C. L. Frazier, J. San Filippo, A. M. Lambowitz, and D. A. Mills (2003)
Appl. Envir. Microbiol. 69, 1121-1128
   Abstract »    Full Text »    PDF »
Group II introns as phylogenetic tools: structure, function, and evolutionary constraints.
S. A. Kelchner (2002)
Am. J. Botany 89, 1651-1669
   Abstract »    Full Text »    PDF »
Compilation and analysis of group II intron insertions in bacterial genomes: evidence for retroelement behavior.
L. Dai and S. Zimmerly (2002)
Nucleic Acids Res. 30, 1091-1102
   Abstract »    Full Text »    PDF »
Effective inhibition of herpes simplex virus 1 gene expression and growth by engineered RNase P ribozyme.
P. Trang, J. Lee, A. F. Kilani, J. Kim, and F. Liu (2001)
Nucleic Acids Res. 29, 5071-5078
   Abstract »    Full Text »    PDF »
Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility.
B. S. Chevalier and B. L. Stoddard (2001)
Nucleic Acids Res. 29, 3757-3774
   Abstract »    Full Text »    PDF »
The Biotin Switch Method for the Detection of S-Nitrosylated Proteins.
S. R. Jaffrey and S. H. Snyder (2001)
Sci. STKE 2001, pl1
   Abstract »    Full Text »    PDF »
Phylogenetic relationships among group II intron ORFs.
S. Zimmerly, G. Hausner, and X.-c. Wu (2001)
Nucleic Acids Res. 29, 1238-1250
   Abstract »    Full Text »    PDF »


ADVERTISEMENT
Click Me!

ADVERTISEMENT

To Advertise     Find Products

Science. ISSN 0036-8075 (print), 1095-9203 (online)