Development of the novel drug releasing system triggered by hybridization with target sequence

We have already established the strategy of synchronous activation by hybridization, in which the highly reactive cross-linking agent, 2-amino-6-vinylpurine nucleoside analog, can be generated from its stable precursors, the phenylsulfide derivatives, by a hybridization-promoted activation process with selectivity to cytosine. In this study, this in situ activation system was applied to the method for the drug releasing system triggered by hybridization with the target sequence.     

Isolation of mesoderm-specific genes expressed in the Drosophila embryo

Tissue differentiation during embryonic development involves activation of specific genes. To isolate genes selectively expressed in mesoderm and nervous system in the Drosophila embryo, we have screened a cDNA library with molecular probes enriched in specific gene sequences from both tissues. In this way, we have isolated six mesoderm-specific genes, as demonstrated by in situ hybridization to embryo sections. Two of these genes, expressed during muscle differentiation, are described here for the first time. These genes have been localized in the 17A region of the first chromosome and in the 60A region of the second chromosome, respectively. No neural-specific genes were identified using this approach, most probably because of the low sensitivity of detection methods which combine filter hybridization techniques with the use of complex probes.     

Influence of secondary structure on kinetics and reaction mechanism of DNA hybridization

Hybridization of nucleic acids with secondary structure is involved in many biological processes and technological applications. To gain more insight into its mechanism, we have investigated the kinetics of DNA hybridization/denaturation via fluorescence resonance energy transfer (FRET) on perfectly matched and single-base-mismatched DNA strands. DNA hybridization shows non-Arrhenius behavior. At high temperature, the apparent activation energies of DNA hybridization are negative and independent of secondary structure. In contrast, when temperature decreases, the apparent activation energies of DNA hybridization change to positive and become structure dependent. The large unfavorable enthalpy of secondary structure melting is compensated for by concomitant duplex formation. Based on our results, we propose a reaction mechanism about how the melting of secondary structure influences the hybridization process. A significant point in the mechanism is that the rate-limiting step switches along with temperature variation in the hybridization process of structured DNA, because the free energy profile of hybridization in structured DNA varies with the variation in temperature.     

Human monoclonal autoantibodies to characterize platelet antigens in immune-mediated thrombocytopenia

Idiopathic thrombocytopenic purpura is characterized by antiplatelet antibodies which mediate the rapid destruction of these cells by the reticuloendothelial cell system. Low serum titers of autoantibodies and the polyclonal nature of human serum make it difficult to identify platelet target antigens with plasma antibodies. To circumvent these problems, we have utilized the techniques of EBV transformation and somatic cell hybridization in order to isolate human monoclonal antibodies from patients with ITP. In this paper we describe the use of human monoclonal autoantibodies to characterize an activation specific antigen on GPIIIa and an autoantigen on the GPIb complex. Ultimately, we hope to determine whether these autoantibodies emerge from a pool of naturally occurring antibodies to activation or senescence antigens, or are triggered by environmental agents such as bacteria or virus, which are comprised of antigens similar to those found on the platelet membrane.     

Hyperexpression of the osmB gene in an acidic phospholipid-deficient Escherichia coli mutant

An Escherichia coli pgsA null mutant deficient in acidic phospholipids shows a thermosensitive cell lysis phenotype because of activation of the Rcs phosphorelay signal transduction system. We conducted a DNA microarray analysis with special attention to the genes affected by growth temperature in the mutant deficient in acidic phospholipids. Among the genes identified as highly expressed at high temperature in the pgsA null mutant, the osmB gene was shown to be dependent on the Rcs system for the high expression by dot blot hybridization. Induction of the cloned osmB in the pgsA null mutant caused the thermosensitive defect even in the absence of the Rcs system. Although the deletion of osmB did not suppress the thermosensitivity in the presence of the Rcs system, indicating a multifactorial nature of the deleterious effect of the Rcs activation, we suggest that the osmB hyperexpression is one of the causes of the Rcs-dependent lysis phenotype of the pgsA null mutant.     

Prothrombin overexpressed in post-natal neurones requires blood factors for activation in the mouse brain

Thrombin is thought to mediate, through protease-activated receptors, both protective as well as cytotoxic effects. As thrombin receptors are expressed in the CNS, an important question arises as to whether the intact nervous system is able to generate thrombin by activation of its precursor prothrombin, derived endogenously or only upon extravasation following brain injury. To address this question, transgenic mice that express C-terminally haemagglutinin tagged human prothrombin in post-mitotic neurones were generated. In situ hybridization and immunohistochemical analysis showed abundant and widespread cerebral expression of the transgene. Amidolytic assays of brain homogenates and hippocampal slice cultures demonstrated that activation of transgenic prothrombin required added factors, such as snake venom or blood components. This strongly suggests that any possible action of thrombin in the adult CNS depends on blood-derived factors that activate prothrombin. Furthermore, the results are consistent with the idea that in the non-pathological situation an as yet unidentified ligand activates thrombin receptors in the nervous system.     

优化菌落原位杂交体系筛选野生稻基因组文库

菌落原位杂交仍是当今筛选基因组文库的重要方法之一,但难以保持背景清晰和阳性杂交点明显的稳定的杂交体系.通过对杂交膜不同处理方法的对比,表明利用溶菌酶处理杂交体系进行菌落原位杂交筛选,其杂交结果背景较干净,且能根据杂交信号的强弱很清晰地显色阳性黑点.采用该法筛选野生稻基因组文库,已筛选出22个阳性克隆,并测序分析均为阳性克隆.     

Concurrent mutations in two different ras genes in acute myelocytic leukemias.

DNA transfection analyses (tumorigenicity assay) and hybridization to mutation specific oligonucleotide probes established point mutations in codon 61 of both, N-ras and Ki-ras genes in fresh leukemic cells of an AML patient. Concurrent activation of N-ras and Ki-ras sequences by point mutations in codons 12 were demonstrated for AML cell line Rc2a. Moreover, using a rapid and sensitive dot-blot screening procedure based on the combination of in vitro amplification of ras specific sequences and oligonucleotide hybridization we could show that ras gene activation was not present in primary leukemic cells of the patient this cell line had been derived from, but rather occurred during later passages of Rc2a.     

Innovative integrated system for real-time measurement of hybridization and melting on standard format microarrays

Despite the great popularity and potential of microarrays, their use for research and clinical applications is still hampered by lengthy and costly design and optimization processes, mainly because the technology relies on the end point measurement of hybridization. Thus, the ability to monitor many hybridization events on a standard microarray slide in real time would greatly expand the use and benefit of this technology, as it would give access to better prediction of probe performance and improved optimization of hybridization parameters. Although real-time hybridization and thermal denaturation measurements have been reported, a complete walk-away system compatible with the standard format of microarrays is still unavailable. To address this issue, we have designed a biochip tool that combines a hybridization station with active mixing capability and temperature control together with a fluorescence reader in a single compact benchtop instrument. This integrated live hybridization machine (LHM) allows measuring in real time the hybridization of target DNA to thousands of probes simultaneously and provides excellent levels of detection and superior sequence discrimination. Here we show on an environmental single nucleotide polymorphism (SNP) model system that the LHM enables a variety of experiments unachievable with conventional biochip tools.     

Single-fluorophore monitoring of DNA hybridization for investigating the effect of secondary structure on the nucleation step

Nucleic acid hybridization is one of the essential biological processes involved in storage and transmission of genetic information. Here we quantitatively determined the effect of secondary structure on the hybridization activation energy using structurally defined oligonucleotides. It turned out that activation energy is linearly proportional to the length of a single-stranded region flanking a nucleation site, generating a 0.18 kcal/mol energy barrier per nucleotide. Based on this result, we propose that the presence of single-stranded segments available for non-productive base pairing with a nucleation counterpart extends the searching process for nucleation sites to find a perfect match. This result may provide insights into rational selection of a target mRNA site for siRNA and antisense gene silencing.     

Solid-phase time-resolved fluorescence detection of human immunodeficiency virus polymerase chain reaction amplification products.

A new assay system for the detection of polymerase chain reaction (PCR) amplification products is presented. This single-pot sandwich assay system employs solid-support oligonucleotide-coated capture beads, a rare earth metal chelate-labeled probe, and a time-resolved fluorescence detection. The new assay system was evaluated for various reaction conditions including, DNA denaturation time, hybridization salt concentration, probe concentration, and hybridization time, all of which are important in designing an assay with a high level of sensitivity for the detection of duplex DNA. This nonisotopic assay system was applied to the detection of purified human immunodeficiency virus (HIV) DNA and sensitivity was compared with agarose gel electrophoresis and slot blot hybridization using a 32P-labeled probe. We were able to detect the amplified product from one copy of HIV DNA after 35 cycles of PCR amplification in less than 30 min using this assay, which compared with one copy by gel electrophoresis after 40 cycles of PCR amplification and one copy by slot blot hybridization after 35 cycles of PCR amplification and an overnight exposure of the autoradiogram. Thus, this assay is rapid, sensitive, and easy to use.     

Highly fluorochrome labeled gene probes for quantitative tracing of RNA in individual cells by in situ hybridization.

A new method is presented for preparing highly fluorochrome labeled gene probes suitable for in situ hybridization. For this purpose fluorochromes were attached to a synthetic polypeptide, which was then coupled covalently to various gene probes. The advantage of the reported method is its high labeling efficiency and the easy coupling procedure. The method allows rapid and quantitative detection of homologous RNA at the single cell level. Optimal conditions for the hybridization of fluorochrome-labeled gene probes were established microfluorimetrically, and the specificity and sensitivity of the method were tested. Quantitation of the RNA with a fluorochrome-labeled gene probe in situ in individual cells allows determination of the degree of gene activation in individual cells and may thus provide a new tool for investigation of normal and malignant cells with respect to activation of genes controlling differentiation and proliferation.     

Early clonality and high-frequency proviral integration into the c-myc locus in AKR leukemias.

Blot hybridization of thymocyte DNA from AKR/J mice was used to detect new proviral junction fragments as markers of clonality at different stages of viral leukemogenesis and to detect DNA rearrangements at the c-myc locus due to proviral insertion. Clonal populations of thymocytes were observed in mink cell focus-forming virus-injected mice as early as 35 days postinjection, at a stage distinguishable from frank leukemia by flow cytometric analysis and transplantation bioassay. Specific proviral integrations in the c-myc locus were detected in 15% of these early clones and in up to 65% of late-developing thymomas and frank leukemias. Thus, in this system c-myc activation appears to be a common mechanism in T-cell leukemogenesis.     

Acridine orange binding to chromatin of individual cells and nuclei under different staining conditions. I. Binding capacity of chromatin.

A study was made to find the optimal conditions for titration of the strong acridine orange binding sites of DNA in situ by an equilibrium staining method. Low concentrations of dye (≈10^?6 M) and an equilibration time of about 1 h were found necessary. Chick erythrocyte nuclei were used as a model system to compare results of this equilibrium method with those of conventional staining. Before staining, nuclei were subjected to acid extraction and denaturation or to biological activation via cell hybridization. Qualitatively similar results were obtained with the two staining methods, but the equilibrium method circumvents the problems of dye-to-dye aggregation and differences in diffusion conditions, and thus gives more easily interpretable data and true quantitative information about the properties of chromatin in situ.     

Identification and characterization of a novel Mt-retrotransposon highly represented in the female mouse germline

The control of primordial follicle recruitment into the growing follicle population is a major limiting process in female reproduction. In order to gain insight into the molecular processes occurring at the time of primordial follicle activation, a subtractive hybridization analysis was performed between cDNAs prepared from temporally distinct mouse neonatal ovarian tissues that differed according to the state of primordial follicle activation. One highly represented clone associated with activation was an Mt retrotransposon-like sequence designated Mtfull, which was subsequently cloned and determined to be novel and restricted in expression to the ovary. The polyadenylated 1684-bp sequence has long terminal repeats, is predicted to be noncoding, and is the predominant Mti-related sequence present in the mouse ovary. In situ hybridization further localized Mtfull expression to the oocyte and confirmed that expression is concomitant with follicle activation. Together with in silico data, we predict Mtfull plays an essential role in folliculogenesis through regulation of gene expression.     

Indiscriminate degradation of RNAs in interferon-treated, vaccinia virus-infected mouse L cells.

In this report we used Northern blot hybridization analysis to characterize the fate of several species of viral RNA transcribed from internal and terminal regions of vaccinia DNA in interferon-treated, infected mouse L cells grown in suspension. All species of viral RNAs were expressed but were reduced in amount. Larger-sized RNAs were reduced more than smaller-sized RNAs. This reduction appears to be related to the activation of the interferon-mediated double-stranded RNA-dependent 2-5A synthetase-endoribonuclease system, as the rRNA cleavage pattern characteristic of this system was observed early in infection and in cell extracts in response to exogenous 2-5A. Thus, in interferon-treated, vaccinia-infected mouse L cells in suspension, there is indiscriminate degradation of viral and cellular RNAs, and this RNA breakdown might play a role in the interferon-mediated inhibition of protein synthesis.     

Multifaceted,Cross-Generational Costs of Hybridization in Sibling Drosophila species

Maladaptive hybridization, as determined by the pattern and intensity of selection against hybrid individuals, is an important factor contributing to the evolution of prezygotic reproductive isolation. To identify the consequences of hybridization between Drosophila pseudoobscura and D. persimilis, we estimated multiple fitness components for F1 hybrids and backcross progeny and used these to compare the relative fitness of parental species and their hybrids across two generations. We document many sources of intrinsic (developmental) and extrinsic (ecological) selection that dramatically increase the fitness costs of hybridization beyond the well-documented F1 male sterility in this model system. Our results indicate that the cost of hybridization accrues over multiple generations and reinforcement in this system is driven by selection against hybridization above and beyond the cost of hybrid male sterility; we estimate a fitness loss of >95% relative to the parental species across two generations of hybridization. Our findings demonstrate the importance of estimating hybridization costs using multiple fitness measures from multiple generations in an ecologically relevant context; so doing can reveal intense postzygotic selection against hybridization and thus, an enhanced role for reinforcement in the evolution of populations and diversification of species.