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Selected Clones (selected + clone)
Selected AbstractsGenetic redundancy in human cervical carcinoma cells: Identification of cells with "normal" propertiesINTERNATIONAL JOURNAL OF CANCER, Issue 10 2007Anastasia Bachmann Abstract Although it is generally assumed that cancer arises from a singular cell, a tumor must be considered as a dynamic and emergent biological structure, whose organizing principle is determined by genetic and epigenetic modifications, occurring variably in response to microenvironmental selection conditions. As previously shown, HPV-positive cervical carcinoma cells have lost their ability to induce IFN-, upon TNF-, treatment. However, regarding cancer as a non-linear system, which may, even in the absence of an apparent selection pressure, fluctuate between different "metastable" phenotypes, we demonstrate that TNF-, mediated IFN-, induction is not irreversibly disturbed in all cells. Using the IFN-, sensitive Encephalomyocarditis virus (EMCV) as a tool to monitor antiviral activity in long-term established malignant HeLa cells, rare IFN-, expressing clones were rescued from a population of non-responsive and EMCV-sensitive cells. Antiviral activity was mediated by the re-expression of IRF-1 and p48 (IRF-9), both key regulatory molecules normally found to be suppressed in cervical carcinoma cells. Upon inoculating of selected clones into immunocompromised animals, a reduced or even an absence of tumorigenicity of initially highly malignant cells could be discerned. These data indicate that both the absence of interferon signaling and the ability to form tumors were reversed in a minority of cells. We provide a paradigm for the existence of innate genetic redundancy mechanisms, where a particular phenotype persists and can be isolated without application of drugs generally changing the epigenetic context. © 2007 Wiley-Liss, Inc. [source] Analysis of the CD2 and spliceosomal Sm B/B, polyproline-arginine motifs defined by a monoclonal antibody using a phage-displayed random peptide libraryJOURNAL OF MOLECULAR RECOGNITION, Issue 6 2006Dimitri Monos Abstract The cytoplasmic region of the CD2 receptor of lymphocytes contains proline-rich motifs, which are involved in T cell activation and interleukin-2 production. An intracellular CD2 binding protein, CD2BP2, interacts with two tandem PPPPGHR segments of the CD2 tail. CD2BP2 contains a GYF (glycine-tyrosine-phenylalanine) domain that confers binding to these proline-rich sequences. Monoclonal antibody 3E10 that was previously raised against a peptide containing the CD2 PPPPGHR segment reacts with the native CD2 molecule and spliceosomal Sm B/B, proteins. To identify the exact epitope on the CD2 peptide recognized by 3E10, a phage-displayed combinatorial peptide library was used. Analysis of the selected clones revealed that the mAb 3E10 binds preferentially to the motif PxxPPGxR. Experiments using amino acid substitutions with synthetic peptides confirmed the reactivity of mAb 3E10 with this motif. In addition, we show that several similarities exist between this motif and the CD2BP2-GFY recognition motif PPGxR/K. Binding of antibody 3E10 indicates some degree of degeneracy, which is consistent with its ability to recognize structurally related polyproline,arginine motifs found in intracellular proteins including Sm B/B, proteins and other RNA binding proteins. Thus, mAb 3E10 can be used to specifically identify a sub-class of proline-rich motifs, and as such can be used to study the potential role of these proline-rich sequences in mediating protein-protein interactions. Copyright © 2006 John Wiley & Sons, Ltd. [source] Cloning and pharmacological characterization of the equine adenosine A2A receptor: a potential therapeutic target for the treatment of equine endotoxemiaJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 4 2006C. I. BRANDON The aim of the current study was to clone the equine adenosine A2A receptor gene and to establish a heterologous expression system to ascertain its pharmacologic profile via radioligand binding and functional assays. An eA2A -R expression construct was generated by ligation of the eA2A cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA2A -R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (KD), and receptor densities (Bmax) of selected clones. Equilibrium competition binding revealed a rank order of agonist potency of ATL > CV-1808 > NECA > 2-CADO > CGS21680, and a rank order of antagonist potency as ZM241385 > 8-phenyltheophylline > p -sulfophenyltheophylline > caffeine. Furthermore, adenylate cyclase assays using selective A2A -R agonists revealed that the eA2A -R functionally coupled to G,s as indicated by an increase in intracellular [3H]cAMP upon receptor activation. Finally, NF- ,B reporter gene assays revealed a CGS21680 concentration-dependent inhibition of NF- ,B activity. These results indicate that the heterologously expressed eA2A -R has a pharmacological profile similar to that of other mammalian A2A receptors and thus can be utilized for further characterization of the eA2A -R to ascertain whether it can serve as a suitable pharmacological target for equine inflammatory disease. [source] Isolation and characterization of microsatellites in Pacific white shrimp Penaeus (Litopenaeus) vannameiMOLECULAR ECOLOGY RESOURCES, Issue 3 2002P. Cruz Abstract Five polymorphic microsatellite loci were characterized for Penaeus (Litopenaeus) vannamei. Loci were isolated using a partial Sau3A1 genomic library by the sequencing of randomly selected clones and by a biotinylated (CT)10 and (GT)10 probes screening procedure. The last strategy resulted in the most useful data. About 40% of the clones showed a previously reported satellite/microsatellite (PVS1), reducing the chance of finding new microsatellite regions. Whereas two of the microsatellite loci with more than 10 alleles will be useful for mating analysis in a breeding program, the others might prove useful for population genetic studies. [source] Trifluralin herbicide-induced resistance of melon to fusarium wilt involves expression of stress- and defence-related genesMOLECULAR PLANT PATHOLOGY, Issue 1 2007MAYA LOTAN-POMPAN SUMMARY To identify genes involved in trifluralin herbicide-induced resistance of melon to Fusarium oxysporum f. sp. melonis, suppression subtractive hybridization (SSH) and cDNA-amplified fragment-length polymorphism (cDNA-AFLP) were used. A total of 123 clones,60 of which have never been isolated from melon,were isolated, sequenced and annotated. A significant proportion (35%) of the total 123 clones exhibited similarity to genes that have been formerly described as stress- or defence-related. Thirty-two selected clones were subjected to a detailed expression analysis, one-third of which were found to be up-regulated in response to trifluralin treatment and/or fusarium inoculation. The putative roles of seven of these clones in stress are discussed. Furthermore, the expression of four stress-related and up-regulated genes was enhanced when the plants were subjected to salinity stress, suggesting that trifluralin induces a general stress response which protects the plant against fusarium wilt. [source] Clonal selection in ,Uzunmusa' hazelnutPLANT BREEDING, Issue 4 2003Abstract Clonal selection was practised in ,Uzunmusa' hazelnut over the past 3 years (1999-2001) to select the highest quality types. Based on an initial assessment of a total of 102 types, 45 were selected for further study. The best types were selection numbers (SN) 397 and 570. The two selected clones have very good characteristics and seem to be superior to the standard clone. The clones had a higher kernel percentage (62.72%), a higher number of nuts per cluster (5.5), thinner shells (0.75 mm) and heavier nuts (2.34 g). On the other hand, the clones seem to be very suitable for the nut industry because of their oil content and size. These types have very thin shells which are suitable for in-shell market. [source] Mapping of expressed sequence tags from a porcine early embryonic cDNA libraryANIMAL GENETICS, Issue 2 2001T. P. L. Smith The goal of this study was to identify and map genes expressed during the elongation phase of embryogenesis in swine. Expressed sequence tags were analysed from a previously described porcine cDNA library prepared from elongating swine embryos. Average insert length of randomly selected clones was approximately 600 bp, with a range from <100 to >2500 bp. Single-pass, coding strand sequences from 1132 independent clones were compared with the GenBank non-redundant (nr) database via BLASTN analysis to identify potential porcine homologous of known genes. Among these sequences, 781 (69%) showed significant (score >300) homology to non- mitochondrial sequences previously deposited in GenBank. Sequences matching interleucin 1 , and thymosin , 10 were most frequently observed (24 and 18 clones, respectively), in addition to matches with 310 other distinct genes. No significant match in the GenBank nr database was obtained for 303 sequences. Analysis demonstrated that 151 (50%) had open reading frames (ORF) extending at least 50 codons from the first base of the clone insert. Genetic markers were developed and used to map a subset of 17 genes, selected on the basis of function or of the ability to design primers that successfully amplified porcine genomic DNA, to 10 different porcine chromosomes, providing a set of mapped markers corresponding to genes expressed during conceptus elongation. [source] Cre recombinase-mediated site-specific modification of a cellular genome using an integrase-defective retroviral vectorBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Shuohao Huang Abstract Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase-mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild-type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase-defective retroviral vectors (IDRV), which contains an ATG-deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418-resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site-specific recombination. The successful substitution of the original viral integration machinery with a non-viral mechanism could expand the application of retroviral vectors. Biotechnol. Bioeng. 2010;107:717,729. © 2010 Wiley Periodicals, Inc. [source] | |||||||||||||