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101.
phbB,phbC基因克隆,序列分析及植物表达载体的构建 总被引:11,自引:0,他引:11
利用聚合酶链式反应技术,从真养产碱杆菌Alcaligenes eutrophus H16染色体DNA中的主增并克隆了调控聚-β-羧基丁酸生物合成的两个关键酶基因;依赖NADPH的乙酰乙酰CoA还原酶基因和PHB合成酶基因。限制性内切酶图谱和核苷酸序列分析证实了克隆结果,并表明所克隆的基因与国外报道的有很高的同源性。 相似文献
102.
Localization of binding site for encephalomyocarditis virus RNA polymerase in the 3'-noncoding region of the viral RNA. 总被引:5,自引:1,他引:4
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We previously showed that encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) binds specifically to 3'-terminal segments of EMC virus RNA. This binding, which depends on both the 3'-noncoding region (3'-NCR) and 3'-poly (A) tail [together denoted 3'-NCR(A)], may be an important step in the initiation of virus replication. In this paper, the 3'-NCR and 3'-poly(A) were separately transcribed then mixed, but no complex with 3Dpol was obtained, showing that covalent attachment of the 3'-poly(A) to the 3'-NCR is essential for complex formation. Mutational and deletion analyses localized a critical determinant of 3Dpol binding to a U-rich sequence located 38-49 nucleotides upstream of the 3'-poly(A). Similar analyses led to the identification of a sequence of A residues between positions +10 and +15 of the 3'-poly(A) which are also critical for 3Dpol binding. As U-rich and A-rich regions are important for 3Dpol binding, a speculative model is proposed in which 3Dpol induces and stabilizes the base-pairing of the 3'-poly(A) with the adjacent U-rich sequence to form an unusual pseudoknot structure to which 3Dpol binds with high affinity. 相似文献
103.
Structural basis for the RNA binding selectivity of oligonucleotide analogues containing alkylsulfide internucleoside linkages and 2'-substituted 3'-deoxyribonucleosides.
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In this report we describe the synthesis of oligonucleotides containing sulfide-linked dinucleoside units, namely rT(2'OH)sdT, rT(2'OMe)sdT, dTsrU(2'OMe) and dT(2'OMe)srU(2'OMe). We also describe the interactions of such oligomers with complementary DNA and RNA targets, and provide the structural basis for their remarkable RNA binding selectivity. In all cases, the Tm values of the S/P-chimera duplexes were lower than those of the corresponding unmodified duplexes. We attribute this to steric interactions between the 5'sulfur and the atoms of the nearby base/sugar residues. The 2'-substituents (i.e., 2'OH or 2'OMe) vicinal to the alkylsulfide internucleoside linkage significantly perturb the structure and stability of the duplexes formed with DNA, and more so than with RNA. The introduction of three rT(2'OH)sdTp (or rT(2'OMe)sdTp) units into an oligodeoxynucleotide sequence was sufficient to abolish binding to complementary DNA but not RNA. The same three substitutions with dTsrU(2'OMe)p and dT(2'OMe)srU(2'OMe)p did not abolish binding to DNA but the resulting complexes had poor thermal stability. The RNA-binding 'selectivity' exhibited by these oligomers is attributed to the tendency of the 2'-substituted (branched) furanoses to adopt the C3'-endo pucker, a conformation that is inconsistent with the B-form structure of helical DNA. The preference of these sugars to exist often exclusively in the C3'-endo form is attributed to stereoelectronic effects, namely gauche and anomeric effects. Our findings support the hypothesis that nucleoside analogues puckered exclusively in the C3'-endo form may result in them being especially good binders of targeted mRNA [S.H. Kawai (1991), Ph.D. Thesis, McGill University; Kawasaki et al. (1993) J. Med. Chem. 36, 831-841]. 相似文献
104.
105.
Parimal C. Sen Zhou Meng Ai Tushar K. Ray 《Archives of biochemistry and biophysics》1980,205(2):340-351
The transversal distribution of the free NH2 groups associated with phosphatidyl ethanolamine and the intrinsic membrane proteins of the purified pig gastric microsomes was quantitated and their relations to the function of the gastric K+-stimulated ATPase was investigated. Three different chemical probes such as 2,4,6-trinitrobenzene sulfonic acid (TNBS), 1-fluoro-2,4-dinitrobenzene (FDNB), and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) were used for the study. The structure-function relationship of the membrane NH2 groups was studied after modification with the probes under various conditions and relating the inhibition of the K+-stimulated ATPase to the ATPase-dependent H+ accumulation by the gastric microsomal vesicles. TNBS (2 mm) inhibits nearly completely the K+-stimulated ATPase and the vesicular dye accumulation, both in presence and absence of valinomycin plus K+. Both the K+-ATPase and dye uptake were largely (about 50%) protected against TNBS inhibition if the treatment with TNBS was carried out in presence of 2 mm ATP. TNBS and FDNB labeled 70% of the total microsomal PE; the intra- and extravesicular orientation being 48 and 22%, respectively. The presence or absence of ATP did not have any effect on the TNBS labeling of microsomal PE. ATP, however, significantly (P < 0.05) reduced the labeling of protein-bound NH2 groups of gastric microsomes by TNBS. The intra- and extravesicular orientation of the protein NH2 groups were 60 and 40%, respectively. Eighteen percent of the total protein-NH2 appeared to be associated with the K+-stimulated ATPase; the rest being associated with non-ATPase proteins of the microsomes. About half (50%) of the total free NH2 groups of the K+-stimulated ATPase were exposed to the vesicle exterior and were found to play critical roles in gastric ATPase function. The generation of florescence after MDPF conjugation of gastric microsomes was largely (50%) inhibited by ATP. ATP also protected completely the MDPF inhibition of gastric K+-stimulated ATPase and dye uptake. 相似文献
106.
茶尺蠖人工饲料的研究 总被引:3,自引:0,他引:3
本报道饲养茶尺蠖(Ectropis obliqua hypulina Wehrli)的5种人工饲料及其饲养方法.5种人工饲料均可用于大规模饲养一代幼虫.其中62号配方可用于续代饲养,在实验室内饲养5代的结果表明。效果良好.在饲料成分的加工.配制及饲养方法等方面.较前人有相当大的改进,因而叶因子用量减少,幼虫历期缩短.用人工饲料饲养的幼虫历期为13-20天。基本接近以茶叶饲养的对照(11—18天).实验结果还表明饲料含水量.饲养方法.饲养密度和添加饲料的次数不同,对茶尺蠖的生长发育有明显的影响.初龄幼虫用平面培养基倒置饲养和高龄幼虫用片状饲料正置饲养为最佳方法. 相似文献
107.
本文报道了[B10,22-Asp,B25-Tyr-NH2]-去B链羧端五肽胰岛素的制备及其生物活性。结果表明,这一类似物的生物活力比去五肽胰岛素(DPI)的活力高一倍,但却比Gerald所报道的[B10-Asp,B25-Tyr-NH_2]-DPI的活力低很多,说明后者的高活性可能依赖于分子中B22-Arg的存在。 相似文献
108.
将单拷贝人α心钠素基因3′端用Ban Ⅱ酶解除去包括终止密码在内的36个碱基对,代之以人工合成的含Glu-Lys-Phe-Glu连接片段与另一单拷贝人α心钠素基因的5′端串连成编码60肽的双拷贝心钠素基因,克隆于大肠杆菌分泌型表达载体pIN-Ⅲ-OmpA_2质粒中,表达生成60肽的双拷贝人α型心钠素衍生物,在信号肽的作用下分泌至胞膜间质并自动切割为60肽的外源基因产物。分子量约8K的表达产物用分子筛或超滤膜分离后再经HPLC纯化,表达产物具有明显的心钠素放免活性和舒张血管活性。 相似文献
109.
110.
用改进的固相磷酰三酯法合成了oligo-d(G-C)_3。以氩离子激光为激发光源,波长488nm.,在室温条件下,分别测定了纯化后的oligo-d(G-C)_3和其组分单体5’-dGMP和5’-dCMP的激光喇曼谱。观察到被测定的物质在300-2500cm~(-1)频率区间,各自都有其特征的谱形和喇曼峰。5’-dGMP和5’-dCMP谱中大多数特征峰在寡聚体的谱中消失,而在oligo-d(G-C)_3谱中出现了几处新的喇曼峰。经查证,峰832,851和899cm~(-1)系糖-磷酸主链的特征喇曼峰,另外几处峰与DNA的构象有关。实验结果表明oligo-d(G-C)_3在水溶液中(室温)主要以B-构象存在。 相似文献