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91.
Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease. 总被引:9,自引:7,他引:2
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Y Shimizu K Yamaji Y Masuho T Yokota H Inoue K Sudo S Satoh K Shimotohno 《Journal of virology》1996,70(1):127-132
In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, we developed an in vitro NS3 protease assay system consisting of three purified viral elements: (i) a recombinant NS3 protease which was expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein (MBP-NS3), (ii) synthetic NS4A fragments, and (iii) a synthetic peptide substrate which mimics the NS5A/5B junction. We showed that the NS3 protease activity of MBP-NS3 was enhanced in a dose-dependent manner by 4A18-40, which is a peptide composed of amino acid residues 18 to 40 of NS4A. The optimal activity was observed at a 10-fold molar excess of 4A18-40 over MBP-NS3. The coefficient for proteolytic efficiency, kcat/Km, of NS3 protease was increased by about 40 times by the addition of a 10-fold molar excess of 4A18-40. Using a series of truncations of 4A18-40, we estimated that amino acid residues 22 to 31 in NS4A (SVVIVGRIIL) constituted the core sequence for the effector activity. Single-substitution experiments with 4A21-34, a peptide composed of amino acid residues 21 to 34 of NS4A, suggested the importance of several residues (Val-23, Ile-25, Gly-27, Arg-28, Ile-29, and Leu-31) for its activity. In addition, we found that some single-amino-acid substitutions in 4A21-34 were able to inhibit the enhancement of NS3 protease activity by 4A18-40. This approach has potential as a novel strategy for inhibiting the NS3 protease activity important for hepatitis C virus proliferation. 相似文献
92.
Hiroyuki Tsunoda Toshio Ohshima Jun Tohyama Masayuki Sasaki Norio Sakuragawa Frank Martiniuk 《Human genetics》1996,97(4):496-499
We report a missense mutation in an adult Japanese patient with acid alpha-glucosidase (GAA) deficiency. A TC to GT transition
at nucleotides 1585–1586, was identified. This transition resulted in an amino acid substitution of Ser-529 to Val (S529V)
in exon 11. We also have demonstrated that the S529V mutation abolishes the catalytic activity of the enzyme. Our data suggest
that this mutation is the cause of the clinical manifestation known as adult-onset GAA deficiency. The missense mutation described
here is a new mutation, and the first identified in Japanese patients with GAA deficiency.
Received: 23 May 1995 相似文献
93.
94.
Recent molecular biological, chemical, physiological and morphological studies indicate that Trichosporon cutaneum and related species should be reclassified. In this study, antigenic characteristics of the species were determined. The results of adsorption experiments revealed that there were at least three serological types: I, II and III. Specific factor sera I, II and III were prepared on the basis of adsorption experiments and isolates were serotyped by cell slide agglutination (CSA). Since the CSA test was difficult to read in some strains, the results of the CSA test were compared with the findings from an enzyme-linked immunosorbent assay (ELISA). For the ELISA, crude polysaccharide antigens prepared from the culture supernatant were used as the antigen. The types determined by ELISA correlated well with those determined by the CSA test. These data suggest that T. cutaneum and related species have at least three serological types, and that the typing can be done by either CSA or ELISA. 相似文献
95.
可育的抗除草剂溴苯腈转基因小麦 总被引:21,自引:0,他引:21
报道了采用微粒轰击(Microprojectile bom bardm ent) 幼胚将除草剂抗性基因导入小麦(Triticumaestivum L.)的转化研究。实验共使用了13 个小麦品种, 从开花后14~18 d 的籽粒中剥取幼胚, 植物表达质粒含有CaMV 35S启动子控制的除草剂溴苯腈抗性基因bxn 以及筛选标记基因NTPⅡ。采用高压放电基因枪,用质粒DNA 包被的钨粒轰击预培养3 d 的幼胚。在含有卡那霉素类似物geneticin G418sulphate 的MS培养基上, 经过多步骤筛选和分化, 从800 多个幼胚中获得了16 株转化苗。除草剂抗性鉴定和Southern 杂交分析证明, 其中4 株为转基因植物,具有溴苯腈抗性, 并且自交可育。转化工作从分离幼胚到转化苗鉴定完毕, 最短时间为6 个月, 因此, 该方法是一项快速有效的基因导入技术 相似文献
96.
Li-Xin Zhang Hou-Guo Liang Jun Wang Wen-Rui Li Tian-Zhi Yu 《Photosynthesis research》1996,48(3):379-384
The 33 kDa protein of Photosystem II has one intrachain disulfide bond. Fluorescence spectroscopy shows that the major groups in the protein that bind to Ca2+ should be the carboxylic side groups of glutamic acid and/or aspartic acid. Fluorescence and Fourier-transform infrared (FTIR) spectroscopic studies indicate that the conformation of the 33 kDa protein is altered upon reduction, while the reduced protein still retains the secondary structure. FTIR spectroscopy also shows that the metal ions induce a relative decrease of unordered structure and -sheet, and a substantial increase of -helix in both the intact and the reduced 33 kDa protein. This indicates that the addition of cations results in a much more compact structure and that both the intact and the reduced 33 kDa proteins have the ability to bind calcium. The above results may suggest that the disulfide bridge is not essential for calcium binding.Abbreviations CD
circular dichroism
- FTIR
Fourier transform infrared
- La
lanthanum
- PS
photosystem
- Tb
terbium 相似文献
97.
Shuji Nakamura David W. Stock Karen L. Wydner Jacques A. Bollekens Kenichi Takeshita Brian M. Nagai Shigeru Chiba Toshio Kitamura Thomas M. Freeland Zhiyong Zhao Jun Minowada Jeanne B. Lawrence Kenneth M. Weiss Frank H. Ruddle 《Genomics》1996,38(3):314
We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt geneNvHBox-5.The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouseDlx7upstream of the homeodomain that may account for some of this divergence. We have mapped humanDLX7distal to the 5′ end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters. 相似文献
98.
Kazutoyo Osoegawa Rie Susukida Saishi Okano Jun Kudoh Shinsei Minoshima Nobuyoshi Shimizu Pieter J. de Jong Juergen Groet Jane Ives Hans Lehrach Dean Nizetic Eiichi Soeda 《Genomics》1996,32(3):375
The major phenotypic features of Down syndrome have been correlated with partial trisomies of chromosome 21, allowing us to define the candidate gene region to a 4-Mb segment on the 21q22.2 band. We present here a high-resolution physical map with megabase-sized cosmid/PAC contigs. This ordered clone library has provided unique material for the integration of a variety of mappable objects, including exons, cDNAs, restriction sites, etc. Furthermore, our results have exemplified a strategy for the completion of the chromosome 21 map to sequencing. 相似文献
99.
100.
控制大豆白花亲本籽粒脐色的基因有带R与r之分,带R基因的白花产本与紫花亲本杂交,F1代籽料出现蓝脐性状,其基因型为I-R-W1-tt。当控制脐色的基因有两对相差时(R、r;W1、w1)F2代籽粒脐色分离蓝脐与无色脐之比为9∶7。 相似文献