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Catalytic mechanism of DNA topoisomerase IB

MOLECULAR CELL 2000年第6期5卷 1035-1041页

作者： Krogh, BO Shuman, S Sloan Kettering Inst Program Mol Biol New York NY 10021 USA

Type IB topoisomerases and tyrosine recombinases are structurally homologous strand transferases that act through DNA-(3'-phosphotyrosyl)-enzyme intermediates. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes transesterification of tyrosine to the scissile phosphodiester. We used 8'-bridging phosphorothiolate-modified DNAs to implicate Lys-167 as a general acid catalyst. The lower pK(a), of the 5'-S leaving group versus 5'-O restored activity to the K167A mutant, whereas there was no positive thio effect for mutants R223A and H265A. The lysine is located atop a flexible hairpin loop, and it shifts into the minor groove upon DNA binding. Coupling of conformational changes in a general acid loop to covalent catalysis of phosphoryl transfer is one of several mechanistic features shared by the topoisomerase/recombinase and protein phosphatase superfamilies.

关键词：氨基酸取代精氨酸/遗传学精氨酸/代谢碱基序列催化作用保守序列/遗传学 DNA/遗传学 DNA/代谢 DNA拓扑异构酶类 Ⅰ型/化学 DNA拓扑异构酶类 Ⅰ型/遗传学 DNA拓扑异构酶类 Ⅰ型/代谢组氨酸/遗传学组氨酸/代谢动力学赖氨酸/遗传学赖氨酸/代谢模型分子分子序列数据突变酪氨酸/代谢痘苗病毒/酶学病毒蛋白质类/化学病毒蛋白质类/遗传学病毒蛋白质类/代谢

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Cross-species homologous recombination in Leishmania donovani reveals the sites of integration

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MOLECULAR AND BIOCHEMICAL PARASITOLOGY 2000年第1期107卷 123-128页

作者： Krobitsch, S Clos, J Bernhard Nocht Inst Trop Med D-20359 Hamburg Germany

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The DIF1 gene of Arabidopsis is required for meiotic chromosome segregation and belongs to the REC8/RAD21 cohesin gene family

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PLANT JOURNAL 1999年第4期19卷 463-472页

作者： Bhatt, AM Lister, C Page, T Fransz, P Findlay, K Jones, GH Dickinson, HG Dean, C Univ Oxford Dept Plant Sci Oxford OX1 3RB England John Innes Ctr Plant Sci Res Norwich NR4 7UH Norfolk England Univ Birmingham Dept Biol Sci Birmingham B15 2TT W Midlands England

Cohesins are a group of conserved proteins responsible for cohesion between replicated sister chromatids during mitosis and meiosis and which are implicated in double-strand break repair and meiotic recombination. We describe here the identification and characterisation of an Arabidopsis gene - DETERMINATE, INFERTILE1 (DIF1), which is a homolog of the Schizosaccharomyces pombe REC8/RAD21 cohesin genes, and is essential for meiotic chromosome segregation. Five independent alleles of the DIF1 gene were isolated by transposon mutagenesis, and the mutants show complete male and female sterility. Pollen mother cells (PMCs) of dif1 mutants show multiple meiotic defects which are represented by univalent chromosomes and chromosome fragmentation at metaphase I, and acentric fragments and chromatin bridges in meiosis I and II. Consequently, chromosome segregation is strongly affected, resulting in meiotic products of uneven size, shape and of variable ploidy. The similarities in phenotype, and the sequence homology between DIF1 and the REC8/RAD21 cohesins suggests that cohesin function is largely conserved between eukaryotes and highlights the essential role cohesins play in plant meiosis.

关键词：等位基因氨基酸序列拟南芥属/细胞学拟南芥属/遗传学拟南芥蛋白质类染色体畸变/遗传学染色体分离/遗传学克隆分子保守序列/遗传学生育力真菌蛋白质类/化学真菌蛋白质类/遗传学基因植物/遗传学减数分裂/遗传学分子序列数据突变核蛋白质类/化学核蛋白质类/遗传学表型磷蛋白类/化学磷蛋白类/遗传学种系发生植物组成部分/细胞学植物组成部分/遗传学植物蛋白质类/化学植物蛋白质类/遗传学植物蛋白质类/生理学花粉/细胞学花粉/遗传学 RNA 信使/分析 RNA 信使/遗传学非洲粟酒人体酵母蛋白质类序列同源性氨基酸动物人类

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Coronavirus-induced membrane fusion requires the cysteine-rich domain in the spike protein

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VIROLOGY 2000年第1期269卷 212-224页

作者： Chang, KW Sheng, YW Gombold, JL Louisiana State Univ Hlth Sci Ctr Dept Microbiol & Immunol Shreveport LA 71130 USA

The spike glycoprotein of mouse hepatitis virus strain A59 mediates the early events leading to infection of cells, including fusion of the viral and cellular membranes. The spike is a type I membrane glycoprotein that possesses a conserved transmembrane anchor and an unusual cysteine-rich (cys) domain that bridges the putative junction of the anchor and the cytoplasmic tail. In this study, we examined the role of these carboxyl-terminal domains in spike-mediated membrane fusion. We show that the cytoplasmic tail is not required for fusion but has the capacity to enhance membrane fusion activity. Chimeric spike protein mutants containing substitutions of the entire transmembrane anchor and cys domain with the herpes simplex virus type 1 glycoprotein D (gD-1) anchor demonstrated that fusion activity requires the presence of the A59 membrane-spanning domain and the portion of the cys domain that lies upstream of the cytoplasmic tail. The cys domain is a required element since its deletion from the wild-type spike protein abrogates fusion activity. However, addition of the cys domain to fusion-defective chimeric proteins was unable to restore fusion activity. Thus, the cys domain is necessary but is not sufficient to complement the gD-1 anchor and allow for membrane fusion. Site-specific mutations of conserved cysteine residues in the cys domain markedly reduce membrane fusion, which further supports the conclusion that this region is crucial for spike function. The results indicate that the carboxyl-terminus of the spike transmembrane anchor contains at least two distinct domains, both of which are necessary for full membrane fusion. (C) 2000 Academic Press.

关键词：氨基酸序列氨基酸取代/遗传学细胞融合细胞系保守序列/遗传学半胱氨酸/遗传学半胱氨酸/代谢细胞质/代谢基因报告/遗传学动力学膜融合膜糖蛋白类/化学膜糖蛋白类/遗传学膜糖蛋白类/代谢分子序列数据肝炎病毒鼠/化学肝炎病毒鼠/遗传学肝炎病毒鼠/代谢肝炎病毒鼠/生理学蛋白质结构三级重组融合蛋白质类/化学重组融合蛋白质类/遗传学重组融合蛋白质类/代谢序列缺失/遗传学转染病毒包膜蛋白质类/化学病毒包膜蛋白质类/遗传学病毒包膜蛋白质类/代谢 β半乳糖苷酶类/遗传学 β半乳糖苷酶类/代谢动物小鼠

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Gonadoblastoma, testicular and prostate cancers, and the TSPY gene

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AMERICAN JOURNAL OF HUMAN GENETICS 1999年第4期64卷 921-927页

作者： Lau, YFC Univ Calif San Francisco Vet Adm Med Ctr Dept Med Div Cell & Dev Genet San Francisco CA 94121 USA

Discusses the role of Y chromosomal loci in oncogenesis. Focus on the putative oncogne TSPY, which may be responsible for tumors of the prostate and testes and for gonadoblastoma, an unusual cancer that is found in phenotypic females who carry a Y chromosome; Evolutionary conservation of TSPY gene on the mammalian Y chromosome; Normal and abnormal expression of TSPY in the testis.

关键词：细胞周期蛋白质类染色体蛋白质类非组蛋白保守序列/遗传学细胞周期蛋白B/代谢 DNA结合蛋白质类/遗传学 DNA结合蛋白质类/代谢基因表达调控肿瘤/遗传学疾病遗传易感性/遗传学性腺母细胞瘤/遗传学组蛋白监控蛋白类核蛋白质类前列腺肿瘤/遗传学前列腺肿瘤/代谢蛋白质类/遗传学性别决定区Y蛋白质睾丸肿瘤/遗传学睾丸肿瘤/代谢转录因子 Y染色体/遗传学动物人类男(雄)性

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ConStruct:: a tool for thermodynamic controlled prediction of conserved secondary structure

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NUCLEIC ACIDS RESEARCH 1999年第21期27卷 4208-4217页

作者： Lück, R Gräf, S Steger, G Univ Dusseldorf Inst Biol Phys D-40225 Dusseldorf Germany

A tool for prediction of conserved secondary structure of a set of homologous single-stranded RNAs is presented, For each RNA of the set the structure distribution is calculated and stored in a base pair probability matrix, Gaps, resulting from a multiple sequence alignment of the RNA set, are introduced into the individual probability matrices. These 'aligned' probability matrices are summed up to give a consensus probability matrix emphasizing the conserved structural elements of the RNA set. Because the multiple sequence alignment is independent of any structural constraints, such an alignment may result in introduction of gaps into the homologous probability matrices that disrupt a common consensus structure, By use of its graphical user interface the presented tool allows the removal of such misalignments, which are easily recognized, from the individual probability matrices by optimizing the sequence alignment with respect to a structural alignment. From the consensus probability matrix a consensus structure is extracted, which is viewable in three different graphical representations. The functionality of the tool is demonstrated using a small set of U7 RNAs, which are involved in 3'-end processing of histone mRNA precursors. Supplementary Material lists further results obtained. Advantages and drawbacks of the tool are discussed in comparison to several other algorithms.

关键词：算法碱基错配碱基配对碱基序列共有序列/遗传学保守序列/遗传学数据库事实型突变/遗传学核酸构象 RNA/化学 RNA/遗传学 RNA/代谢 RNA 小核/遗传学序列比对序列同源性核酸软件热力学用户计算机接口动物人类

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Structural equivalence in the transcribed spacers of pre-rRNA transcripts in Schizosaccharomyces pombe

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NUCLEIC ACIDS RESEARCH 1999年第15期27卷 3071-3078页

作者： Lalev, AI Nazar, RN Univ Guelph Dept Mol Biol & Genet Guelph ON N1G 2W1 Canada

The structure of the internal transcribed spacer 2 (ITS2) in Schizosaccharomyces pombe was re-evaluated with respect to phylogenetically conserved features in yeasts, features in other transcribed spacer regions as well as the binding of transacting factors which potentially play a role in ribosomal maturation, Computer analyses and probes for nuclease protection indicate a very simple core structure consisting of a single extended hairpin which includes the interacting termini of the mature 5.8S and 25S rRNAs, Comparisons with ITS2 sequences in greatly diverging organisms indicate that the same feature also can be recognized, This is especially clear in organisms that contain very short sequences in which the putative structures are much less ambiguous, Diversity between organisms is the result of changes in hairpin length as well as the addition of branched helices, Protein binding and gel retardation studies with the S. pombe ITS2 further indicate that, as observed in the 3' external transcribed spacer (ETS) and ITS1 regions, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing but also a site for specific interactions with one or more soluble factors. Taken together with other analyses on transcribed spacer regions, the present data suggest that the spacer regions all may act in a similar fashion, not only to organize the maturing terminal sequences, but also serve to organize specific soluble factors possibly acting with snoRNAs or in a manner which is analogous with that of the free snoRNPs.

关键词：碱基配对碱基序列结合竞争性保守序列/遗传学内切核糖核酸酶类/代谢进化分子分子序列数据核酸构象种系发生 RNA前体/化学 RNA前体/遗传学 RNA前体/代谢 RNA加工转录后/遗传学 RNA 真菌/化学 RNA 真菌/遗传学 RNA 真菌/代谢 RNA 核糖体/化学 RNA 核糖体/遗传学 RNA 核糖体/代谢 RNA结合蛋白质类/代谢调控序列核酸/遗传学裂殖酵母菌属/遗传学构效关系转录遗传/遗传学人类

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The narrow sheath duplicate genes:: Sectors of dual aneuploidy reveal ancestrally conserved gene functions during maize leaf development

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GENETICS 2000年第3期155卷 1379-1389页

作者： Scanlon, MJ Chen, KD McKnight, CC Univ Georgia Dept Bot Athens GA 30602 USA Univ Calif Berkeley Dept Plant & Microbial Biol Berkeley CA 94720 USA

The narrow sheath mutant of maize displays a leaf and plant stature phenotype controlled by the duplicate factor mutations narrow sheath1 and narrow sheath2. Mutant leaves fail to develop a lateral domain that includes the leaf margins. Genetic data are presented to show that the narrow sheath mutations map to duplicated chromosomal regions, reflecting an ancestral duplication of the maize genome. Genetic and cytogenetic evidence indicates that the original mutation at narrow sheath2 is associated with a chromosomal inversion on the long arm of chromosome 4. Meristematic sectors of dual aneuploidy were generated, producing plants genetically mosaic for NARROW SHEATH function. These mosaic plants exhibited characteristic half-plant phenotypes, in which leaves from one side of the plant were of nonmutant morphology and leaves from the opposite side were of narrow sheath mutant phenotype. The data suggest that the narrow sheath duplicate genes may perform ancestrally conserved, redundant functions in the development of a lateral domain in the maize leaf.

关键词：等位基因非整倍性染色体图保守序列/遗传学 DNA 植物/分析 DNA 植物/辐射效应基因重复/遗传学镶嵌现象/遗传学诱变表型植物叶/化学植物叶/遗传学植物叶/生长和发育花粉/细胞学花粉/遗传学多态性限制性片段长度重组遗传种子/遗传学种子/辐射效应易位遗传 X线玉米/化学玉米/遗传学玉米/生长和发育

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Vnd/nkx, ind/gsh, and msh/msx:: conserved regulators of dorsoventral neural patterning?

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CURRENT OPINION IN NEUROBIOLOGY 2000年第1期10卷 63-71页

作者： Cornell, RA Von Ohlen, T Univ Oregon Inst Neurosci Eugene OR 97403 USA

Expression of vnd in ventral, ind in intermediate, and msh in dorsal columns of fly neurectoderm, and of homologous gene families in corresponding domains of vertebrate neurectoderm, suggests that elements of dorsoventral neural patterning have been evolutionarily conserved. However, upstream signaling pathways regulating this columnar gene expression pattern appear to have diverged significantly throughout evolution. In addition, while recent loss-of-function studies in flies and mice indicate that these three genes may have a conserved role in regional specification, there is no obvious conservation of the particular cell fates deriving from corresponding domains. The three-column expression pattern may thus represent a developmental mechanism that is more resistant to evolutionary changes than genetic events upstream or downstream of it.

关键词：躯体发育模式中枢神经系统/细胞学中枢神经系统/胚胎学中枢神经系统/代谢保守序列/遗传学保守序列/生理学果蝇蛋白质类黑腹果蝇基因表达调控发育期同源盒结构域蛋白质类/遗传学同源盒结构域蛋白质类/生理学信号传导转录因子脊椎动物动物

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CAPN 8: Isolation of a new mouse calpain-isoenzyme

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1999年第3期260卷 671-675页

作者： Braun, C Engel, M Theisinger, B Welter, C Seifert, M Univ Saar Inst Human Genet D-66421 Homburg Germany

Recent molecular biological approaches indicate that calpain, also named CANP for calcium-activated neutral protease and originally characterized as an intracellular cytoplasmatic nonlysosomal cysteine protease that requires calcium ions for activity, constitutes a large superfamily consisting of ubiquitous and tissue specific homologues, which are widely distributed in cells of various organisms from human to fungus. Due to the increasing number of substrates along with the involvement of calpain isoenzymes in mammalian diseases, especially in malignancies, members of the calpain superfamily seem to be important biomodulators in physiological as well as pathological cell function. Here we report the characterisation of a new calpain, named CAPN 8 with a different C-terminal domain, implicating a putative new regulatory mechanism. Northern blot analysis revealed an ubiquitous expression with different RNA levels in all tissues examined. Highest levels were found in brain, kidney, and digestive tract, suggesting a specific regulatory function of CAPN 8 in these tissues. (C) 1999 Academic Press.

关键词：氨基酸序列印迹法 RNA 脑/代谢卡配因/化学卡配因/遗传学卡配因/分离和提纯保守序列/遗传学物理图谱消化系统/代谢表达的序列标记同工酶类/化学同工酶类/遗传学同工酶类/分离和提纯肾/代谢分子序列数据开放读码框架/遗传学 RNA 信使/遗传学 RNA 信使/代谢序列比对动物小鼠

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