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71.
C. Mark Fletcher David N. M. Jones Robert Diamond David Neuhaus 《Journal of biomolecular NMR》1996,8(3):292-310
Summary Two modifications to the commonly used protocols for calculating NMR structures are developed, relating to the treatment of NOE constraints involving groups of equivalent protons or nonstereoassigned diastereotopic protons. Firstly, a modified method is investigated for correcting for multiplicity, which is applicable whenever all NOE intensities are calibrated as a single set and categorised in broad intensity ranges. Secondly, a new set of values for pseudoatom corrections is proposed for use with calculations employing centre-averaging. The effect of these protocols on structure calculations is demonstrated using two proteins, one of which is well defined by the NOE data, the other less so. It is shown that failure to correct for multiplicity when using r-6 averaging results in overly precise structures, higher NOE energies and deviations from geometric ideality, while failure to correct for multiplicity when using r-6 summation can cause an avoidable degradation of precision if the NOE data are sparse. Conversely, when multiplicities are treated correctly, r-6 averaging, r-6 summation and centre averaging all give closely comparable results when the structure is well defined by the data. When the NOE data contain less information, r-6 averaging or r-6 summation offer a significant advantage over centre averaging, both in terms of precision and in terms of the proportion of calculations that converge on a consisten result.Abbreviations HMG
high mobility group
- NOE
nuclear Overhauser enhancement
- NOESY
nuclear Overhauser enhancement spectroscopy
- rmsd
root-mean-square deviation
- YASAP
yet another simulated-annealing protocol 相似文献
72.
Lili Bao Le Deng Lihua Nie Shouzhuo Yao Wanzhi Wei 《Biosensors & bioelectronics》1996,11(12):1193-1198
A rapid method for microorganism detection using a piezoelectric quartz crystal sensor (PQC) coated with a thin liquid culture medium film was developed and applied to detect the cell number of Proteus vulgaris. This method employed the viscosity and density response of PQC and utilized the coagulation of gelatine medium solution in which the microorganisms had grown to determine the microorganism indirectly. Three time points (TT1, DT, TT2) were obtained from the coagulation curve and were found to be in good linear relationship with the logarithm of the initial number of P. vulgaris in the range 1·3 × 102−1·3 × 105 cells/ml. The detection was rapid and accurate because the coagulation of the thin liquid culture medium film was quick and the time points in the response curve were sharp and so were easy to determine accurately. The detection time was less than 4 h and only a micro sample was needed. A 5 h preincubation was needed before detection. Some experimental conditions are discussed in detail. 相似文献
73.
采用逆转录聚合酶链反应(RT-PCR)从广东省一例慢性丙型肝炎病人血清中获得丙型肝炎病毒(HCV)5'端非编码区(5'NCR)302bp的cDNA片段,经补齐和提纯后插入pUC19质粒,获得的重组体pUN进行序列测定。将pUN的目的基因亚克隆进体外转录载体pSPORTI多克隆位点的EooRI和PstI切点之间,所得重组体pSN线性化后由T_7RNA多聚酶及SP6RNA多聚酶引导体外转录反应,产物经凝胶电泳及特异引物RT-PCR,证实SP6引导的是正义RNA,T7合成的是反义RNA,其大小分别力429bp和362bp。并证实所得RNA力HCV5'NCRcDNA转录而来。获得的HCV5'NCRcDNA和RNA在常规逆转录和PCR步骤中用于设立有效的模板对照,对消除假用性及评估试剂有重要意义。同时,HCV5'NCR体外转录载体的构建可用于制各RNA探针和反义RNA,改进后还可作为定量PCR的竞争性模板。 相似文献
74.
核糖核酸(RNA)与稀土铽离子在pH5.0~6.5条件下能形成具有较强荧光的络合物,其激发峰在288nm处,发射峰为494nm及545nm处.RNA在0.1~10mg/L范围内与其荧光强度呈线性关系,其检出限为6.0×10 ̄(-8)mol/L.腺苷酸,尿苷酸,胞苷酸对RNA的干扰比较小,因此可在其存在下选择性地测定RNA. 相似文献
75.
M. Yamagishi 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1995,91(6-7):830-835
Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of Lilium species and inter-specific hybrids. The optimum annealing temperature of the polymerase chain reaction (PCR) for the RAPD assay in Lilium was 54 °C, which is relatively higher than the temperature used for other genera reported by previous researchers. Among 76 primers used to amplify genomic DNA by PCR, 18 primers (24%) generated polymorphic DNA fragments in Lilium species and hybrids. Cultivars were also identified by RAPD markers. Some amplified fragments were unique to species of each section and to hybrids derived from these species; that is, they were the section-specific DNA markers. Sections, Sinomartagon, Leucolirion b, Leucolirion a and Archelirion could be identified by 6 section-specific markers amplified with five primers. Seven inter-section hybrids showed the section-specific bands of both parental sections, indicating that these markers would be useful for identifying the parental sections of inter-section hybrids. 相似文献
76.
Mixed-phase plants of Griffithsia japonica Okamura spontaneously occurred in a laboratory culture. Four female plants produced tetrasporangia and spermatangia in addition to their normal female reproductive structures (bisexual/mixed-phase plants), and four male plants produced tetrasporangia as well as spermatangia (male/mixed-phase plants). To determine the nuclear ploidy level of these mixed-phase plants, relative nuclear sizes of male, female, tetrasporangial, and mixed-phase plants were measured using a microscopic image analysis system. Haploid gametophytes could be distinguished from diploid tetrasporophytes by relative nuclear sizes, with the later having nuclei twice the size of the former. Relative nuclear sizes of the mixed-phase plants were similar to those of the haploid plants. Thus, the mixed-phase plants were determined to be haploid. Haploid mixed-phase plants of G. japonica have a potential to produce male, female and tetrasporangial reproductive structures. Sex determination models are discussed to explain "haploid" mixed-phase phenomena in red algae . 相似文献
77.
Barbara C. Boyer 《Hydrobiologia》1995,305(1-3):217-222
In spiralian embryos determination of the axes of bilateral symmetry is associated with D quadrant specification. This can occur late through equal cleavage and cell interactions (conditional specification) or by the four-cell stage through unequal cleavage and cytoplasmic localization (autonomous specification). Freeman & Lundelius (1992) suggest that in spiralian coelomates the former method is ancestral and the latter derived, with evolutionary pressure to shorten metamorphosis resulting in early D quadrant determination through unequal cleavage and appearance of adult features in the larvae. Because of the key phylogenetic position of the turbellarian platyhelminthes, understanding the method of axis specification in this group is important in evaluating the hypothesis. Polyclad development, with equal quartet spiral cleavage, is believed to represent the most primitive condition among living turbellarians and has been examined experimentally in Hoploplana inquilina. Blastomere deletions at the two and four-cell stage produce larvae that are abnormal in morphology and symmetry, indicating that early development is not regulative, and also establish that the embryo does not have an invariant cell lineage. Deletions of micromeres and macromeres at the eight-cell stage indicate that cell interactions are involved in dorso-ventral axis determination, with cross-furrow macromeres playing a more significant role than non-cross-furrow cells. The results support the idea that conditional specification is the primitive developmental mode that characterized the common ancestor of the turbellarians and spiralian coelomates. Evolutionary trends in development in polyclads and other turbellarian orders are discussed. 相似文献
78.
N-terminal sequence analysis of atrial granule serine proteinase purified by affinity chromatography
Atrial granule serine proteinase is considered the leading candidate endoproteolytic processing enzyme of pro-atrial natriuretic factor. Its cleavage specificity is directed toward a monobasic amino acid processing site, and as such, the atrial enzyme is distinguished from the family of prohormone convertases which act at dibasic amino acid processing sites. To delineate the molecular mechanisms which distinguish monobasic from dibasic amino acid-directed processing enzymes, pure atrial enzyme is needed for sequence determination leading to molecular cloning, and for preparation of antisera. An affinity chromatography purification scheme seemed a logical modification of our established procedures to yield suitable amounts of enzyme for further studies. Surprisingly, pseudo-peptide bond inhibitors of the atrial enzyme [Damodaran and Harris (1995),J. Protein Chem., this issue] formed ineffective affinity ligands, even though these compounds contain essential residues on either side of what would be the scissile bond in a peptide substrate. On the other hand, tripeptide aldehydes (based on the substrate recognition sequence of the atrial enzyme) linked to Sepharose formed effective affinity matrices, permitting purification of the enzyme in a single step from a subcellular fraction enriched for atrial granules and lysosomes. Hence, the enzyme was purified 2000-fold in 90% overall yield, and subjected to N-terminal sequence analysis through 26 residues. The sequence determined, XXPEAAGLPG[R, L]GNPVP[F, G]R[Q, I]XY[G, E]XR(N, A]V, indicates that the atrial enzyme is unique, showing little sequence homology to other proteins in the database.Abbreviations AGSP
atrial granule serine proteinase
- ANF
atrial natriuretic factor
- BSA
bovine serum albumin
-
Bz
benzoyl
- EACA
6()-aminocaproic acid
- HEPES
N-2-hydroxyethylpiperazine-N'-propanesulfonic acid
- HPLC
high-performance liquid chromatography
- PEG
polyethylene glycol-3350
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Single-letter abbreviations are used to denote amino acids 相似文献
79.
Maurizio Pellecchia Gerhard Wider Hideo Iwai Kurt Wüthrich 《Journal of biomolecular NMR》1997,10(2):193-197
A novel 2D NMR experiment, 2D HE(NE)HGHH, is presented for the assignment ofarginine side chain 1H and 15N resonances inuniformly 15N-labeled proteins. Correlations between1H, 1Hand 1H are established on the basis of3J(15N,1H) heteronuclear scalarcoupling constants, and sequence-specific assignments are obtained by overlapof these fragments with 1H chemical shiftsobtained by assignment procedures starting from the polypeptide backbone.Since guanidino protons exchange quite rapidly with the bulk water, the 2DHE(NE)HGHH pulse scheme has been optimized to avoid saturation and dephasingof the water magnetization during the course of the experiment. As anillustration, arginine side chain assignments are presented for two uniformly15N-labeled proteins of 7 and 23 kDa molecular weight. 相似文献
80.
The NMR side-chain assignments and solution structure of enzyme IIBcellobiose of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli. 总被引:1,自引:1,他引:0 下载免费PDF全文
E. Ab G. Schuurman-Wolters J. Reizer M. H. Saier K. Dijkstra R. M. Scheek G. T. Robillard 《Protein science : a publication of the Protein Society》1997,6(2):304-314
The assignment of the side-chain NMR resonances and the determination of the three-dimensional solution structure of the C10S mutant of enzyme IIBcellobiose (IIBcel) of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli are presented. The side-chain resonances were assigned nearly completely using a variety of mostly heteronuclear NMR experiments, including HCCH-TOCSY, HCCH-COSY, and COCCH-TOCSY experiments as well as CBCACOHA, CBCA(CO)NH, and HBHA(CBCA)(CO)NH experiments. In order to obtain the three-dimensional structure, NOE data were collected from 15N-NOESY-HSQC, 13C-HSQC-NOESY, and 2D NOE experiments. The distance restraints derived from these NOE data were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In an iterative procedure, additional NOE assignments were derived from the calculated structures and new structures were calculated. The final set of structures, calculated with approximately 2000 unambiguous and ambiguous distance restraints, has an rms deviation of 1.1 A on C alpha atoms. IIBcel consists of a four stranded parallel beta-sheet, in the order 2134. The sheet is flanked with two and three alpha-helices on either side. Residue 10, a cysteine in the wild-type enzyme, which is phosphorylated during the catalytic cycle, is located at the end of the first beta-strand. A loop that is proposed to be involved in the binding of the phosphoryl-group follows the cysteine. The loop appears to be disordered in the unphosphorylated state. 相似文献