A constitutively expressed Myc-like gene involved in anthocyanin biosynthesis from Perilla frutescens: molecular characterization,heterologous expression in transgenic plants and transactivation in yeast cells

The coordinate expression of anthocyanin biosynthetic genes in leaves and stems of a red forma of Perilla frutescens is presumably controlled by regulatory gene(s). A Myc-like gene (Myc-rp) was isolated from a cDNA library prepared from the leaves of red P. frutescens, and its deduced amino acid sequence shows 64% identity with that of delila from snapdragon. The Myc-rp gene was expressed in leaves and roots of both red and green P. frutescens equally. Comparison of deduced amino acid sequence of Myc-rp with that of Myc-gp, the second allele isolated from a green forma of P. frutescens, indicates that the 132nd amino acid, alanine, existing in MYC-RP was changed to serine in MYC-GP. The heterologous expression of these two alleles of Myc-like gene in tobacco and tomato resulted in an increase of the anthocyanin contents in flowers of tobacco and vegetative tissues and flowers of tomato. However, the flowers of transgenic tobacco expressing the fragment with a partial deletion (encoding 1–115 amino acids deleted) of Myc-gp gave no change in anthocyanin accumulation, but some morphological changes of the flower were observed. In yeast, the MYC-RP/GP and Delila protein exhibited transactivation activity on the GAL-1 promoter from yeast and the promoter of dihydroflavonol 4-reductase (DFR) gene from P. frutescens. A transactivation domain of MYC-RP/GP and Delila could be located in the region between the 193rd and the 420th amino acid of MYC-RP/GP proteins. Our data indicate that this Myc-like gene presumably functions in the regulation of anthocyanin biosynthesis similarly in different tissues of dicot plants.     

Molecular cloning and biochemical characterization of a novel anthocyanin 5-O-glucosyltransferase by mRNA differential display for plant forms regarding anthocyanin

UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT) is responsible for the modification of anthocyanins to more stable molecules in complexes for co-pigmentation, supposedly resulting in a purple hue. The cDNA encoding 5-GT was isolated by a differential display applied to two different forms of anthocyanin production in Perilla frutescens var. crispa. Differential display was carried out for mRNA from the leaves of reddish-purple and green forms of P. frutescens, resulting in the isolation of five cDNA clones predominantly expressed in the red form. The cDNA encoded a polypeptide of 460 amino acids, exhibiting a low homology with the sequences of several glucosyltransferases including UDP-glucose: anthocyanidin 3-O-glucosyltransferase. By using this cDNA as the probe, we also isolated a homologous cDNA clone from a petal cDNA library of Verbena hybrida. To identify the biochemical function of the encoded proteins, these cDNAs were expressed in Saccharomyces cerevisiae cells. The recombinant proteins in the yeast extracts catalyzed the conversion of anthocyanidin 3-O-glucosides into the corresponding anthocyanidin 3,5-di-O-glucosides using UDP-glucose as a cofactor, indicating the identity of the cDNAs encoding 5-GT. Several biochemical properties (optimum pH, Km values, and sensitivity to inhibitors) were similar to those reported previously for 5-GTs. Southern blot analysis indicated the presence of two copies of 5-GT genes in the genome of both red and green forms of P. frutescens. The mRNA accumulation of the 5-GT gene was detected in the leaves of the red form but not in those of the green form and was induced by illumination of light, as observed for other structural genes for anthocyanin biosynthesis in P. frutescens.     

Genetic screen for signal peptides in Hydra reveals novel secreted proteins and evidence for non-classical protein secretion

We have screened a Hydra cDNA library for sequences encoding N-terminal signal peptides using the yeast invertase secretion vector pSUC [Jacobs et al., 1997. A genetic selection for isolating cDNAs encoding secreted proteins. Gene 198, 289–296]. We isolated and sequenced 907 positive clones; 88% encoded signal peptides; 12% lacked signal peptides. By searching the Hydra EST database we identified full-length sequences for the selected clones. These encoded 37 known proteins with signal peptides and 40 novel Hydra-specific proteins with signal peptides. Localization of two signal peptide-containing sequences, VEGF and ferritin, to the secretory pathway was confirmed with GFP fusion proteins. In addition, we isolated 105 clones which lacked signal peptides but which supported invertase secretion from yeast. Isolation of plasmids from these clones and retransformation in invertase-negative yeast cells confirmed the phenotype. A GFP fusion protein of one such clone encoding the foot morphogen pedibin was localized to the cytoplasm in transfected Hydra cells and did not enter the ER/Golgi secretory pathway. Secretion of pedibin and other proteins lacking signal peptides appears to occur by a non-classical protein secretion route.     

大豆类钙调磷酸酶B亚基GmCBL1互作候选蛋白的筛选

Ca2+是非生物胁迫信号转导途径中的重要信号分子,植物类钙调磷酸酶B亚基蛋白(CBL,calcineurin B-like proteins)是一类重要的钙信号受体蛋白,主要通过与其他蛋白的特异结合传递信号,使植物形成对非生物胁迫的响应。本实验室已经获得大豆Gm CBL1基因,功能鉴定显示Gm CBL1增强了转基因拟南芥对非生物胁迫的耐性。为了进一步研究Gm CBL1的作用机理,本研究构建诱饵载体p GBKT7::Gm CBL1,利用酵母双杂交技术筛选大豆Gm CBL1的互作蛋白。通过对筛选获得的106个蛋白基因测序和Blast比对分析,并根据其可能的生理功能对这些候选蛋白归类,整理得到4类蛋白:能量代谢相关蛋白、修饰蛋白、防御蛋白、钙信号转导相关蛋白。筛选得到候选蛋白的功能预测初步表明,大豆Gm CBL1参与多条信号途径,为进一步研究探索大豆CBL介导的抗逆信号转导途径奠定了基础。     

A Novel Human Gene (WDR25) Encoding a 7-WD40-Containing Protein Maps on 14q32

Members of the large family of WD-repeat proteins are involved in diverse functions such as RNA-procession, signal transduction, vesicular trafficking, cytoskeletal assembly, and cell cycle control. By large-scale-sequencing analysis of a human fetal brain cDNA library, we isolated a novel human cDNA encoding a 7-WD40-repeat protein. This cDNA is 2004 bp in length and it codes for a 544-amino-acid protein. We term it human WD40-repeat-containing gene 25 (WDR25) and this gene shows significant similarity with human pre-mRNA splicing factor 17. The WDR25 gene is mapped to chromosome 14q32 and contains seven exons. RT-PCR analysis shows that the WDR25 gene is widely expressed in human tissues and the expression levers in heart, muscle, testis, ovary, uterus, and prostate are relatively high.     

利用酵母双杂交系统筛选与黄瓜花叶病毒外壳蛋白互作的寄主蛋白

利用酵母双杂交系统,以黄瓜花叶病毒(Cucumber mosaic virus,CMV)的外壳蛋白(coat protein,CP)为诱饵,从番茄叶片c DNA文库中筛选与其互作的蛋白。结果显示,诱饵载体pBT3-SUC-CMV-CP均能在酵母细胞中正确表达,无自激活活性而且对酵母无毒性;通过对酵母双杂交文库的筛选和回转验证,共获得了98个阳性克隆,分别编码67个可能与CMV-CP相互作用的蛋白,分别参与植物防御反应、光合作用、物质转运、信号转导、能量代谢、氨基酸代谢、细胞壁的形态建成、植物的激素代谢等。本研究结果表明,CMV CP可同时调控寄主的多个代谢过程,在CMV的致病过程中有多重功能。     

The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions.

Mutations of the lethal(1)discs large-1 (dlg) tumor suppressor gene of Drosophila cause neoplastic overgrowth of the imaginal discs. Sequencing of a near full-length cDNA predicts a protein containing a domain homologous to yeast guanylate kinase and a region homologous to SH3, a putative regulatory motif in nonreceptor protein tyrosine kinases and other signal transduction proteins. Immunofluorescence analysis using antibodies directed against fusion peptides shows that the dlg gene product is localized in an apical belt of the lateral cell membrane, at the position of the septate junction. The results suggest that a signal transduction process involving guanine nucleotides occurs at the septate junction and is necessary for cell proliferation control in Drosophila epithelia.     

OsFY, a Homolog of AtFY, Encodes a Protein that Can Interact with OsFCA-γ in Rice （Oryza sativa L.）

FCA and FY are flowering time related genes involved in the autonomous flowering pathwayin Arabidopsis.FCA interacts with FY to regulate the alternative processing of FCA pre-mRNA.The FCA/FY interaction is also required for the regulation of FLC expression,a major floral repressor in Arabidopsis.However,it is not clear if the regulation of this autonomous flowering pathway is also present in monocotplants,such as rice.Recently,alternative RNA processing of OsFCA was observed in rice,which stronglysuggested the existence of an autonomous flowering pathway in rice.In this work,we cloned the cDNA ofthe autonomous flowering pathway gene OsFY from rice.The predicted OsFY protein contained a conserved7 WD-repeat region and at least two Pro-Pro-Leu-Pro motifs compared to Arabidopsis FY.The protein-protein interaction between OsFY and OsFCA-γ,the key feature of their gene function,was also demon-strated using the yeast two-hybrid system.The GenBank database search provided evidence of expressionfor other autonomous pathway gene homologs in rice.These results indicate that the autonomous floweringpathway is present in monocots,and the regulation through FY and FCA interaction is conserved betweenmonocots and dicots.     

Genetic screen for signal peptides in Hydra reveals novel secreted proteins and evidence for non-classical protein secretion

We have screened a Hydra cDNA library for sequences encoding N-terminal signal peptides using the yeast invertase secretion vector pSUC [Jacobs et al., 1997. A genetic selection for isolating cDNAs encoding secreted proteins. Gene 198, 289-296]. We isolated and sequenced 907 positive clones; 88% encoded signal peptides; 12% lacked signal peptides. By searching the Hydra EST database we identified full-length sequences for the selected clones. These encoded 37 known proteins with signal peptides and 40 novel Hydra-specific proteins with signal peptides. Localization of two signal peptide-containing sequences, VEGF and ferritin, to the secretory pathway was confirmed with GFP fusion proteins. In addition, we isolated 105 clones which lacked signal peptides but which supported invertase secretion from yeast. Isolation of plasmids from these clones and retransformation in invertase-negative yeast cells confirmed the phenotype. A GFP fusion protein of one such clone encoding the foot morphogen pedibin was localized to the cytoplasm in transfected Hydra cells and did not enter the ER/Golgi secretory pathway. Secretion of pedibin and other proteins lacking signal peptides appears to occur by a non-classical protein secretion route.     

WD-重复蛋白

 WD基元又称Trp-ASP或WD40,由40个左右的氨基酸残基组成,具有保守的GH和WD序列.WD基元存在于很多具有调控功能的蛋白质中,介导蛋白质之间的相互作用,在信号转导、蛋白运输、染色体修饰、转录或RNA加工等过程中具有重要作用.WD重复蛋白(WD-repeat protein)是含有多个保守的WD基元的蛋白质.近年发现,WD-repeat基因突变与人的几种疾病相关.本文对真核生物WD-重复蛋白的研究进展进行了综述,阐明了WD 重复蛋白的β-propeller结构特征及其作用机制,并对WD-重复蛋白的未来研究方向进行展望.     

Interactions between secreted GRA proteins and host cell proteins across the paratitophorous vacuolar membrane in the parasitism of Toxoplasma gondii

Interactions between GRA proteins of dense granules in Toxoplasma gondii and host cell proteins were analyzed by yeast two-hybrid technique. The cMyc-GRA fusion proteins expressed from pGBKT7 plasmid in Y187 yeast were bound to host cell proteins from pGADT7-Rec-HeLa cDNA library transformed to AH109 yeast by mating method. By the selection procedures, a total of 939 colonies of the SD/-AHLT culture, 348 colonies of the X-alpha-gal positive and PCR, 157 colonies of the X-beta-gal assay were chosen for sequencing the cDNA and finally 90 colonies containing ORF were selected to analyze the interactions. GRA proteins interacted with a variety of host cell proteins such as enzymes, structural and functional proteins of organellar proteins of broad spectrum. Several specific bindings of each GRA protein to host proteins were discussed presumptively the role of GRA proteins after secreting into the parasitophorous vacuoles (PV) and the PV membrane in the parasitism of this parasite.     

Identification and Characterization of MT-1X as a Novel FHL3-Binding Partner

Four and a half LIM domain protein 3 (FHL3) is a member of the FHL protein family that plays roles in the regulation of cell survival, cell adhesion and signal transduction. However, the mechanism of action for FHL3 is not yet clear. The aim of present study was to identify novel binding partner of FHL3 and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, FHL3 was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Methionine-1X was identified as a novel FHL3 binding partner. The interaction between FHL3 and the full length MT-1X was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore,the result demonstrated that MT-1X knockdown promoted the FHL3-induced inhibitory effect on HepG2 cells by regulating FHL3-mediated Smad signaling and involving in the modulation the expression of G2/M phase-related proteins through interaction with FHL3. These findings suggest that functional interactions between FHL3 and MT-1X may provide some clues to the mechanisms of FHL3-regulated cell proliferation.     

生物钟PRR蛋白促进拟南芥幼苗中花青素的合成

生物钟(circadian clock)是激发植物生理特征节律性表达，并使之维持稳定的保守内源调节机制。PRR(PSEUDO-RESPONSE REGULATOR)蛋白家族是生物钟中央振荡器的重要组成部分，调控植物的种子萌发、下胚轴伸长和开花等多种生命过程。花青素(anthocyanin)是植物次生代谢产物，对植物的繁衍、生长发育和抵抗逆境胁迫具有重要作用。该研究以拟南芥(Arabidopsis thaliana)为对象，探讨生物钟PRR蛋白对花青素生物合成的调控功能和分子机制。结果表明：(1)在PRR基因单突变体及多突变体幼苗中，花青素的积累明显降低，某些花青素合成相关基因的表达也显著降低。(2)相反，在PRR5过表达幼苗中，花青素的积累以及某些花青素合成相关基因的表达则显著升高。(3)蛋白相互作用结果显示，PRR5蛋白能与MYB75、TT8、MYB90及MYB113等花青素调控蛋白相互作用，并形成复合物。(4)遗传学分析结果显示，拟南芥PRR5诱导幼苗中花青素的合成依赖于MYB家族花青素调控蛋白。综上认为，生物钟PRR蛋白可能通过PRR5与MYB75、TT8等相互作用，促进拟南芥幼...