| 1. |
- Ahlgren-Berg, Alexandra, et al.
(författare)
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A comparative analysis of the bifunctional Cox proteins of two heteroimmune P2-like phages with different host integration sites
- 2009
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Ingår i: Virology. - : Elsevier BV. - 0042-6822 .- 1096-0341. ; 385:2, s. 303-12
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Tidskriftsartikel (refereegranskat)abstract
- The Cox protein of the coliphage P2 is multifunctional; it acts as a transcriptional repressor of the Pc promoter, as a transcriptional activator of the P(LL) promoter of satellite phage P4, and as a directionality factor for site-specific recombination. The Cox proteins constitute a unique group of directionality factors since they couple the developmental switch with the integration or excision of the phage genome. In this work, the DNA binding characteristics of the Cox protein of WPhi, a P2-related phage, are compared with those of P2 Cox. P2 Cox has been shown to recognize a 9 bp sequence, repeated at least 6 times in different targets. In contrast to P2 Cox, WPhi Cox binds with a strong affinity to the early control region that contains an imperfect direct repeat of 12 nucleotides. The removal of one of the repeats has drastic effects on the capacity of WPhi to bind to the Pe-Pc region. Again in contrast to P2 Cox, WPhi Cox has a lower affinity to attP compared to the Pe-Pc region, and a repeat of 9 bp can be found that has 5 bp in common with the repeat in the Pe-Pc region. WPhi Cox, however, is essential for excisive recombination in vitro. WPhi Cox, like P2 Cox, binds cooperatively with integrase to attP. Both Cox proteins induce a strong bend in their DNA targets upon binding.
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| 2. |
- Cardoso-Palacios, Carlos, et al.
(författare)
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A structure-function analysis of P2 integrase
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Bacteriophage P2 integrase catalyzes site-specific recombination between the phage DNA and the host chromosome thereby promoting integration or excision of the phage genome. P2 integrase belongs to the large tyrosine family of integrases that shows little sequence identity besides some conserved boxes and patches in the catalytic domain. However, the overall structure of the tyrosine family of integrases seems to be similar. Phage integrases have the potential as tools for site-specific gene insertions into eukaryotic genomes provided that target sequences are available. To elucidate the possibility of evolving the P2 integrase to accept new targets, we have in this work initiated a structure-function analysis of the P2 integrase using two approaches based on a comparison of the predicted secondary structure of P2 integrase with that determined for the lambda integrase. First, we have made hybrids between P2 integrase and the related WΦ integrase that has a different host DNA target, to locate the region promoting specificity between the integrases. This, however, has not been possible, the N-terminal domains can be exchanged without losing biological activity and this will not affect the specificity. All other hybrids made were biological inactive. Next we have made an alanine scanning of the alpha helices believed to be involved in specific interactions with the target, and four amino acids have been identified as candidates for sequence-specific interactions with the core.
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| 3. |
- Carlsson Almlöf, Jonas, et al.
(författare)
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Novel risk genes for systemic lupus erythematosus predicted by random forest classification
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Genome-wide association studies have identified risk loci for SLE, but a large proportion of the genetic contribution to SLE still remains unexplained. To detect novel risk genes, and to predict an individual's SLE risk we designed a random forest classifier using SNP genotype data generated on the "Immunochip" from 1,160 patients with SLE and 2,711 controls. Using gene importance scores defined by the random forest classifier, we identified 15 potential novel risk genes for SLE. Of them 12 are associated with other autoimmune diseases than SLE, whereas three genes (ZNF804A, CDK1, and MANF) have not previously been associated with autoimmunity. Random forest classification also allowed prediction of patients at risk for lupus nephritis with an area under the curve of 0.94. By allele-specific gene expression analysis we detected cis-regulatory SNPs that affect the expression levels of six of the top 40 genes designed by the random forest analysis, indicating a regulatory role for the identified risk variants. The 40 top genes from the prediction were overrepresented for differential expression in B and T cells according to RNA-sequencing of samples from five healthy donors, with more frequent over-expression in B cells compared to T cells.
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| 4. |
- Frumerie, Clara, et al.
(författare)
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Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells
- 2008
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Ingår i: Journal of Applied Microbiology. - : Oxford University Press (OUP). - 1364-5072 .- 1365-2672. ; 105:1, s. 290-299
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Tidskriftsartikel (refereegranskat)abstract
- AIMS: To investigate if the site-specific tyrosine integrase (Int) from phage P2 has features that would make it interesting for use of gene transfer into eukaryotic cells. These include the possibility of promoting recombination with a nonphage sequence, abolishing the requirement for the bacterial DNA-binding and -bending protein integration host factor (IHF), and localization to the nucleus of eukaryotic cells. METHODS AND RESULTS: We show that the Int protein catalyzes site-specific recombination using a human sequence in Escherichia coli and in vitro although not as efficiently as with the wild-type bacterial sequence, and that insertion of high mobility group recognition boxes in the phage attachment site substrate abolish the requirement of IHF and allows efficient recombination in vitro in a eukaryotic cell extract. Furthermore, we show by fluorescence that the Int protein contains a functional intrinsic nuclear localization signal, localizing it to the nucleus in both HeLa and 293 cells. CONCLUSIONS: We conclude that P2 Int may be a potential tool for site-specific integration of genes into the human chromosome. SIGNIFICANCE AND IMPACT OF THE STUDY: The study implies the possibility of using multiple prokaryotic Int proteins with different specific integration sites in human cells for future gene therapy programmes.
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| 5. |
- Frumerie, Clara, et al.
(författare)
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Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox
- 2005
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Ingår i: Virology. - : Elsevier BV. - 0042-6822 .- 1096-0341. ; 332:1, s. 284-294
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Tidskriftsartikel (refereegranskat)abstract
- Bacteriophage P2 integrase (Int) mediates site-specific recombination leading to integration or excision of the phage genome in or out of the bacterial chromosome. Int belongs to the large family of tyrosine recombinases that have two different DNA recognition motifs binding to the arm and core sites, respectively, which are located within the phage attachment sites (attP). In addition to the P2 integrase, the accessory proteins Escherichia coli IHF and P2 Cox are needed for recombination. IHF is a structural protein needed for integration and excision by bending the DNA. As opposed to lambda, only one IHF site is found in P2 attP. P2 Cox controls the direction of recombination by inhibiting integration but being required for excision. In this work, the effects of accessory proteins on the capacity of Int to bind to its DNA recognition sequences are analyzed using electromobility shifts. P2 Int binds with low affinity to the arm site, and this binding is greatly enhanced by IHF. The arm binding domain of Int is located at the N-terminus. P2 Int binds with high affinity to the core site, and this binding is also enhanced by IHF. The fact that the cooperative binding of Int and IHF is strongly reduced by lengthening the distance between the IHF and core binding sites indicates that the distance between these sites may be important for cooperative binding. The Int and Cox proteins also bind cooperatively to attP.
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| 6. |
- Grunewald, Johan, et al.
(författare)
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T-cell receptor-HLA-DRB1 associations suggest specific antigens in pulmonary sarcoidosis
- 2016
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Ingår i: European Respiratory Journal. - : European Respiratory Society (ERS). - 0903-1936 .- 1399-3003. ; 47:3, s. 898-909
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Tidskriftsartikel (refereegranskat)abstract
- In pulmonary sarcoidosis, CD4(+) T-cells expressing T-cell receptor V alpha 2.3 accumulate in the lungs of HLA-DRB1*03(+) patients. To investigate T-cell receptor-HLA-DRB1*03 interactions underlying recognition of hitherto unknown antigens, we performed detailed analyses of T-cell receptor expression on bronchoalveolar lavage fluid CD4(+) T-cells from sarcoidosis patients. Pulmonary sarcoidosis patients (n=43) underwent bronchoscopy with bronchoalveolar lavage. T-cell receptor alpha and beta chains of CD4(+) T-cells were analysed by flow cytometry, DNA-sequenced, and three-dimensional molecular models of T-cell receptor-HLA-DRB1*03 complexes generated. Simultaneous expression of V alpha 2.3 with the V beta 22 chain was identified in the lungs of all HLA-DRB1*03(+) patients. Accumulated V alpha 2.3/V beta 22-expressing T-cells were highly clonal, with identical or near-identical V alpha 2.3 chain sequences and inter-patient similarities in V beta 22 chain amino acid distribution. Molecular modelling revealed specific T-cell receptor-HLA-DRB1*03-peptide interactions, with a previously identified, sarcoidosis-associated vimentin peptide, (Vim)(429-443) DSLPLVDTHSKRTLL, matching both the HLA peptide-binding cleft and distinct T-cell receptor features perfectly. We demonstrate, for the first time, the accumulation of large clonal populations of specific V alpha 2.3/V beta 22 T-cell receptor-expressing CD4(+) T-cells in the lungs of HLA-DRB1*03(+) sarcoidosis patients. Several distinct contact points between V alpha 2.3/V beta 22 receptors and HLA-DRB1*03 molecules suggest presentation of prototypic vimentin-derived peptides.
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| 7. |
- Mandali, Sridhar, et al.
(författare)
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Characterization of the site-specific recombination system of phage ΦD145, and its capacity to promote recombination in human cells
- 2010
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Ingår i: Virology. - : Elsevier BV. - 0042-6822 .- 1096-0341. ; 408:1, s. 64-70
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Tidskriftsartikel (refereegranskat)abstract
- Phage integrases have the potential of becoming tools for safe site-specific integration of genes into unmodified human genomes. The P2-like phages have been found to have different bacterial host integration sites and consequently they have related integrases with different sequence specificities. In this work the site-specific recombination system of the P2-like phage ΦD145 is characterized. The minimal attB site is determined to 22 nt with 18 nt identity to the core region of attP. A non-coding sequence on the human chromosome 13 is shown to be a rather good substrate for recombination in vivo in bacteria as well as in a plasmid system in HeLa cells when HMG protein recognition sequences are inserted between the left arm-binding site and the core in the complex phage attachment site attP. Thus ΦD145 integrase that belongs to the tyrosine family shows potential as a tool for site-specific integration into the human genome.
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| 8. |
- Sylwan, Lina, et al.
(författare)
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Identification of bases required for P2 integrase core binding and recombination
- 2010
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Ingår i: Virology. - : Elsevier. - 0042-6822 .- 1096-0341. ; 404, s. 240-245
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Tidskriftsartikel (refereegranskat)abstract
- Temperate coliphage P2 integrates its genome into the host chromosome upon lysogenization via a site-specific recombination event mediated by an integrase belonging to the complex family of tyrosine recombinases. The host integration site attB (BOB′) is localized in the end of the cyaR gene and shares 27 nucleotides with the core of attP (COC′). In the present study we determine the minimal attB site using an in vivo recombination assay. Ten nt on the left side (B) are found to be nonessential for recombination. We show that the integrase has higher affinity for the right side (B′) compared to B and that artificial B′OB′ and an attP site with a matching core (C′OC′) are efficient substrates for recombination in vitro. We have analyzed single nucleotides in attB and find that sequence homology within a non-centrally located quadruplet in the hypothetical overlap region is essential for efficient recombination in vivo.
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| 9. |
- Sylwan, Lina, et al.
(författare)
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Identification of bases required for P2 Integrase core-binding and recombination
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Temperate coliphage P2 integrates its genome into the host chromosome upon lysogenization via a site-specific recombination event mediated by an integrase belonging to the complex family of tyrosine recombinases. The host integration site attB (BOB´) is localized in the end of the cyaR gene and share 27 nucleotides with the core of attP (COC´). In the present study we determine the minimal attB site using an in vivo recombination assay and 10 nt on the left side (B) are found to be nonessential for recombination. We show that the integrase has higher affinity for the right side (B´) compared to B and that artificial B´OB´ and an attP site with a matching core (C´OC´) are efficient substrates for recombination in vitro. We have analyzed single nucleotides in attB and find that sequence homology within a non-centrally located quadruplet in the hypotetical overlap region is essential for efficient recombination in vivo.
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| 10. |
- Sylwan, Lina, 1975-
(författare)
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Site-Specific Recombination : Integrases, Accessory Factors and DNA Targets of P2-like Coliphages
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The temperate coliphage P2 and its family members integrate their genomes into the host Escherichia coli chromosome by a site-specific recombination mechanism to form lysogeny. Integration takes place between the complex phage attP site and the simple bacterial attB site and is catalyzed by the phage encoded integrase (Int). Similar to the archetype λ Int, the P2-like phage integrases are heterobivalent tyrosine recombinases which possess the ability to simultaneously bind two different and distant types of DNA sequences within the attP region. To bridge the core and the flanking arm-binding sites in attP, the integrase requires the assistance of accessory factors that bend the DNA; the host encoded IHF and the phage encoded Cox protein. Cox acts as a directionality factor by being required for integration but is inhibitory for the excisive reaction. The purpose of this doctoral thesis has been to gain a more detailed knowledge of the site-specific recombination systems of phages P2 and WΦ, which are close relatives but integrate into different host targets. The future aim is to develop these systems for targeted integration into the genome of higher eukaryotes. The P2 Int and an N-terminal truncation of the integrase were shown to bind cooperatively together with IHF or Cox to the DNA targets, however the N-truncated protein lost its ability to bind to the arm sequence. WΦ Cox was shown to bind cooperatively with WΦ Int to attP whereas the opposite was evident for WΦ Cox and IHF. The 27 nucleotides that are identical between the core and attB of phage P2 were investigated for their importance in binding and recombination. The right part of the core was shown to be the primary Int binding site where one single base substitution was shown to abolish P2 Int binding and recombination. An alanine scanning of the two predicted alpha-helices in the presumed core-binding domain of P2 Int was carried out in order to identify amino acids involved in binding to the core. An in vivo excisive assay and an in vivo integrative assay were used resulting in the identification of four amino acids as candidates for core-binding. The fact that the recombination reaction shows directionality renders the site-specific recombination systems of the P2-like phages attractive to develop as tools for safe and efficient non-viral gene delivery in humans. The wild-type P2 integrase was shown to accept a human attB sequence and localizes to the nucleus in human cell lines. The work presented in this thesis has increased our understanding of the site-specific recombination systems of the phages P2 and WΦ and provides a basis for further characterization and development for future use in a eukaryotic context.
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