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Serine integrase chimeras with activity in E. coli and HeLa cells.

Farruggio AP, Calos MP - Biol Open (2014)

Bottom Line: The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms.Our work is the first to demonstrate chimeric serine integrase activity.This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5120, USA.

No MeSH data available.


Related in: MedlinePlus

Chimeric att-sites: organization and nomenclature.(A) Overview of parental and hybrid att-site structure. Serine integrase att-sites consist of a dinucleotide core (black bar) that is flanked by two half-sites. Checkered patterns are used here to indicate attB half-sites; solid colors are used for attP. Drawings are not to scale. See the text for an explanation of our hybrid att-site naming system. (B) Detailed overview of parental and hybrid attB seqences. Mismatches in the B0–B3 and B0–B6 alignments are underlined. CbC B0 and TcT B0 are equivalent to the phiC31 and TG1 attB sites, respectively. (C) Detailed overview of parental and hybrid attP seqences. Mismatches in the P0–P3 and P0–P6 alignments are underlined. CbC P0 and TcT P0 are equivalent to the phiC31 and TG1 attP sites, respectively. In (B) and (C), only a central 26 nucleotide window is shown; see supplementary material Table S2 for the full sequences of all att-sites used in our study.
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f03: Chimeric att-sites: organization and nomenclature.(A) Overview of parental and hybrid att-site structure. Serine integrase att-sites consist of a dinucleotide core (black bar) that is flanked by two half-sites. Checkered patterns are used here to indicate attB half-sites; solid colors are used for attP. Drawings are not to scale. See the text for an explanation of our hybrid att-site naming system. (B) Detailed overview of parental and hybrid attB seqences. Mismatches in the B0–B3 and B0–B6 alignments are underlined. CbC B0 and TcT B0 are equivalent to the phiC31 and TG1 attB sites, respectively. (C) Detailed overview of parental and hybrid attP seqences. Mismatches in the P0–P3 and P0–P6 alignments are underlined. CbC P0 and TcT P0 are equivalent to the phiC31 and TG1 attP sites, respectively. In (B) and (C), only a central 26 nucleotide window is shown; see supplementary material Table S2 for the full sequences of all att-sites used in our study.

Mentions: Chimeric att-sites were named using the format “WvW QR”. In this scheme, W and v refer to the source of the outer and inner att-site sequence, respectively (Fig. 3A). Parental sources are indicated using the same ‘B’, ‘C’ and ‘T’ code established for integrase hybrids (phiBT1, phiC31 and TG1, respectively). Q indicates the type of att-site; the letters ‘P’ and ‘B’ are used to specify attP and attB, respectively. R is the number of “core” bases in each W half-site that have been derived from the integrase v site (Fig. 3A–C). The dinucleotide crossover bases are not included in the tally for R. Thus, “CbC B3” is a phiC31-phiBT1-phiC31 hybrid attB sequence where the three core bases in each half-site have been taken from phiBT1 attB, and all remaining bases are from phiC31 attB (supplementary material Table S2; Fig. 3B). All “P0” and “B0” sites have wild-type half-sites and a ‘TT’ dinucleotide crossover core. All hybrid att-site sequences have been provided in supplementary material Table S2.


Serine integrase chimeras with activity in E. coli and HeLa cells.

Farruggio AP, Calos MP - Biol Open (2014)

Chimeric att-sites: organization and nomenclature.(A) Overview of parental and hybrid att-site structure. Serine integrase att-sites consist of a dinucleotide core (black bar) that is flanked by two half-sites. Checkered patterns are used here to indicate attB half-sites; solid colors are used for attP. Drawings are not to scale. See the text for an explanation of our hybrid att-site naming system. (B) Detailed overview of parental and hybrid attB seqences. Mismatches in the B0–B3 and B0–B6 alignments are underlined. CbC B0 and TcT B0 are equivalent to the phiC31 and TG1 attB sites, respectively. (C) Detailed overview of parental and hybrid attP seqences. Mismatches in the P0–P3 and P0–P6 alignments are underlined. CbC P0 and TcT P0 are equivalent to the phiC31 and TG1 attP sites, respectively. In (B) and (C), only a central 26 nucleotide window is shown; see supplementary material Table S2 for the full sequences of all att-sites used in our study.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197438&req=5

f03: Chimeric att-sites: organization and nomenclature.(A) Overview of parental and hybrid att-site structure. Serine integrase att-sites consist of a dinucleotide core (black bar) that is flanked by two half-sites. Checkered patterns are used here to indicate attB half-sites; solid colors are used for attP. Drawings are not to scale. See the text for an explanation of our hybrid att-site naming system. (B) Detailed overview of parental and hybrid attB seqences. Mismatches in the B0–B3 and B0–B6 alignments are underlined. CbC B0 and TcT B0 are equivalent to the phiC31 and TG1 attB sites, respectively. (C) Detailed overview of parental and hybrid attP seqences. Mismatches in the P0–P3 and P0–P6 alignments are underlined. CbC P0 and TcT P0 are equivalent to the phiC31 and TG1 attP sites, respectively. In (B) and (C), only a central 26 nucleotide window is shown; see supplementary material Table S2 for the full sequences of all att-sites used in our study.
Mentions: Chimeric att-sites were named using the format “WvW QR”. In this scheme, W and v refer to the source of the outer and inner att-site sequence, respectively (Fig. 3A). Parental sources are indicated using the same ‘B’, ‘C’ and ‘T’ code established for integrase hybrids (phiBT1, phiC31 and TG1, respectively). Q indicates the type of att-site; the letters ‘P’ and ‘B’ are used to specify attP and attB, respectively. R is the number of “core” bases in each W half-site that have been derived from the integrase v site (Fig. 3A–C). The dinucleotide crossover bases are not included in the tally for R. Thus, “CbC B3” is a phiC31-phiBT1-phiC31 hybrid attB sequence where the three core bases in each half-site have been taken from phiBT1 attB, and all remaining bases are from phiC31 attB (supplementary material Table S2; Fig. 3B). All “P0” and “B0” sites have wild-type half-sites and a ‘TT’ dinucleotide crossover core. All hybrid att-site sequences have been provided in supplementary material Table S2.

Bottom Line: The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms.Our work is the first to demonstrate chimeric serine integrase activity.This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5120, USA.

No MeSH data available.


Related in: MedlinePlus