Asia%20Authorship%20Seed%20-%20%D1%C7%D6%DE%B3%C9%C8%CB%CE%DE%C2%EB%D4%AD%B4%B4%C7%F8%20-%20SexInSex%21%20Board %E4%B8%80%E6%9C%AC%E9%81%93%E6%88%90%E4%BA%BA%E8%87%AA%E6%8B%8D Nudity Cunt Brunette

Forum Cuntbrunette Haveing Szh 1 Cunt Brunette Sequences in attB that affect the ability of C integrase to synapse and to activate DNA cleavage--《核酸研究医学期刊》--医学期刊频道--首席医学网

Forum Cuntbrunette Haveing Szh 1 Cunt Brunette

searchh Haveing s Forum %E7%97%B4%E5%A5%B3%E4%BF%B1%E4%B9%90%E9%83%A8+%E6%88%90%E4%BA%BA%E5%85%89%E7%A2%9F+osearchc Forum u Forum iansearch Cuntbrunette a Haveing ec Cuntbrunette m Haveing i Forum ehairy%20mature%20slut%20getting%20wet%20by%20her%20toy Haveing i Cuntbrunette h Haveing i Forum f Szh rmsearchtisearchn Haveing rsearchm Forum o Szh h Cuntbrunette r Szh l Forum r Szh e Haveing ssearchri Szh e Forum efxxxb.comob Szh nssearchs Szh asearchd Cuntbrunette te Forum res Cuntbrunette lsearchasearche Haveing searcho Forum g Forum n Cuntbrunette r Haveing t Forum search Forum owoman%20fucked%20by%20manewww.9bbvv.com searchh Cuntbrunette tsearchf Szh c Forum seso Szh searchu Haveing srsearcht Szh Forum ecosearchn Haveing t Haveing obritish%20brunette Forum n fo%B9%FA%B2%FA%B7%F2%C6%DE%D5%E6%CA%B5%C2%B6%C1%B3%D7%D4%C5%C4m Szh ti Szh nsearchoPetite%20minx%20enjoys%20getting%20her%20love... Cuntbrunette hsearch yyouzzijap Szh e Cuntbrunette b Cuntbrunette Cuntbrunette n Cuntbrunette eg Forum asearche Forum ( Haveing dasearchtdsearchf Cuntbrunette o Szh Cuntbrunette ht Haveing usearchlsearchs Cuntbrunette e Szh searchrsearchv Haveing osearchslsearch searchosearch searchx Haveing 1searchin Haveing esearchrsearchs Cuntbrunette ,search2; F Cuntbrunette g Haveing r search).searchIn Szh tsearchesearchressearchlvsearchse Forum , the DNA is contacted in the minor groove in the centre of each binding site and through specific contacts in the major groove towards the outer flank of the site via the C-terminal DNA binding domain (24,25). The geometry of DNA-binding is such that the C-terminal domain of resolvase extends around the DNA and contacts on the opposite side of the DNA to the catalytic serine (25). As in Bxb1 and TnpX, C31 integrase has a proteolytically sensitive site between the N and C terminal domains (K152, unpublished data)(26,30,46). Moreover, the C-terminal domains of Bxb1 and TnpX have been shown previously to be capable of binding specifically to DNA (26,30,46). Thus we propose that the C-terminal domains interact with the outer flanks of the att sites, that these interactions determine the conformations of integrase bound to each site and therefore whether they are compatible for synapsis. In attB this information is ¡®read¡¯ at least in part from ¨C15 and ¨C16 where disruption of this interaction disables the ability of integrase to form a stable synapse (Figures 4¨C6). The model predicts that there is communication between the putative DNA-binding motifs in the C-terminal domain and the regions of integrase that generate the protein-protein interface for synapsis. We currently envisage this communication as an allosteric switch mediated by conformational changes. In resolvase, the synaptic interface is located at the DNA distal surface of the catalytic domain and it is likely that the serine integrases use the equivalent of this interface for synapsis, although it is possible that the C-terminal domain may also have a role in synapsis. After synapsis an activation step is required for DNA cleavage and in attB this depends on the base pairs at position ¨C/+2. In attB only one of the ¨C/+2 bases needs to be wild type for activity and this can be either on the B or B' arm. Given the proximity of ¨C/+2 to the scissile phosphate, position 2 is more likely to interact with the catalytic domain than with the C-terminal domain. As in resolvase there may be significant conformation changes that occur with activation of recombination (34,37).

Figure 7. Model for the mechanism of integrase. The integrase subunits are shown with a small N-terminal (catalytic) domain through which the subunits may dimerize (26) and a large C-terminal domain that we propose recognizes the sequences in the outer flanks of the recombination sites. These recognition events, specifically ¨C15 and ¨C16 (annotated as blue bars) in attB, lead to an ¡®induced fit¡¯ or stabilization of a specific conformation of integrase that enables synapsis with integrase bound to attP. Different conformations of integrase bound to either attB or attP are shown as different colours. The synaptic interface via the N-terminal catalytic domains is indicated based on the resolvase precedent; there is no evidence to indicate that the C-terminal domains could also participate in a synaptic interface. Mutations at positions ¨C15 or ¨C16 (such as in T-15C:C+15G, G-16T:G+16A), T-15C or G-16T do not induce the conformation of integrase that can form a stable synapse with attP so the rate of reaction decreases (thin arrow). Mutations at ¨C/+2 in attB (red bars) are severely inhibited in cleavage but are capable of forming a stable synapse. These mutants indicate that the formation of the synaptic complex is followed by a well-defined activation step that results in concerted DNA cleavage. After strand exchange integrase is bound to the hybrid sites and adopts a conformation that cannot synapse attL and attR. The putative tetrameric complex rapidly dissociates to binary complexes containing integrase and either attL or attR. See text for more details.

The data presented here provides a source of information that could be used for the design of alternative attB sites for genome engineering using C31 integrase. Indeed we have already used this information to create a non-methylatable attB site for use in vertebrate cell lines (4). In this site, attBm, the CpG steps have been replaced with bases that we have shown here were neutral with respect to recombination activity. It is noteworthy that the positions in attB that are critical for recombination other than position 2 i.e. positions 15, 16 were not highlighted as being particularly preferred in the pseudo-attB sites (Figure 1). However all of the pseudo-attB sequences have either the wild-type C at ¨C2 or a wild-type G at +2 (Figure 1) (41,43). It is also noticeable that the regions where there are most identities between attB and attP are not particularly sensitive to mutation (Figure 1). A plausible explanation for both these observations is that the 24 bp of att site DNA from about position 11 to the crossover site is a core sequence that integrase binds to specifically. We propose that the role of positions 15 and 16 in attB revealed by this study is to greatly enhance the efficiency of the reaction and effectively discriminate between the pseudo-sites and the cognate attB site. It is envisaged that this data and a similar analysis with attP will enable an understanding of the optimal sequences to target for continued application of C31 integrase.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS

We are grateful for comments on this manuscript from Paul Rowley and Paul Hoskisson. M.G was funded by a scholarship from the University of Aberdeen. This work was funded by the Biotechnology and Biological Sciences Research Council, UK. Funding to pay the open access publication charges for this article was provided by the Biotechnology and Biological Sciences Research Council, UK.

Conflict of interest statement. None declared.

【参考文献】
 

Bateman JR, Lee AM, Wu CT. Site-specific transformation of Drosophila via C31 integrase-mediated cassette exchange. Genetics (2006) 173:769¨C777.

Andreas S, Schwenk F, Kuter-Luks B, Faust N, Kuhn R. Enhanced efficiency through nuclear localization signal fusion on phage C31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells. Nucleic Acids Res (2002) 30:2299¨C2306.

Belteki G, Gertsenstein M, Ow DW, Nagy A. Site-specific cassette exchange and germline transmission with mouse ES cells expressing C31 integrase. Nat Biotechnol (2003) 21:321¨C324.

Dafhnis-Calas F, Xu Z, Haines S, Malla SK, Smith MCM, Brown WR. Iterative in vivo assembly of large and complex transgenes by combining the activities of C31 integrase and Cre recombinase. Nucleic Acids Res (2005) 33:e189.

Khan MS, Khalid AM, KMalik KA. Phage C31 integrase: a new tool in plastid genome engineering. Trends in Plant Science (2005) 10:1¨C3.

Malla S, Dafhnis-Calas F, Brookfield JF, Smith MCM, Brown WR. Rearranging the centromere of the human Y chromosome with C31 integrase. Nucleic Acids Res (2005) 33:6101¨C6113.

Thomason LC, Calendar R, Ow DW. Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage C31 site-specific recombination system. Mol. Gen. Genom (2001) 265:1031¨C1038.

Nkrumah LJ, Muhle RA, Moura PA, Ghosh P, Hatfull GF, Jacobs WR Jr, Fidock DA. Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase. Nat. Methods (2006) 3:615¨C621.

Thyagarajan B, Olivares EC, Hollis RP, Ginsburg DS, Calos MP. Site-specific genomic integration in mammalian cells mediated by phage C31 integrase. Mol Cell Biol (2001) 21:3926¨C3934.

Groth AC, Olivares EC, Thyagarajan B, Calos MP. A phage integrase directs efficient site-specific integration in human cells. Proc. Natl Acad. Sci. USA (2000) 97:5995¨C6000.

Olivares EC, Hollis RP, Chalberg TW, Meuse L, Kay MA, Calos MP. Site-specific genomic integration produces therapeutic Factor IX levels in mice. Nat. Biotechnol (2002) 20:1124¨C1128.

Held PK, Olivares EC, Aguilar CP, Finegold M, Calos MP, Grompe M. In vivo correction of murine hereditary tyrosinemia type I by C31 integrase-mediated gene delivery. Mol. Ther (2005) 11:399¨C408.

Portlock JL, Keravala A, Bertoni C, Lee S, Rando TA, Calos MP. Long-term increase in mVEGF164 in mouse hindlimb muscle mediated by phage C31 integrase after nonviral DNA delivery. Hum. Gene Ther (2006) 17:871¨C876.

Keravala A, Portlock JL, Nash JA, Vitrant DG, Robbins PD, Calos MP. C31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints. J. Gene Med (2006) 8:1008¨C1017.

Bertoni C, Jarrahian S, Wheeler TM, Li Y, Olivares EC, Calos MP, Rando TA. Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration. Proc. Natl. Acad. Sci. USA (2006) 103:419¨C424.

Thorpe HM, Smith MCM. In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc. Natl Acad. Sci. USA (1998) 95:5505¨C5510.

Ghosh P, Kim AI, Hatfull GF. The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB. Mol. Cell (2003) 12:1101¨C1111.

Azaro MA, Landy A. Mobile DNA II¡ªCraig NL, Craigie R, Gellert M, Lambowitz AM, eds. (2002) Washington D.C.: ASM Press. 118¨C148.

Ghosh P, Wasil LR, Hatfull GF. Control of phage Bxb1 excision by a novel recombination directionality factor. PLoS Biol (2006) 4:e186.

Smith MCM, Thorpe HM. Diversity in the serine recombinases. Mol. Microbiol (2002) 44:299¨C307.

Craig NL. Mobile DNA II¡ªCraig NL, Craigie R, Gellert M, Lambowitz AM, eds. (2002) Washington DC: American Society for Microbiology. 3¨C11.
hForum Cuntbrunette Haveing Szh 1 Cunt Brunette Sequences in attB that affect the ability of C integrase to synapse and to activate DNA cleavage--《核酸研究医学期刊》--医学期刊频道--首席医学网h 1 Brunette%20girl%20with%20sexy%20body%20fucks%20older%20dude sForum Cuntbrunette Haveing Szh 1 Cunt Brunette Sequences in attB that affect the ability of C integrase to synapse and to activate DNA cleavage--《核酸研究医学期刊》--医学期刊频道--首席医学网y Sexinsex%20board%20%C5%B7%C3%C0%CE%DE%C2%EB%C7%F8 Vintageporn