Capturing genes encoding membrane and secreted proteins important for mouse development.

A strategy based on the gene trap was developed to prescreen mouse embryonic stem cells for insertional mutations in genes encoding secreted and membrane-spanning proteins. The "secretory trap" relies on capturing the N-terminal signal sequence of an endogenous gene to generate an active beta-galactosidase fusion protein. Insertions were found in a cadherin gene, an unc6-related laminin (netrin) gene, the sek receptor tyrosine kinase gene, and genes encoding two receptor-linked protein-tyrosine phosphatases, LAR and PTP kappa. Analysis of homozygous mice carrying insertions in LAR and PTP kappa showed that both genes were effectively disrupted, but neither was essential for normal embryonic development.     

Selection for genes encoding secreted proteins and receptors.

Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.     

Identification of metastasis-associated breast cancer genes using a high-resolution whole genome profiling approach

Purpose  

We employed a whole genome tumor profiling approach in an attempt to identify DNA copy number alterations (CNAs) and new candidate genes that are correlated with the metastatic potential of a primary breast carcinoma and with progression at the metastatic site.     

Antiestrogen stimulation of the production of a 37,000 molecular weight secreted protein and estrogen stimulation of the production of a 32,000 molecular weight secreted protein in MCF-7 human breast cancer cells

Antiestrogens (AE), such as tamoxifen, selectively increase the production of a secreted protein of 37,000 mol wt (Mr) in estrogen receptor-containing human breast cancer cells (MCF-7, but not in estrogen receptor-negative MDA-MB-231 cells), and the production of this protein by AE is inhibited by concomitant estradiol (E) treatment. Likewise, E increases the production of a 32,000 Mr secreted protein whose synthesis is inhibited by AE. Proteins were detected by [35S]methionine and [35S]cysteine labeling of cells and analysis of proteins by sodium dodecyl sulfate-polyacrylamide gels and fluorography. Enhanced production of the 37,000 Mr protein is observed within 6 h of AE treatment, with maximal synthesis seen at 1-2 days when this protein represents about 6% of the total radiolabeled secreted proteins. This protein is stimulated maximally (approximately 4-fold) by 10(-8) M trans-hydroxytamoxifen or LY117018 or 10(-6) M tamoxifen, and its AE specificity is seen by the fact that trans-tamoxifen increases this protein, whereas cis-tamoxifen, an estrogen, does not. In addition to stimulating the synthesis of previously identified 160,000 and 52,000 Mr secreted proteins, E increases the production of a 32,000 Mr secreted protein. When cells are grown in estrogen-free conditions, i.e. in charcoal-dextran-treated serum in medium lacking the estrogen phenol red, the basal level of the 32,000 Mr protein is extremely low, and E stimulation results in a 10-fold increase in the production of this protein, with increases observable by 6 h and maximal stimulation at 2 days. Interestingly, the basal level of synthesis of the 37,000 Mr protein is high in the absence of E and is then stimulated only minimally by the addition of AE, suggesting that this protein is clearly produced as an estrogen-antagonistic protein. Amino acid incorporation conducted in the presence of tunicamycin and endoglycosidase H indicates that both of these proteins are glycoproteins. These proteins should serve as useful markers for AE and E action and may be involved in AE and E modulation of cell proliferation and/or cell function.     

寄生虫分泌/膜蛋白的生物信息学特征及研究策略

分泌/膜蛋白足寄生虫与宿主相互作用的主要分子,由于其直接暴露于宿主免疫系统及体内药物,因此是寄生虫诊断试剂、疫苗及药物作用靶标主要的候选分子,对其系统深入的研究有助于阐明寄生虫与宿主相瓦作用的分子机制.该文概述了寄生虫分泌/膜蛋白的牛物信息学特征及研究策略,并用生物信息学方法对部分寄生虫一些重婴的诊断及疫苗候选分子进行了分泌/膜篮白特征的分析.     

寄生虫分泌/膜蛋白的生物信息学特征及研究策略

分泌/膜蛋白足寄生虫与宿主相互作用的主要分子,由于其直接暴露于宿主免疫系统及体内药物,因此是寄生虫诊断试剂、疫苗及药物作用靶标主要的候选分子,对其系统深入的研究有助于阐明寄生虫与宿主相瓦作用的分子机制.该文概述了寄生虫分泌/膜蛋白的牛物信息学特征及研究策略,并用生物信息学方法对部分寄生虫一些重婴的诊断及疫苗候选分子进行了分泌/膜篮白特征的分析.     

寄生虫分泌/膜蛋白的生物信息学特征及研究策略

分泌/膜蛋白足寄生虫与宿主相互作用的主要分子,由于其直接暴露于宿主免疫系统及体内药物,因此是寄生虫诊断试剂、疫苗及药物作用靶标主要的候选分子,对其系统深入的研究有助于阐明寄生虫与宿主相瓦作用的分子机制.该文概述了寄生虫分泌/膜蛋白的牛物信息学特征及研究策略,并用生物信息学方法对部分寄生虫一些重婴的诊断及疫苗候选分子进行了分泌/膜篮白特征的分析.     

寄生虫分泌/膜蛋白的生物信息学特征及研究策略

分泌/膜蛋白足寄生虫与宿主相互作用的主要分子,由于其直接暴露于宿主免疫系统及体内药物,因此是寄生虫诊断试剂、疫苗及药物作用靶标主要的候选分子,对其系统深入的研究有助于阐明寄生虫与宿主相瓦作用的分子机制.该文概述了寄生虫分泌/膜蛋白的牛物信息学特征及研究策略,并用生物信息学方法对部分寄生虫一些重婴的诊断及疫苗候选分子进行了分泌/膜篮白特征的分析.     

寄生虫分泌/膜蛋白的生物信息学特征及研究策略

分泌/膜蛋白足寄生虫与宿主相互作用的主要分子,由于其直接暴露于宿主免疫系统及体内药物,因此是寄生虫诊断试剂、疫苗及药物作用靶标主要的候选分子,对其系统深入的研究有助于阐明寄生虫与宿主相瓦作用的分子机制.该文概述了寄生虫分泌/膜蛋白的牛物信息学特征及研究策略,并用生物信息学方法对部分寄生虫一些重婴的诊断及疫苗候选分子进行了分泌/膜篮白特征的分析.     

寄生虫分泌/膜蛋白的生物信息学特征及研究策略

分泌/膜蛋白足寄生虫与宿主相互作用的主要分子,由于其直接暴露于宿主免疫系统及体内药物,因此是寄生虫诊断试剂、疫苗及药物作用靶标主要的候选分子,对其系统深入的研究有助于阐明寄生虫与宿主相瓦作用的分子机制.该文概述了寄生虫分泌/膜蛋白的牛物信息学特征及研究策略,并用生物信息学方法对部分寄生虫一些重婴的诊断及疫苗候选分子进行了分泌/膜篮白特征的分析.     

寄生虫分泌/膜蛋白的生物信息学特征及研究策略

分泌/膜蛋白足寄生虫与宿主相互作用的主要分子,由于其直接暴露于宿主免疫系统及体内药物,因此是寄生虫诊断试剂、疫苗及药物作用靶标主要的候选分子,对其系统深入的研究有助于阐明寄生虫与宿主相瓦作用的分子机制.该文概述了寄生虫分泌/膜蛋白的牛物信息学特征及研究策略,并用生物信息学方法对部分寄生虫一些重婴的诊断及疫苗候选分子进行了分泌/膜篮白特征的分析.     

寄生虫分泌/膜蛋白的生物信息学特征及研究策略

分泌/膜蛋白足寄生虫与宿主相互作用的主要分子,由于其直接暴露于宿主免疫系统及体内药物,因此是寄生虫诊断试剂、疫苗及药物作用靶标主要的候选分子,对其系统深入的研究有助于阐明寄生虫与宿主相瓦作用的分子机制.该文概述了寄生虫分泌/膜蛋白的牛物信息学特征及研究策略,并用生物信息学方法对部分寄生虫一些重婴的诊断及疫苗候选分子进行了分泌/膜篮白特征的分析.     

寄生虫分泌/膜蛋白的生物信息学特征及研究策略

分泌/膜蛋白足寄生虫与宿主相互作用的主要分子,由于其直接暴露于宿主免疫系统及体内药物,因此是寄生虫诊断试剂、疫苗及药物作用靶标主要的候选分子,对其系统深入的研究有助于阐明寄生虫与宿主相瓦作用的分子机制.该文概述了寄生虫分泌/膜蛋白的牛物信息学特征及研究策略,并用生物信息学方法对部分寄生虫一些重婴的诊断及疫苗候选分子进行了分泌/膜篮白特征的分析.     

寄生虫分泌/膜蛋白的生物信息学特征及研究策略

分泌/膜蛋白足寄生虫与宿主相互作用的主要分子,由于其直接暴露于宿主免疫系统及体内药物,因此是寄生虫诊断试剂、疫苗及药物作用靶标主要的候选分子,对其系统深入的研究有助于阐明寄生虫与宿主相瓦作用的分子机制.该文概述了寄生虫分泌/膜蛋白的牛物信息学特征及研究策略,并用生物信息学方法对部分寄生虫一些重婴的诊断及疫苗候选分子进行了分泌/膜篮白特征的分析.     

Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion.

We have constructed a series of plasmids containing a modified form of the phoA gene of Escherichia coli K-12 that have general utility for studies of protein secretion. In these plasmids, the promoter and signal sequence-encoding region of the phoA gene have been deleted; thus, expression of the gene, giving rise to active alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1], is absolutely dependent upon fusion in the correct reading frame to DNA containing a promoter, a translational start site, and a complete signal sequence-encoding region. Alkaline phosphatase, which is normally located in the periplasm of E. coli, is efficiently secreted to the periplasm when fused either to a signal sequence from another periplasmic protein, beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6), or to signal sequences from the outer membrane proteins LamB and OmpF. These heterologous signal sequences are processed during secretion. In the absence of a complete signal sequence, phosphatase becomes localized in the cytoplasm and is inactive. Phosphatase fusion proteins lacking up to 13 amino-terminal amino acids beyond the signal sequence show the same specific activity as that of the wild-type enzyme. However, a significant decrease in activity is seen when 39 or more amino-terminal amino acids are deleted. Addition of approximately 150 amino acids from the enzyme beta-lactamase to the amino terminus of alkaline phosphatase has little effect on the specific activity of the enzyme. The ability to change the amino terminus of phosphatase without altering its activity makes the enzyme particularly useful for construction of protein fusions. The fact that phosphatase is designed for transport across the cytoplasmic membrane makes it an ideal tool for study of protein secretion.     

Ethylene-regulated gene expression: molecular cloning of the genes encoding an endochitinase from Phaseolus vulgaris.

A full-length copy of bean leaf chitinase mRNA has been cloned. The 1146-base-pair insert of pCH18 encodes the 27-residue amino-terminal signal peptide of the precursor and 301 residues of the mature protein. Utilizing pCH18 as a hybridization probe, we have shown that the increase in translatable chitinase mRNA seen upon ethylene treatment of bean seedlings is due to a 75- to 100-fold increase in steady-state mRNA levels. Southern blot analysis of bean genomic DNA revealed that chitinase is encoded by a small, multigene family consisting of approximately four members. From our nucleotide sequence analysis of five additional chitinase cDNA clones, it appears that at least two of these genes are expressed. Three of the bean chitinase genes have been isolated from a Sau3A genomic library and partially characterized.     

三叶因子2相互作用蛋白基因在胃癌细胞cDNA文库中的筛选

目的:筛选并克隆人胃癌细胞eDNA文库中与三叶因子2(trefoil factor family 2,TFF2)蛋白相互作用的蛋白基因.方法:RT-PCR方法验证胃癌细胞存在TFF2mRNA表达:PCR法扩增TFF2基因,Not I与Sal I双酶切后定向克隆到酵母表达载体pDEST32,构建TFF2的诱饵质粒;Western blot验证TFF2诱饵质粒在酵母细胞中相应融合蛋白的表达:将诱饵质粒和人胃癌-cDNA文库猎物质粒共同转化MaV203酵母感受态细胞,通过营养缺陷型培养基(ura、His)与x-gal进行3重筛选阳性克隆,提取阳性克隆质粒测序,应用蛋白质数据库及生物信息学技术,对测序结果进行分析.结果:RT-PCR法证实胃癌细胞中存在TFF2mRNA表达;构建pDEST32-TFF2诱饵表达质粒成功:Western blot法证实诱饵质粒转化酵母细胞后正确表达TFF2-GAL4DBD融合蛋白:通过酵母双杂交筛选胃癌细胞cDNA文库,共获得35个表达His、Ura及x-gal报告基因的阳性克隆,其中测序成功为16个克隆,包含12个已知蛋白基因与4个未知功能基因.结论:从胃癌细胞cDNA文库中筛选出多种TFF2相互作用蛋白基因,其可能与胃癌的发生发展密切相关.