我国两株登革2型病毒5′和3′端非编码区序列测定及二级结构分析

 为测定我国两株临床症状、乳鼠神经毒力不同的登革 2型病毒流行株 5′和 3′端非编码区序列 (untranslated region,UTR) ,分析二级结构差异与毒力变化的关系 ,分别从 D2 - 0 4、D2 - 44株感染的 C6/ 36细胞及鼠脑中提取总 RNA.以该 RNA为模板 ,利用 RACE法 ,分别扩增了 D2 - 0 4、D2 -44株的 5′和 3′末端 c DNA片段 .将其分别与 p GEM- T载体连接得到重组质粒 ,测定上述 c DNA插入片段的序列 .用 RNAdraw软件预测 D2 - 0 4、D2 - 44株 5′和 3′端非编码区的二级结构 .D2 - 0 4、D2 -44株 5′端和 3′端非编码区分别有 96和 454个核苷酸 .其中 5′非编码区 59位 C(D2 - 0 4 )→T(D2 -44 ) ,使 D2 - 44二级结构稳定性下降 ;3′端非编码区有 1 5个核苷酸不同 ,其中 T(355)→ A,T(32 6)→ G引起了所在位置二级结构自由能变化 ,且分别位于两个保守序列区 (conserved sequence,CS)CS1、CS2 A.这些位点变化可能与毒力有关 .     

A simultaneous protein-binding assay for adenosine 3':5'-monophosphate in biological materials.

Adenosine 3′:5′-monophosphate (cyclic AMP) and guanosine 3′:5′-monophosphate (cyclic GMP) have been determined simultaneously by combining individual protein binding assays using different isotopically labeled cyclic nucleotides. Preparations of cyclic AMP-binding protein from beef adrenal cortex and cyclic GMP-binding protein from the fat body of silkworm pupae ($\text{Bombyx mori}$) have been used for the assay. The method allows the analysis of cyclic AMP and cyclic GMP levels in crude extracts without any purification. The assay has been applied to hormone-stimulated Mouse liver and phorbol ester-treated Rat embryo cells.     

Partial characterization of 5′(3′)-ribonucleotide and 3′-ribonucleotide phosphohydrolases from Tradescantia albiflora leaves

Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn²⁺ and Fe²⁺ were inhibitors for both by 20–80%; while Mg²⁺, Ca²⁺, Co²⁺, K⁺, Na⁺ at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes.     

Oxidation Chemistry of Adenosine-3′, 5′-Cyclic Monophosphate at Pyrolytic Graphite Eletrode

The voltammetric oxidation of adenosine-3′,5′-cyclic monophosphate (3′,5′-CAMP) has been studied in the pH range 2.13–10.07 using pyrolytic graphite electrode (PGE). Voltammetric, coulometric, spectral studies, and product characterization indicate that the oxidation of 3′,5′-CAMP occurs in an EC reaction involving a 6H⁺, 6e process at pH 7.24. Electrooxidized products were seperated by semipreparative high performance liquid chromatography (HPLC) and were characterized by mp, ¹HNMR, FTIR, and GC-mass as allantoin cyclic ribose monophosphate and 3 dimers as the major products. A detailed interpretation of the redox mechanism of 3′,5′-CAMP also has been presented to account for the formation of various products.     

Non-dependence on native structure of pig liver pyruvate kinase when used as a substrate for cyclic 3',5'-AMP-stimulated protein kinase.

Alkali-inactivated pig liver pyruvate kinase, type L, and a cyanogen bromide fragment from the same enzyme were shown to be phosphorylated by (³²P)ATP and cyclic 3′,5′-AMP-stimulated protein kinase. In both cases the rate of phosphorylation was higher than with the native enzyme. Pyruvate kinases types A and M were not phosphorylated under the same conditions. From the ³²P-labelled cyanogen bromide fragment (³²P)phosphorylserine was isolated. The electrophoretic pattern of (³²P)phosphopeptides obtained on partial acid hydrolysis of the fragment indicated that the phosphorylated site of the fragment was identical with that of the native pyruvate kinase.     

Mass spectrometric identification of adenosine 3′:5′-cyclic monophosphate isolated from a higher plant tissue

Mass spectrometric evidence is presented confirming the identification of the adenosine nucleotide previously isolated from tissues of Phaseolus vulgaris as adenosine 3′: 5′-cyclic monophosphate.     

Cyclic guanosine 3',5'-monophosphate and phosphodiesterase activity in mitogen-stimulated human lymphocytes.

The cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase from human leukemic lymphocytes differes from the normal cell enzyme in having a much higher activity and a loss of inhibition by cyclic guanosine 3′,5′-monophosphate (cyclic GMP). In an effort to determine the mechanism of these alterations, we have studied this enzyme in a model system, lectin-stimulated normal human lymphocytes. Following stimulation of cells with concanavalin A (con A) the enzyme activity gradually becomes altered, until it fully resembles the phosphodiesterase found in leukemic lymphocytes. The changes in the enzyme parallel cell proliferation as measured by increases in thymidine incorporation into DNA. The addition of a guanylate cyclase inhibitor preparation from the bitter melon prevents both the changes in the phosphodiesterase and the thymidine incorporation into DNA. This blockage can be partially reversed by addition of 8-bromo cyclic guanosine 3′,5′-monophosphate (8-bromo cyclic GMP) to the con A-stimulated normal lymphocytes. These results indicate a possible role of cyclic GMP in a growth related alteration of cyclic AMP phosphodiesterase.     

Study on Disulfur-Backboned Nucleic Acids: Part 3. Efficient Synthesis of 3′,5′-Dithio-2′-Deoxyuridine and Deoxycytidine

A general method is described for synthesizing 3′,5′-dithio-2′-deoxypyrimidine nucleosides 6 and 13 from normal 2′-deoxynucleosides. 2,3′-Anhydronucleosides 2 and 9 are applied as intermediates in the process to reverse the conformation of 3′-position on sugar rings. The intramolecular rings of 2,3′-anhydrothymidine and uridine are opened by thioacetic acid directly to produce 3′-S-acetyl-3′-thio-2′-deoxynucleosides 3 or 5. To cytidine, OH^? ion exchange resin was used to open the ring and 2′-deoxycytidine 10 was abtained in which 3′-OH group is in threo-conformation. The 3′-OH is activated by MsCl, and then substituted by potassium thioacetate to form the S,S′-diacetyl-3′,5′-dithio-2′-deoxycytidine 12. The acetyl groups in 3′,5′ position are removed rapidly by EtSNa in EtSH solution to afford the target molecules 6 and 13. The differences of synthetic routes between uridine and cytidine are also discusssed.     

Visualization of drug-nucleic acid interactions at atomic resolution. IV. Structure of an aminoacridine--dinucleoside monophosphate crystalline complex, 9-aminoacridine--5-iodocytidylyl (3'--5') guanosine

9-Aminoacridine forms a crystalline complex with the dinucleoside monophosphate, 5-iodocytidylyl(3′–5′)guanosine (iodoCpG). These crystals are monoclinic, space group P2₁ with $\text{a = 13.98}\text{A}\text{?}\text{, b = 30.58}\text{A}\text{?}\text{, c = 22.47}\text{A}\text{?}$ and β = 113.9 °. The structure has been solved to atomic resolution by Patterson and Fourier methods, and refined by a combination of Fourier and sum-function Fourier methods. The asymmetric unit contains four 9-aminoacridine molecules, four iodoCpG molecules and 21 water molecules, a total of 245 atoms. 9-Aminoacridine demonstrates two different intercalative binding modes and, along with these, two slightly different intercalative geometries in this model system.The first of these is very nearly symmetric, the 9-amino group lying in the narrow groove of the intercalated base-paired nucleotide structure. The second shows grossly asymmetric binding to the dinucleotide, the 9-amino group lying in the wide groove of the structure. Associated with these two different intercalative binding modes is a difference in geometries in the structures. Although both structures demonstrate C3′ endo (3′–5′) C2′ endo mixed sugar puckering patterns (i.e. both cytidine residues have C3′ endo sugar conformations, while both guanosine residues have C2′ endo sugar conformations), with corresponding twist angles between base-pairs of about 10 °, they differ in the magnitude of the helical screw axis dislocation accompanying intercalation (Sobell et al., 1977a,b). In the pseudosymmetric intercalative structure, this value is about +0.5 Å, whereas in the asymmetric intercalative structure this value is about +2.7 Å. These conformational differences can be best described as a “sliding” of base-pairs on the intercalated acridine molecule.Although the pseudosymmetric intercalative structure can be used in 9-aminoacridine-DNA binding, the asymmetric intercalative structure cannot since this poses stereochemical difficulties in connecting neighboring sugar-phosphate chains to the intercalated dinucleotide. It is possible, however, that the asymmetric binding mode is related to the mechanism of 9-aminoacridine-induced frameshift mutagenesis (Sakore et al., 1977), and we discuss this possibility here in further detail.     

The Three-Dimensional Structure of 3′-Deoxycytidine

Abstract

Structural analysis of 3′-deoxycytidine and comparison with 2′-deoxynucleosides reveals no noticeable effect on the conformation of the molecule due to the lack of 3′-oxygen atom. There are two crystallographically independent molecules and both adopt the anti conformation with C3′-endo sugar puckering. A ‘head-to-tail’ packing of the molecules along the b axis results in a virtual ‘2′-5′ polycytidylic acid chain.     

紫色马铃薯类黄酮3，′5′-羟化酶基因（StF3′5′H）的克隆及分析

类黄酮3′,5′羟-化酶（ flavonoid 3′,5′-hydroxylase, F3′5′H）是植物花青素生物合成途径中的一个关键酶,紫色土豆（ Solanum tueb or sum） F3′5′H基因的克隆将为花青素合成调控和花青素代谢工程研究提供优质基因资源。研究采用RACE技术克隆了紫色土豆F3′5′H基因的cDNA全长序列,用生物信息学方法对其核苷酸和蛋白质序列进行了分析,并用半定量PCR 技术分析了F3′5′H基因在不同组织中的表达情况,同时研究了赤霉素和蔗糖处理后F3′5′H基因表达与花青素积累之间的相关性。研究结果表明,克隆的紫色土豆F3′5′H的cDNA全长为1854 bp,包含一个1530 bp的完整ORF,共编码509个氨基酸。生物信息学分析表明,StF3′5′H基因推测编码的氨基酸序列与其它植物的F3′5′H蛋白的相似性很高。 StF3′5′H基因的表达具有组织特异性,在紫色土豆根、茎和叶柄中都有表达,其中在叶柄中表达最强,而在块茎、叶轴和叶片中几乎检测不到StF3′5′H基因的表达。赤霉素和蔗糖能促进紫色土豆StF3′5′H基因的表达,进而促进花青素的积累。     

Guanosine 3′:5′-cyclic monophosphate changes during germination of Hordeum vulgare

Guanosine 3′: 5′-cyclic monophosphate (cGMP) isolated from barley seeds and seedlings was purified using neutral alumina and anion-exchange column chromatography, then descending paper chromatography, and finally estimated by means of radioimmunoassay. The putative compound was identified on cellulose chromatography in three solvent systems.During the early phase of the germination, the cGMP content decreased steadily from 30 fmol/g of dry seeds to undetectable amounts in seeds after 18 h of germination. The process of seedling growth was associated with the increase of cGMP concentration. Nine-day-old seedlings contained 147 and 200 fmol/g of fresh weight in the roots and in the coleoptiles plus leaves, respectively.     

Anomalous electrophoretic migration of oligodeoxynucleotides with terminal OH groups: Applications for DNA exonuclease characterization

Oligodeoxynucleotides with a terminal OH group on both the 5′ and 3′ ends migrate anomalously in 23% polyacrylamide-7 m urea gels. This migration anomaly can be exploited to characterize nuclease digestion products. Thus, using specific substrates and the methods described, several types of DNA exonuclease activity can be readily distinguished.     

A NEW APPROACH TO OLIGONUCLEOTIDE N3′⇉P5′ PHOSPHORAMIDATE BUILDING BLOCKS

A new synthetic approach to 5′-phosphoramidites of 3′-aminonucleosides was developed. The methodology relies upon the use of 3′-amino-2′,3′-dideoxy nucleosides as the key starting materials. The final phosphoramidite products were obtained with high yields via 2–3-step efficient chemical transformations using selective introduction of orthogonal protective groups to the 3′-aminonucleoside sugar and base moieties.     

Comparison of cyclic nucleotide specificity of guanosine 3′,5′-monophosphate-dependent protein kinase and adenosine 3′,5′-monophosphate-dependent protein kinase

Guanosine 3′,5′-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3′,5′-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-folds less than that of cylic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic. AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophophorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

Stimulation by ATP of [3H]dopamine binding to synaptic membrane fractions of canine caudate nucleus

The rate of [³H]dopamine binding to crude synaptic membranes from canine caudate nucleus was considerably increased by 2 mM ATP, 5′-adenylylimidodiphosphate and GTP or by 1 mM 5′-guanylyl-imidodiphosphate, while strongly inhibited by 2 mM ADP and GDP. Half maximal concentrations of [³H]dopamine to bind to the membranes were 1.11 × 10^?7M and 8.75 × 10^?6M in the absence of 4 mM ATP, indicating a negative cooperativity of the dopamine receptor, and 9.25 × 10^?7 M in its presence. Hill coefficient was increased from 0.70 to 1.04 by addition of 4 mM ATP. The optimal concentration of ATP for [³H]dopamine binding was in the range of 0.5 to 5 mM.     

Occurrence of adenosine 3′:5′-cyclic monophosphate in plant tissues

A procedure is described which unequivocally demonstrates the presence of adenosine 3′:5′-cyclic monophosphate in Phaseolus vulgaris. Its concentration was determined spectrophotometrically at 2·6–9·2 nmol g^?1 of tissue (dry wt) for 6-day-old seedlings and about one-tenth of this in 13-day-old plants.     

Antagonistic effect of methadone on steroidogenesis and the adenylate cyclase system in isolated rat adrenocortical cells

Methadone exhibits an antagonistic effect toward steroidogenesis which lies prior to progesterone in the biosynthetic pathway in isolated rat adrenal cells. Levels of adenosine cyclic 3′–5′ monophosphate are depressed in a dose dependent fashion in ACTH stimulated cells as is steroidogenesis in cells stimulated with N⁶O²-dibutyryl adenosine cyclic 3′–5′ monophosphate. Stimulation produced by the ACTH analog, O-nitrophenyl sulfenyl ACTH, is also inhibited by methadone. The participation of adenosine cyclic 3′–5′ monophosphate as an obligatory messenger in ACTH stimulated steroidogenesis is discussed with respect to the pharmacological properties of methadone in this system.     

Subcellualr localizations of guanylate cyclase and 3′,5′-cyclic nucleotide phophodiesterase in sea urchin sperm

The subcellular localizations of guanylate cyclase and 3′,5′-cyclic nucleotide phophodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella (tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 980% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucelotide phosphodiesterase in sperm flagella were also briefly described.