Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp SLP-76 was 2007 bp, consisting of a 5'-terminal untranslated region (UTR) of 285 bp, a 3'-terminal UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homologues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts.     

猴副流感病毒SV5的PCR检测方法建立及初步应用

[目的]建立检测SV5的PCR方法并加以初步应用。[方法]根据Genbank中报道的SV5序列,针对其中的SH基因设计引物进行PCR反应,扩增产物进行测序并用BLAST软件进行同源性比对,同时利用限制性内切酶的酶切反应以证实此PCR反应的特异性。在此基础上设计巢式PCR提高此方法的灵敏度。利用此方法对20份猴肾源细胞培养物和40份血清标本进行检测。[结果]利用设计的引物扩增出的序列测序结果证实与报道的SV5SH基因相对位置的序列一致。AccIII限制性内切酶可对PCR产物进行特异性酶切。巢式PCR比一次PCR的敏感度有所提高。用此方法检测的20份猴肾源细胞培养物和40份血清标本结果为阴性。[结论]本文首次初步建立了检测SV5病毒的PCR方法,排除实验室用20份猴肾源细胞培养物和40份血清标本SV5的污染。     

Prokaryotic Suppression Subtractive Hybridization PCR cDNA Subtraction, a Targeted Method To Identify Differentially Expressed Genes

Molecular biology tools can be used to monitor and optimize biological treatment systems, but the application of nucleic acid-based tools has been hindered by the lack of available sequences for environmentally relevant biodegradation genes. The objective of our work was to extend an existing molecular method for eukaryotes to prokaryotes, allowing us to rapidly identify differentially expressed genes for subsequent sequencing. Suppression subtractive hybridization (SSH) PCR cDNA subtraction is a technique that can be used to identify genes that are expressed under specific conditions (e.g., growth on a given pollutant). While excellent methods for eukaryotic SSH PCR cDNA subtraction are available, to our knowledge, no methods previously existed for prokaryotes. This work describes our methodology for prokaryotic SSH PCR cDNA subtraction, which we validated using a model system: Pseudomonas putida mt-2 degrading toluene. cDNA from P. putida mt-2 grown on toluene (model pollutant) or acetate (control substrate) was subjected to our prokaryotic SSH PCR cDNA subtraction protocol to generate subtraction clone libraries. Over 90% of the sequenced clones contained gene fragments encoding toluene-related enzymes, and 20 distinct toluene-related genes from three key operons were sequenced. Based on these results, prokaryotic SSH PCR cDNA subtraction shows promise as a targeted method for gene identification.     

微量RNA的cDNA PCR文库的构建

使用PCR（polymerase chain reaction)技术,调制了mRNA的cDNA PCR文库,实验证明,cDNA PCR文库能使原cDNA的量放大数百倍,同时,使用人体K562培养细胞的总RNA,对cDNA PCR文库法和反转录中的β-Actin的cDNA量进行了比较,cDNA PCR文库法中的β-Actin的cDNA量大于高于反转录中的β-Actin的cDNA量,使用75pg的人体K562培养细胞的总RNA,调制成50ul的CDNA PCR文库,使用1ul的CDNA PCR文库进行了PCR反应时,可对文库中β-Actin的CDNA进行PCR检测,因此,CDNA PCR文库显示了良好的信息放大性能。     

Isolation and characterization of rat 3Y1 fibroblast clones overexpressing the src homology region of phospholipase C-gamma 2.

To examine the regulatory function of the src-related SH2 and SH3 (SH2/SH3) region of phospholipase C-gamma 2 (PLC-gamma 2), we expressed this region of rat PLC-gamma 2 cDNA in rat 3Y1 fibroblasts and isolated and characterized a number of clones (approximately 20 clones). An increase of endogenous tyrosine kinase activity was observed in all cell clones that highly expressed a translational product of the SH2/SH3 domain. Moreover, endogenous phosphatidylinositol 4,5-bisphosphate hydrolyzing activity was also enhanced in these clones, and PLC-gamma 1 seemed to be preferentially activated among endogenous PLC isozymes. Genistein, an inhibitor of tyrosine kinase, inhibited this activation of PLC-gamma 1, and tyrosine phosphorylation was observed on PLC-gamma 1 molecules, indicating the involvement of tyrosine kinases in the PLC-gamma 1 activation. These results suggest that the SH2/SH3 region of PLC-gamma would function as a multidirectional regulator which controls at least two major signaling pathways: tyrosine kinase and phosphatidylinositol 4,5-bisphosphate hydrolysis.     

猪SOCS-3 cDNA序列克隆及在各组织中表达

 根据大鼠细胞因子信号转导抑制因子（suppressor of cytokine signaling,SOCS)-3设计并合成一对引物,从八眉猪皮下脂肪组织提取总mRNA,RT-PCR扩增获得猪SOCS-3 cDNA;利用半定量（semi-quantitative, SQ）RT-PCR技术检测大白猪各组织中SOCS-3 mRNA的表达量.扩增获得SOCS-3 基因GenBank 登陆号DQ644577,用CDD软件进行SOCS-3 基因结构分析,该序列具有SOCS-3 特有的保守结构域SH2和SOCS-box. 用Clustal W软件进行同源性分析发现,猪SOCS-3 基因与人、大鼠和小鼠的同源性分别为92%、88%和88%.SQ RT-PCR组织表达检测表明：SOCS-3 基因在心脏、肝脏、脾脏、肺脏、肾脏、肌肉、皮下脂肪和内脏脂肪中均有表达,其中肺脏和脾脏的表达丰度最高,肝脏和内脏脂肪中的表达量最低.     

一步法扩增克隆IBDV上海超强毒VP2—4—3基因

分离、纯化了鸡传染性法氏囊病病毒超强毒 (vvIBDV)上海株SH95的病毒核酸dsRNA ,应用随机引物将RNA反转录成cDNA ,以此为模板一步扩增出A片段前体融合蛋白基因即VP2 4 3基因 ,将其克隆入 pGEM T载体 ,并进行序列分析 ,其与超强毒株HK4 6的核苷酸序列的同源性达 98% ,整个基因有 5个氨基酸差异 ,同源性达99.5 1% (10 0 7/ 10 12 )。     

cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor.

Using immobilized PDGF receptor as an affinity reagent, we purified an 85 kd protein (p85) from cell lysates and we cloned its cDNA. The protein contains an SH3 domain and two SH2 domains that are homologous to domains found in several receptor-associated enzymes. Recombinant p85 overexpressed in mammalian cells inhibited the binding of endogenous p85 and a 110 kd protein to the receptor and also blocked the association of PI3-kinase activity with the receptor. Experiments with receptor mutants and with short peptides derived from the kinase insert region of the PDGF receptor showed that the recombinant p85 binds to a well-defined phosphotyrosine-containing sequence of the receptor. p85 appears to be the subunit of PI3-kinase that links the enzyme to the ligand-activated receptor.     

Purification of recombinant SH2/SH3 proteins of phospholipase C-gamma 1 and -gamma 2 and their inhibitory effect on PIP2-hydrolysis induced by both types of phospholipase C-gamma.

In order to examine physiological function of the SH2/SH3 region of phospholipase C-gamma (Z region), we independently expressed cDNA fragments corresponding to the SH2/SH3 region of PLC-gamma 1 and PLC-gamma 2 in Escherichia coli. Although these recombinant proteins were recovered in particulate fractions by centrifugation of cell extracts, they were successfully solubilized by guanidium hydrochloride and then purified to homogeneity by heparin column chromatography. The molecular mass of the proteins was 45 kDa (derived from PLC-gamma 1 and designated as rP45Z) and 38 kDa (derived from PLC-gamma 2 and designated as rP38Z), which was consistent with that as expected from inserted cDNA. We determined the effect of purified rP45Z or rP38Z on PIP2-hydrolyzing activity of either PLC-gamma 1 or PLC-gamma 2 and found that these proteins strongly suppressed the rate of PLC-dependent PIP2-hydrolysis. Furthermore, both rP45Z and rP38Z were phosphorylated at tyrosine residue by epidermal growth factor receptors and their inhibitory effect on PIP2-hydrolysis was significantly decreased by this phosphorylation. These results indicate that the Z region might be involved in autoregulation of PLC-gamma as intrinsic negative regulator.     

一个新的鼠STE20家族激酶的克隆和鉴定

从鼠肝cDNA文库克隆了一个新的STE20类蛋白激酶,Mess1.其cDNA长1.7 kb,编码了一个497个氨基酸残基的多肽,与人MST2具有95%的氨基酸相同.Mess1蛋白氨基末端激酶催化区的序列与STE20同源,其羧基末端包含了一簇丝氨酸/苏氨酸和谷氨酸丰富的序列,被认为具有介导与SH2功能区结合的作用.MESS1可能通过与含有SH2功能区的蛋白质相互作用参与细胞内信号转导.     

一步PCR快速扩增辽宁碱蓬甜菜碱醛脱氢酶cDNA 3'末端序列

根据已获得的辽宁碱蓬甜菜碱醛脱氢酶cDNA的部分序列,设计一条基因特异性引物,与通用引物并用,一步PCR成功地克隆了辽宁碱蓬甜菜碱醛脱氢酶cDNA 3′末端。与常规的3′RACE法相比,一步PCR法具有快速、简便、经济等优点,是一种非常快捷的扩增cDNA 3′末端序列的方法。 Abstract:Based on part of a known cDNA sequence of Suaeda liaotungensis betaine aldehyde dehydrogenase,we successfully cloned the 3′cDNA end of S.lianotungensis betaine aldehyde dehydrogenase using one step PCR with a gene specific primer and universal primer.Compared with the typical 3′ RACE,one step PCR is rapid,simple and inexpensive.It is very rapid to amplify an unknown cDNA 3′end using this method.     

A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.

A new SH2-containing sequence, SHC, was isolated by screening cDNA libraries with SH2 representative DNA probes. The SHC cDNA is predicted to encode overlapping proteins of 46.8 and 51.7 kd that contain a single C-terminal SH2 domain, and an adjacent glycine/proline-rich motif with regions of homology with the alpha 1 chain of collagen, but no identifiable catalytic domain. Anti-SHC antibodies recognized three proteins of 46, 52, and 66 kd in a wide range of mammalian cell lines. These SHC proteins complexed with and were phosphorylated by activated epidermal growth factor receptor. The physical association of SHC proteins with activated receptors was recreated in vitro by using a bacterially expressed SHC SH2 domain. NIH 3T3 mouse fibroblasts that constitutively overexpressed SHC acquired a transformed phenotype in culture and formed tumors in nude mice. These results suggest that the SHC gene products couple activated growth factor receptors to a signaling pathway that regulates the proliferation of mammalian cells.     

SPIN90 (SH3 protein interacting with Nck, 90 kDa), an adaptor protein that is developmentally regulated during cardiac myocyte differentiation

In the yeast two-hybrid screening, we have isolated a cDNA clone from a human heart library using Nck Src homology 3 (SH3) domains as bait. The full-length cDNA, which encoded 722 amino acids, was identified as a VIP54-related gene containing an SH3 domain, proline-rich motifs, a serine/threonine-rich region, and a long C-terminal hydrophobic region. We refer to this protein as SPIN90 (SH3 Protein Interacting with Nck, 90 kDa). The amino acid sequence of the SH3 domain has the highest homology with those of Fyn, Yes, and c-Src. SPIN90 was broadly expressed in human tissues; in particular, it was highly expressed in heart, brain, and skeletal muscle, and its expression was developmentally regulated during cardiac myocyte differentiation. SPIN90 is able to bind to the first and third SH3 domains of Nck, in vitro, and is colocalized with Nck at sarcomere Z-discs within cardiac myocytes. Moreover, treatment with antisera raised against SPIN90 disrupted sarcomere structure, suggesting that this protein may play an important role in the maintenance of sarcomere structure and/or in the assembly of myofibrils into sarcomeres.