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11.
Enzymatic labeling of nucleic acids is a fundamental tool in molecular biology with virtually every aspect of nucleic acid
hybridization technique involving the use of labeled probes. Different methods for enzymatic labeling of DNA, RNA and oligonucleotide
probes are available today. In this review, we will describe both radioactive and nonradioactive labeling methods, yet the
choice of system for labeling the probe depends on the application under study. 相似文献
12.
We have developed a nonradioactive oligonucleotide multilocus DNA fingerprinting method for Cynoglossum officinale . Of the 19 probes tested, six probes yielded banding patterns for all restriction enzymes used. All but one of the informative probes are repeats with a four-base motif. Approximately 60% of the loci appeared to be polymorphic. The sensitivity of the nonradioactive method was equal to that of the radioactive method. In addition, a new simple calculation method is presented to estimate selfing rates and approximate 95% confidence limits from the DNA fingerprint profiles avoiding 'between-gel' comparisons. The selfing rates differed significantly (as determined from 95% confidence intervals) between naturally pollinated individuals of C. officinale within the experimental population. The estimates ranged from 0 to 70% selfing. 相似文献
13.
Crystallization and preliminary X-ray diffraction studies of aSFP, a bovine seminal plasma protein with a single CUB domain architecture. 下载免费PDF全文
J. M. Dias A. L. Carvalho I. Klln J. J. Calvete E. Tpfer-Petersen P. F. Varela A. Romero C. Urbanke M. J. Romo 《Protein science : a publication of the Protein Society》1997,6(3):725-727
Bovine acidic seminal fluid protein (aSFP) is a 1.29 kDa polypeptide of the spermadhesin family built by a single CUB domain architecture. The CUB domain is an extracellular module present in 16 functionally diverse proteins. To determine the three-dimensional structure of aSFP, the protein was crystallized at 21 degrees C by vapor diffusion in hanging drops, using ammonium sulfate, pH 4.7, and polyethyleneglycol 4,000 as precipitants, containing 10% dioxane to avoid the formation of clustered crystals. Elongated prismatic crystals with maximal size of 0.6 x 0.3 x 0.2 mm3 diffract to beyond 1.9 A resolution and belong to space group P2(1)2(1)2(1), with cell parameters a = 52.4 A, b = 41.5 A, c = 48.2 A. There is one aSFP molecule per asymmetric unit, which corresponds to a crystal volume per unit molecular mass of 2.04 A3/Da, and analytical ultracentrifugation analysis show that aSFP is a monomeric protein. 相似文献
14.
Crystallization and preliminary X-ray crystallographic analysis of squalene-hopene cyclase from Alicyclobacillus acidocaldarius. 下载免费PDF全文
K. U. Wendt C. Feil A. Lenhart K. Poralla G. E. Schulz 《Protein science : a publication of the Protein Society》1997,6(3):722-724
The membrane-associated protein squalene-hopene cyclase from Alicyclobacillus acidocaldarius was overexposed in Escherichia coli and purified by ion exchange and gel permeation chromatography. Crystals of three interrelated forms were grown by vapor diffusion under identical conditions. The crystals diffract to about 2.3 A resolution, but they are unstable in the X-ray beam. An interpretable heavy-atom derivative was obtained. 相似文献
15.
HIV-1 Nef protein: purification, crystallizations, and preliminary X-ray diffraction studies. 总被引:1,自引:1,他引:0 下载免费PDF全文
P. Franken S. Arold A. Padilla M. Bodeus F. Hoh M. P. Strub M. Boyer M. Jullien R. Benarous C. Dumas 《Protein science : a publication of the Protein Society》1997,6(12):2681-2683
Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HIV-1LAI Nef protein in the unliganded form and in complex with the wild-type SH3 domain of the P59fyn protein-tyrosine kinase. One-dimensional NMR experiments show that full-length protein and truncated fragment corresponding to the product of HIV-1 protease cleavage have a well-folded compact tertiary structure. The ligand-free HIV-1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 A. The Nef-Fyn SH3 cocrystals belong to the space group P6(1)22 or its enantiomorph, P6(5)22, with unit cell dimensions of a = b = 108.2 A and c = 223.7 A. Both crystal forms diffract to a resolution limit of 3.0 A resolution using synchrotron radiation, and are thus suitable for X-ray structure determination. 相似文献
16.
Characterization and crystallization of human uroporphyrinogen decarboxylase. 总被引:4,自引:0,他引:4 下载免费PDF全文
J. D. Phillips F. G. Whitby J. P. Kushner C. P. Hill 《Protein science : a publication of the Protein Society》1997,6(6):1343-1346
The cytosolic enzyme uroporphyrinogen decarboxylase (URO-D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. Recombinant human URO-D has been expressed in Escherichia coli with a histidine tag and has been purified to homogeneity. Purified protein was determined to be a monodisperse dimer by dynamic light scattering. Equilibrium sedimentation analysis confirmed that the protein is dimeric, with a dissociation constant of 0.1 microM. URO-D containing an amino-terminal histidine tag was crystallized in space group P3(1)21 or its enantiomer P3(2)21 with unit cell dimensions a = b = 103.6 A, c = 75.2 A. There is one molecule in the asymmetric unit, consistent with generation of the dimer by the twofold axis of this crystallographic operator. Native data have been collected to 3.0 a resolution. 相似文献
17.
Crystallization and preliminary crystallographic analysis of recombinant human P38 MAP kinase. 下载免费PDF全文
S. Pav D. M. White S. Rogers K. M. Crane C. L. Cywin W. Davidson J. Hopkins M. L. Brown C. A. Pargellis L. Tong 《Protein science : a publication of the Protein Society》1997,6(1):242-245
The recombinant human p38 MAP kinase has been expressed and purified from both Escherichia coli and SF9 cells, and has been crystallized in two forms by the hanging drop vapor diffusion method using PEG as precipitant. Both crystal forms belong to space group P2(1)2(1)2(1). The cell parameters for crystal form 1 are a = 65.2 A, b = 74.6 A and c = 78.1 A. Those for crystal form 2 are a = 58.3 A, b = 68.3 A and c = 87.9 A. Diffraction data to 2.0 A resolution have been collected on both forms. 相似文献
18.
The Fv fragment of a monoclonal antibody, 7E2 (IgG1, κ, murine), which is directed against the integral membrane protein cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans, was cloned and produced in Escherichia coli. Crystals suitable for highresolution X-ray analysis were obtained by microdialysis under low salt conditions. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of a = 51.51 Å, b = 56.15 Å, c = 99.86 Å (1 Å = 0.1 nm) and contain one F v fragment per asymmetric unit. Using synchrotron radiation diffraction data were collected up to 1.28 Å resolution. This high resolution is very unusual for a heterodimeric protein. The crystals should open the way for refining not only the atomic positions, but also for obtaining information about internal dynamics. © 1995 Wiley-Liss, Inc. 相似文献
19.
Christiane Chine Michael Fountoulakis Heinz Dbeli Brigitte D'Arcy Fritz Winkler Allan D'Arcy 《Proteins》1995,23(4):591-594
A complex of human interferon-γ (IFN- γ) with the soluble extracellular domain of the IFN- γ receptor α-chain (IFN-γ-R) has been crystallised. Crystals of the complex were grown using PEG 4000 as the precipitating agent in the presence of β-octyl glucoside. The receptor-ligand complex crystallizes in a monoclinic space group and diffracts to about 3.0 Å resolution. Isomorphous crystals have been obtained with complex containing selenomethionine and cysteine mutants of IFN-γ, which may facilitate the ongoing X-ray structure determination. © 1995 Wiley-Liss, Inc. 相似文献
20.
Fluorescently labelled, rRNA-targeted oligonucleotide probes in the study of microbial ecology 总被引:4,自引:0,他引:4
R. I. AMANN 《Molecular ecology》1995,4(5):543-554