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Gene Fused (gene + fused)
Selected AbstractsStructure and promoter analysis of the mouse membrane-bound transferrin-like protein (MTf) geneFEBS JOURNAL, Issue 5 2001Kazuko Nakamasu Recently, we purified membrane-bound transferrin-like protein (MTf) from the plasma membrane of rabbit chondrocytes and showed that the expression levels of MTf protein and mRNA were much higher in cartilage than in other tissues [Kawamoto T, Pan, H., Yan, W., Ishida, H., Usui, E., Oda, R., Nakamasu, K., Noshiro, M., Kawashima-Ohya, Y., Fujii, M., Shintani, H., Okada, Y. & Kato, Y. (1998) Eur. J. Biochem.256, 503,509]. In this study, we isolated the MTf gene from a constructed mouse genomic library. The mouse MTf gene was encoded by a single-copy gene spanning ,,26 kb and consisting of 16 exons. The transcription-initiation site was located 157 bp upstream from the translation-start codon, and a TATA box was not found in the 5, flanking region. The mouse MTf gene was mapped on the B3 band of chromosome 16 by fluorescence in situ hybridization. Using primary chondrocytes, SK-MEL-28 (melanoma cell line), ATDC5 (chondrogenic cell line) and NIH3T3 (fibroblast cell line) cells, we carried out transient expression studies on various lengths of the 5, flanking region of the MTf gene fused to the luciferase reporter gene. Luciferase activity in SK-MEL-28 cells was higher than in primary chondrocytes. Although no luciferase activity was detectable in NIH3T3 cells, it was higher in chondrocytes than in ATDC5 chondrogenic cells. These findings were consistent with the levels of expression of MTf mRNA in these cells cultured under similar conditions. The patterns of increase and decrease in the luciferase activity in chondrocytes transfected with various 5, deleted constructs of the MTf promoter were similar to that in ATDC5 cells, but differed from that in SK-MEL-28 cells. The findings obtained with primary chondrocytes suggest that the regions between ,693 and ,444 and between ,1635 and ,1213 contain positive and negative cis -acting elements, respectively. The chondrocyte-specific expression of the MTf gene could be regulated via these regulatory elements in the promoter region. [source] Inhibition of Fas-mediated apoptotic cell death of murine T lymphocytes in a mouse model of immunosenescence in linkage to deterioration in cell membrane raft functionIMMUNOLOGY, Issue 1 2004Toshihiro Yokoyama Summary We previously developed a transgenic mouse line into which a rabbit protein kinase C, (PKC,) gene fused to a human CD2 promoter/enhancer was introduced, and we found that immunosenescence was facilitated in these transgenic mice. In this study, we found that along with age-dependent increase in the level of protein expression of PKC, and its translocation to the membrane, activated T cells became less sensitive to apoptosis-inducing anti-Fas antibody. The capacity of T cells to express Fas antigen on their surfaces in response to anti-CD3 and interleukin-2 was impaired in PKC,-transgenic mice of relatively advanced age, although background Fas expression levels on T cells from those mice were high. We then found that out of proportion to a high level of cell surface Fas expression the density of cholera toxin B (CTx)-binding raft elements decreased in PKC,-transgenic mice of relatively advanced age and to a lesser extent in normal mice of advanced age. Correspondingly, the expression level of raft-associating Lck was decreased in these mice. These findings suggest for the first time that immunosenescence of T cells involves a decrease in density of cell surface CTx-binding raft elements, which might underlie a deterioration in T-cell signal pathway for either cell death or cell activation. [source] Structure of the Mouse Glutamate Decarboxylase 65 Gene and Its PromoterJOURNAL OF NEUROCHEMISTRY, Issue 4 2000Preferential Expression of Its Promoter in the GABAergic Neurons of Transgenic Mice Abstract: GABA is synthesized by glutamate decarboxylase (GAD), which has two forms, GAD65 and GAD67. To elucidate the molecular mechanisms of mouse GAD65 (mGAD65) gene expression, we isolated and characterized the mGAD65 gene. The mGAD65 gene was found to be divided into 16 exons and spread over 75 kb. The sequence of the first exon and the 5,-flanking region indicated the presence of potential neuron-specific cis -regulatory elements. We used transgenic mice to examine the expression pattern conferred by a 9.2-kb promoter-proximal DNA fragment of the mGAD65 gene fused to the bacterial lacZ reporter gene. Transgenic mice showed high ,-galactosidase activity specifically in brain and testis. They also showed characteristic patterns of transgene expression in olfactory bulb, cerebellar cortex, and spinal cord, a similar expression pattern to that of endogenous mGAD65. However, no transgene expression was observed in the ventral thalamus or hypothalamus, in which high mGAD65 gene expression levels have been observed. These results suggest that the 9.2-kb DNA fragment of the mGAD65 gene is associated with its tissue-specific expression and its targeted expression in GABAergic neurons of specific brain regions but that additional regulatory elements are necessary to obtain fully correct expression. [source] Sigma factor selectivity in Borrelia burgdorferi: RpoS recognition of the ospE/ospF/elp promoters is dependent on the sequence of the ,10 regionMOLECULAR MICROBIOLOGY, Issue 6 2006Christian H. Eggers Summary Members of the ospE/ospF/elp lipoprotein gene families of Borrelia burgdorferi, the Lyme disease agent, are transcriptionally upregulated in response to the influx of blood into the midgut of an infected tick. We recently have demonstrated that despite the high degree of similarity between the promoters of the ospF (PospF) and ospE (PospE) genes of B. burgdorferi strain 297, the differential expression of ospF is RpoS-dependent, while ospE is controlled by ,70. Herein we used wild-type and RpoS-deficient strains of B. burgdorferi and Escherichia coli to analyse transcriptional reporters consisting of a green fluorescent protein (gfp) gene fused to PospF, PospE, or two hybrid promoters in which the ,10 regions of PospF and PospE were switched [PospF (E , 10) and PospE,(F , 10) respectively]. We found that the PospF,10 region is both necessary and sufficient for RpoS-dependent recognition in B. burgdorferi, while ,70 specificity for PospE is dependent on elements outside of the ,10 region. In E. coli, sigma factor selectivity for these promoters was much more permissive, with expression of each being primarily due to ,70. Alignment of the sequences upstream of each of the ospE/ospF/elp genes from B. burgdorferi strains 297 and B31 revealed that two B31 ospF paralogues [erpK (BBM38) and erpL (BBO39)] have ,10 regions virtually identical to that of PospF. Correspondingly, expression of gfp reporters based on the erpK and erpL promoters was RpoS-dependent. Thus, the sequence of the PospF,10 region appears to serve as a motif for RpoS recognition, the first described for any B. burgdorferi promoter. Taken together, our data support the notion that B. burgdorferi utilizes sequence differences at the ,10 region as one mechanism for maintaining the transcriptional integrity of RpoS-dependent and -independent genes activated at the onset of tick feeding. [source] Interaction between two cis -acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stressesTHE PLANT JOURNAL, Issue 2 2003Yoshihiro Narusaka Summary Many abiotic stress-inducible genes contain two cis -acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions. We precisely analyzed the 120 bp promoter region (,174 to ,55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences. Deletion and base substitution analyses of this region showed that the DRE-core motif functions as DRE and that the DRE/DRE-core motif could be a coupling element of ABRE. Gel mobility shift assays revealed that DRE-binding proteins (DREB1s/CBFs and DREB2s) bind to both DRE and the DRE-core motif and that ABRE-binding proteins (AREBs/ABFs) bind to ABRE in the 120 bp promoter region. In addition, transactivation experiments using Arabidopsis leaf protoplasts showed that DREBs and AREBs cumulatively transactivate the expression of a GUS reporter gene fused to the 120 bp promoter region of rd29A. These results indicate that DRE and ABRE are interdependent in the ABA-responsive expression of the rd29A gene in response to ABA in Arabidopsis. [source] Expression of antibodies using single-open reading frame vector design and polyprotein processing from mammalian cellsBIOTECHNOLOGY PROGRESS, Issue 3 2009Yune Z. Kunes Abstract We describe a novel polyprotein precursor-based approach to express antibodies from mammalian cells. Rather than expressing heavy and light chain proteins from separate expression units, the antibody heavy and light chains are contained in one single-open reading frame (sORF) separated by an intein gene fused in frame. Inside mammalian cells this ORF is transcribed into a single mRNA, and translated into one polypeptide. The antibody heavy and light chains are separated posttranslationally, assembled into the functional antibody molecule, and secreted into culture medium. It is demonstrated that Pol I intein from P. horikoshii mediates protein splicing and cleavage reactions in mammalian cells, in the context of antibody heavy and light chain amino acid sequences. To allow the separation of antibody heavy chain, light chain, and the intein, we investigated a number of intein mutations designed to inhibit intein-mediated splicing but preserve cleavage reactions. We have also designed constructs in which the signal peptide downstream from intein has altered hydrophobicity. The use of some of these mutant constructs resulted in more efficient antibody secretion, highlighting areas that can be further explored in improving such an expression system. An antibody secreted using one of the sORF constructs was characterized. This antibody has correct N-terminal sequences for both of its heavy and light chains, correct heavy and light chain MW as well as intact MW as measured by mass spectrometry. Its affinity to antigen, as measured by surface plasmon resonance (SPR), is indistinguishable from that of the same antibody produced using conventional method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | ||||||||||