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Own Promoter (own + promoter)
Selected AbstractsThe VpreB1 enhancer drives developmental stage-specific gene expression in vivoEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2003Steve Licence Abstract In adult mice, the VpreB genes are expressed in bone marrow progenitor (pro-) and precursor (pre-) B,cells. As part of the pre-B,cell receptor, the proteins are crucial for the proliferation of these cells and consequently normal B,lymphocyte development. Using cell lines, we identified a lineage- and developmental-stage-specific VpreB1 enhancer. Here, we analyze its specificity in vivo by generating transgenic mice in which expression of a reporter gene (human CD122) is regulated by the VpreB1 enhancer in the context of its own promoter. All transgenic lines expressed thereporter gene in the bone marrow in a copy number-independent manner, whereas expression levels were integration site-dependent. While the enhancer is not tissue specific, within the B,cell lineage the expression pattern of human CD122 mimicked that of endogenous VpreB1. Thus, low levels were detected in pro-B,cells, high levels in pre-BI and slightly lower levels in pre-BII cells; no expression was detected in immature/mature B,cells. Furthermore, when in vitro cultured transgenic pre-B,cells differentiated into immature B,cells there was concomitant down-regulation of human CD122 andendogenous VpreB1. Thus the VpreB1 enhancer is sufficient to ensure developmental stage-specific expression of a reporter gene in B,lymphocytes in vivo. [source] NFAT expression in human osteoclastsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005Christopher J. Day Abstract Nuclear factor of activated T-cells cytoplasmic (NFATc) is a family of transcription factors originally identified in T-cells. The gene family is currently known to have four members (NFATc1 through NFATc4) which have roles both within and outside the immune system. We show that NFATc1 is the major induced NFAT in human osteoclasts, with expression greatly exceeding that of NFATc2 through NFATc4. In macrophage-like cells in culture, NFATc1 through NFATc4 are expressed at similar low levels. NFATc1 is comprised of five mRNA transcript variants known to encode three different protein isoforms. The mRNA encoding isoform C (mRNA variant 3) was the most expressed with 38 copies per nanogram followed by isoform B (mRNA variant 5) with 17 copies per nanogram of total RNA. Isoform A (mRNA variant 1) and mRNA variants 2 and 4 made up less than 1% of the total NFATc1 expressed. NFATc1 is activated by calcineurin after calcium-calmodulin signalling. The induction of NFATc1 in osteoclasts was not altered in the presence of cyclosporin A, an inhibitor of calcineurin, suggesting that NFATc1 does not participate in autoregulatory activation of its own promoter. The NFATc1 variants expressed by human osteoclasts are not those normally expressed by effector T-cells but are similar to those seen in naïve T-cells. © 2005 Wiley-Liss, Inc. [source] CPMK2, an SLT2-homologous mitogen-activated protein (MAP) kinase, is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis-related MAP kinase cascade in phytopathogenic fungiMOLECULAR MICROBIOLOGY, Issue 2 2002Géraldine Mey Summary Cpmk2 , encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea , is an orthologue of SLT2 from Saccharomyces cerevisiae , the first isolated from a biotrophic, non-appressorium-forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea , that have been deprived of their MAP kinase gene mps1 , the ability of cpmk2 to complement the defects of , mps1 was investigated. Interestingly, the C. purpurea gene, under the control of its own promoter, was able to complement the M. grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2 / MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity. [source] Immunolocalization of the PmSUC1 Sucrose Transporter in Plantago major Flowers and Reporter-Gene Analyses of the PmSUC1 Promoter Suggest a Role in Sucrose Release from the Inner IntegumentPLANT BIOLOGY, Issue 3 2007C. Lauterbach Abstract: This paper presents a detailed analysis of the PmSUC1 gene from Plantago major, of its promoter activity in Arabidopsis, and of the tissue specific localization of the encoded protein in Plantago. PmSUC1 promoter activity was detected in the innermost layer of the inner integument (the endothel) of Arabidopsis plants expressing the gene of the green fluorescent protein (GFP) under the control of the PmSUC1 promoter. This promoter activity was confirmed with a PmSUC1-specific antiserum that identified the PmSUC1 protein in the endothel of Plantago and of Arabidopsis plants expressing the PmSUC1 gene under the control of its own promoter. PmSUC1 promoter activity and PmSUC1 protein were also detected in pollen grains during maturation inside the anthers and in pollen tubes during and after germination. These results demonstrate that PmSUC1 is involved in sucrose partitioning to the young embryo and to the developing pollen and growing pollen tube. In the innermost cell layer of the inner integument, a tissue that delivers nutrients to the endosperm and the embryo, PmSUC1 may catalyze the release of sucrose into the apoplast. [source] Transcriptional activation of the ,- catenin gene at the invasion front of colorectal liver metastases,THE JOURNAL OF PATHOLOGY, Issue 3 2009Obul R Bandapalli Abstract ,-Catenin is a pivotal molecule of the Wnt-signalling pathway, involved in regulation of developmental and oncogenic processes as well as in intercellular adhesion. So far, ,-catenin has been thought to be regulated mainly at the protein level. Here, we provide evidence for a transcriptional mechanism of ,-catenin regulation at the invasion front of colorectal liver metastases. In a nude mouse/LS174T cell xenograft model of colorectal liver metastases, we observed ,-catenin up-regulation at the mRNA and protein levels and a 13.7-fold increase of ,-catenin promoter activity in the cancer cells of the invasion front. In addition, the promoter activity was five-fold higher in the interior of the tumour than in cells growing in cell culture. In vitro studies revealed binding of TCF-4 to the ,-catenin promoter and reduced promoter activity by over-expression of dominant negative TCF-4, or ,-catenin knock-down and increased activity by ,-catenin over-expression, indicating that ,-catenin acts as co-transcription factor of its own promoter. In 55% (7/13) of clinical specimens, ,-catenin mRNA was markedly elevated in the cancer cells of the invasion front. Elevation of mRNA was paralleled by increased nuclear and cytoplasmic ,-catenin protein concentrations. These data indicate that transcriptional regulation contributes to the dynamic changes of ,-catenin levels upon the confrontation of tumour cells with the host microenvironment. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Rice cellulose synthase-like D4 is essential for normal cell-wall biosynthesis and plant growthTHE PLANT JOURNAL, Issue 6 2009Ming Li Summary Cellulose synthase-like (CSL) proteins of glycosyltransferase family 2 (GT2) are believed to be involved in the biosynthesis of cell-wall polymers. The CSL D sub-family (CSLD) is common to all plants, but the functions of CSLDs remain to be elucidated. We report here an in-depth characterization of a narrow leaf and dwarf1 (nd1) rice mutant that shows significant reduction in plant growth due to retarded cell division. Map-based cloning revealed that ND1 encodes OsCSLD4, one of five members of the CSLD sub-family in rice. OsCSLD4 is mainly expressed in tissues undergoing rapid growth. Expression of OsCSLD4 fluorescently tagged at the C- or N-terminus in rice protoplast cells or Nicotiana benthamiana leaves showed that the protein is located in the endoplasmic reticulum or Golgi vesicles. Golgi localization was verified using phenotype-rescued transgenic plants expressing OsCSLD4,GUS under the control of its own promoter. Two phenotype-altered tissues, culms and root tips, were used to investigate the specific wall defects. Immunological studies and monosaccharide compositional and glycosyl linkage analyses explored several wall compositional effects caused by disruption of OsCSLD4, including alterations in the structure of arabinoxylan and the content of cellulose and homogalacturonan, which are distinct in the monocot grass species Oryza sativa (rice). The inconsistent alterations in the two tissues and the observable structural defects in primary walls indicate that OsCSLD4 plays important roles in cell-wall formation and plant growth. [source] Comparative biochemical and functional studies of family I soluble inorganic pyrophosphatases from photosynthetic bacteriaFEBS JOURNAL, Issue 15 2007María R. Gómez-García Soluble inorganic pyrophosphatases (inorganic diphosphatases, EC 3.6.1.1) were isolated and characterized from three phylogenetically diverse cyanobacteria , Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Pseudanabaena sp. PCC 6903 , and one anoxygenic photosynthetic bacterium, Rhodopseudomonas viridis (purple nonsulfur). These enzymes were found to be family I soluble inorganic pyrophosphatases with c. 20 kDa subunits with diverse oligomeric structures. The corresponding ppa genes were cloned and functionally validated by heterologous expression. Cyanobacterial family I soluble inorganic pyrophosphatases were strictly Mg2+ -dependent enzymes. However, diverse cation cofactor dependence was observed for enzymes from other groups of photosynthetic bacteria. Immunochemical studies with antibodies to cyanobacterial soluble inorganic pyrophosphatases showed crossreaction with orthologs of other main groups of phototrophic prokaryotes and suggested a close relationship with the enzyme of heliobacteria, the nearest photosynthetic relatives of cyanobacteria. A slow-growing Escherichia coli JP5 mutant strain, containing a very low level of soluble inorganic pyrophosphatase activity, was functionally complemented up to wild-type growth rates with ppa genes from diverse photosynthetic prokaryotes expressed under their own promoters. Overall, these results suggest that the bacterial family I soluble inorganic pyrophosphatases described here have retained functional similarities despite their genealogies and their adaptations to diverse metabolic scenarios. [source] Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA libraryGENES TO CELLS, Issue 3 2000Da-Qiao Ding Background Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green fluorescent protein (GFP)-fusion genomic DNA library of S. pombe. Results We constructed the S. pombe GFP-fusion genomic DNA library by fusing, in all three reading frames, random fragments of genomic DNA to the 5, end of the GFP gene in such a way that expression of potential GFP-fusion proteins would be under the control of the own promoters contained in the genomic DNA fragments. Fission yeast cells were transformed with this plasmid library, and microscopic screening of 49 845 transformants yielded 6954 transformants which exhibited GFP fluorescence, of which 728 transformants showed fluorescence localized to distinct intracellular structures such as the nucleus, the nuclear membrane, and cytoskeletal structures. Plasmids were isolated from 516 of these transformants, and a determination of their DNA sequences identified 250 independent genes. The intracellular localizations of the 250 GFP-fusion constructs was categorized as an image database; using this database, DNA sequences can be searched for based on the localizations of their products. Conclusions A number of new intracellular structural components were found in this library. The library of GFP-fusion constructs also provides useful fluorescent markers for various intracellular structures and cellular activities, which can be readily used for microscopic observation in living cells. [source] Functional analysis of lung tumor suppressor activity at 3p21.3GENES, CHROMOSOMES AND CANCER, Issue 12 2006Arja ter Elst The early and frequent occurrence of deletions at 3p21.3 in lung cancer has led to the consideration of this chromosomal region as a lung cancer (LUCA) critical region with tumor suppressor activity. We covered this 19 genes-containing region with overlapping P1 artificial chromosomes (PACs), in which genes are likely accompanied by their own promoters or other regulatory sequences. With these PACs we transfected cells from a small cell lung cancer (SCLC) cell line which readily caused tumors in nude mice. Per PAC we selected two cell clones with a low number of PAC copies integrated at a single genomic site. The selected clones were s.c. injected into nude mice to investigate whether the integrated genes suppressed the tumor-inducing capacity of the original SCLC cell line. We could demonstrate PAC-specific gene expression in the transfected cells. All of the PAC integration sites were different. It appeared that introduction of a PAC or even an empty PAC vector causes some chromosomal instability, which in principle may either promote or inhibit cell growth. However, both cell clones with integration of the same PAC from the centromeric part of the LUCA region in different genomic sites were the sole pair of clones that caused smaller tumors than did the original SCLC cell line. This suggests that rather than the induced chromosomal instability, the DNA sequence of that PAC, which in addition to two protein-encoding genes contains at least one potential miRNA gene, is responsible for the tumor suppressor activity. © 2006 Wiley-Liss, Inc. [source] |