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51.
The reaction mechanism of nuclease P1 from Penicillium citrinum has been investigated using single-stranded dithiophosphorylated di-, tetra-, and hexanucleotides as substrate analogs. The complexes crystallize in tetragonal and orthorhombic space groups and have been solved by molecular replacement. The high resolution structures give a clear picture of base recognition by P1 nuclease at its two nucleotide-binding sites, especially the 1.8 Å structure of a P1-tetranucleotide complex which can be considered a P1-product complex. The observed binding modes are in agreement with a catalytic mechanism where the two closely spaced zinc ions activate the attacking water while the third, more exposed zinc ion stabilizes the leaving 03' oxyanion. Stacking as well as hydrogen bonding interactions with the base 5' to the cleaved phosphodiester bond are important elements of substrate binding and recognition. Modelling of a productive P1-substrate complex based on the solved structures suggests steric hindrance as the likely reason for the resistance of Rp-phosphorothioates and phosphorodithioates. Differences with the highly homologous nuclease S1 from Aspargillus oryzae are discussed. Proteins 32:414–424, 1998. © 1998 Wiley-Liss, Inc. 相似文献
52.
Zeno Fldes-Papp Gerd Baumann Eckhard Birch-Hirschfeld Holger Eickhoff Karl Otto Greulich Albrecht K. Kleinschmidt Hartmut Seliger 《Biopolymers》1998,45(5):361-379
In this paper we put forward improved mathematical methods for detecting synthesis parameters in connection with analyzing crude products of chemically synthesized oligonucleotides. The crude products experimentally sampled are separated by high-performance capillary electrophoresis and ion-exchange high-performance liquid chromatography. The measured separation profiles of experimental syntheses can be expressed as target and nontarget yields; they are characterized by a few parameters. These parameters account for nonlinear synthesis equations that are solvable by employing iteration procedures. We provide here a theoretical as well as computational analysis based upon specific models for stepwise chain growth. Under nonconstant (nonuniform) conditions we use here an exponential form of growth, with different expressions for calculating the fractal dimension of the biochemical process under study. Step lengths of parameter variations in an interval of finite length have to be adjusted properly to find convergent solutions in a mathematical, regularly four-dimensional parameter space. It is conceivable to have most, if not all, of the calculating and plotting carefully done by a computer. This analysis represents the experimental situation up to 65-mer target oligonucleotides analyzed so far. We thus obtain the dynamics of the polymerization process limited in number by fractal models. The advantage, calculating these new methods as compared to qualitatively judged experimental methods, lies in the satisfactory evaluation of crude products, also of large amounts, of syntheses of these biopolymers. © 1998 John Wiley & Sons, Inc. Biopoly 45: 361–379, 1998 相似文献
53.
Peter Nilsson Bjrn Persson Anita Larsson Mathias Uhln Per-ke Nygren 《Journal of molecular recognition : JMR》1997,10(1):7-17
Two different strategies for scanning and screening of mutations in polymerase chain reaction (PCR) products by hybridization analysis are described, employing real-time biospecific interaction analysis (BIA) for detection. Real-time BIA was used to detect differences in hybridization responses between PCR products and different 17-mer oligonucleotide probes. For the analysis using a biosensor instrument, two different experimental formats were investigated based on immobilization of either biotinylated PCR products or oligonucleotide probes onto a sensor chip. Applied on the human tumour suppressor p53 gene, differences in hybridization levels for full-match and mismatch situations employing both formats allowed the detection of point mutations in exon 6 PCR products, derived from a breast tumour biopsy sample. In addition, a mutant sample sequence could be detected in a 50/50 background of wild type exon 6 sequence. The suitability of the different formats for obtaining a regenerable system and a high throughput of samples is discussed. © 1997 John Wiley & Sons, Ltd. 相似文献
54.
Stephen Neidle 《Biopolymers》1997,44(1):105-121
This review surveys the crystal structures between minor groove drugs and oligonucleotides, of which over thirty have now been determined. The various factors that are involved in the observed A/T sequence selectivity of these drugs are examined in structural terms. The roles of, in particular, hydrogen-bond recognition and sequence-dependent groove width, are assessed, and as a consequence the minor groove drugs have been classified into two categories, dependent on the relative roles played by these two factors in sequence recognition. Implications for the recognition of non-A/T sequences are discussed. © 1997 John Wiley & Sons, Inc. Biopoly 44: 105–121, 1997 相似文献
55.
A. S. Levina E. A. Mikhaleva M. N. Repkova V. F. Zarytova 《Russian Journal of Bioorganic Chemistry》2008,34(1):80-86
A simple and efficient method of synthesis of polyamine-oligonucleotide conjugates (PA-oligos) in high yields (up to 95%) was suggested. The terminal phosphate group of deprotected oligonucleotides was selectively activated with the redox pair triphenylphosphine-dipyridyl disulfide in the presence of a nucleophilic catalyst, and the activated oligonucleotide derivative was subjected to the reaction with a polyamine. 相似文献
56.
57.
Eric Wickstrom Mathew L. Thakur Edward R. Sauter 《International journal of peptide research and therapeutics》2003,10(3-4):191-214
Summary Genomic sequencing makes it possible to identify all the genes of an organism, now includingHomo sapiens. Yet measurement of the expression of each gene of interest still presents a daunting prospect. Northern blots, RNase protection
assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from
cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living
cells and tissues. Unfortunately, no reliable method has been available to measure levels of specific mRNAsin vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression,
and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the
absence of conjugated ligands. Nevertheless, we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides
designed to bind to IGF-1 receptors on malignant cells are taken up specifically and concentrated in nuclei. We have postulated
that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and
will permit scintigraphic imaging of oncogene mRNAs in human cancer xenografts in a mouse model. The oncogenes cyclin D1,ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experiments provide a proof-of-principle for noninvasive detection
of oncogene expression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular
gene of interest in a cell or tissue type with characteristic receptors. 相似文献
58.
Jeannette P. Staheli Jonathan T. Ryan A. Gregory Bruce Richard Boyce Timothy M. Rose 《Methods (San Diego, Calif.)》2009,49(1):32
Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) have proven to be a powerful tool for the identification of novel genes. CODEHOPs are designed from highly-conserved regions of multiply-aligned protein sequences from members of a gene family and are used in PCR amplification to identify distantly-related genes. The CODEHOP approach has been used to identify novel pathogens by targeting amino acid motifs conserved in specific pathogen families. We initiated a program utilizing the CODEHOP approach to develop PCR-based assays targeting a variety of viral families that are pathogens in non-human primates. We have also developed and further improved a computer program and website to facilitate the design of CODEHOP PCR primers. Here, we detail the method for the development of pathogen-specific CODEHOP PCR assays using the papillomavirus family as a target. Papillomaviruses constitute a diverse virus family infecting a wide variety of mammalian species, including humans and non-human primates. We demonstrate that our pan-papillomavirus CODEHOP assay is broadly reactive with all major branches of the virus family and show its utility in identifying a novel non-human primate papillomavirus in cynomolgus macaques. 相似文献
59.
A. S. Pavlova P. E. Vorobyev V. F. Zarytova 《Russian Journal of Bioorganic Chemistry》2009,35(2):197-206
Monofunctional conjugates of 15-mer triplex-forming oligonucleotide (TFO) with covalently attached bleomycin A5 residue at the 5′-end (Blm-p15) were synthesized. Bifunctional conjugates of TFO containing, in addition to Blm, the residues of intercalator 6-chloro-2-methoxy-9-aminoacridine (Acr) or N-(2-hydroxyethyl)phenazinium (Phn) were obtained for the first time. The Acr and Phn residues were attached to the 3′-phosphate group of TFO through L1 and L2 linkers, respectively, resulting in the compounds Blmp15pL1-Acr and Blm-p15pL2-Phn. The values of dissociation constants of the corresponding triplexes were evaluated using the gel retardation method. The Acr residue in Blm-p15pL1-Acr was shown to enhance the stability of the formed triplex by one order of magnitude. It was demonstrated that all synthesized conjugates are capable of specifically and nonspecifically damaging a target DNA, forming direct breaks and alkaline-labile sites. The extent of the specific cleavage of the target DNA was 15% in the case of a fivefold excess of the conjugates over the DNA duplex. The site-specific triplex-mediated cleavage of a target DNA was shown for the first time to occur predominantly (>90%) with the formation of the direct breaks of both DNA strands. The results show the availability of bleomycin-containing oligonucleotides as antigene compounds. 相似文献
60.
Hua-qi PAN Nan WANG Li LIU Lei LIU Jiang-chun HU Pu-yan CHEN Shu-jin WANG Rui-bing CAO 《中国病毒学》2009,24(3)
Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses. 相似文献