New mammalian chloride channel identified by expression cloning.

Ion channels selectively permeable to chloride ions regulate cell functions as diverse as excitability and control of cell volume. Using expression cloning techniques, a complementary DNA from an epithelial cell line has been isolated, sequenced and its putative structure examined by site-directed mutagenesis. This cDNA, encoding a 235-amino-acid protein, gave rise to a chloride-selective outward current when expressed in Xenopus oocytes. The expressed, outwardly rectifying chloride current was calcium-insensitive and was blocked by nucleotides applied to the cell surface. Mutation of a putative nucleotide-binding site resulted in loss of nucleotide block but incurred dependence on extracellular calcium concentration. The unusual sequence of this putative channel protein suggests a new class of ion channels not related to other previously cloned chloride channels.     

Cell-free expression of functional Shaker potassium channels.

The functional activity of ion channels and other membrane proteins requires that the proteins be correctly assembled in a transmembrane configuration. Thus, the functional expression of ion channels, neurotransmitter receptors and complex membrane-limited signalling mechanisms from complementary DNA has required the injection of messenger RNA or transfection of DNA into Xenopus oocytes or other target cells that are capable of processing newly translated protein into the surface membrane. These approaches, combined with voltage-clamp analysis of ion channel currents, have been especially powerful in the identification of structure-function relationships in ion channels. But oocytes express endogenous ion channels, neurotransmitter receptors and receptor-channel subunits, complicating the interpretation of results in mRNA-injected eggs. Furthermore, it is difficult to control experimentally the membrane lipids and post-translational modifications that underlie the regulation and modulation of ion channels in intact cells. A cell-free system for ion channel expression is ideal for good experimental control of protein expression and modulatory processes. Here we combine cell-free protein translation, microsomal membrane processing of nascent channel proteins, and reconstitution of newly synthesized ion channels into planar lipid bilayers to synthesize, glycosylate, process into membranes, and record in vitro the activity of functional Shaker potassium channels.     

cDNA cloning of bovine substance-K receptor through oocyte expression system

The neuropeptide receptors which are present in very small quantities in the cell and are embedded tightly in the plasma membrane have not been well characterized. Mammals contain three distinct tachykinin neuropeptides, substance P, substance K and neuromedin K, and it has been suggested that there are multiple tachykinin receptors. By electrophysiological measurement, we have previously shown that Xenopus oocytes injected with brain and stomach mRNAs faithfully express mammalian substance-P and substance-K receptors, respectively. Here we report the isolation of the cDNA clone for bovine substance-K receptor (SKR) by extending this method to develop a new cloning strategy. We constructed a stomach cDNA library with a cloning vector that allowed in vitro synthesis of mRNAs and then identified a particular cDNA clone by testing for receptor expression following injection of the mRNAs synthesized in vitro into the oocyte system. Because oocytes injected with exogenous mRNAs can express numerous receptors and channels, our new strategy will be applicable in the general molecular cloning of these proteins. The result provides the first indication that the neuropeptide receptor has sequence similarity with rhodopsin-type receptors (the G-protein-coupled receptor family) and thus possesses multiple membrane-spanning domains.     

Coexpression of two distinct muscle acetylcholine receptor alpha-subunits during development

The nicotinic acetylcholine receptor is a ligand-gated channel that mediates signalling at the vertebrate neuromuscular junction. It is a pentameric complex of four different subunits, assembled with a stoichiometry of alpha 2 beta gamma delta. Muscle-like alpha-subunits have been cloned from Torpedo, mouse, calf, rat, chicken, human and Xenopus, and only a single alpha-subunit complementary DNA from each species has been detected. We report here the cloning and characterization of a second muscle alpha-subunit cDNA from Xenopus, and show that this and a previously reported Xenopus alpha-subunit cDNA are encoded by distinct genes. The novel alpha-subunit reported here is expressed uniquely in oocytes; but both types of alpha-subunit are coexpressed throughout muscle development. This latter observation indicates that the expression of these two alpha-subunits is different from a previously reported developmental 'subunit-switch' mechanism used to generate channel diversity.     

Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel

In cardiac muscle, where Ca2+ influx across the sarcolemma is essential for contraction, the dihydropyridine (DHP)-sensitive L-type calcium channel represents the major entry pathway of extracellular Ca2+. We have previously elucidated the primary structure of the rabbit skeletal muscle DHP receptor by cloning and sequencing the complementary DNA. An expression plasmid carrying this cDNA, microinjected into cultured skeletal muscle cells from mice with muscular dysgenesis, has been shown to restore both excitation-contraction coupling and slow calcium current missing from these cells, so that a dual role for the DHP receptor in skeletal muscle transverse tubules is suggested. We report here the complete amino-acid sequence of the rabbit cardiac DHP receptor, deduced from the cDNA sequence. We also show that messenger RNA derived from the cardiac DHP receptor cDNA is sufficient to direct the formation of a functional DHP-sensitive calcium channel in Xenopus oocytes. Furthermore, higher calcium-channel activity is observed when mRNA specific for the polypeptide of relative molecular mass approximately 140,000 (alpha 2-subunit) associated with skeletal muscle DHP receptor is co-injected.     

Cloning and enzymology analysis of farnesyl pyrophosphate synthase gene from a superior strain of<Emphasis Type="Italic">Artemisia annua</Emphasis> L.

A cDNA(af1) encoding farnesyl pyrophosphate synthase AaFPS1(FPS,EC2.5.1.1/EC2.5.1.10) from a high yield Artemisia annua strain 025 has heen cloned from its cDNA library .Sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with molecular weight of 39 kD.Deduced aa sequence of the cDNA was similar to FPS from other plants,yeast and mammals,containing 5 conserved domains found in both prenyl trans-ferase and polyprenyl synthase,The expression of the cDNA in Escherichia coli showed measurable specific activity of FPS in vitro.The enzyme was purified by ion exchange chromatography and its kinetics was measured ,These results would further promote the molecular regulation of artemisnin biosynthesis.     

分泌型Survivin真核表达载体的构建及其在HeLa细胞中的表达

目的:构建含6个组氨酸标签(His6)的小鼠存活素(Survivin)融合蛋白的真核表达载体,及其在真核细胞中的表达。方法:根据小鼠Survivin序列设计引物,经过RT-PCR克隆Survivin的cDNA并进一步构建分泌型Sur-vivin的真核表达载体,经测序鉴定后在大肠杆菌中扩增在HeLa细胞中表达;以金属离子螯合层析富集,再用免疫印迹法鉴定。结果:克隆到Survivin编码区全长序列,经DNA测序后证明与已报道序列相同。构建氨基端融合小鼠IL-2信号肽,羧基端带有His6标签的Survivin融合蛋白的真核表达载体,在HeLa细胞中转染成功并且表达;细胞上清经HisTrap HP柱层析富集后,进行免疫印迹分析,表明该融合蛋白以分泌型方式在HeLa细胞中表达。结论:成功克隆Survivin的cDNA,并构建其分泌型真核表达载体。     

B→O blood conversion

To maintain a constant supply of “universal blood type”,or group O red blood cells,has benefit in specialized transfusion condition.In this study, α-galactosidase cDNA was cloned from Catimor coffee bean grown in Hainan island,China,by the RT-PCR method.We have constructed a vector for α-galactosidase cDNA expression and transferred α-galactosidase cDNA into Pichia pastoris GS115 cells by electroporation.The recombinant α-galactosidase(r-α-GalE)was purified by cation ion exchange chromatography.After studying the biochemical characters of r-α-GalE we have succeeded in converting human erythrocytes from group B to group O.The animal experiment showed that transfusion of enzymetically converted group O red blood cells(ECORBC)was perfectly safe.     

α6/α3β2β3乙酰胆碱受体在非洲爪蟾卵母细胞中的表达及其条件优化

为建立有效的α6/α3β2β3 nAChRs(α6/α3β2β3，或简写为α6β2*，*代表其他亚基)体外重组表达实验模型，利用非洲爪蟾卵母细胞表达体系，采用显微注射RNA的方法，利用双电极电压钳技术检测电流，对α6/α3β2β3 nAChRs亚型进行体外重组表达研究，并对其表达条件进行优化，获得了α6/α3β2β3 nAChRs优化的重组表达体系.烟碱型乙酰胆碱受体(nAChRs)是一类重要的配体门控离子通道，其亚型众多且结构相似，但各亚型的生理学和药理学功能截然不同.天然的α6β2* nAChRs很难体外表达，体外表达模型的表达电流非常小.因此, 本实验拟研究优化该亚型的表达，进行优化后的体外表达模型，产生的激发电流显著提高，为以该亚型为靶点的新药筛选提供实验模型和基础.     

卵母细胞在离子通道研究中的应用

由于非洲爪蟾卵母细胞体积较大，故其蛋白质和RNA合成活性很高，、同时，卵母细胞本身仅有很少几种内源性通道和受体，因此可以忠实、高效地表达多种类型的外源性通道和受体、非洲爪蟾卵母细胞现已成为一种研究外源性离子通道基因的工具，在分析多亚基通道翻译后的加工和组装、检测突变通道特性、比较不同组织来源的通道特性、检测第二信使系统对通道功能的调制、分析和鉴定编码通道蛋白的克隆基因等方面有暑广泛的应用。     

水牛卵母细胞孤雌激活及孤雌胚与体外受精胚发育的比较

目的对MII期水牛卵母细胞进行人工诱导激活,可以间接判断体外成熟卵母细胞质量的优劣,并且,卵母细胞的充分激活也是提高核移植效率的关键因素之一。比较了水牛卵母细胞体外成熟时间对孤雌激活胚发育能力的影响以及不同化学激活方法对水牛卵母细胞孤雌激活效果的影响,并在相同条件下,对孤雌激活胚与体外受精胚的发育能力进行了比较。结果表明,水牛卵母细胞体外成熟27 h或30 h的囊胚发育率(19.0%,17.7%)明显高于体外成熟21 h或24 h的囊胚发育率(12.3%,13.8%);Ion联合6_DMAP激活水牛卵母细胞的效果优于其他几组激活方法;在相同培养条件下,孤雌激活胚与体外受精胚的发育能力存在着差异,其中卵裂率差异不显著,但孤雌激活胚的囊胚发育率显著高于体外受精胚(21.7%,13.0%)。     

Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs.

The glutamate receptor (GluR) channel plays a key part in brain function. Among GluR channel subtypes, the NMDA (N-methyl-D-aspartate) receptor channel which is highly permeable to Ca2+ is essential for the synaptic plasticity underlying memory, learning and development. Furthermore, abnormal activation of the NMDA receptor channel may trigger the neuronal cell death observed in various brain disorders. A complementary DNA encoding a subunit of the rodent NMDA receptor channel (NMDAR1 or zeta 1) has been cloned and its functional properties investigated. Here we report the identification and primary structure of a novel mouse NMDA receptor channel subunit, designated as epsilon 1, after cloning and sequencing the cDNA. The epsilon 1 subunit shows 11-18% amino-acid sequence identity with rodent GluR channel subunits that have been characterized so far and has structural features common to neurotransmitter-gated ion channels. Expression from cloned cDNAs of the epsilon 1 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional GluR channels with high activity and characteristics of the NMDA receptor channel. Furthermore, the heteromeric NMDA receptor channel can be activated by glycine alone.     

Location of functional regions of acetylcholine receptor alpha-subunit by site-directed mutagenesis

The availability of cloned cDNAs encoding the four subunits of the Torpedo acetylcholine receptor, which can be expressed to make functional receptors in Xenopus oocytes, has made possible a detailed investigation of the functions of the different structural components of the receptor. The functional analysis of receptors with alpha-subunits altered at specific sites by site-directed mutagenesis of the cDNA has allowed the location of specific regions of the alpha-subunit molecule involved in acetylcholine binding and forming a transmembrane ionic channel.     

Cloning of a cDNA for a glutamate receptor subunit activated by kainate but not AMPA.

Fast excitatory transmission in the vertebrate central nervous system is mediated mainly by L-glutamate. On the basis of pharmacological, physiological and agonist binding properties, the ionotropic glutamate receptors are classified into NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate) and kainate subtypes. Sequence homology between complementary DNA clones encoding non-NMDA glutamate receptor subunits reveals at least two subunit classes: the GluR1 to GluR4 class and the GluR5 class. Here we report the cloning and expression of a functional rat glutamate receptor subunit cDNA, GluR6, which has a very different pharmacology from that of the GluR1-GluR4 class. Receptors generated from the GluR1-GluR4 class have a higher apparent affinity for AMPA than for kainate. When expressed in Xenopus oocytes the homomeric GluR6 receptor is activated by kainate, quisqualate and L-glutamate but not by AMPA, and the apparent affinity for kainate is higher than for receptors from the GluR1-GluR4 class. Desensitization of the receptor was observed with continuous application of agonist. The homomeric GluR6 glutamate receptor exhibits an outwardly rectifying current-voltage relationship. In situ hybridizations reveal a pattern of GluR6 gene expression reminiscent of the binding pattern obtained with [3H]kainate.     

Expression of functional potassium channels from Shaker cDNA in Xenopus oocytes

The Shaker gene of Drosophila melanogaster encodes a potassium-selective ion channel, the 'A' channel, or one of its subunits. A single Shaker messenger RNA species suffices to direct the synthesis of functional A channels in Xenopus oocytes. Physiological characteristics of the A currents induced by two different mRNA species are compared.     

Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes

The c-mos proto-oncogene is expressed as a maternal mRNA in oocytes and early embryos of Xenopus laevis, but its translation product pp39mos is detectable only during progesterone-induced oocyte maturation. Microinjection of mos-specific antisense oligonucleotides into oocytes not only prevents expression of pp39mos, but also blocks germinal vesicle breakdown, indicating that it functions during reinitiation of meiotic division.     

胞内型磷脂酶A2基因亚克隆与基因表达

目的 研究花生四稀酸在细胞中的释放及前列腺素的合成,构建与花生四稀酸释放相关的表达系统。即胞内型磷脂酶Ａ２（ｃｙｔｏｓｏｌｉｃ ｐｈｏｓｐｈｏｌｉｐａｓｅ ｃＰＬＡ２）ｃＤＮＡ－ｐＲＣ／ＣＭＶ。方法 从克隆载体ｐＭＴ２用Ｓａｌ１制备ｃＰＡＬ２ｃＤＮＡ;平末端连接ｃＰＡＬ２－ｐＲＣ／ＣＭＶ;转染鼠巨噬细胞及筛选正向阳性克隆;ｎｏｒｔｈｅｒｎ ｂｌｏｔ检测ｃＰＬＡ２ ｃＤＮＡ基因表达。结果 人ｃＰＬＡ     

Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family

Amino-acid sequences derived from complementary DNAs encoding the alpha- and beta-subunits of the GABA/benzodiazepine receptor from bovine brain show homology with other ligand-gated receptor subunits, suggesting that there is a super-family of ion-channel-containing receptors. Co-expression of the in vitro-generated alpha-subunit and beta-subunit RNAs in Xenopus oocytes produces a functional receptor and ion channel with the pharmacological properties characteristic of the GABAA receptor.