Molecular and phenotypic analysis of the S. cerevisiae MNN10 gene identifies a family of related glycosyltransferases

The Saccharomyces cerevisiae mnn10 mutant is defective in thesynthesis of N-linked oligosaccharides (Ballou et al., 1989).This mutation has no effect on O-linked sugars, but resultsin the accumulation of glycoproteins that contain severely truncatedN-linked outer-chain oligosaccharides. We have cloned the MNN10gene by complementation of the hygromycin B sensitivity conferredby the mutant phenotype. Sequence analysis predicts that Mnn10pis a 46.7 kDa type II membrane protein with structural featurescharacteristic of a glycosyltransferase. Subcellular fractionationdata indicate that most of the Mnn10 protein cofractionateswith Golgi markers and away from markers for the endoplasmicreticulum (ER), suggesting Mnn10p is localized to the Golgicomplex. A comparison of the Mnn10 protein sequence to proteinsin the two different databases identified five proteins thatare homologous to Mnn10p, including a well characterized Schizosaccharomycespombe     

中国萤火虫云南窗萤荧光素酶cDNA的克隆表达和序列分析(英文）

从一种来自中国日行性萤火虫（云南窗萤）发光器官mRNA中克隆、测序并表达了有功能的荧光素酶。云南窗萤荧光素酶的cDNA序列有1 647个碱基,编码548个氨基酸残基。从推测得到的氨基酸序列的比对分析得出：云南窗萤的荧光素酶与来自Lampyris noctiluca, L. turkestanicus和Nyctophila cf. caucasica三种萤火虫的荧光素酶有97.8%的序列一致性。从推测得出的氨基酸序列进行系统发育分析,其结果表明：云南窗萤和Lampyris+Nyctophila聚在一起, 与同属的发光强夜行性的萤火虫不形成的单系。云南窗萤荧光素酶在大肠杆菌中表达的条带大约70 kDa,并且在有荧光素存在时发出黄绿色荧光。对荧光素酶的结构模拟和分析表明,云南窗萤荧光素酶基因的氨基端和羧基端结构域之间的裂沟处存在这5个多肽环,这正是从其他荧光素酶推测得到的催化荧光反应时的底物结合位点。云南窗萤和窗萤属的其他3种萤火虫的荧光素酶相比,有13个不同氨基酸位点,位于模拟分子结构的表面。对于这些多肽环、不同氨基酸残基和晶体结构的进一步研究有利于解释日行和夜行性萤火虫荧光素酶的差异。     

Structure and expression of a cDNA encoding a putative neoxanthin cleavage enzyme (NCE), isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library

Oxidative cleavage of a 9-cis xanthophyll probably representsthe key regulatory step in abscisic acid (ABA) biosynthesis.A transposon tagged maize (Zea mays) mutant vp14, provided theoriginal DNA sequence data needed to design a VP14 fusion proteincapable of catalysing this reaction in vitro. A cDNA encodinga similar protein has now been isolated from a wilt-relatedtomato (Lycopersicon esculentum Mill.) library. The tomato cDNAand derived amino acid sequence have been compared to thoseof maize and of other enzymes catalysing broadly similar oxidativecleavage reactions. The results of Northern analysis in tomatoindicated that mRNA levels of this vp14 homologue increaseddramatically in response to water stress. Key words: ABA biosynthesis, oxidative cleavage step, tomato     

Structure and expression of a cDNA encoding zeaxanthin epoxidase, isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library

Zeaxanthin epoxldase (ZE) catalyses two early steps in the abscisicacid (ABA) biosynthetic pathway. The sequence of a cDNA cloneencoding ZE from Nicotiana plumbaginifolia was reported In 1996and represented the first DNA sequence data on an ABA biosyntheticenzyme. The N. plumbaginifolia cDNA has been used to providea heterologous probe to isolate a ZE cDNA from tomato (Lycopersiconesculentum Mill.). DNA and amino acid sequence differences areconsidered in relation to putative functional domans withinthe enzyme. The results of northern analysis in tomato are discussedin relation to the effects of water stress on ZE mRNA levels. Key words: ABA biosynthesis, zeaxanthin epoxidase, tomato     

Abolishment of N-glycan mannosylphosphorylation in glyco-engineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes

Mannosylphosphorylated glycans are found only in fungi, including yeast, and the elimination of mannosylphosphates from glycans is a prerequisite for yeast glyco-engineering to produce human-compatible glycoproteins. In Saccharomyces cerevisiae, MNN4 and MNN6 genes are known to play roles in mannosylphosphorylation, but disruption of these genes does not completely remove the mannosylphosphates in N-glycans. This study was performed to find unknown key gene(s) involved in N-glycan mannosylphosphorylation in S. cerevisiae. For this purpose, each of one MNN4 and five MNN6 homologous genes were deleted from the och1Δmnn1Δmnn4Δmnn6Δ strain, which lacks yeast-specific hyper-mannosylation and the immunogenic α(1,3)-mannose structure. N-glycan profile analysis of cell wall mannoproteins and a secretory recombinant protein produced in mutants showed that the MNN14 gene, an MNN4 paralog with unknown function, is essential for N-glycan mannosylphosphorylation. Double disruption of MNN4 and MNN14 genes was enough to eliminate N-glycan mannosylphosphorylation. Our results suggest that the S. cerevisiae och1Δmnn1Δmnn4Δmnn14Δ strain, in which all yeast-specific N-glycan structures including mannosylphosphorylation are abolished, may have promise as a useful platform for glyco-engineering to produce therapeutic glycoproteins with human-compatible N-glycans.

     

Functional characterization of high-affinity Na(+)/dicarboxylate cotransporter found in Xenopus laevis kidney and heart

The SLC13 gene family includes sodium-coupled transporters for citric acid cycle intermediates and sulfate. The present study describes the sequence and functional characterization of a SLC13 family member from Xenopus laevis, the high-affinity Na⁺/dicarboxylate cotransporter xNaDC-3. The cDNA sequence of xNaDC-3 codes for a protein of 602 amino acids that is 70% identical to the sequences of mammalian NaDC-3 orthologs. The message for xNaDC-3 is found in the kidney, liver, intestine, and heart. The xNaDC-3 has a high affinity for substrate, including a K_m for succinate of 4 µM, and it is inhibited by the NaDC-3 test substrates 2,3-dimethylsuccinate and adipate. The transport of succinate by xNaDC-3 is dependent on sodium, with sigmoidal activation kinetics, and lithium can partially substitute for sodium. As with other members of the family, xNaDC-3 is electrogenic and exhibits inward substrate-dependent currents in the presence of sodium. However, other electrophysiological properties of xNaDC-3 are unique and involve large leak currents, possibly mediated by anions, that are activated by binding of sodium or lithium to a single site. SLC13 gene family; citric acid cycle intermediate; lithium     

Cloning and Functional Expression of a Novel Geranylgeranyl Pyrophosphate Synthase Gene from Arabidopsis thaliana in Escherichia coli

A gene encoding a novel geranylgeranyl pyrophosphate (GGPP)synthase from Arabidopsis thaliana has been identified and termedGGPS5. The gene has been sequenced and expressed in Escherichiacoli. The deduced amino acid sequence showed 64.5% and 57.5%identity with a putative GGPP synthase from Arabidopsis andCapsicum annuum, respectively. GGPP enzymatic activity was detectedin E. coli cells expressing the GGPS5 gene in two differentways. One was the direct measurement of GGPP synthase activityin cell extracts and the other was the yellow color productionof cells when the GGPS5 gene was co-expressed with crtB, crtI,crtX, crtY and crtZ genes derived from Erwinia uredovora. (Received May 20, 1996; Accepted December 14, 1996)     

Primary Structure and Properties of Glutathione Reductase from Arabidopsis thaliana

A cDNA encoding glutathione reductase (EC 1.6.4.2 [EC] ) from Arabidopsisthaliana was cloned by immunoscreening. The amino acid sequencededuced from the nucleotide sequence agrees with the N-terminalamino acid sequence of the major isozyme (GR II) purified fromleaves of A. thaliana. The predicted polypeptide comprises anN-terminal leader sequence of 74 amino acids, which has featuresof chloroplast-targeting peptides, and a mature polypeptideof 491 residues with a molecular mass of 52.7 kDa, which showshomology with glutathione reductases from other species. TheK_m for GSSG was 44 µM and that for NADPH was 5.0 µMfor GR II at 25C. The pH optimum for GR II was 7.5 to 8.0.The native molecular mass of GR II was 110 kDa, indicating thatGR II is a homodimer. GR II had an isoelectric point of 4.8.The cDNA hybridizes with a 2.1-kb poly(A)⁺RNA from leaves ofA. thaliana. Genomic Southern analysis indicates that the genecorresponding to the cDNA is likely a single-copy gene. (Received July 1, 1993; Accepted September 8, 1993)     

The Polyphosphate Kinase Gene of Pseudomonas aeruginosa

We have cloned and sequenced a gene encoding polyphosphate kinase(PPK) from Pseudomonas aeruginosa PAO1. The gene immediatelyfollows the hemB gene encoding porphobilinogen synthase responsiblefor heme synthesis. The predicted amino acid sequence of P.aeruginosa PPK is similar to those of PPKs previously characterizedexcept that it possesses an extra stretch of 46 amino acidsat its N-terminus, which has significant similarity to the Ras-relatedprotein ARA5 of Arabidopsis thaliana. When P. aeruginosa PPKwas overproduced in Escherichia coli, ATP-dependent polyphosphate-synthesizingactivity was drastically enhanced, confirming that the proteinis a PPK.     

家蝇细胞凋亡起始酶Caspase-1基因的克隆及在不同虫态的表达

为了研究Caspases家族在昆虫发育变态中的作用,通过RT-PCR扩增并结合RACE技术,克隆得到家蝇Musca domestica Caspase-1基因1条,命名为Mdom-Caspase-1（GenBank中cDNA序列号为EU854472, 氨基酸序列号为ACF71490）。该基因全长1 295 bp,阅读框序列870 bp,共编码289个氨基酸,理论分子量32.83 kDa,等电点8.67。 Mdom-Caspase-1蛋白有5个保守的半胱氨酸位点QACQG, 具有Caspase的典型特征; 整个分子呈现亲水性, 有8个区域共89个氨基酸为亲酯性, 蛋白质二级结构主要由11个α螺旋区、7个β-折叠区、17个β-转角区组成。昆虫间Caspase-1分子具有明显的保守性, Mdom-Caspase-1与黑腹果蝇Drosophila melanogaster、埃及伊蚊Aedes aegypti和致倦库蚊Culex quinquefasciatus的Caspase-1氨基酸序列相似性为65%~77%。RT-PCR半定量分析结果表明, Mdom-Caspase-1基因在家蝇各个虫态中均有表达, 但在卵期、3龄幼虫、预蛹、蛹和羽化5 d的雌虫中的表达量明显高于其他虫态。这些结果提示Caspase-1可能与昆虫发育变态关系密切, 为进一步研究昆虫Caspase-1功能、设计Caspase-1抑制剂提供了分子基础。     

Somatic Embryogenesis and Plant Regeneration from Freely-suspended Cells and Cell Groups of Panicum maximum Jacq

Embryogenic calluses derived from cultured immature embryosand young inflorescences of Panicum maximum Jacq. were placedin Murashige and Skoog's liquid medium supplemented with 1 mg1^–1 2, 4- dichlorophenoxyacetic acid (2, 4-D) and 2.5per cent coconut water, to initiate suspension cultures. Suspensionsconsisted of two types of cells: small, richly-cytoplasmic andoften starch-containing embryogenic cells, and large, vacuolatednon-embryogenic cells. A presumed sequence of developmentalstages from single embryogenic cells to globular and heart-shapedstages of embyrogenesis was observed in the suspension cultures.Plantlets were produced from the embryoids when the suspensionswere plated in an agar medium without any hormone or with only0.2 mg 1^–12, 4-D or naphthalene acetic acid. Embryogenicsuspension cultures derived from immature embryos as well asfrom inflorescence segments gave rise to plants which showedthe normal somatic chromosome number of 2n = 4x = 32. Panicum maximum Jacq., Guinea grass, embryogenesis, regeneration, suspension culture     

The Regulation of Proton/Amino Acid Symport in Riccia fluitans L. by Cytosolic pH and Proton Pump Activity

In the aquatic liverwort Riccia fluitans the regulation of theplasma membrane H⁺/amino acid symport has been investigated.Cytosolic pH (pH_c), membrane potential (E_m) and membrane conductancehave been measured and related to transport data, (i) The releaseof [¹⁴C]amino acids is strongly stimulated by cytosolic acidification,induced by the external addition of acetic acid, a decreasein external K⁺, and in the change from light to dark. On average,a decrease in pHc of 0.5 to 0.6 units corresponded with a 4-foldstimulation in amino acid efflux. (ii) External pH changes havefar less effect on substrate transport than the cytosolic pHshifts of the same order. (iii) The inwardly directed positivecurrent, induced by amino acids, is severely inhibited by cytosolicacidification. (iv) Fusicoccin (FC) stimulates amino acid uptakewithout considerable change in proton motive force. (v) Whenthe proton motive force is kept constant, the uptake of aminoacids into Riccia thalli is much lower than when the pump isdeactivated. It is suggested that both the proton pump activityand cytosolic pH are the dominant factors in the regulationof the H⁺/amino acid symport across the plasma membrane of Ricciafluitans, and it is concluded that the proton motive force isnot a reliable quantity to predict and interpret transport kinetics. Key words: Amino acid, cytosolic pH, pH-sensitive electrode, proton motive force, regulation, Riccia fluitans     

C-ABI3, the Carrot Homologue of the Arabidopsis ABI3, is Expressed during Both Zygotic and Somatic Embryogenesis and Functions in the Regulation of Embryo-Specific ABA-Inducible Genes

A carrot gene homologous to the ABI3 gene of Arabidopsis wasisolated from a carrot somatic embryo cDNA library and designatedC-ABI3. The sequence of C-ABI3 was very similar to those ofABI3 of Arabidopsis and VP1 of maize in certain conserved regions.The expression of C-ABI3 was detected specifically in embryogeniccells, somatic embryos and developing seeds. Thus, expressionof C-ABI3 was limited to tissues that acquired desiccation tolerancein response to endogenous or exogenous abscisic acid (ABA).Endogenous levels of ABA in seeds increased transiently andthen desiccation of seeds started. The expression of C-ABI3in developing seeds was observed prior to the increase in levelsof endogenous ABA that was followed by desiccation of seeds.In transgenic mature leaves in which C-ABI3 was ectopicallyexpressed, expression of ECP31, ECP63 and ECP40 was inducedby treatment with ABA, which indicates that the expression ofECP genes was controlled by the pathway(s) that involved C-ABI3and ABA. This suggests that C-ABI3 has the same function asVP1/ABI3 factor in carrot somatic embryos. (Received March 4, 1998; Accepted September 4, 1998)     

Cloning, Nucleotide Sequences and Differential Expression of the nifH and nifH-Like (frxC) Genes from the Filamentous Nitrogen-Fixing Cyanobacterium Plectonema boryanum

The frxC gene, found in liverwort chloroplast DNA, encodes aprotein of unknown function. The deduced amino acid sequenceof the protein shows significant homology to that of ni-trogenaseFe-protein encoded by the nifH gene. We have cloned the frxCand nifH genes from the nitrogen-fixing cyanobacterium Plectonemaboryanum, using frxC- and nifH-specific probes, and have determinedtheir nucleotide sequences. The amino acid sequence deducedfrom the frxC gene of P. boryanum exhibits 83% homology to thatof the protein encoded by the/rxCgene from liverwort, whereasit exhibits only 34% homology to that encoded by the nifH genefrom the same organism, namely, P. boryanum. Northern blot analysisshowed that the frxC gene was transcribed more actively undernitrogenase-repressed conditions than under nitrogenase-inducedconditions, suggesting that the FrxC protein has a functiondistinct from nitrogen fixation. These results, together withthe phylogenetic relationship between the nifH and frxC genes,indicate that the frxC and nifH genes are derived from a commonancestral gene but have evolved independently to encode proteinswith different functions. (Received April 27, 1991; Accepted August 12, 1991)     

The Mnn2 Mannosyltransferase Family Modulates Mannoprotein Fibril Length,Immune Recognition and Virulence of Candida albicans

The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans.