• Vaughn Ewing posted an update 2 months ago

    At the same time, homologous recombination is generally as highthroughput- compatible as LIC. The modified primer design of the yeast-based DREAM mutagenesis method is simple and like the classic SDM covers all aspects of mutagenesis: mutation, MLN4924 insertion and deletion. The DREAM mutagenesis primer pair can easily be designed by the following basic rules: each oligonucleotide should be around 50 bases long, with 20 bases of 59 primer-toprimer overlap for an efficient recombination of the mutated plasmid ends in yeast, carrying the mutation, deletion or insertion in their middle, and 30 bases of 39-sequence for template annealing. The 20 bp overlap on both ends of the linear mutagenesis product results in efficient homology-based gap repair in S. cerevisiae to form the circular, mutated plasmid. To minimize the occurrence of errors, the number of thermal cycles is restricted to 18 as recommended for the conventional SDM kit. Accordingly, all analyzed DREAM clones so far replicated the template plasmid sequence i.e. apart from the introduced mutation the whole BSEP coding sequence was found to be unchanged. The new mutagenesis method permits the rapid realization of patient-derived BSEP mutations for immediate study in cell cultures. Using DREAM, we could show that a BSEP mutation identified in a patient with progressive familial intrahepatic cholestasis type 2 results in a trafficking defect of the mutant protein that prevents BSEP from being correctly incorporated into the plasma membrane. Future mutations can be generated quickly for their study in mammalian cell lines and/or in vitro on the isolated recombinant protein. This is a major advantage since the realization of for example BSEP mutations was previously a workintensive and time-consuming task. Glycosylation has been shown to be irrelevant for the function of other human ABC transporters expressed in yeast in the past. Furthermore, BSEP expressed in Sf9 cells, which also harbors a glycosylation pattern different from the human pattern, was functional. Thus, it is very likely that the glycosylation state and/or pattern of BSEP is not relevant for its function. The pattern was similar for WT and KO mice, however the intensity of CLU bands in KO mice was significantly reduced. In order to assess cellular distribution of sCLU in splenic stroma, we used immunohistochemical staining of frozen spleen sections with commercial polyclonal anti-CLU antibodies raised against recombinant mouse CLU Glu22-Glu448. Polyclonality and usage of almost full-length protein as immunogen ensured that this antibody would recognize different CLU isoforms in different applications. AF2747 specificity was confirmed by specific staining of HEK293 cells transiently transfected with full-length CLU. This expression pattern is broader than previously reported, though the brightest staining was still observed in B-cell areas, especially in GCs after immunization, and is attributed to FDC for which clusterin is used as one of differential markers. An important difference with the previous observations consists in the clear absence of marginal zone staining in spleen. Diffuse staining was observed in spleen red pulp, MLN medulla and lumen of high endothelial venules, which can be explained by the high amount of sCLU in blood. GC staining also had a diffuse appearance, not resembling stromal cell contours, which may be indicative of active secretion of sCLU in this area. Previously, sCLU secretion by FDC was shown by Verbrugghe et al. who detected clusterin immunoreactivity in the endoplasmic reticulum, Golgi apparatus, and on the plasma membrane of FDC in human Payer’s patches by electron microscopy. In contrast to the wild type pattern, only faint staining of few stromal cells could be seen in disorganized white pulp of the spleens of LTbR-KO mice. Diffuse staining of red pulp was not affected. This may reflect not only the absence of FDC, which contribute to the bright staining of B-cell follicles in WT mice spleen, but also downregulation of CLU in other stromal cell types in the absence of LTbR signal. Adherent bacteria profoundly differ from planktonic bacteria in physiology and gene expression. From this collective surfaceattached life mode, the bacteria gain significant adaptive advantages and exhibit increased resistance to many biocides. This adhesion-induced physiological shift was suggested very early on by scientists studying bacterial populations in aqueous receptacles and has since been confirmed on the basis of molecular biology data. Recently, abundant information on gene expression and metabolic pathway alterations in established biofilms has emerged due to the increasing spread of molecular genetics. However, the mechanisms of such a transition are not known. The data, obtained on a several hour or day time scale, depict interfering biochemical cascades upor downregulated in the surface-attached mode of growth compared to the free-floating mode. This reinforces the idea of a surface-attached specific mode of life, but does not enable distinguishing triggering events from further developmental stages that drive biological changes on surfaces. In particular, the respective contributions of the various factors prevailing in biofilms -actual cell surface contact, cell-cell interactions, secreted soluble molecules or extracellular matrix synthesis, together with modifications in the physical and chemical environment due to confinement of cells in a 3D viscoelastic architecture – have not been identified, and their causality remains elusive. In this paper, we focused on the early stage of cell-surface contact formation. Evidence of a direct cell response upon initial adhesion is scarce. Using reporter gene technology and microscope observation in Pseudomonas aeruginosa individual cells, Davies and Geesey concluded that attachment of the cell to a glass surface induced algC upregulation as early as the first 15 min of contact.