透明质酸生产菌溶血素S基因缺失突变菌株的构建及其特性北大核心CSCD

【目的】马链球菌兽疫亚种是工业上生产透明质酸的主要菌种,该菌能产生引起宿主细胞溶血的链球菌溶血素S(streptolysin S,SLS)毒素,因而其产品的安全性一直是人们所担心的问题。本实验的目的就是通过基因敲除的方法构建不产SLS的透明质酸生产工程菌,同时探讨溶血素sag A基因缺失对菌株透明质酸合成和其他毒力因子的影响。【方法】利用温度敏感/自杀性质粒p JR700载体系统,构建马链球菌兽疫亚种sag A基因缺失突变株;通过PCR扩增,溶血平板和SLS含量测定等方法确定sag A基因缺失;采用分光光度、SDS-PAGE和细胞毒性试验等分析方法,对野生菌株和sag A基因缺失突变菌株透明质酸含量、透明质酸分子量、溶血素Hylc、透明质酸分解酶、甘油醛-3-磷酸脱氢酶和菌体表面蛋白等相关毒力因子进行对比研究。【结果】获得了透明质酸产量提高30%而溶血活性极低的马链球菌兽疫亚种sag A基因缺失突变株。该突变株与野生菌株相比较,透明质酸分解酶活性增加而透明质酸相对分子量降低,此外,与毒力相关的表面蛋白含量、溶血素Hylc和甘油醛-3-磷酸脱氢酶活性也显著降低。细胞毒性实验结果表明,野生菌株与sag A基因缺失突变菌株的培养物上清液,对细胞活性的影响存在显著差异。【结论】在马链球菌兽疫亚种中sag A不仅是表达溶血素SLS的基因,同时sag A基因对菌株透明质酸合成、透明质酸分解酶、菌体表面蛋白、溶血素Hylc和甘油醛-3-磷酸脱氢酶等都具有调节作用。     

生于蔷薇科植物上的链格孢属真菌一新种

报道生于蔷薇科植物枇杷Eriobotrya japonica叶片上的链格孢属一新种,对该种结合形态描述及分子系统学分析明晰其分类地位。命名为枇杷链格孢Alternaria eriobotryae。研究标本保藏于贵州大学植物病理学实验室（HGUP）和中国科学院菌物标本馆（HMAS）。     

不同启动子调控外源基因在斑玉蕈中的表达分析

启动子是控制基因转录的重要顺式元件,也是遗传转化实验中驱动外源基因表达的重要工具。在同一真菌中,不同启动子驱动外源基因表达水平可能存在明显差异。因此,选择合适的启动子是提高外源基因表达水平的关键。本研究分别应用花椰菜病毒35S RNA（cauliflower mosaic virus 35S RNA,CaMV35S）和斑玉蕈甘油醛-3-磷酸脱氢酶（Hypsizygus marmoreus glyceraldehyde-3-phosphate dehydrogenase,HmGPD）基因的启动子构建了两个遗传转化质粒,在斑玉蕈中分别驱动外源的植物花青素合成基因表达,并利用来自刺芹侧耳的萎锈灵抗性基因进行转基因筛选。两个质粒通过农杆菌介导转化斑玉蕈单核菌株后,对具有萎锈灵抗性的转化子经PCR方法进行转基因验证,并运用实时荧光定量PCR对阳性转化子中外源基因的表达水平进行比较分析。结果表明,CaMV35S和HmGPD基因的启动子均成功驱动了植物花青素合成基因在斑玉蕈中转录,为增强基因表达而引入的内含子在转录过程中均被正确切割。其中,HmGPD启动子驱动外源基因表达水平比CaMV35S启动子驱动外源基因表达水平强22-36倍。     

Differential expression of the three yeast glyceraldehyde-3-phosphate dehydrogenase genes

Utilizing yeast strains containing insertion mutations in each of the three glyceraldehyde-3-phosphate dehydrogenase structural genes, the level of expression of each gene was determined in logarithmically growing cells. The contribution of the TDH1, TDH2, and TDH3 gene products to the total glyceraldehyde-3-phosphate dehydrogenase activity in wild type cells is 10-15, 25-30, and 50-60%, respectively. The relative proportions of expression of each gene is the same in cells grown in the presence of glucose or ethanol as carbon source although the total glyceraldehyde-3-phosphate dehydrogenase activity in cells grown in the presence of glucose is 2-fold higher than in cells grown on ethanol. The polypeptides encoded by each of the structural genes were identified by two-dimensional polyacrylamide gel electrophoresis. The TDH3 structural gene encodes two resolvable forms of glyceraldehyde-3-phosphate dehydrogenase which differ by their net charge. The apparent specific activity of glyceraldehyde-3-phosphate dehydrogenase encoded by the TDH3 structural gene is severalfold lower than the enzymes encoded by TDH1 or TDH2. The polypeptides encoded by the TDH2 or TDH3 structural genes form catalytically active homotetramers. The apparent Vmax for the homotetramer encoded by TDH3 is 2-3-fold lower than the homotetramer encoded by TDH2. Evidence is presented that isozymes of glyceraldehyde-3-phosphate dehydrogenase exist in yeast cells, however, the number of different isozymes formed was not established. These data confirm that the three yeast glyceraldehyde-3-phosphate dehydrogenase genes encode catalytically active enzyme and that the genes are expressed at different levels during logarithmic cell growth.     

Structure of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase complexed with chalepin,a natural product inhibitor,at 1.95 A resolution

The structure of the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) from Trypanosoma cruzi complexed with chalepin, a natural product from Pilocarpus spicatus, has been determined by X-ray crystallography to 1.95 Å resolution. The structure is in the apo form without cofactors in the subunits of the tetrameric gGAPDH in the asymmetric unit. Unequivocal density corresponding to the inhibitor was clearly identified in one monomer. The final refined model of the complex shows extensive conformational changes when compared with the native structure. The mode of binding of chalepin to gGAPDH and its implications for inhibitor design are discussed.     

Nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene from the mesophilic methanogenic archaebacteria Methanobacterium bryantii and Methanobacterium formicicum. Comparison with the respective gene structure of the closely related extreme thermophile Methanothermus fervidus

The genes for glyceraldehyde-3-phosphate dehydrogenase (gap genes) from the mesophilic methanogenic archaebacteria Methanobacterium formicicum and Methanobacterium bryantii were cloned and sequenced. The deduced amino acid sequences show 95% identity to each other and about 70% identity to the glyceraldehyde-3-phosphate dehydrogenase from the thermophilic methanogenic archaebacterium Methanothermus fervidus. Although the sequence similarity between the archaebacterial glyceraldehyde-3-phosphate dehydrogenase and the homologous enzyme of eubacteria and eukaryotes is low, an equivalent secondary-structural arrangement can be deduced from the profiles of the physical parameters hydropathy, chain flexibility and amphipathy. In order to find possible thermophile-specific structural features of the enzyme from M. fervidus, a comparative primary-sequence analysis was performed. Amino acid exchanges leading, to a stabilization of the main-chain conformation, could be found throughout the sequence of the thermophile enzyme. Striking features of the thermophile sequence are the preference for isoleucine, especially in beta-sheets, and a low arginine/lysine ratio of 0.54.     

蚁巢伞属真菌Termitomyces clypeatus实时荧光定量PCR内参基因的筛选

蚁巢伞属真菌尖盾蚁巢伞Termitomyces clypeatus是一类与大白蚁亚科昆虫共生的野生食用真菌,因味道鲜美备受消费者喜爱。为进一步对蚁巢伞属真菌开展相关生物学和遗传学研究,本试验选取8个候选内参基因[3-磷酸甘油醛脱氢酶(GAPDH)、磷酸葡萄糖变异酶(PGM)、β微管蛋白(TUB)、β肌动蛋白(ACT)、翻译延长因子1-α (EF1)、蛋白磷酸酶2A (PP2A)、聚泛素(UBQ)和翻译延伸因子2 (EF2)],对其在T. clypeatus菌株不同生长发育时期(菌丝体、巢内萌发期及成熟子实体)的表达稳定性进行评估,通过4种软件(geNorm、NormFinder、BestKeeper以及RefFinder)进行数据分析,结果表明：在供试条件下,ACT、EF1和TUB的相对表达量处于较稳定的状态,可作为蚁巢伞属真菌功能基因转录水平分析的内参基因。     

Differentiation of Staphylococcus spp. by terminal-restriction fragment length polymorphism analysis of glyceraldehyde-3-phosphate dehydrogenase-encoding gene

Classical phenotypic and biochemical testing do not lead to correct identification of the distinct Staphylococcus species. Therefore, the aim of our study was to develop a method for the reliable and accurate determination of distinct Staphylococcus species.

In the present study, the 931–934-bp partial sequences of the glyceraldehyde-3-phosphate dehydrogenase-encoding (gap) gene of 28 validly described Staphylococcus species were amplified and sequenced. By using the respective sequence information we performed a terminal-restriction fragment length polymorphism (T-RFLP) analysis. For T-RFLP the partial gap gene was amplified with double-fluorescently labelled primers and digested with the restriction enzymes DdeI, BspHI and TaqI. Distinctive T-RFLP patterns were rendered by the use of capillary electrophoresis with laser-induced fluorescence detection. This molecular method allowed us to identify all 28 Staphylococcus species with high specificity. This was validated by analysis of 34 Staphylococcus epidermidis and 28 Staphylococcus haemolyticus isolates.

These results demonstrate the feasibility and applicability of the T-RFLP method based on the partial gap gene sequences for rapid and accurate species identification.     

Lifetimes of mRNAs for clock-regulated proteins in a dinoflagellate

Both pulsed and continuous applications of the RNA polymerase II inhibitor thiolutin cause a dramatic but reversible loss of bioluminescence and its overt rhythmicity in cells of the dinoflagellate Lingulodinium polyedrum (formerly Gonyaulax polyedra). Such cells remain alive, and the rhythm resumes after an interval, the length of which depends on the concentration of thiolutin used. The period and phase of the resumed rhythm were not systematically altered following such treatments, and the effects were not different at different circadian phases. For three different genes, luciferin binding protein (lbp), luciferase (lcf), and glyceraldehyde-3-phosphate dehydrogenase (gapdh), which are circadian-regulated at the level of translation, the amounts of their mRNAs were determined by Northern blots for times up to 12.5h following the addition of 1.5 µM thiolutin. Consistent with previous reports that their abundances do not change with circadian time, their levels remained high for several hours after thiolutin addition, but then did diminish.     

Mechanism of action and fate of the fungicide chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) in biological systems: 2. In vitro reactions

The reaction characteristics of chlorothalonil with glyceraldehyde-3-phosphate dehydrogenase (GPDH), from yeast, (EC 1.2.1.12) were studied in vitro. Enzyme inhibition was related to the amount of [¹⁴C]chlorothalonil bound to the protein. Kinetics of enzyme inhibition was non-competitive for the substrate glyceraldehyde-3-phosphate (GAP) (K_i = 0.42 μM). Reversal of enzyme inhibition could not be demonstrated with the low molecular thiol dithiothreitol (DTT), although the thiol did protect the protein against the toxic action of the fungicide. Because 5,5' dithiobis-(2-nitrobenzoic) acid (DTNB) reduced the binding of ¹⁴C-labeled fungicide by approximately 90% it is postulated that chlorothalonil affects catalytic activity by reacting with the 4 sulfhydryl sites (cysteine-149) responsible for the binding of GAP. Certain reaction characteristics of the trichloromethyl sulfenyl fungicides with GPDH were found to be similar to those of chlorothalonil. However, chlorothalonil differed from those fungicides in that it did not react with non-thiol groups of either GPDH or -chymotrypsin (CT) and had a slower reaction rate with the GPDH. It is suggested that the differences in reaction rates of the fungicides are due to the molecular size and the chemical nature of the reactive toxiphores.     

Coat protein of Chinese wheat mosaic virus upregulates and interacts with cytosolic glyceraldehyde-3-phosphate dehydrogenase,a negative regulator of plant autophagy,to promote virus infection

Autophagy is an intracellular degradation mechanism involved in antiviral defense, but the strategies employed by plant viruses to counteract autophagy-related defense remain unknown for the majority of the viruses. Herein,we describe how the Chinese wheat mosaic virus(CWMV, genus Furovirus) interferes with autophagy and enhances its infection in Nicotiana benthamiana. Yeast two-hybrid screening and in vivo/in vitro assays revealed that the 19 k Da coat protein(CP19 K) of CWMV interacts with cyt...     

实时定量PCR评估金针菇的内参基因稳定性

金针菇在中国和日本是最受人喜爱的食用菌之一,在重要栽培食用菌中产销量排名第四。近年来,已在活性物质、分子标记及基因鉴定方面开展了大量工作,但未见有金针菇内参基因稳定性研究的报道,导致金针菇基因表达研究无内源参考基因稳定性数据作为参考。本研究采用geNorm、NormFinder、BestKeeper和RefFinder 4种软件评估18S核糖体RNA（18S）、28S核糖体RNA（28S）、60S核糖体蛋白L18（Rpl18）、肌动蛋白1（Act1）、3-磷酸甘油醛脱氢酶（Gapdh）、翻译延伸因子EF1-alpha（Ef1A）、DNA指导的RNA聚合酶亚基2（Rpb2）、细胞色素C氧化酶亚基1（Cox1）及细胞色素b（CytB）等9个内参基因的稳定性。综合分析结果显示,单个基因中Act1基因最稳定,CytB表达稳定性最差,但单个候选内参基因的稳定性不能够满足定量PCR实验要求;根据geNorm软件分析结果,CytB和Rpb2是金针菇定量PCR的稳定内参基因组合。本研究首次评估了金针菇的内参基因稳定性,将为后续的基因表达研究提供参考。     

An enigmatic GAPDH gene in the symbiotic dinoflagellate genus Symbiodinium and its related species (the order Suessiales): possible lateral gene transfer between two eukaryotic algae, dinoflagellate and euglenophyte

A group of unicellular eukaryotic algae, the dinoflagellates, are known to possess two types of gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). An enzyme encoded by one type of gene possibly plays a key role in the glycolytic pathway of the cytosol and the other in the Calvin cycle of plastids. In the present study, an additional type of GAPDH gene (GapC3) was found in the symbiotic dinoflagellates, Symbiodinium spp. and their related species, Gymnodinium simplex and Polarella glacialis, all of which belong to the order Suessiales. Since no intracellular translocation signal is found at both amino- and carboxy-termini of its deduced amino acid sequence, the protein is predicted to function in the cytosol. However, it may not be involved in glycolysis due to the presence of an amino acid signature that allows binding for NADP⁺. It is likely that dinoflagellate species, other than Suessiales investigated in this study, lack this type of GAPDH. Phylogenetic analysis placed GapC3 from the Suessialean species firmly in the clade composed of GAPDH from spirochetes, euglenophytes (cytosolic type) and kinetoplastids (glycosomal type). Specifically, this enigmatic GAPDH gene in dinoflagellates was closely related to its cytosolic counterpart in euglenophytes. It has been previously reported that plastid-targeted (Calvin cycle) GAPDH genes of the dinoflagellates Pyrocystis spp. and that of the euglenophyte Euglena gracilis also seem to share a common ancestor. It appears highly likely that at least two genes (cytosolic and plastid-targeted GAPDH genes) have been laterally transferred between these two eukaryotic algal groups.     

Age-related effects in enzyme catalysis

Summary Rat muscle glyceraldehyde-3-phosphate dehydrogenase is one of several enzymes which have been found to undergo age-related modifications. While the amount of this enzyme in muscle tissue does not change with age, both its specific activity and affinity towards its co-enzyme are significantly reduced in the old tissue.Age-related structural changes were found to exist in the nicotinamide binding site of the enzyme and the reactions leading to the activity loss in old glyceraldehyde-3-phosphate dehydrogenase were shown to involve a reversible modification of the essential cysteine-149 residue at the active site of the enzyme. The aging effects were simulated by a controlled oxidation of cys-149 in samples of young glyceraldehyde-3-phosphate dehydrogenase and subsequent reduction of this residue by 2-mercaptoethanol. The enzyme modified in this way closely resembles native old glyceraldehyde-3-phosphate dehydrogenase, indicating that the structural modifications in the latter enzyme are indeed introduced by a post-translational process. The mechanism for aging of glyceraldehyde-3-phosphate dehydrogenase which is proposed, based on these observations, thus assumes an oxidation of cys-149 as its first step followed by irreversible conformational changes in the enzyme molecule. The aging of glyceraldehyde-3-phosphate dehydrogenase may thus be triggered by the reduced ability of old muscle tissue to protect its constituents against oxidation.Abbreviations CPL circular polarization of luminescence - DTNB 5,5-dithiobis (2-nitrobenzoic acid) - GPDH D-glyceraldehyde-3-phosphate dehydrogenase - ENAD⁺ nicotinamide 1,N⁶-ethenoadenine dinucleotide     

木犀科植物叶绿体基因组结构特征和系统发育关系

木犀科11属19个种叶绿体基因组的一般特征和变异特征的比较分析显示, 结果表明, 该科叶绿体基因组大小为154-165 kb, 其差异主要是大单拷贝(LSC)长度的差异所致。Jasminum属3个物种的叶绿体基因组长度与其余物种有较大差异, 该属clpP基因内含子和accD基因丢失。共线性分析表明, Jasminum属3个物种多个基因出现基因重排现象, 倒位可能是重排的主要原因。Jasminum属在IRb/SSC和SSC/IRa边界的基因均与其它物种不同; 重复序列与SSR数量检测结果表明, Jasminum属与其余物种在数量及重复长度上差异较大。基于CDS数据构建的系统发育树表明, Abeliophyllum distichum和Forsythia suspensa为木犀科中较早分化的类群。     

木犀科植物叶绿体基因组结构特征和系统发育关系

木犀科11属19个种叶绿体基因组的一般特征和变异特征的比较分析显示, 结果表明, 该科叶绿体基因组大小为154-165 kb, 其差异主要是大单拷贝(LSC)长度的差异所致。Jasminum属3个物种的叶绿体基因组长度与其余物种有较大差异, 该属clpP基因内含子和accD基因丢失。共线性分析表明, Jasminum属3个物种多个基因出现基因重排现象, 倒位可能是重排的主要原因。Jasminum属在IRb/SSC和SSC/IRa边界的基因均与其它物种不同; 重复序列与SSR数量检测结果表明, Jasminum属与其余物种在数量及重复长度上差异较大。基于CDS数据构建的系统发育树表明, Abeliophyllum distichum和Forsythia suspensa为木犀科中较早分化的类群。     

Identification of the mammalian DNA-binding protein P8 as glyceraldehyde-3-phosphate dehydrogenase.

The DNA-binding protein P8 from transformed hamster fibroblasts (line NIL-1-hamster sarcoma virus) has been purified to homogeneity by DNA-cellulose and phosphocellulose chromatography. The molecular weight of dissociated P8 is 36000, the same as that reported for the subunits of glyceraldehyde-3-phosphate dehydrogenase, and the mobility of these proteins in polyacrylamide gels is identical. The amino acid composition of P8 is very similar to that of glyceraldehyde-3-phosphate dehydrogenase. When assayed for glyceraldehyde-3-phosphate dehydrogenase activity the P8 preparation had a specific activity of 54.6 units/mg, a value comparable to that of the crystalline enzyme from several sources. Furthermore, serum prepared against P8 crossreacts with glyceraldehyde-3-phosphate dehydrogenase from hamster muscle. These results show that P8 is glyceraldehyde-3-phosphate dehydrogenase. The interaction of P8 from transformed fibroblasts and glyceraldehyde-3-phosphate dehydrogenase from hamster and rabbit muscle with DNA has been studied using a Millipore filtration technique. These proteins have affinity for single-stranded DNA but not for double-stranded DNA.     

Identification of the ATP-binding heat-inducible protein of MR = 37,000 as glyceraldehyde-3-phosphate dehydrogenase

We previously found a novel ATP-binding heat-inducible protein of Mr = 37,000 in BALB/c 3T3 cells. Here, we found that the peptide mapping of this 37-kDa protein was similar to that of rabbit glyceraldehyde-3-phosphate dehydrogenase. Therefore, we biochemically compared the 37-kDa protein with a product translated from mRNA which was hybrid-selected using a cDNA for encoding chick glyceraldehyde-3-phosphate dehydrogenase and found that these two proteins were very similar. Northern blotting analysis using its cDNA as a probe revealed that glyceraldehyde-3-phosphate dehydrogenase was a heat-inducible protein in BALB/c 3T3 cells and that it was induced by stresses including treatment with alpha, alpha'-dipyridyl.     

Isolation and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocyte membranes.

A rapid and convenient procedure for isolating human glyceraldehyde-3-phosphate dehydrogenase from erythrocytes has been developed and yields enzyme with a specific activity of 33–52. The physical and catalytic properties of the enzyme are similar to those of rabbit muscle enzyme. Reassociation of freshly isolated human glyceraldehyde-3-phosphate dehydrogenase with washed erythrocyte membranes increases the specific activity and stability of the enzyme suggesting that enzyme-membrane interactions may have an important effect on the conformation and catalytic activity. That the human enzyme behaves as a dimer of dimers, similar to the behavior or rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, is suggested by its half-of-the-sites reactivity toward 4-iodoacetamido-1-naphthol. The human enzyme binds nicotinamide hypoxanthine dinucleotide, a structural analog of NAD⁺, with negative cooperativity, further indicating its similarity to rabbit muscle enzyme.