褐飞虱吞蛋白的基因克隆、多克隆抗体制备及表达定位

【目的】探析吞蛋白(endophilin)在褐飞虱Nilaparvata lugens生长繁殖过程中的作用。【方法】克隆褐飞虱吞蛋白基因,并进行生物信息学分析;在大肠杆菌Escherichia coli Rosetta中诱导表达褐飞虱吞蛋白基因,融合蛋白经Ni柱亲和层析法纯化后,免疫新西兰兔子,获得相应的多克隆抗体;将所得的抗体用于检测褐飞虱卵巢中吞蛋白的表达和定位。【结果】克隆得到褐飞虱两个吞蛋白基因endophilin A(Endo A)和endophilin B(Endo B),GenBank登录号分别为KY126096和KY126095,分别编码387个和352个氨基酸,都含有吞蛋白典型的BAR结构域和SH3结构域,但它们在结构上存在差异。ELISA和Western blot检测结果表明,制备的兔抗血清效价达1∶1 000 000,并表现出较好特异性。将获得的抗体应用于免疫荧光实验表明,Endo A和Endo B蛋白在褐飞虱卵巢中普遍表达,在卵巢滤泡细胞的细胞间隙、细胞膜、细胞质中广泛分布,而且与脂类物质的分布模式类似,与正在侵入褐飞虱卵巢的类酵母共生菌共定位。【结论】获得了褐飞虱吞蛋白基因Endo A和Endo B序列,并明确了其生物信息学特征,成功制备了Endo A和Endo B多克隆抗体,分析了Endo A和Endo B在褐飞虱卵巢中的表达情况,认为其可能与褐飞虱卵巢发育和成熟以及类酵母共生菌入侵褐飞虱卵巢有关。这些结果为进一步研究Endo A和Endo B在褐飞虱中的生物学功能奠定了基础。     

水稻EPSP合酶基因的克隆、结构分析和定位

5-烯醇丙酮莽草酸-3-磷酸(EPSP)合酶是芳香族氨基酸合成途径中的一个关键酶, 该基因在植物抗除草剂基因工程中具有重要的应用价值. 根据水稻EPSP合酶基因的EST序列设计探针, 在水稻TAC基因组文库中筛选到16个阳性克隆. 对阳性克隆E11进行亚克隆, 由此获得了由3661核苷酸组成的水稻EPSP合酶基因全序列. 序列分析和同源性比较揭示, 该基因由8个外显子和7个内含子组成. 以窄叶青8号/京系17组合构建的DH群体和分子图谱将水稻EPSP合酶基因定位于水稻第6条染色体的上端.     

鸡传染性支气管炎病毒变异株(793/B)核蛋白基因的克隆与序列分析

根据GenBank已经发表的传染性支气管炎病毒(IBV)N全基因组序列设计引物,对IBV 793/B分离毒株N基因进行克隆与序列分析.结果表明,IBV 793/B的N基因由1229bp组成,与GenBank已发表的11株IBV的N基因相比较,IBV 793/B的N基因共有88处点突变,在第991位发生了一个核苷酸的缺失.N基因的核苷酸同源性为86.9%～91.4%,氨基酸同源性为75.8%～77.5%.表明IBV 93/B的N基因存在着较大的变异性.     

水稻中一个NBS-LRR抗病同源基因家族的克隆和分析

利用克隆的抗病基因同源序列RS13作为探针,从水稻IR64的BAC文库中筛选到4个阳性克隆,其中一个克隆14E19能够覆盖其余3个克隆。对14E19进行全序列测定和分析,获得了73kb的全长DNA序列,基因预测显示其上有4个编码NBS-LRR结构域的基因(NL),分别命名为NL-A,B,C和D。对具有相同基因组背景的IRBB56同一染色体位置上跨度更大的BAC克隆106P13进行分析,发现其上有10个NL同源拷贝,其中4个同14E19上的NL一样。搜索日本晴、93—11、广陆矮4号的序列,发现三者有类似的同源序列。但与已知的NBS-LRR抗病基因同源性较低,说明NL是一个至少由10个成员(分别命名为NL-A至J)组成的新基因家族。对NL家族进行RT-PCR和cDNA库筛选分析,发现NL-B基因能够在抗白叶枯病品系IRBB4中表达,暗示该基因参与了抗病反应。     

地衣芽孢杆菌α-淀粉酶基因的克隆和及其启动子功能鉴定

根据已知α-淀粉酶编码基因保守区核苷酸序列,通过PCR和反向PCR技术克隆出Bacillus licheniformisCICIM B0204α-淀粉酶编码基因amyL全长序列及其上下游序列。B.licheniformisCICIM B0204amyL由1539bp组成,其上游180bp为启动子序列,下游160bp为终止子序列;成熟肽由512个氨基酸残基组成,氨基端的29个氨基酸残基为α-淀粉酶的信号肽。通过基因及其氨基酸序列比对发现,amyL及其编码产物与芽孢杆菌来源的α-淀粉酶具有高度相似性。将amyL的结构基因在PT7介导下于大肠杆菌中诱导表达,获得具有α-淀粉酶活性的表达产物。将amyL的启动子序列和信号肽序列与B.licheniformisCICIM B2004的β-甘露聚糖酶结构基因进行读框内重组,在大肠杆菌中获得了β-甘露聚糖酶的分泌表达,重组大肠杆菌表达295U/mL的β-甘露聚糖酶酶活。     

高糖土壤微生物宏基因组文库的构建及β-葡萄糖苷酶基因鉴定

采用宏基因组技术构建了高糖土壤微生物的DNA文库,该文库约含9万个克隆,文库外源DNA总容量为3.1×10~9bp。利用活性筛选策略,对文库进行筛选,获得11个β-葡萄糖苷酶的阳性克隆,并对其中2个表达β-葡萄糖苷酶的克隆进行亚克隆和序列分析,获得两个编码新型β-葡萄糖苷酶的基因分别命名为:unbgl3A和unbgl3B。生物信息学分析表明:unbgl3A基因由2241个碱基对组成,unbgl3B基因由2292个碱基对组成。在核苷酸水平上,unbgl3A、unbgl3B与已知数据库中的β-葡萄糖苷酶基因没有任何相似性。在氨基酸水平上,与GenBank数据库中已知β-葡萄糖苷酶的相似性分别为73%和69%。     

葡萄白藜芦醇合酶基因的克隆及序列分析

目的:从葡萄中克隆白藜芦醇合酶基因vrs1并对其序列进行生物信息学分析.方法:利用葡萄总RNA为模板,采用RT - PCR技术克隆白藜芦醇合酶基因vrs1并亚克隆进T- Vector.利用生物信息学工具对其核酸和蛋白序列进行分析.结果:测序结果显示其cDNA序列全长为1 257bp,含有一个1 179bp的开放阅读框.生物信息学分析表明葡萄白藜芦醇合酶基因编码392个氨基酸,分子量为42.9kDa,理论等电点为5.97,具有芪合酶家族固有的氨基酸保守结构域,二级结构主要由α-螺旋、无规则卷曲、延伸链和β-转角组成.结论:该基因的克隆、生物信息学分析为进一步研究其功能奠定了基础.     

青藏高原高原鼠兔Na+,K+-ATP酶β2亚基基因编码区的克隆与分析

目的:克隆青藏高原高原鼠兔Na ,K -ATP酶β2亚基(ATP1B2)的基因编码区,并分析其序列特征,以揭示高原鼠兔低氧适应的分子基础。方法:采用RT-PCR技术从高原鼠兔脑组织中扩增出ATP1B2基因编码区cDNA序列并进行序列测定,采用生物信息学技术对其进行分析。结果:ATP1B2基因编码区由873bp组成,编码290个氨基酸残基。序列分析结果显示,高原鼠兔ATP1B2编码区的核酸序列与兔、人、牛、大鼠、小鼠及狗分别有99%、93%、91%、91%、90%和90%的同源性。结论:克隆出青藏高原高原鼠兔ATP1B2基因编码区,为进一步了解高原鼠兔低氧适应的分子机制提供了基础。     

鸡传染性支气管炎病毒变异株（793／B）核蛋白基因的克隆与序列分析

根据GenBank已经发表的传染性支气管炎病毒(IBV)N全基因组序列设计引物,对IBV793/B分离毒株N基因进行克隆与序列分析。结果表明,IBV793/B的N基因由1229bp组成,与GenBank已发表的11株IBV的N基因相比较,IBV793/B的N基因共有88处点突变,在第991位发生了一个核苷酸的缺失。N基因的核苷酸同源性为86.9%～91.4%,氨基酸同源性为75.8%～77.5%。表明IBV93/B的N基因存在着较大的变异性。     

变色栓菌漆酶基因的克隆与序列分析

本研究以PCR扩增的方法,从变色栓(菌Trametes versicolor)的基因组DNA中扩增出了一预期大小的DNA片断,并将其克隆到了pUCM-T载体上。经筛选、酶切、PCR鉴定,序列分析,证明该片断为变色栓菌漆酶基因的克隆。该基因的开放阅读框由1560个核苷酸组成,编码一个由519个氨基酸组成的多肽。与GeneBank中发表的Laccase基因(AY081188)序列比较发现,编码的氨基酸序列同源性为96%,而核苷酸序列的同源性为92%。     

Preimplantation mouse embryos and liver express the same type I keratin gene product

The cytoskeletal B protein isolated from extraembryonic endodermal cells (Endo B) is a 50-kDa subunit of intermediate filaments that is expressed in trophoblast and extraembryonic endoderm of early mouse embryos. Endo B was compared to cytokeratin D of adult mouse liver by immunoprecipitation, two-dimensional gel electrophoresis, and peptide mapping. The two proteins were indistinguishable. A cDNA probe for Endo B mRNA identified mRNA species of similar size in liver and endoderm, and primer extension analysis indicates that the Endo B mRNAs from the two cell types have similar 5' ends. An internal fragment of the Endo B cDNA was found to cross-hybridize with a conservative domain of a human type I keratin cDNA under low stringency conditions, demonstrating that Endo B is related to type I keratins. However, under stringent conditions necessary for genomic Southern analysis, mouse and human genomic fragments homologous to the Endo B cDNA were distinct from those defined by hybridization with the type I keratin cDNA. These results indicate that Endo B is related to the type I keratin family and expands the number of type I keratin genes identified in both the mouse and human genomes. It is likely that extraembryonic endoderm, one of the first differentiated cell types of the mammalian embryo, and adult liver express the same Endo B gene.     

Differential distribution of cytokeratins after microinjection of anti-cytokeratin monoclonal antibodies

In order to investigate the relationship of different cytokeratins within one cell, monoclonal antibodies directed against three trophectoderm cytokeratins TROMA 1, 2 and 3 were microinjected into mouse teratocarcinoma-derived trophoblastoma cells and indirect immunofluorescence tests were used to follow the subsequent localization of their respective antigens Endo A, B and C. Microinjection of TROMA 1 or 2 resulted in the perinuclear collapse of Endo A, B and C-containing filaments. Microinjection of TROMA 3 resulted in the perinuclear collapse of filaments containing Endo A and B, whereas Endo C condensed into cytoplasmic aggregates which appear as speckles in the fluorescence microscope. The speckles were electron microscopically located using indirect gold-labeling techniques and had a dense, granulous structure. They were often found to be associated with microtubules, although colchicine treatment before microinjection did not interfere with speckle formation. These experiments demonstrate that cytokeratins can become differentially distributed within the cytoplasm after microinjection of an anti-cytokeratin monoclonal antibody. Since Endo A is a type II cytokeratin and Endo B and C are type I cytokeratins, these results suggest that different members of one cytokeratin subfamily may be associated with cytokeratin filaments which have different functions within the same cell.     

An endonuclease with multiple cutting sites, Endo.SceI, initiates genetic recombination at its cutting site in yeast mitochondria.

Endo.SceI is a mitochondrial sequence-specific endonuclease which has multiple cutting sites. In order to examine the possible role of Endo.SceI in homologous recombination, we analyzed the mode of recombination upon mating using antibiotic resistance markers on the mitochondrial genome. The segregation of a marker located very close to one of the Endo.SceI cutting sites showed a disparity (polarized segregation, i.e. gene conversion). This gene conversion depended on the presence of the functional Endo.SceI gene. In vivo cutting of mitochondrial DNA upon mating was detected at the cutting site in the antibiotic marker region, which also depended on the Endo.SceI activity. These results suggest that mitochondrial recombination is induced by cleavage of mitochondrial DNA by this sequence-specific endonuclease. This is the first demonstration that a sequence-specific endonuclease with multiple cutting sites induces genetic recombination.     

Murine endodermal cytokeratins Endo A and Endo B are localized in the same intermediate filament

The intracellular distribution of extra-embryonic endodermal, cytoskeletal proteins A (Endo A) and B (Endo B) was investigated by double-label immunofluorescent microscopy and double-label immunoelectron microscopy. In parietal endodermal cells, the immunofluorescent distribution of Endo B was always coincident with that of Endo A and could be distinguished from vimentin, particularly at the periphery of the cell. At the electron microscopic level, antibodies against both Endo A and Endo B recognized both bundles and individual intermediate filaments. Double-label immunoelectron microscopy was achieved by use of two sizes of colloidal gold particles (5 nm and 20 nm) that were stabilized with secondary antibodies. These results show that Endo A and B are found in the same intermediate filament and probably co-polymerize to form such structures.     

Intermediate filament protein synthesis in preimplantation murine embryos

The synthesis of two extraembryonic endodermal cytoskeletal proteins (Endo B, Mr = 50,000; Endo A, Mr = 55,000) was detected by immunoprecipitation at the 4- to 8-cell stage of preimplantation mouse development. The first detectable synthesis of both proteins occurs at about the same time as the earliest allocation of cells to the trophectodermal lineage. Both Endo A and B were identified in the two-dimensional gel pattern of blastocyst cytoskeletal proteins prepared by nonionic detergent and high-salt extraction. Endo A and B were identified as the y and x blastocyst cytoskeletal proteins, respectively, previously described by other investigators. Antibodies to Endo B are shown to react with intermediate filaments at the electron microscopic level, confirming that Endo B is an authentic intermediate filament protein. Previously, the TROMA 1 monoclonal antibody prepared by other investigators was shown to react specifically with Endo A and to decorate trophoblast cytoskeletons but did not react with the inner cell mass of blastocysts. Endo B antibodies are now also shown to decorate trophoblast cytoskeletons.     

Cloning of the human keratin 18 gene and its expression in nonepithelial mouse cells.

Human keratin 18 (K18) and the homologous mouse protein, Endo B, are intermediate filament subunits of the type I keratin class. Both are expressed in many simple epithelial cell types including trophoblasts, the first differentiated cell type to appear during mouse embryogenesis. The K18 gene was identified and cloned from among the 15 to 20 similar sequences identified within the human genome. The identity of the cloned gene was confirmed by comparing the sequence of the first two exons to the K18 cDNA sequence and transfecting the gene into various murine cell lines and verifying the encoded protein as K18 by immunoprecipitation and partial peptide mapping. The transfected K18 gene was expressed in mouse HR9 parietal endodermal cells and mouse fibroblasts even though the fibroblasts fail to express endogenous Endo B. S1 nuclease protection analysis indicated that mRNA synthesized from the transfected K18 gene is initiated at the same position as authentic K18 mRNA found in both BeWo trophoblastoma cells and HeLa cells. Pulse-chase experiments indicated that the human K18 protein is stable in murine parietal endodermal cells (HR9) which express EndoA, a complementary mouse type II keratin. Surprisingly, however, K18 was degraded when synthesized in cells which lack a type II keratin. This turnover of K18 may be an important mechanism by which epithelial cells maintain equal molar amounts of both type I and II keratins. In addition, the levels of the endogenous type I Endo B in parietal endodermal cells were compensatingly down regulated in the presence of the K18 protein, while the levels of the endogenous type II Endo A were not affected in any of the transfected cell lines.     

Bax Activates Endophilin B1 Oligomerization and Lipid Membrane Vesiculation

Endophilins participate in membrane scission events that occur during endocytosis and intracellular organelle biogenesis through the combined activity of an N-terminal BAR domain that interacts with membranes and a C-terminal SH3 domain that mediates protein binding. Endophilin B1 (Endo B1) was identified to bind Bax, a Bcl-2 family member that promotes apoptosis, through yeast two-hybrid protein screens. Although Endo B1 does not bind Bax in healthy cells, during apoptosis, Endo B1 interacts transiently with Bax and promotes cytochrome c release from mitochondria. To explore the molecular mechanism of action of Endo B1, we have analyzed its interaction with Bax in cell-free systems. Purified recombinant Endo B1 in solution displays a Stokes radius indicating a tetrameric quarternary structure. However, when incubated with purified Bax, it assembles into oligomers more than 4-fold greater in molecular weight. Although Endo B1 oligomerization is induced by Bax, Bax does not stably associate with the high molecular weight Endo B1 complex. Endo B1 oligomerization requires its C-terminal Src homology 3 domain and is not induced by Bcl-xL. Endo B1 combined with Bax reduces the size and changes the morphology of giant unilamellar vesicles by inducing massive vesiculation of liposomes. This activity of purified Bax protein to induce cell-free assembly of Endo B1 may reflect its activity in cells that regulates apoptosis and/or mitochondrial fusion.     

Molecular cloning and characterization of the Endo B cytokeratin expressed in preimplantation mouse embryos

A cDNA clone of a keratin-related, intermediate filament protein, designated Endo B, was constructed from size-fractionated parietal endodermal mRNA and characterized. The 1466-nucleotide cDNA insert contains an open reading frame of 1272 nucleotides that would result in 5' and 3' noncoding sequences of 54 and 60 nucleotides, respectively. The predicted amino acid composition, molecular weight (47,400), and peptide pattern correlate well with data obtained on the isolated protein. The predicted amino acid sequence fits easily into the general domain structure suggested for all intermediate filament proteins with a unique amino-terminal head domain, a large conserved central domain of predominantly alpha-helical structure, and a relatively unique carboxyl-terminal or tail domain. Over the entire molecule, Endo B is 43% identical with human 52-kDa epidermal type I keratin. However, over two of the three regions contained in the central domain that are predicted to form coiled-coil structures, the Endo B is 54-68% identical with other type I keratin sequences. This homology, along with the presence of the completely conserved sequence DNARLAADDFR-KYE, which is found in all type I keratins, permits the unambiguous identification of Endo B as a type I keratin. Comparison of the Endo B sequence to other intermediate filament proteins reveals 22 residues which are identical in all intermediate filament proteins regardless of whether filament formation requires only one type of protein subunit (vimentin, desmin, glial fibrillar acidic protein, or a neurofilament protein) or two dissimilar types (type I and type II keratins). Endo B mRNA was detectable in RNA isolated from F9 cells treated with retinoic acid for 48 h. Approximately three to five genes homologous to Endo B were detected in the mouse genome.     

Co-expression of specific acid and basic cytokeratins in teratocarcinoma-derived fibroblasts treated with 5-azacytidine

Epithelial cells always co-express acidic and basic keratin polypeptides. Mesenchymal cells, which do not normally contain keratins, can be induced by the inhibitor of DNA methylation 5-azacytidine to synthesize the basic keratin Endo A. In the present paper we show that the acidic keratins Endo B and Endo C can also be induced by 5-azacytidine in teratocarcinoma-derived fibroblasts. Furthermore, individual cells in which Endo B and/or Endo C keratins are found, always co-express the basic polypeptide Endo A. Other cytokeratins are not or very rarely found. Interestingly, Endo A, B, and C are usually associated in vivo and are known to be the first keratin polypeptides appearing during the development of the mouse embryo.