Site-specific recombination in human cells catalyzed by the wild-type integrase protein of coliphage HK022

Background. Telomeres in Drosophila melanogaster are similar to those of other eukaryotes in terms of their function, although they are formed by non-LTR retrotransposons instead of telomerase-based short repeats. The length of the telomeres in Drosophila depends on the number of copies of these transposable elements. A dominant mutation, Tel1, causes a several-fold elongation of telomeres. Methods. In this study we identified the Tel1 mutation by a combination of transposon-induced, site-specific recombination and next generation sequencing. Results. Recombination located Tel1 to a 15 kb region in 92A. Comparison of the DNA sequence in this region with the Drosophila Genetic Reference Panel of wild type genomic sequences delimited Tel1 to a 3 bp deletion inside intron 8 of Ino80. Discussion. The mapped Tel1 mutation (3-bp deletion found in Ino80) did not appear to affect the quantity or length of the Ino80 transcript. Tel1 causes a significant reduction in transcripts of CG18493, a gene nested in an intron 8 of Ino80, which is expressed in ovaries and expected to encode a serine-type peptidase.

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Characterization of Holliday structures in FLP protein-promoted site-specific recombination

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.235-242.1990 ◽

1990 ◽

Vol 10 (1) ◽

pp. 235-242

Author(s):

L Meyer-Leon ◽

R B Inman ◽

M M Cox

Keyword(s):

Reaction Rate ◽

Reaction Pathway ◽

Reaction Intermediates ◽

Wild Type ◽

Site Specific ◽

Site Specific Recombination ◽

Isomerization Process ◽

Previous Demonstration ◽

Rate Limiting

Holliday structures are formed in the course of FLP protein-promoted site-specific recombination. Here, we demonstrate that Holliday structures are formed in reactions involving wild-type substrates and that they are kinetically competent with respect to the overall reaction rate. Together with a previous demonstration of chemical competence (L. Meyer-Leon, L.-C. Huang, S. W. Umlauf, M. M. Cox, and R. B. Inman, Mol. Cell. Biol. 8:3784-3796, 1988), Holliday structures therefore meet all criteria necessary to establish that they are obligate reaction intermediates in FLP-mediated site-specific recombination. In addition, kinetic evidence suggests that two distinct forms of the Holliday intermediate are present in the reaction pathway, interconverted in an isomerization process that is rate limiting at 0 degree C.

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Site-Specific Integrative Elements of Rhizobiophage 16-3 Can Integrate into Proline tRNA (CGG) Genes in Different Bacterial Genera

Journal of Bacteriology ◽

10.1128/jb.184.1.177-182.2002 ◽

2002 ◽

Vol 184 (1) ◽

pp. 177-182 ◽

Cited By ~ 21

Author(s):

Szabolcs Semsey ◽

Béla Blaha ◽

Krisztián Köles ◽

László Orosz ◽

Péter P. Papp

Keyword(s):

Rhizobium Meliloti ◽

Trna Genes ◽

Target Sequence ◽

Integrase Gene ◽

Site Specific ◽

Site Specific Recombination ◽

Tyrosine Recombinases ◽

Recombination System ◽

Integrase Protein ◽

Site Specific Recombination System

ABSTRACT The integrase protein of the Rhizobium meliloti 41 phage 16-3 has been classified as a member of the Int family of tyrosine recombinases. The site-specific recombination system of the phage belongs to the group in which the target site of integration (attB) is within a tRNA gene. Since tRNA genes are conserved, we expected that the target sequence of the site-specific recombination system of the 16-3 phage could occur in other species and integration could take place if the required putative host factors were also provided by the targeted cells. Here we report that a plasmid (pSEM167) carrying the attP element and the integrase gene (int) of the phage can integrate into the chromosomes of R. meliloti 1021 and eight other species. In all cases integration occurred at so-far-unidentified, putative proline tRNA (CGG) genes, indicating the possibility of their common origin. Multiple alignment of the sequences suggested that the location of the att core was different from that expected previously. The minimal attB was identified as a 23-bp sequence corresponding to the anticodon arm of the tRNA.

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Site-specific recombination in human cells catalyzed by phage λ integrase mutants 1 1E. Lorbach and N. Christ contributed equally to this work. 2 2Edited by M.Yaniv

Journal of Molecular Biology ◽

10.1006/jmbi.2000.3532 ◽

2000 ◽

Vol 296 (5) ◽

pp. 1175-1181 ◽

Cited By ~ 30

Author(s):

Elke Lorbach ◽

Nicole Christ ◽

Micha Schwikardi ◽

Peter Dröge

Keyword(s):

Human Cells ◽

Site Specific ◽

Site Specific Recombination

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Phage TP901-1 Site-Specific Integrase Functions in Human Cells

Journal of Bacteriology ◽

10.1128/jb.184.13.3657-3663.2002 ◽

2002 ◽

Vol 184 (13) ◽

pp. 3657-3663 ◽

Cited By ~ 53

Author(s):

Stephanie M. Stoll ◽

Daniel S. Ginsburg ◽

Michele P. Calos

Keyword(s):

Mammalian Cells ◽

Time Course ◽

Human Cells ◽

Partial Purification ◽

Site Specific ◽

E Coli ◽

Phage Integrase ◽

Mammalian Genomes ◽

Integrase Protein

ABSTRACT We demonstrate that the site-specific integrase encoded by phage TP901-1 of Lactococcus lactis subsp. cremoris has potential as a tool for engineering mammalian genomes. We constructed vectors that express this integrase in Escherichia coli and in mammalian cells and developed a simple plasmid assay to measure the frequency of intramolecular integration mediated by the integrase. We used the assay to document that the integrase functions efficiently in E. coli and determined that for complete reaction in E. coli, the minimal sizes of attB and attP are 31 and 50 bp, respectively. We carried out partial purification of TP901-1 integrase protein and demonstrated its functional activity in vitro in the absence of added cofactors, characterizing the time course and temperature optimum of the reaction. Finally, we showed that when expressed in human cells, the TP901-1 integrase carries out efficient intramolecular integration on a transfected plasmid substrate in the human cell environment. The TP901-1 phage integrase thus represents a new reagent for manipulating DNA in living mammalian cells.

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Analysis by Mutagenesis of a Chromosomal Integron Integrase from Shewanella amazonensis SB2BT

Journal of Bacteriology ◽

10.1128/jb.01537-08 ◽

2009 ◽

Vol 191 (6) ◽

pp. 1933-1940 ◽

Cited By ~ 2

Author(s):

André Larouche ◽

Paul H. Roy

Keyword(s):

Antibiotic Resistance Genes ◽

Cysteine Residue ◽

Wild Type ◽

Recombination Mechanism ◽

Integrase Gene ◽

Site Specific ◽

Site Specific Recombination ◽

Environmental Bacterium ◽

Target Sites ◽

A Site

ABSTRACT Integrons are mobile genetic elements that can integrate and disseminate genes as cassettes by a site-specific recombination mechanism. Integrons contain an integrase gene (intI) that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a cassette. The plasmid-specified IntI1 excises a greater variety of cassettes (principally antibiotic resistance genes), and has greater activity, than chromosomal integrases. The aim of this study was to analyze the capacity of the chromosomal integron integrase SamIntIA of the environmental bacterium Shewanella amazonensis SB2BT to excise various cassettes and to compare the properties of the wild type with those of mutants that substitute consensus residues of active integron integrases. We show that the SamIntIA integrase is very weakly active in the excision of various cassettes but that the V206R, V206K, and V206H substitutions increase its efficiency for the excision of cassettes. Our results also suggest that the cysteine residue in the β-5 strand is essential to the activity of Shewanella-type integrases, while the cysteine in the β-4 strand is less important for the excision activity.

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Characterization of Holliday structures in FLP protein-promoted site-specific recombination.

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.235 ◽

1990 ◽

Vol 10 (1) ◽

pp. 235-242 ◽

Cited By ~ 19

Author(s):

L Meyer-Leon ◽

R B Inman ◽

M M Cox

Keyword(s):

Reaction Rate ◽

Reaction Pathway ◽

Reaction Intermediates ◽

Wild Type ◽

Site Specific ◽

Site Specific Recombination ◽

Isomerization Process ◽

Previous Demonstration ◽

Rate Limiting

Holliday structures are formed in the course of FLP protein-promoted site-specific recombination. Here, we demonstrate that Holliday structures are formed in reactions involving wild-type substrates and that they are kinetically competent with respect to the overall reaction rate. Together with a previous demonstration of chemical competence (L. Meyer-Leon, L.-C. Huang, S. W. Umlauf, M. M. Cox, and R. B. Inman, Mol. Cell. Biol. 8:3784-3796, 1988), Holliday structures therefore meet all criteria necessary to establish that they are obligate reaction intermediates in FLP-mediated site-specific recombination. In addition, kinetic evidence suggests that two distinct forms of the Holliday intermediate are present in the reaction pathway, interconverted in an isomerization process that is rate limiting at 0 degree C.

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