926 resultados para Nucleic acid detection tests
Resumo:
In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assays.
Resumo:
UDP-glucuronosyltransferase (UGT) 1A1 (UGT1A1) catalyzes the glucuronidation of bilirubin in liver. Among all UGT isoforms identified to date, it is the only relevant bilirubin-glucuronidating enzyme in human. Because glucuronoconjugation is the major route of bilirubin elimination, any genetic alteration that affects bilirubin glucuronosyltransferase activity may result in a more or less severe hyperbilirubinemia. In this study, we report the cloning and characterization of the transcriptional regulation of the mouse UGT1A1 gene. Primary-structure analysis of the mouse Thymidine Adevice promoter revealed marked differences with its human homolog. First, the mouse promoter lacks the highly polymorphic thymidine/adenine repeat occurring in the human promoter, which has been associated with some forms of hyperbilirubinemia. Second, an L1 transposon element, which is absent in the human promoter, is found 480 bp upstream of the transcription start site in mouse. Using the electromobility shift and DNase I footprinting experiments, we have identified a hepatocyte nuclear factor 1-binding site in the mouse UGT1A1 promoter that confers responsiveness to both factors HNF1alpha and HNF1beta in HEK293 cells. Furthermore, we show that this element, which is conserved in the human promoter, also confers strong HNF1 responsiveness to the human UGT1A1 gene. Together, these results provide evidence for a major regulatory function of this liver-enriched transcription factor in UGT1A1 activity in both rodents and human.
Resumo:
Progressive destruction of the insulin-producing beta cells in nonobese diabetic mice is observed after infiltration of the pancreas with lymphocytes [Makino, S., Kunimoto, K., Muraoka, Y., Mizushima, Y., Katagiri, K. & Tochino, Y. (1980) Exp. Anim. (Tokyo) 29, 1-13]. We show that the genes for tumor necrosis factor alpha and granzyme A, a serine protease associated with cytoplasmic granules of cytotoxic cells, are expressed during the development of spontaneous diabetes mellitus in the nonobese diabetic mouse. Granzyme A-positive cells are found both in and surrounding the islets, implying induction prior to islet infiltration. Tumor necrosis factor alpha expression is exclusively observed in the intra-islet infiltrate, predominantly in lymphocytes adjacent to insulin-producing beta cells, the targets of the autoimmune destruction, implying that tumor necrosis factor alpha expression is induced locally--i.e., in the islet. A considerable portion of cells expressing tumor necrosis factor alpha appear to be CD4+ T cells. This T-cell subset was previously shown to be necessary for development of the disease. Thus, these findings may be important for understanding the pathogenesis of autoimmune diabetes mellitus and potentially also for that of other T-cell-mediated autoimmune diseases.
Resumo:
In recent years there has been growing interest in the question of how the particular topology of polymeric chains affects their overall dimensions and physical behavior. The majority of relevant studies are based on numerical simulation methods or analytical treatment; however, both these approaches depend on various assumptions and simplifications. Experimental verification is clearly needed but was hampered by practical difficulties in obtaining preparative amounts of knotted or catenated polymers with predefined topology and precisely set chain length. We introduce here an efficient method of production of various single-stranded DNA knots and catenanes that have the same global chain length. We also characterize electrophoretic migration of the produced single-stranded DNA knots and catenanes with increasing complexity.
Resumo:
Background: Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells. Results: The study of 13C distribution of Jukat cells exposed to low edelfosine concentration, which induces apoptosis in ¿5% of cells, revealed metabolic changes previous to the development of apoptotic program. Specifically, it was found that low dose of edelfosine stimulates the TCA cycle. These metabolic perturbations were coupled with an increase of nucleic acid synthesis de novo, which indicates acceleration of biosynthetic and reparative processes. The further increase of the TCA cycle fluxes, when higher doses of drug applied, eventually enhance reactive oxygen species (ROS) production and trigger apoptotic program. Conclusion: The application of Isodyn to the analysis of mechanism of edelfosine-induced apoptosis revealed primary drug-induced metabolic changes, which are important for the subsequent initiation of apoptotic program. Initiation of such metabolic changes could be exploited in anticancer therapy.
Resumo:
The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.
Resumo:
Eukaryotic transcription is tightly regulated by transcriptional regulatory elements, even though these elements may be located far away from their target genes. It is now widely recognized that these regulatory elements can be brought in close proximity through the formation of chromatin loops, and that these loops are crucial for transcriptional regulation of their target genes. The chromosome conformation capture (3C) technique presents a snapshot of long-range interactions, by fixing physically interacting elements with formaldehyde, digestion of the DNA, and ligation to obtain a library of unique ligation products. Recently, several large-scale modifications to the 3C technique have been presented. Here, we describe chromosome conformation capture sequencing (4C-seq), a high-throughput version of the 3C technique that combines the 3C-on-chip (4C) protocol with next-generation Illumina sequencing. The method is presented for use in mammalian cell lines, but can be adapted to use in mammalian tissues and any other eukaryotic genome.
Resumo:
Endogenous and infectious mouse mammary tumor viruses (MMTVs) encode in their 3' long terminal repeat a protein that exerts superantigen activity; that is, it is able to interact with T cells via the variable domain of the T cell receptor (TCR) beta chain. We show here that transmission of an infectious MMTV is prevented when superantigen-reactive cells are absent through either clonal deletion due to the expression of an endogenous MTV with identical superantigen specificity or exclusion due to expression of a transgenic TCR beta chain that does not interact with the viral superantigen. A strict requirement for superantigen-reactive T cells is also seen for a local immune response following MMTV infection. This immune response locally amplifies the number of MMTV-infected B cells, most likely owing to their clonal expansion. Collectively, our data indicate that a superantigen-induced immune response is critical for the MMTV life cycle.
Resumo:
The human melanoma-associated antigen identified by the monoclonal antibody (mAb) Me14-D12 is a cell surface protein whose expression is induced by interferon-gamma (IFN-gamma). We have recently reported the molecular cloning of a genomic probe specific for the gene and mRNA of this protein. By screening with the genomic probe, we have now isolated a full length 3.0 kb cDNA from a Raji cell line-derived lambda-gt10 library. Sequence analysis of this cDNA showed a 99.8% homology with the intercellular adhesion molecule-1 (ICAM-1). Mouse Ltk- cells stably transfected with the human cDNA clone were found to express the ICAM-1 antigenic determinants detected by mAb Me14-D12 and a reference anti-ICAM-1 mAb, as judged by surface immunofluorescence. Immunoprecipitation of surface-iodinated proteins with mAb Me14-D12 revealed the presence of a 90 kD molecule with identical mobility to ICAM-1. In addition, mAb Me14-D12 could inhibit the phorbolester-stimulated aggregation of U937 cells. The findings show that the human melanoma-associated Me14-D12 antigen is the adhesion molecule ICAM-1.
Simulations of action of DNA topoisomerases to investigate boundaries and shapes of spaces of knots.
Resumo:
The configuration space available to randomly cyclized polymers is divided into subspaces accessible to individual knot types. A phantom chain utilized in numerical simulations of polymers can explore all subspaces, whereas a real closed chain forming a figure-of-eight knot, for example, is confined to a subspace corresponding to this knot type only. One can conceptually compare the assembly of configuration spaces of various knot types to a complex foam where individual cells delimit the configuration space available to a given knot type. Neighboring cells in the foam harbor knots that can be converted into each other by just one intersegmental passage. Such a segment-segment passage occurring at the level of knotted configurations corresponds to a passage through the interface between neighboring cells in the foamy knot space. Using a DNA topoisomerase-inspired simulation approach we characterize here the effective interface area between neighboring knot spaces as well as the surface-to-volume ratio of individual knot spaces. These results provide a reference system required for better understanding mechanisms of action of various DNA topoisomerases.
Resumo:
The ecdysone-responsive DNA sequence of the Drosophila hsp27 gene promoter contains four direct and inverted repeats reminiscent of those that compose the vertebrate palindromic estrogen response element (ERE) and the thyroid hormone/retinoic acid response element (TRE/RRE). Interestingly, a 3 bp substitution in the wild-type Hsp27 ecdysone response element (EcdRE) increases both its similarity with the vertebrate ERE and TRE/RRE and its capacity to confer ecdysone responsiveness to a heterologous promoter. Remarkably, increasing the spacing between the inverted repeats of this strong EcdRE by two nucleotides converts it into an ERE. Inversely, decreasing the spacing between the two inverted repeats of the vertebrate consensus palindromic ERE, from three to one nucleotide, converts it into a functional EcdRE. Thus, the only difference between an invertebrate EcdRE and a vertebrate palindromic ERE or TRE/RRE is in the spacing between the conserved inverted repeated motifs forming these palindromic HREs. The finding that the sequence motif 5'-GGTCA-3' present in the vertebrate ERE and TRE/RRE is also a functionally important characteristic of an invertebrate HRE, suggests that a common ancestor regulatory DNA sequence gave rise to all HREs known so far. We discuss the possibility that this progenitor motif is the GGTCA sequence.
Resumo:
Marie Unna hereditary hypotrichosis (MUHH) is an autosomal dominant form of genetic hair loss. In a large Chinese family carrying MUHH, we identified a pathogenic initiation codon mutation in U2HR, an inhibitory upstream ORF in the 5' UTR of the gene encoding the human hairless homolog (HR). U2HR is predicted to encode a 34-amino acid peptide that is highly conserved among mammals. In 18 more families from different ancestral groups, we identified a range of defects in U2HR, including loss of initiation, delayed termination codon and nonsense and missense mutations. Functional analysis showed that these classes of mutations all resulted in increased translation of the main HR physiological ORF. Our results establish the link between MUHH and U2HR, show that fine-tuning of HR protein levels is important in control of hair growth, and identify a potential mechanism for preventing hair loss or promoting hair removal.
Resumo:
The SOS screen, as originally described by Perkins et al. (1999) [7], was setup with the aim of identifying Arabidopsis functions that might potentially be involved in the DNA metabolism. Such functions, when expressed in bacteria, are prone to disturb replication and thus trigger the SOS response. Consistently, expression of AtRAD51 and AtDMC1 induced the SOS response in bacteria, even affecting E. coli viability. 100 SOS-inducing cDNAs were isolated from a cDNA library constructed from an Arabidopsis cell suspension that was found to highly express meiotic genes. A large proportion of these SOS(+) candidates are clearly related to the DNA metabolism, others could be involved in the RNA metabolism, while the remaining cDNAs encode either totally unknown proteins or proteins that were considered as irrelevant. Seven SOS(+) candidate genes are induced following gamma irradiation. The in planta function of several of the SOS-inducing clones was investigated using T-DNA insertional mutants or RNA interference. Only one SOS(+) candidate, among those examined, exhibited a defined phenotype: silenced plants for DUT1 were sensitive to 5-fluoro-uracil (5FU), as is the case of the leaky dut-1 mutant in E. coli that are affected in dUTPase activity. dUTPase is essential to prevent uracil incorporation in the course of DNA replication.
Resumo:
We describe the transcriptional potentiation in estrogen responsive transcription extracts of the Xenopus vitellogenin B1 gene promoter through the formation of a positioned nucleosome. Nuclease digestion and hydroxyl radical cleavage indicate that strong, DNA sequence-directed positioning of a nucleosome occurs between -300 and -140 relative to the start site of transcription. Deletion of this DNA sequence abolishes the potentiation of transcription due to nucleosome assembly. The wrapping of DNA around the histone core of the nucleosome positioned between -300 and -140 creates a static loop in which distal estrogen receptor binding sites are brought close to proximal promoter elements. This might facilitate interactions between the trans-acting factors themselves and/or RNA polymerase. Such a nucleosome provides an example of how chromatin structure might have a positive effect on the transcription process.