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Wild-type Background (wild-type + background)
Selected AbstractsOligodendrocyte-specific ceramide galactosyltransferase (CGT) expression phenotypically rescues CGT-deficient mice and demonstrates that CGT activity does not limit brain galactosylceramide levelGLIA, Issue 3 2005Inge Zöller Abstract Galactosylceramide (GalC) is the major sphingolipid of the myelin membrane. Mice lacking GalC due to ceramide galactosyltransferase (CGT) deficiency form unstable and functionally affected myelin and exhibit a progressive demyelination, accompanied by severe motor coordination deficits. In addition to oligodendrocytes, CGT is also expressed in other cells, e.g., neurons and astrocytes. We examined the possibility that lack of CGT in these cells contributes to the phenotype of CGT-deficient mice. Toward this aim, we generated transgenic mice expressing CGT under the control of oligodendrocyte-specific proteolipid protein (PLP) promoter and examined the possibility of a transgenic rescue of CGT-deficient mice. CGT-deficient mice expressing the PLP-CGT transgene did not show any behavioral abnormalities, normal myelin structure, and MBP levels. CGT activity as well as GalC and sulfatide levels of rescued mice were not significantly different from wild-type controls. Thus, transgenic rescue with the PLP-CGT transgene was apparently complete. In contrast to wild-type and rescued mice, PLP-CGT transgenic mice on a wild-type background exhibited significantly elevated CGT activity which directly correlated with an increase in non-hydroxy fatty acid (NFA)-GalC, but not ,-hydroxy fatty acid (HFA)-GalC. HFA-GalC decreased in adult transgenic mice, indicating that NFA-GalC, but not HFA-GalC levels are limited by CGT activity. As a consequence, the total amount of GalC is unchanged over a rather wide range of CGT expression levels in the mouse brain. Our results indicate that loss of CGT in oligodendrocytes is exclusively responsible for the myelin structural deficits, demyelination, and behavioral abnormalities in CGT-deficient mice. © 2005 Wiley-Liss, Inc. [source] Several distinct localization patterns for penicillin-binding proteins in Bacillus subtilisMOLECULAR MICROBIOLOGY, Issue 3 2004Dirk-Jan Scheffers Summary Bacterial cell shape is determined by a rigid external cell wall. In most non-coccoid bacteria, this shape is also determined by an internal cytoskeleton formed by the actin homologues MreB and/or Mbl. To gain further insights into the topological control of cell wall synthesis in bacteria, we have constructed green fluorescent protein (GFP) fusions to all 11 penicillin-binding proteins (PBPs) expressed during vegetative growth of Bacillus subtilis. The localization of these fusions was studied in a wild-type background as well as in strains deficient in FtsZ, MreB or Mbl. PBP3 and PBP4a localized specifically to the lateral wall, in distinct foci, whereas PBP1 and PBP2b localized specifically to the septum. All other PBPs localized to both the septum and the lateral cell wall, sometimes with irregular distribution along the lateral wall or a preference for the septum. This suggests that cell wall synthesis is not dispersed but occurs at specific places along the lateral cell wall. The results implicate PBP3, PBP5 and PBP4a, and possibly PBP4, in lateral wall growth. Localization of PBPs to the septum was found to be dependent on FtsZ, but the GFP,PBP fluorescence patterns were not detectably altered in the absence of MreB or Mbl. [source] Enhanced biocontrol activity of Trichoderma virens transformants constitutively coexpressing ,-1,3- and ,-1,6-glucanase genesMOLECULAR PLANT PATHOLOGY, Issue 4 2007SLAVICA DJONOVI SUMMARY Evidence for the role of chitinases, proteases and ,-1,3- and ,-1,6-glucanases in mycoparasitism by Trichoderma species has been well documented. Moreover, constitutive over-expression of genes encoding individual cell-wall-degrading enzymes (CWDEs) has been shown to improve the potential of biological agents. In this study, we generated transformants of T. virens in which ,-1,3- and ,-1,6-glucanase genes, TvBgn2 and TvBgn3, respectively, were constitutively coexpressed in the same genetic T. virens Gv29.8 wild-type background. The double over-expression transformants (dOEs) grow and sporulate slower than the wild-type (WT). However, the reduction in growth did not seem to affect their mycoparasitic and biocontrol capabilities, as dOEs displayed much higher levels of total ,-1,3- and ,-1,6-glucanase activity than the WT. This higher enzymatic activity of dOEs positively correlated with observed in vitro inhibition of Pythium ultimum and Rhizoctonia solani mycelia, and with enhanced bioprotection of cotton seedlings against P. ultimum, R. solani and Rhizopus oryzae. Besides effective biocontrol of all pathogens at an original inoculum level, the performance of dOEs was highly enhanced (up to 312% of WT performance) when pathogen pressure was greater (i.e. concentration of inoculum was higher or pathogens applied in combination). These results demonstrate that the strategy of introducing multiple lytic enzyme-encoding genes through transformation of a given biocontrol strain can be successfully used to achieve better biocontrol. [source] DNA hypomethylation reduces homologous pairing of inserted tandem repeat arrays in somatic nuclei of Arabidopsis thalianaTHE PLANT JOURNAL, Issue 4 2005Koichi Watanabe Summary Fluorescent chromatin tagging makes possible tracking of specific loci in vivo and in situ. Loci tagged by the lac operator (lacO)/GFP-LacI/Nuclear Localization Signal (NLS) system show rapid motility and constrained chromatin dynamics in somatic nuclei of a transgenic line, designated EL702C, in Arabidopsis thaliana. The tagged loci associated with each other significantly more often than expected at random, due to homologous pairing of the lacO tandem repeat arrays. Furthermore, these arrays associated significantly more often than average euchromatic regions with heterochromatic chromocenters (CCs). We show now that the inserted lacO array in this transgenic line became strongly methylated at CG sites in the T3 generation, which can be reversed upon transfer into the mutant backgrounds of decrease in DNA methylation 1 (ddm1) and methyltransferase 1 (met1). Concomitantly, the tagged loci showed lower association frequencies as compared with the transgenics in wild-type background, which is correlated with a significant decrease in allelic and ectopic pairing of the lacO repeat arrays as visualized by fluorescence in situ hybridization. In contrast, the preferential association of the lacO arrays with heterochromatin, locus mobility in somatic nuclei and transcription of neighboring transgenes were not altered by reduced DNA methylation in ddm1 and met1 backgrounds. Our results show that repeat arrays can activate hypermethylation of the inserted locus that correlates with high frequencies of homologous pairing in somatic cells. In contrast, the preferential association of these inserted arrays with CCs in plant cells occurs through another mechanism. [source] Phosphate availability regulates biosynthesis of two antibiotics, prodigiosin and carbapenem, in Serratia via both quorum-sensing-dependent and -independent pathwaysMOLECULAR MICROBIOLOGY, Issue 2 2003Holly Slater Summary Serratia sp. ATCC 39006 produces two secondary metabolite antibiotics, 1-carbapen-2-em-3-carboxylic acid (Car) and the red pigment, prodigiosin (Pig). We have previously reported that production of Pig and Car is controlled by N -acyl homoserine lactone (N -AHL) quorum sensing, with synthesis of N -AHLs directed by the LuxI homologue SmaI, and is also regulated by Rap, a member of the SlyA family. We now describe further characterization of the SmaI quorum-sensing system and its connection with other regulatory mechanisms. We show that the genes responsible for biosynthesis of Pig, pigA,O, are transcribed as a single polycistronic message in an N -AHL-dependent manner. The smaR gene, transcribed convergently with smaI and predicted to encode the LuxR homologue partner of SmaI, was shown to possess a negative regulatory function, which is uncommon among the LuxR-type transcriptional regulators. SmaR represses transcription of both the pig and car gene clusters in the absence of N -AHLs. Specifically, we show that SmaIR exerts its effect on car gene expression via transcriptional control of carR, encoding a pheromone-independent LuxR homologue. Transcriptional activation of the pig and car gene clusters also requires a functional Rap protein, but Rap dependency can be bypassed by secondary mutations. Transduction of these suppressor mutations into wild-type backgrounds confers a hyper-Pig phenotype. Multiple mutations cluster in a region upstream of the pigA gene, suggesting this region may represent a repressor target site. Two mutations mapped to genes encoding pstS and pstA homologues, which are parts of a high-affinity phosphate transport system (Pst) in Escherichia coli. Disruption of pstS mimicked phosphate limitation and caused concomitant hyper-production of Pig and Car, which was mediated, in part, through increased transcription of the smaI gene. The Pst and SmaIR systems define distinct, yet overlapping, regulatory circuits which form part of a complex regulatory network controlling the production of secondary metabolites in Serratia ATCC 39006. [source] | ||||||||||