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。     

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。     

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。     

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为模板,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ＧＡＤ基因序列,有一处碱基的差别,但并不涉及氨基酸的改变。同时还对用ＰＣＲ扩增长片段ＤＮＡ进行了方法学上的探讨。     

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     

人端粒酶逆转录酶（hTERT）基因第3内含子的克隆 …

从端粒酶活性呈性阳的水生细胞株人肺ＳＣＰ－Ａ－１中分离了总ＲＮＡ,以此为模板,结合ＲＴ－ＰＣＲ技术和长模板ＰＣＲ技术,用ｈＴＥＲＴ基因特异性引物扩增到一长约２．２ｋｂ的ｃＤＮＡ片段。将该片纯化后克隆到通用测序本Ｔ－ｅａｓｙ ｖｅｃｔｏｒ上得到重组质粒。用测引物ＳＰ６和Ｔ７对该片段进行部分双向测序。经序列分析和同源比较推测该片段包含了ｈＴＥＲＴ基因的第３内含子。该结果提示了ＲＴ－ＰＣＲ技术和长模板Ｐ     

应用循环逆转录PCR技术检测丙型肝炎病毒RNA

循环逆转录（ｃｉｒｃｕｌａｔｏｒｙ ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ,ＣＲＴ）是线性增长逆转录ｃＤＮＡ产量的一种新技术。为了将该技术用于检测ＨＣＶ ＲＮＡ,通过改变ＣＲＴ的循环次数,结合竞争ＰＣＲ,作出标准曲线。采用１６次ＣＲＴ加３４次循环ＰＣＲ检测了１３６例ＨＣＶ ＥＬＩＳＡ阳性、５４例ＨＣＶ ＥＬＩＳＡ阴性和１０８例临床可疑病人全血标本,并与逆转录ＰＣＲ（ＲＴ－ＰＣＲ）和巢式ＰＣＲ（     

大鼠4.5S RNAs的cDNA克隆与序列分析

利用ＲＴ－ＰＣＲ方法,首次从大鼠肝脏细胞总ＲＮＡ中扩增出４．５Ｓ ＲＮＡｓ的ｃＤＮＡ。该ｃＤＮＡ被克隆到ｐＧＥＭ３Ｚｆ（＋）质粒上,经酶切电泳鉴定,然后测序。与报道的小鼠和仓鼠４．５Ｓ ＲＮＡｓ序列进行了比较研究,并对该分子的结构特点进行了初步分析。     

HCV5＇端非编码区cDNA的体外转录

采用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）从广东省一例慢性丙型肝炎病人血清中获得丙型肝炎病毒（ＨＣＶ）５＇端非编码区（５＇ＮＣＲ）３０２ｂｐ的ｃＤＮＡ片段,经补齐和提纯后插入ｐＵＣ１９质粒,获得的重组体ｐＵＮ进行序列测定。将ｐＵＮ的目的基因亚克隆进体外转录载体ｐＳＰＯＲＴＩ多克隆位点的ＥｏｏＲＩ和ＰｓｔＩ切点之间,所得重组体ｐＳＮ线性化后由Ｔ＿７ＲＮＡ多聚酶及ＳＰ６ＲＮＡ多聚酶引导体外转录反应,产物经凝胶电泳及特异引物ＲＴ－ＰＣＲ,证实ＳＰ６引导的是正义ＲＮＡ,Ｔ７合成的是反义ＲＮＡ,其大小分别力４２９ｂｐ和３６２ｂｐ。并证实所得ＲＮＡ力ＨＣＶ５＇ＮＣＲｃＤＮＡ转录而来。获得的ＨＣＶ５＇ＮＣＲｃＤＮＡ和ＲＮＡ在常规逆转录和ＰＣＲ步骤中用于设立有效的模板对照,对消除假用性及评估试剂有重要意义。同时,ＨＣＶ５＇ＮＣＲ体外转录载体的构建可用于制各ＲＮＡ探针和反义ＲＮＡ,改进后还可作为定量ＰＣＲ的竞争性模板。     

简并引物PCR技术研究洋水仙PVY组病毒初报

利用简并引物ＲＴ－ＰＣＲ扩增技术,结合ＲＴ－ＰＣＲ扩增片段ＲＥＬＰ分析,对洋水仙１４个病毒病样品进行分组检测,结果表明洋水仙植物上存在有四种以上ＰＶＹ组病毒侵染。它们可能是ＴＢＶ、ＮＹＳＶ、ＮＳＳＶ和ＯｎＹＤＶ等。     

Molecular cloning of a novel human gene on chromosome 4p11 by immunoscreening of an ovarian carcinoma cDNA library

In our efforts to identify immunoreactive antigens in ovarian cancer, we used the method of immunoscreening of an ovarian carcinoma cDNA expression library with ascites fluid from ovarian cancer patients. Among many positive clones, one was found to contain partial sequence of a novel gene. By searching expressed sequence tags (ESTs) and human genome project databases as well as by screening other cDNA libraries and by RT-PCR strategies, we were able to obtain the full-length cDNA sequence (1.4 kb) and establish the genomic organization of this new gene. We also identified two alternatively spliced forms, encoding for slightly different proteins. The longer form (1.4 kb) is predicted to encode for a 27.6 kDa protein of 245 amino acids. The shorter form (1.3 kb) encodes for a truncated protein of 20.7 kDa and 208 amino acids. These proteins are not significantly homologous to any known protein in the GenBank database. This gene is composed of nine exons and eight introns. By fluorescence in situ hybridization (FISH), it was mapped to chromosome 4p11. This gene is highly expressed in many tissues, including testis, brain, placenta, ovary, prostate, and mammary gland. The high level expression of the shorter form is restricted to the central nervous system, including brain, cerebellum, and spinal cord, suggesting that this form may have a unique function in the central nervous system.     

Cloning and expression of neuron-specific enolase in the corpus luteum of dairy goats

Neuron-specific enolase (NSE) is the key molecular marker for diffuse neuroendocrine system (DNES) cells, its expression in the pregnant corpus luteum (CL) of dairy goats was studied by the immunofluorescence method and the ultra structural characteristics of luteal cells were detected by the electron microscopy to identify the existence of DNES cells in the pregnant CL of dairy goats. Besides, the coding sequence of dairy goats NSE gene was cloned and its biological information was analyzed. Results revealed that NSE immunopositive cells exhibited widespread cytoplasmic staining throughout the whole pregnant CL. In addition, these cells showed typical characteristics of DNES cells in the electron microscopy. These results suggested that many DNES cells exist in the pregnant CL of dairy goats. Meanwhile, we identified the coding sequence of dairy goats NSE (GenBank Accession No. JN887466). Its nucleotide sequence homology was found to be 97.9%, 89.3%, 90% and 92.6%, respectively, compared with that of Bos taurus, Rattus norvegicus, Mus musculus and Homo sapiens, while the amino acid sequence homology was 99.1%, 97%, 97.2% and 98.2% respectively. These results first showed that the functional amino acids coded by the NSE gene were highly conserved in Caprine, B. taurus, R. norvegicus, M. musculus and H. sapiens. It was implied that the gene NSE in dairy goats had close homology to that of NSE of other species. Our findings demonstrated the possible existence of DNES cells in pregnant CL, providing new clue for further understanding of interactions between the neuroendocrine and reproductive systems. Characterization of gene sequence of dairy goats NSE will enable us to synthesize interference RNA for further study on the role of NSE on the formation, function and apoptosis of pregnant CL in dairy goats.     

Characterization of a novel mouse brain gene (mbu-1) identified by digital differential display

Using in silico approaches, we cloned a novel mouse gene (mbu-1) that was strictly expressed in the central nervous system. mbu-1 was first identified as an EST after carrying out digital differential display for unigene libraries from various mouse tissues. The full-length cDNA sequence was obtained by extending the ends of EST by RACE. The cDNA sequence was 2611 bp long and contained an ORF of 597 AA. A positive cis-acting region was found in the neuroblastomaxglioma hybrid, NG108-15, and in human embryonic kidney HEK293 cell lines. RT-PCR and in situ hybridization analysis showed that the mbu-1 gene was only expressed in the brain and spinal cord during the embryonic stages, and throughout all regions of the adult brain, showing higher levels in the hippocampus and hypothalamus.     

Neuronal Survival Factor from Bovine Brain Is Identical to Neuron-Specific Enolase

Neuronal survival factors in the central nervous system were investigated by using a primary culture of embryonic rat neocortical neurons. Bovine hippocampus was homogenized, and the supernatant from high-speed centrifugation was used as the starting material. At the step of DE-52 ion-exchange chromatography, neuronal survival activity was recovered in two fractions, fraction 14 (F14) and fraction 23 (F23). Antisera to the crude F14 and F23 fractions were raised in rabbits. These two antisera completely inhibited the neurotrophic activity of both fractions. Western blotting analysis revealed that anti-F14 antiserum recognized mainly a 30-kDa protein in F14 and anti-F23 antiserum recognized mainly a 44-kDa protein in F23. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of F23, the 44-kDa protein was cut out from the gel and partial amino acid sequences of the protein fragments were determined. A GenBank data bank indicated that the amino acid sequence of the fragment was identical to that of neuron-specific enolase (NSE). In our assay system, commercially available NSE itself possessed neuronal survival activity for the cultured neocortical neurons. The effects of NSE and F23 were inhibited completely by anti-NSE polyclonal antibody. Furthermore, highly purified NSE supported the survival of cultured neurons in a dose-dependent manner, and the neurotrophic effect was inhibited by monoclonal antibody to the NSE. These results strongly suggest that NSE is one of the neuronal survival factors in the central nervous system.     

Kirrel2, a novel immunoglobulin superfamily gene expressed primarily in beta cells of the pancreatic islets

A novel immunoglobulin superfamily (Igsf) protein gene was discovered by computational analysis of human draft genomic DNA, and multiple cDNA clones were obtained. The protein encoded by this gene contains five Ig domains, one transmembrane domain, and an intracellular domain. It has significant similarity with several known Igsf proteins, including Drosophila RST (irregular chiasm C-roughest) protein and mammalian KIRREL (kin of irregular chiasm C-roughest), NEPH1, and NPHS1 (nephrin) proteins. All these proteins have multiple Ig domains, possess properties of cell adhesion molecules, and play important roles in organ development. RT-PCR and Northern blot results indicate this gene is predominantly expressed in pancreas, and public sequence databases indicate there is also expression in the nervous system. We have named this gene Kirrel2 (kin of irregular chiasm-like 2), to reflect its similarity to irregular chiasm C-roughest and Kirrel. Four splice forms of Kirrel2 were observed, including two that we cloned from pancreas mRNA as well as two GenBank entries, one from the brain and one from a retinoblastoma cell line. A partial cDNA clone of the mouse orthologue was obtained by RT-PCR from mouse brain, and the inferred protein sequence has 85% sequence identity to the human protein. Immunohistochemical staining results indicate that the KIRREL2 protein is conserved from rodents to primates, and it is highly expressed in pancreatic islets. RT-PCR results on mouse pancreatic cell lines indicate that expression in the pancreas is restricted to beta cells. Thus, KIRREL2 protein is a beta-cell-expressed Ig domain protein and may be involved in pancreas development or beta cell function.     

A novel second myostatin gene is present in teleost fish.

We report on the isolation and characterisation of the complete cDNA sequence encoding a novel bone morphogenetic protein-like protein (sbMSTN-b) in the teleost fish Sparus aurata. The encoded protein is 68% identical to S. aurata MSTN at the amino acid level, and homologues were also found in Umbrina cirrosa and Tetraodon nigroviridis. Phylogenetic analysis suggests that the MSTN-b gene may be present in most, perhaps all, teleost fish species. RT-PCR on different tissues/stages indicates that MSTN-b is expressed almost exclusively in the central nervous system, starting from late larval stages. Quantitative analyses indicate an increase of sbMSTN-b expression in the brain associated with metamorphosis, at the same time as completion of nervous system differentiation.