大鼠脑谷氨酸脱羧酶基因的cDNA克隆及序列分析

胰岛β－细胞自身抗原蛋白之一是脑中谷氨酸脱羧酶（Ｇｌｕｔａｍｉｃａｃｉｄｄｅｃａｒｂｏｘｙｌａｓｅ,ＧＡＤ,ＥＣ４．１．１．１５）同源物,以双链ｃＤＮＡ为模权,用ＰＣＲ方法快速克隆了Ｗｉｓｔａｒ大鼠脑ＧＡＤ基因的ｃＤＮＡ将此包括编码５９３个氨基酸的全长ＤＮＡ片段重组入ｐＵＣ质粒并用双脱的氧末端终止法测定了全部序列,证明其全长为１７７９ｂｐ,经比较发现Ｗｉｓｔａｒ大鼠脑与Ｒｕｓｓｅｌｌ报导的大鼠脑Ｇ     

大鼠脑神经元特异性烯醇化酶基因的cDNA克隆及序列分析

用ＲＴ－ＰＣＲ方法快速克隆了Ｗｉｓｔａｒ大鼠脑神经元特异性烯醇化酶（ＮＳＥ）的ｃＤＮＡ,将此包括编码全长ＮＳＥ４３３个氨基醚的ＤＮＡ片段重组入ｐＵＣ质粒,并用ＰＣＲ方法测定了全部顺序,经重复实验,发现Ｗｉｓｔａｒ大鼠与Ｆｏｒｓｓ－Ｐｅｔｔｅｒ报导的ＳＤ大鼠ＮＳＥ基因顺序,有两外单碱基的差别,其中一个涉及氨基酸的改变。同时还对ＲＮＡ的提取及长片段ＤＮＡ的ＲＴ－ＰＣＲ扩增进行了方法学的探讨。     

大鼠脑α1型甲状腺激素受体基因cDNA克隆及序列分析

使用ＲＴ－ＰＣＲ方法克隆了Ｗｉｓｔａｒ大鼠脑α１型甲状腺激素受体的ｃＤＮＡ,得到包含起始及终止密码子共１２３３ｂｐ、编码４０９个氨基酸的受体全长编码离列,酶切分析后,将此特异ＤＮＡ片段重组入质粒ｐＵＣ系统,得重组质料ｐＴＲＡ。双脱氧末端终止法测定了全部核苷酸顺序。     

人蛋白C cDNA基因的克隆及序列分析

为实现人蛋白C cDNA在哺乳动物细胞中的表达以及研究其生物学特性,针对人蛋白C cDNA序列设计引物,运用逆转录聚合酶链反应(RT-PCR)从人胎肝总RNA中钓取人蛋白C cDNA,将其克隆入pIRES neo载体中,通过酶切和PCR鉴定出重组体并进行测序分析。结果表明,获得大小为1386bp的人蛋白C cDNA基因,成功构建人蛋白C cDNA载体pIRES/hPC,为进一步进行人蛋白C cDNA的表达和活性鉴定奠定了基础。     

大鼠脑cDNA文库的构建

采用简单高效的cDNA合成技术制备Wistar大鼠脑cDNA基因文库,以纯化的poly( A)⁺－RNA为模板,含Not I切点的oligo－（dT）15为引物,在反转录酶的作用下,合成第一股单链cDNA;用E.coli RNase H除去模板RNA,并以E.coli DNA聚合酶Ｉ,E.coli DNA连接酶和T4 DNA聚合酶催化合成cDNA第二条链,即成为双链cDNA;此双股cDNA除0．5μg用于插入pSPORT I载体,转入E.coli DH5a,建成cDNA文库外,其余保存在－20℃,以此cDNA为模板,应用PCR方法,先后克隆了谷氨酸脱羧酶（GAD,1800bp）、神经元特异性烯醇化酶（NSE,1340bp）,甲状腺激素受体（T3－receptor,1230bp）、胆囊收缩素（CCK,345bp）的全编码基因．     

大鼠脑α_1型甲状腺激素受体基因的cDNA克隆及序列分析

使用ＲＴ－ＰＣＲ方法克隆了Ｗｉｓｔａｒ大鼠脑α_１型甲状腺激素受体的ｃＤＮＡ,得到包含起始及终止密码子共１２３３ｂｐ、编码４０９个氨基酸的受体全长编码序列．酶切分析后,将此特异ＤＮＡ片段重组入质粒ｐＵＣ系统,得重组质粒ｐＴＲＡ．双脱氧末端终止法测定了全部核苷酸顺序,结果与文献报导的ＳＤ大鼠的结果一致,同时对长片段ＤＮＡ的ＲＴ－ＰＣＲ扩增进行了方法学的探讨。     

人TRAIL基因cDNA的克隆与序列测定

为研究人TRAIL的基因组结构,生物学性能和用于肿瘤生物治疗的可能性,利用反转录聚合酶链反应(RT-PCR)从人急性早幼粒白血病细胞系HL-60细胞总RNA中扩增出人TRAIL基因编码区cDNA序列,将其克隆至pGEM-T载体中,序列测定表明,克隆片段与文献报道的人TRAIL基因编码区cDNA序列完全一致.     

人TRALL基因cDNA的克隆与序列测定

为研究人 TRALL的基因组结构 ,生物学性能和用于肿瘤生物治疗的可能性 ,利用反转录聚合酶链反应 (RT- PCR)从人急性早幼粒白血病细胞系 HL - 6 0细胞总 PNA中扩增出人 TRALL基因编码区 c DNA序列 ,将其克隆至 p GEM- T载体中 ,序列测定表明 ,克隆片段与文献报道的人TRALL基因编码区 c DNA序列完全一致。     

大鼠脑神经元特异性烯醇化酶基因的cDNA克隆及序列分析

用ＲＴ－ＰＣＲ方法快速克隆了Ｗｉｓｔａｒ大鼠脑神经元特异性烯醇化酶的ｃＤＮＡ,将此包括编码全长ＮＳＥ４３３个氨基酸的ＤＮＡ片段重组入ｐＵＣ质粒,并用ＰＣＲ方法测定了全部顺序,经重复实验,发现Ｗｉｓｔａｒ大鼠与Ｆｏｒｓｓ－Ｐｅｔｔｅｒ报导的ＳＤ大鼠ＮＳＥ基因顺序,有两处单碱基的差别,其中一个涉及氨基酸的改变,同时还对ＲＮＡ的提取及长片段ＤＮＡ的ＲＴ－ＰＣＲ扩增进行了方法学的探讨。     

Comparative expression and purification of human glutamic acid decarboxylase from Saccharomyces cerevisiae and Pichia pastoris

The yeast cell factory is a potentially useful source of proteins in general. They include glutamic acid decarboxylase (GAD), which is one of the major autoantigens for Type 1 diabetes. We have created a hybrid form of GAD consisting of amino acids 1–101 of the human GAD67 protein fused to amino acids 96–585 of the human GAD65 protein, and have modified this to include a C-terminal hexa-Histidine (H6) tag sequence. This hybrid GAD67/65-H6 was expressed in two yeast hosts: constitutively under the control of the plasmid phosphoglycerate kinase promoter (PGK1) in Saccharomyces cerevisiae, and inducibly under the control of the chromosomal alcohol oxidase promoter (AOX1) in Pichia pastoris. Enzymatically active hybrid GAD was prepared from yeast lysates by purification either on an affinity column based on the GAD-1 monoclonal antibody, or by metal-affinity chromatography. The purified GAD67/65-H6 was radiolabelled with iodine-125 and tested with Type 1 diabetes sera in a radioimmunoprecipitation assay, and results were compared with those using untagged GAD67/65 and those using porcine brain GAD. The results of enzymatic and immunological assays show hybrid GAD67/65 is isolated at high specific activity and moderate yield, and the addition of the H6 tag sequences or the choice of yeast strain did not appreciably affect enzyme activity, percentage recovery of GAD, protein purification, or the utility in diagnosis of diabetes in terms of specificity and sensitivity to the various sera.     

Sex difference and response to testosterone in gabaergic cells of the medial preoptic nucleus and ventral bed nuclei of the stria terminalis in gerbils

The medial preoptic nucleus (MPN) and ventral bed nuclei of the stria terminalis (BST) are needed to maintain mating in sexually experienced male gerbils and rats. The gerbil ventral BST is also activated with mating, as assessed by Fos expression, as is the medial MPN (MPNm) of both species. In gerbils, many of those mating-activated cells contain glutamic acid decarboxylase (GAD), the enzyme that synthesizes γ-aminobutyric acid (GABA). Some of those cells are projection neurons, but others may release GABA locally. Through actions in the medial preoptic area, GABA inhibits and testosterone (T) promotes male sex behavior. Thus, T may promote mating, in part, by decreasing GAD in MPNm or ventral BST cells. In rats, T increases GAD mRNA in the central MPN (MPNc), where MPN GABAergic cells are densest, but mating behavior does not change in sexually experienced males when the MPNc is ablated. Therefore, this study focused on the MPNm and ventral BST to ask whether their GABAergic cells respond to T or are sexually dimorphic. This was done by visualizing cells immunoreactive (IR) for GAD₆₇, an isoform found primarily in cell bodies, in male and female gerbils and in castrated males with and without T. At both sites, males had more GAD₆₇-IR cells than females, and T decreased GAD₆₇-IR cell numbers in males. Thus, the MPNm and ventral BST have GABAergic cells that are sexually dimorphic and in which T decreases GAD, consistent with local effects of T and GABA on mating.     

Evidence for Two Distinct Forms of Native Glutamic Acid Decarboxylase in Rat Brain Soluble Extract: An Immunoblotting Study

Immunoblots of the soluble proteins from a rat brain high-speed supernatant dissociated under reducing conditions showed two monomers (molecular weights, 59,000 and 62,000 +/- 2,000) immunolabeled by a glutamic acid decarboxylase (GAD) antiserum. In this extract, a GAD monoclonal antibody trapped the same two monomers, thus confirming that they are both constitutive subunits of GAD. Without treatment under reducing conditions, two additional bands were stained by immunoblotting. Their molecular weights were estimated to be 115,000 and 122,000 +/- 5,000. These results demonstrate the presence, in rat brain soluble extract, of two distinct forms of native GAD. They further support our previous hypothesis that each form is composed by the homodimeric association of each constitutive subunit through disulfide bridges.     

短乳杆菌谷氨酸脱羧酶的生物信息学分析

以短乳杆菌(Lactobacillus brevis)Lb-2菌株cDNA为模板克隆了谷氨酸脱羧酶(Glutamate decarboxylase,GAD)基因。采用在线分析工具及相应软件分析预测了GAD基因核苷酸和氨基酸序列的组成、理化性质、信号肽以及高级结构等,并构建系统发育树。该基因序列全长1 407 bp,为一个完整的阅读框,编码468个氨基酸。GAD相对分子量理论预测值和等电点分别是53 517.8 u和5.42,没有跨膜区,没有其他亚细胞定位序列,为亲水性蛋白,与植物乳杆菌(Lactobacillus plantarum)和德氏乳酸杆菌(Lactobacillus delbrueckii)的GAD进化关系最近。     

Sulfur Amino Acid Metabolism in the Developing Rhesus Monkey Brain: Subcellular Studies of Taurine, Cysteinesulfinic Acid Decarboxylase, γ-Aminobutyric Acid, and Glutamic Acid Decarboxylase

Abstract: Taurine, cysteinesulfinic acid decarboxylase (CSAD), glutamate, γ-aminobutyric acid (GABA), and glutamic acid decarboxylase (GAD) were measured in subcellular fractions prepared from occipital lobe of fetal and neonatal rhesus monkeys. In addition, the distribution of [³⁵S]taurine in subcellular fractions was determined after administration to the fetus via the mother, to the neonate via administration to the mother prior to birth, and directly to the neonate at various times after birth. CSAD, glutamate, GABA, and GAD all were found to be low or unmeasurable in early fetal life and to increase during late fetal and early neonatal life to reach values found in the mother. Taurine was present in large amounts in early fetal life and decreased slowly during neonatal life, arriving at amounts found in the mother not until after 150 days of age. Significant amounts of taurine, CSAD, GABA, and GAD were associated with nerve ending components with some indication that the proportion of brain taurine found in these organelles increases during development. All subcellular pools of taurine were rapidly labeled by exogenously administered [³⁵S]taurine. The subcellular distribution of all the components measured was compatible with the neurotransmitter or putative neuro-transmitter functions of glutamate, GABA, and taurine. The large amount of these three amino acids exceeds that required for such function. The excess of glutamate and GABA may be used as a source of energy. The function of the excess of taurine is still not clear, although circumstantial evidence favors an important role in the development and maturation of the CNS.     

Stress-Induced Alterations in Metabolism of Γ-Aminobutyric Acid in Rat Brain

The effect of a stressful manipulation on the metabolism of gamma-aminobutyric acid (GABA) in the rat brain was studied. Application of an immobilized stress to animals induced a significant increase in the striatal and hypothalamic GABA contents without affecting those in other central structures examined. It was also found that the increase in striatal GABA level preceded that in the hypothalamus. This increase in steady-state levels of GABA in the striatum and hypothalamus disappeared at 12 h after the termination of the application of stress for 3 h, which exhibited a maximal stimulatory action on the GABA contents in both central areas. The activity of L-glutamic acid decarboxylase was found to be significantly elevated in the striatum and hypothalamus following the stress application with a concomitant decrease in the content of L-glutamic acid, which is converted to GABA by the catalytic action of the latter enzyme. The in vivo turnover of GABA in the brain was estimated by taking advantages of the postmortem accumulation of GABA following decapitation and of the selective inhibitory action of a low dose of aminooxyacetic acid on the GABA degrading system, respectively. Analysis using these two different methods revealed that the cerebral turnover of GABA in vivo was not significantly altered under stressful situations despite of the increase in its steady-state level. These results suggest that central GABA system may respond to the input of painful stimuli resulting from the application of a severe physical and psychological stressor, in addition to the well-known functional alterations in catecholamine neurons. The functional significance of these alterations in the central GABA neurons is also discussed.     

The structural and functional heterogeneity of glutamic acid decarboxylase: A review

Studies of the GABA-synthetic enzyme glutamate decarboxylase (glutamic acid decarboxylase; GAD; E.C.4.1.1.15) began in 1951 with the work of Roberts and his colleagues. Since then, many investigators have demonstrated the structural and functional heterogeneity of brain GAD. At least part of this heterogeneity derives from the existence of two GAD genes.In honor of the 70th birthday of Dr. Eugene Roberts     

Drosophila GABAergic systems II. Mutational analysis of chromosomal segment 64AB, a region containing the glutamic acid decarboxylase gene

The Drosophila melanogaster Gad gene maps to region 64A3-5 of chromosome 3L and encodes glutamic acid decarboxylase (GAD), the rate-limiting enzyme for the synthesis of the inhibitory neurotransmitter -aminobutyric acid (GABA). Because this neurotransmitter has been implicated in developmental functions, we have begun to study the role of GABA synthesis during Drosophila embryogenesis. We show that Gad mRNA is expressed in a widespread pattern within the embryonic nervous system. Similarly, GAD-immunoreactive protein is present during embryogenesis. These results prompted us to screen for embryonic lethal mutations that affect GAD activity. The chromosomal region to which Gad maps, however, has not been subjected to an extensive mutational analysis, even though it contains several genes encoding important neurobiological, developmental, or cellular functions. Therefore, we have initially generated both chromosomal rearangements and point mutations that map to the Drosophila 64AB interval. Altogether, a total of 33 rearrangements and putative point mutations were identified within region 64A3-5 to 64B12. Genetic complementation analysis suggests that this cytogenetic interval contains a minimum of 19 essential genes. Within our collection of lethal mutations are several chromosomal rearrangements, two of which are in the vicinity of the Gad locus. One of these rearrangements, Df(3L)C175, is a small deletion that removes the Gad locus and at least two essential genes; the second, T(2;3)F10, is a reciprocal translocation involving the second and third chromosomes with a break within region 64A3-5. Both of these rearrangements are associated with embryonic lethality and decreased GAD enzymatic activity.     

Phylogenesis of Brain Glutamic Acid Decarboxylase from Vertebrates: Immunochemical Studies

Brain high-speed supernatants from various lower and higher vertebrates were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, electroblot on nitrocellulose membranes, and immunolabelling using an anti-glutamic acid decarboxylase (anti-GAD) antiserum prepared from rat antigen. Rat brain extracts showed two distinct immunolabelled bands (MW 59,000 and 62,000 daltons). The molecular weight of the native enzyme was 120,000 daltons. The immunoblot pattern was not affected by a 3-h incubation of the homogenate. In the substantia nigra, the decrease in the immunolabelling of both bands corresponded very closely to the decrease of GAD activity following lesioning of the striato-nigral pathway. Moreover, experiments with preadsorbed antiserum showed that both subunits have common antigenic determinants. The immunolabelling was consistently more intense over the lightest band. The autoradiography of immunoprecipitated rat brain GAD, iodinated prior to electrophoresis, revealed two radiolabelled bands corresponding to the two immunolabelled ones. Their radioactivity was found in a one-to-five ratio which closely paralleled their respective immunolabelling intensity. Thus, the two subunits recognized by the antiserum are not present in stoichiometric proportions in the rat brain high-speed supernatant. These findings suggest the existence of two homodimeric GAD with common antigenic determinants which are present in different amounts. Immunoprecipitation curves of brain GAD from rat, mouse, rabbit, monkey, human, quail, frog, and trout were similar, with a less than 10-fold maximum shift in affinity for GAD. GAD immunoblots from the various higher vertebrates showed a pattern similar to that obtained in rat.(ABSTRACT TRUNCATED AT 250 WORDS)