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Genetics and Biosynthesis of Lipopolysaccharide O-Antigens
Published in Helmut Brade, Steven M. Opal, Stefanie N. Vogel, David C. Morrison, Endotoxin in Health and Disease, 2020
Wendy J. Keenleyside, Chris Whitfield
Although there has been much speculation regarding the role of lateral gene transfer in the generation of antigenic diversity, one area about which there is very little information is the mechanism of DNA transfer. Logically, DNA sequences could be mobilized either through plasmid transfer, transduction by bacteriophage, or by DNA transformation with free DNA. Integration into the recipient cluster could then be mediated by homologous recombination or possibly transposition. The relative involvement of each of these processes is unknown. As previously discussed, lysogenic phage conversion is one source of O-PS diversity, however the phage-encoded changes characterized to date all map outside of the O-PS biosynthesis gene cluster. As discussed above, plasmids are known to be involved to varying extents in O-PS expression, but lateral transfer has only been shown for the O:54 antigen of S. enterica serovar borreze (131). The ColE1-type plasmid carrying the O:54 biosynthesis gene cluster can be mobilized to confer O:54 serospecificity on recipient strains. However, while the various members of the O:54 serogroup are heterogeneous in terms of their endogenous chromosomal O polysaccharide biosynthesis clusters (44,132), the unique structure of the O:54 antigen compared to other Salmonella (or indeed enteric) O antigens (44) and the absence of associated IS or IS-like elements suggest that this plasmid is not a likely source of homologous recombination with chromosomal O-PS biosynthesis sequences.
rDNA: Evolution Over a Billion Years
Published in S. K. Dutta, DNA Systematics, 2019
The overall structure of the rDNA is described by a spacer region and a region giving rise to a primary precursor transcript. The precursor rRNA consists of an external transcribed spacer (ETS) preceding the 18S rRNA gene at the 5′ end of the transcript, a 5.8S rRNA gene separated from both the 18S and 28S rRNA by internal transcribed spacers (ITS 1 and ITS 2), and the 28S rRNA gene at the 3′ end of the transcript.174 The structure of the rDNA has been determined at the nucleotide sequence level. The two major EcoRI fragments of the rDNA were first cloned in the bacterial plasmid pSC1013 to produce CD18, containing mainly the 28S gene and CD30 containing the rest of the rDNA unit. The large EcoRI fragment in CD30 was transferred to the colicinegenic plasmid El (ColE1176) and renamed pXlr12.175 Another spacer region EcoRI fragment (pX1r4) was cloned by Wellauer et al.,167 transferred to the Col El plasmid, and renamed pXlr14.177 These two clones, pXlr12 and pXlr14, provided the basis for most of the early work on the structural mapping of X. laevis rDNA (Figure 9).
Vector Technology of Relevance to Nitrogen Fixation Research*
Published in Peter M. Gresshoff, Molecular Biology of Symbiotic Nitrogen Fixation, 2018
Reinhard Simon, Ursula B. Priefer
The plasmid pRK2013 arose from work undertaken to study replication functions of the plasmid RK2.97 It contains the entire segment of the broad host-range transfer genes of RK2 linked with a Km resistance determinant and cloned into the narrow host-range replicon ColE1. The selective marker is located on Tn903, and this transposon has been introduced into a number of plant-associated pseudomonads using pRK2013 as a suicide vector.98 Derivatives of pRK2013 have also been used as suicidal carrier replicons for other transposons. After pRK2013:: Tn7 transfer, site-specific transposition of Tn7 into a megaplasmid of R. meliloti has been demonstrated.33 The Tn5 derivatives Tn5-233 and Tn5-235 have also been inserted into the R. meliloti genome via pRK2013 transfer.29
Differential efficacy of genetically swapping GAL4
Published in Journal of Neurogenetics, 2019
Ying-Jun Chen, Hao-Hsin Chang, Shih-Han Lin, Tzi-Yang Lin, Ting-Han Wu, Hsin-Ju Lin, Nan-Fu Liou, Chi-Jen Yang, Yuh-Tarng Chen, Kai Hsiang Chang, Cen-You Li, Ya-Hui Chou
We conducted six genetic swap experiments to replace GAL4 with LexA in five GAL4 lines. From each of the five swap experiments, we successfully identified one to five true integrations. The frequencies of uncovering LexA-swap candidates and true integrations were 68.4 and 84.6%, respectively (Figure 3(A,B), Table 1). However, only 564-LexA partially recapitulated the expression patterns of 564-GAL4 in the optic lobe (arrows in Figure 3(D)), but it did not recapitulate the GAL4 expression patterns in the central brain (Figure 3(C)). The other four examined LexA swap lines did not show expression patterns similar to the original GAL4 lines (Figure 3(E), Table 1 and data not shown). To address whether the LexA reporters would have any effect on the observed expression patterns, we used 670-lexA to drive two different reporters, LexAop2-mCD8GFP and LexAop-rCD2GFP, which contain LexA binding sites that were respectively derived from the binding motifs of colE1 and sulA (Lai & Lee, 2006; Pfeiffer et al., 2010) (Figure 3(E)). Neither of these reporters showed expression patterns that were similar to 670-GAL4. Therefore, the properties of reporters are unlikely to cause issues with expression reproducibility found in LexA swaps.
Fab is the most efficient format to express functional antibodies by yeast surface display
Published in mAbs, 2018
Coline Sivelle, Raphaël Sierocki, Kelly Ferreira-Pinto, Stéphanie Simon, Bernard Maillere, Hervé Nozach
Cloning were performed with the SLiCE recombination cloning method46 or classic restriction/ligature method using E.coli DH5α strain (Invitrogen). For each anti-TNF antibody, optimized synthetic genes (VH and VL) were ordered from Eurofins Genomics based on the IMGT sequences. All expression plasmids are derived from previously described pCT-L7.5.126 and share the CEN/ARS replication origin, TRP auxotrophy marker, colE1 replication origin, Ampicillin resistance gene and galactose inducible promoter GAL1. Plasmid pCT-L7.5.1 was a gift from Prof. K. Dane Wittrup (Addgene plasmid # 42900).