蛋白质组表达图谱用于基因功能提示的可行性研究

本文以ＥＣＯ２ＤＢＡＳＥ（Ｅｄｉｔｉｏｎ６）为研究材料,探讨了利用蛋白质组表达图谱提供的生命动态活动信息提高基因组功能提示效果的可行性,在设计出一套较为完整的细胞功能簇（ＣＲＣ）聚类方案的基础上,经考察,７９个蛋白质聚成４个不同的ＣＲＣ。结果显示出功能相关的蛋白质趋向于聚集在相同的ＣＲＣ中,如９种氨酰,ｔＲＮＡ合成酶和４种热休克蛋白分别准确地聚合到ＣＲＣ２和ＣＲＣ３中,这些结果提示,在蛋白质组研究     

应用RAPD技术对不同基因组组合生物型遗传多态性的研究(英文)

利用ＲＡＰＤ技术对不同基因组合的鱼类进行了基因组指纹图谱构建,在ＤＮＡ水平上对基因组成分进行了分析,探讨了其遗传多态性。ＲＡＰＤ结果发现,在２６个随机引物扩增的产物中,平均每个个体观察到约１４２个ＲＡＰＤ标记,单个引物获得的标记平均为５．４。其中４个引物扩增的图谱可将不同的生物型区分开：Ｓ－２６引物的扩增图谱（Ｆｉｇ．１）可将红鲫（ＲＡ）与其它组合区分开,还可将鲤鲫杂种一倍体（ＣＡ）与鲫鲤杂种三倍体（ＣＡＡ）和人工复合三倍体鲤（ＣＣＡ）区分开;Ｓ－８引物（Ｆｉｇ．２）可区分开红鲤（ＲＣ）和镜鲤（ＭＣ）;Ｓ－４５引物（Ｆｉｇ．３）可区分开ＲＣ和ＣＡ;Ｓ－２２引物则可区分开ＣＡＡ和ＣＣＡ。六种生物型均存在基因组特异性的图谱即各自独特的“诊断性”图谱,作者由此建立了详细的分子标记检索表（Ｔａｂｌｅ１）。通过对ＲＡＰＤ图谱的量化分析,利用ＵＰＧＭＡ构建了不同生物型的遗传关系树图;反映了鲤鲫及各种组合生物型之间的遗传相似关系：ＲＣ和ＭＣ属同一种系,聚为一族;ＣＡＡ和ＣＡ基因组类型相同,聚为一族;ＣＣＡ虽自成一体,但可与ＣＡＡ和ＣＡ聚为一族;而ＲＡ与其它组合遗传距离较远,自成一族。ＲＡＰＤ的结果也表明各种生物型内个体间     

破伤风毒素C部分的克隆及在大肠杆菌中的表达

用ＣＲ方法从破伤风杆菌基因组ＤＮＡ上坟增出破伤风毒素Ｃ部分（ｔｅｔａｎｕｓ ｔｏｘｉｎ ｆｒａｇｍｅｎｔ Ｃ,ＴＴＣ）,经测序其基因片段由１３５６ｂｐ组成,可编码４５１个氨基酸残基的多肽。将其插入原核表达载体ｐＥＴ－２８ａ（＋）中,并在大肠杆力ＢＬ２１９ＤＥ３）中表达,其表达量占可溶性总蛋白质的８．２％;ＳＤＳ－ＰＡＧＥ和免疫印迹分析表明,ＴＴＣ基因（该基因ＧｅｎＢａｎｋ登录号为ＡＦ１５４８２）表     

应用微卫星标记对不同基因组组合生物型遗传多态性的研究(英文)

微卫星ＤＮＡ在真核基因组中分布频率为每２０～３０ｋｂ一个,具有片段长度短、序列 高度重复、种类多以及高度多态的特点,极适于遗传变异研究。本文报道：采用微卫星 标记对不同基因组组合的鱼类进行了基因组指纹图谱构建。受试材料为红鲤（ＲＣ）、红鲫 （ＲＡ）、镜鲤（ＭＣ）、鲤鲫杂种二倍体（ＣＡ）鲫鲤杂种三倍体（ＣＡＡ）,人工复合三倍体（ＣＣＡ） 等六种生物型。微卫星ＤＮＡ座位（探针）ＭＦＷ２、ＭＦＷ８和ＭＦＷ１６各自存在１２、１６和 １０个等位基因（Ｆｉｇ．１,２,＆３）。通过对微卫星标记图谱的量化分析,利用ＵＰＧＭＡ构建了 不同生物型的遗传关系树系图（Ｔａｂｌｅ　１,Ｆｉｇ．４）。本研究发现,徽卫星和ＲＡＰＤ分析两种 手段反映六种生物型之间的聚类模式完全一致。然而由微卫星标记获得的生物型内和生 物型之间的遗传距离均大于ＲＡＰＤ的,此结果表明微卫星标记在揭示群体内个体间差 异上有独到之处。     

重组人EPO真核表达质粒在大鼠骨骼肌中的表达及其对贫血的治疗作用

目的和方法构建ＥＰＯ真核细胞表达载体（ｐｃＤ２ＥＰＯ）,用缝线法和注射法将其直接导入肾性贫血大鼠股四头肌,观察ＥＰＯ在骨骼肌细胞中的表达及其对贫血的治疗作用。结果：ｐｃＤ２ＥＰＯ导入股四头肌２周后,骨骼肌细胞内出现ＥＰＯｍＲＮＡ,提示被导入的ＥＰＯ基因在肌细胞内得到表达。贫血动物被治疗１周后,缝线组和注射组动物的ＨＧＢ和ＲＢＣ均显著升高;在第３周,缝线组的ＨＧＢ和ＲＢＣ接近健康大鼠的水平;至第４周,两个治疗组的ＨＧＢ和ＲＢＣ仍显著高于对照组。结论：经缝线法导入的ｐｃＤ２ＥＰＯ在骨骼肌中的表达效率及对贫血的治疗效果明显高于直接注射法,而两种治疗方法对改善肾性贫血动物肾脏清除ＢＵＮ的功能无明显差异     

用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒（ＣＡＶ）环形基因组ＤＮＡ（全长２．３ｋｂ）的ＥｃｏＲＩ位点和ＢａｍＨＩ位点的两侧选择适当序列合成两对引物,用ＰＣＲ技术,从斑点杂交检测到病毒核酸的ＣＡＶ感染的ＭＤＣＣＲＰ１细胞基因组ＤＮＡ中,分别扩增出包含ＥｃｏＲＩ和ＢａｍＨＩ分割开的病毒基因组两部分（１．５ｋｂ和０．８ｋｂ）约１．５ｋｂ和约１．２５ｋｂ的两个片段。再将其中相应序列拼接克隆进ｐＵＣ１８载体,获得包含ＣＡＶ全基因组序列ＤＮＡ片段的克隆质粒ｐＣＡＶ２．４。酶切分析表明,该质粒具有预期的ＢａｍＨＩ位点、ＰｓｔＩ位点、ＨｉｎｄⅢ位点,而预期的ＥｃｏＲＩ位点消失。重组质粒插入ＤＮＡ片段的两端序列分析表明,质粒ｐＣＡＶ２．４是包含ＣＡＶ全基因组序列的重组质粒,插入ＤＮＡ片段序列中的ＥｃｏＲＩ位点序列发生了一个碱基突变。     

血管紧张素Ⅱ对大鼠心肌肌浆网Ca~(2+),Mg~(2+)-ATPase基因转录调节的影响

观察血管紧张素Ⅱ（ＡｎｇⅡ）对心肌肌浆网Ｃａ２＋,Ｍｇ２＋－ＡＴＰａｓｅ基因（ＳＥＲＣＡ２ａ）转录调节的影响,评价ＤＭＰ８１１对此效应的干预作用．６周龄雄性ＳＤ大鼠随机分为３组,每组６只．组１：生理盐水输注;组２：ＡｎｇⅡ输注＋ＤＭＰ８１１管饲（３ｍｇ·ｄ－１·ｋｇ－１）;组３：ＡｎｇⅡ输注（２００ｎｇ·ｍｉｎ－１·ｋｇ－１．１周后称其体重,取心脏并称重,提取心脏总ＲＮＡ后采用Ｎｏｒｔｈｅｒｎｂｌｏｔ的方法检测ＳＥＲ－ＣＡ２ａ的转录水平,采用ＲＴ－ＰＣＲ检测ＡｎｇⅡ１型受体（ＡＴ１）ｍＲＮＡ水平．实验后,组３心重（ＣＷ）、心重／体重（Ｃ／Ｂ）、ＡＴ１受体转录水平均高于组１（分别增加４．７±０．４％,４．９±０．９％和２４．７±３．５％;Ｐ＜０．０１）,而ＳＥＲＣＡ２ａ基因转录水平显著低于组１（降低２０．１±３．０％,Ｐ＜０．０１）,并且ＳＥＲＣＡ２ａｍＲＮＡ水平与ＡＴ１受体ｍＲＮＡ水平呈负相关（ｒ＝－０．７４,Ｐ＜０．０１）．ＡｎｇⅡ导致的上述改变能被ＤＭＰ８１１完全阻断．ＡｎｇⅡ通过其Ⅰ型受体的介导,诱导了ＳＥＲＣＡ２ａ的转录下调     

4α－PDD、PMA对去甲肾上腺素促进大鼠腹腔巨噬细胞Ⅰa抗原表达的影响

本文采用Ｃａ~２＋指示剂的分光光谱法测定巨噬细胞（Ｍφ）内Ｃａ~２＋浓度（［Ｃａ~２＋］ｉ）、ＡＰＡＡＰ桥联酶标法检测Ｍφ膜上Ⅰａ抗原的表达,研究肌醇磷脂代谢中第二信使分子甘油二酯（ＤＧ）在去甲肾上腺素（ＮＥ）促进ＭφⅠａ表达效应中的作用,以进一步探讨ＮＥ效应的跨膜信息传递机制。结果表明：蛋白激酶Ｃ（ＰＫＣ）抑制剂４αＰＤＤ（２５μｇ／ｍｌ）虽不影响ＮＥ（１０￣－８ｍｏｌ／Ｌ）升高Ｍφ［Ｃａ￣２＋］ｉ的效应,却显著减弱了ＮＥ促进ＭφⅠａ抗原表达的效应;而ＰＫＣ激动剂ＰＭＡ（１０ｎｍｏｌ／Ｌ）本身促进ＭφⅠａ抗原表达的作用不明显,也不能进一步增强ＮＥ促进ＭφⅠａ抗原表达的效应。结果提示：ＤＧ激活的ＰＫＣ系统也参与了ＮＥ促进ＭφⅠａ抗原表达的信息传递过程,并与另一第二信使分子肌醇－１,４,５－三磷酸（ＩＰ＿３）介导的Ｃａ￣２＋途径协同发挥作用。     

乳腺癌组织中ER、PR和CEA相关性研究

应用免疫组化技术亲和组化法和ＡＢＣ法,检测了１０６例乳腺癌组织中ＥＲ、ＰＲ和ＣＥＡ水平。其阳性率依次为８３％、８１．１％和８８．７％。其中７９．２％的乳腺癌ＥＲ和ＰＲ表达一致。在９８例ＥＲ和／或ＰＲ阳性乳腺癌中有９２例（９３．９％）呈ＣＥＡ阳性,８例ＥＲ和ＰＲ阴性乳腺癌中６例（７５％）为ＣＥＡ阴性,８９％乳腺癌ＣＥＡ与ＥＲ和ＰＲ表达一致。在癌的分级表达中,随着癌的组织学分级增高ＣＥＡ阳性率增高,而ＥＲ和ＰＲ阳性率减低。结果表明,乳腺癌ＥＲ、ＰＲ和ＣｈＡ表达可反映肿瘤的不同生物学特征。同时检测三者对选择内分泌治疗及判断预后更有意义。     

药物诱导后的人大肠癌细胞的[Ca^2＋]i的变化

采用荧光分光光度计法检测维甲酸（ＲＡ）、１,２５（ＯＨ）２ＶＤ３及佛波酯（ＰＭＡ）诱导ＣＣＬ２２９细胞分化后［Ｃａ２＋］ｉ变化,并观察内质网（ＥＲ）特异的Ｃａ２＋－ＡＴＰａｓｅ抑制剂Ｔｈａｐｓｉｇａｒｇｉｎ（ＴＧ）、ＩＰ３受体抑制剂Ｈｅｐａｒｉｎ对ＲＡ诱导［Ｃａ２＋］ｉ变化的影响,从而探讨ＲＡ诱导［Ｃａ２＋］ｉ变化与ＥＲ的关系。结果显示：ＲＡ和１,２５（ＯＨ）２ＶＤ３在数秒内引起［Ｃａ２＋］ｉ显著升高。在ＥＧＴＡ和Ｖｅｒａｐａｍｉｌ预处理细胞条件下,ＴＧ不能抑制ＲＡ引起Ｃａ２＋从细胞内钙池中外流,ＲＡ作用后ＴＧ仍能升高［Ｃａ２＋］ｉ。另外,Ｈｅｐａｒｉｎ也不能完全抑制ＲＡ升高［Ｃａ２＋］ｉ。提示ＲＡ诱导大肠癌细胞升高［Ｃａ２＋］ｉ可能通过ＥＲ上ＩＰ３敏感性和非敏感性钙池,亦可能细胞内存在除ＥＲ外对ＲＡ敏感的钙池。     

Expression profiling of more than 3500 proteins of MSS-type colorectal cancer by stable isotope labeling and mass spectrometry

An efficient means of identifying protein biomarkers is essential to proper cancer management. A well-characterized proteome resource holds special promise for the discovery of novel biomarkers. However, quantification of the differences between physiological conditions together with deep down profiling has become increasingly challenging in proteomics. Here, we perform expression profiling of the colorectal cancer (CRC) proteome by stable isotope labeling and mass spectrometry. Quantitative analysis included performing mTRAQ and cICAT labeling in a pooled sample of three microsatellite stable (MSS) type CRC tissues and a pooled sample of their matched normal tissues. We identified and quantified a total of 3688 proteins. Among them, 1487 proteins were expressed differentially between normal and cancer tissues by higher than 2-fold; 1009 proteins showed increased expression in cancer tissue, whereas 478 proteins showed decreased expression. Bioinformatic analysis revealed that our data were largely consistent with known CRC relevant signaling pathways, such as the Wnt/β-catenin, caveolar-mediated endocytosis, and RAN signaling pathways. Mitochondrial dysfunction, known as the Waburg hypothesis, was also confirmed. Therefore, our data showing alterations in the proteomic profile of CRC constitutes a useful resource that may provide insights into tumor progression with later goal of identifying biologically and clinically relevant marker proteins. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Proteomic analysis of advanced colorectal cancer by laser capture microdissection and two-dimensional difference gel electrophoresis

The emergence of laser capture microdissection (LCM) and two-dimensional difference gel electrophoresis (2D-DIGE) has been shown to greatly improve the accuracy and sensitivity of global protein expression analysis. However, their combined use in profiling tumour proteome has rarely been reported. In this study, we applied these techniques to profile the protein expression changes of the late stage colorectal cancer (CRC) and its liver metastases. The study revealed that both the primary and secondary tumours showed a distinct protein expression profile compared to normal tissues, but were indistinguishable from each other. Differential analysis between the primary tumour and patient-matched normal colon mucosa identified a total of 71 proteins to be altered in CRC. Over 40% of these proteins have been previously reported as CRC-related proteins, validating the accuracy of the current analysis. We have also identified many previously unknown changes including overexpression of ACY1, HSC70, HnRNP I, HnRNP A3, SET, ANP32A and TUFM in CRC, which have been further verified by western blotting and immunohistochemistry. This study demonstrated that LCM in combination with 2D-DIGE is a powerful tool to analyse the proteome of tumour tissues and may lead to the identification of potential novel protein markers and therapeutic targets for cancer.     

The human proteomics initiative (HPI)

The availability of the human genome sequence has enabled the exploration and exploitation of the human genome and proteome to begin. Research has now focussed on the annotation of the genome and in particular of the proteome. With expert annotation extracted from the literature by biologists as the foundation, it has been possible to expand into the areas of data mining and automatic annotation. With further development and integration of pattern recognition methods and the application of alignments clustering, proteome analysis can now be provided in a meaningful way. These various approaches have been integrated to attach, extract and combine as much relevant information as possible to the proteome. This resource should be valuable to users from both research and industry.     

Functional modelling of an equine bronchoalveolar lavage fluid proteome provides experimental confirmation and functional annotation of equine genome sequences

The equine genome sequence enables the use of high-throughput genomic technologies in equine research, but accurate identification of expressed gene products and interpreting their biological relevance require additional structural and functional genome annotation. Here, we employ the equine genome sequence to identify predicted and known proteins using proteomics and model these proteins into biological pathways, identifying 582 proteins in normal cell-free equine bronchoalveolar lavage fluid (BALF). We improved structural and functional annotation by directly confirming the in vivo expression of 558 (96%) proteins, which were computationally predicted previously, and adding Gene Ontology (GO) annotations for 174 proteins, 108 of which lacked functional annotation. Bronchoalveolar lavage is commonly used to investigate equine respiratory disease, leading us to model the associated proteome and its biological functions. Modelling of protein functions using Ingenuity Pathway Analysis identified carbohydrate metabolism, cell-to-cell signalling, cellular function, inflammatory response, organ morphology, lipid metabolism and cellular movement as key biological processes in normal equine BALF. Comparative modelling of protein functions in normal cell-free bronchoalveolar lavage proteomes from horse, human, and mouse, performed by grouping GO terms sharing common ancestor terms, confirms conservation of functions across species. Ninety-one of 92 human GO categories and 105 of 109 mouse GO categories were conserved in the horse. Our approach confirms the utility of the equine genome sequence to characterize protein networks without antibodies or mRNA quantification, highlights the need for continued structural and functional annotation of the equine genome and provides a framework for equine researchers to aid in the annotation effort.     

BiomarkerDigger: A versatile disease proteome database and analysis platform for the identification of plasma cancer biomarkers

We have developed a proteome database (DB), BiomarkerDigger ( http://biomarkerdigger.org ) that automates data analysis, searching, and metadata‐gathering function. The metadata‐gathering function searches proteome DBs for protein–protein interaction, Gene Ontology, protein domain, Online Mendelian Inheritance in Man, and tissue expression profile information and integrates it into protein data sets that are accessed through a search function in BiomarkerDigger. This DB also facilitates cross‐proteome comparisons by classifying proteins based on their annotation. BiomarkerDigger highlights relationships between a given protein in a proteomic data set and any known biomarkers or biomarker candidates. The newly developed BiomarkerDigger system is useful for multi‐level synthesis, comparison, and analyses of data sets obtained from currently available web sources. We demonstrate the application of this resource to the identification of a serological biomarker for hepatocellular carcinoma by comparison of plasma and tissue proteomic data sets from healthy volunteers and cancer patients.     

Determination and comparison of the Francisella tularensis subsp.novicida U112 proteome to other bacterial proteomes

The proteins expressed by Francisella tularensis subsp. novicida U112 grown to midexponential phase were surveyed by nanoLC-tandem mass spectrometry (LC-MS/MS). To improve annotation of the genome and develop a technology to provide high-throughput analysis of the Francisella proteome in multiple conditions, we sought to establish a fast and simple analysis that would reduce as much as possible the false discovery rate. Our survey detected expression of 63.0% of the predicted proteome from the stable condition of growth in rich medium available at (www.francisella.org). On the basis of detection of essential proteins, we estimated coverage to be approximately 80% of the actual expressed proteome. This suggests that no less than 70% of the proteins could be expressed in this condition. This analysis revealed two previously unidentified protein coding open reading frames and validated 50% of the proteins annotated as hypothetical. On the basis of results of the screen to detect essential proteins, not all proteins expressed provide a measurable contribution to F.t. novicida growth in this condition. Comparison of this protein profile with other profiles previously published suggested that the genome size and number of genes involved in regulation have little effect on the number of proteins expressed in a given stable condition.     

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).     

A high-quality catalog of the Drosophila melanogaster proteome

Understanding how proteins and their complex interaction networks convert the genomic information into a dynamic living organism is a fundamental challenge in biological sciences. As an important step towards understanding the systems biology of a complex eukaryote, we cataloged 63% of the predicted Drosophila melanogaster proteome by detecting 9,124 proteins from 498,000 redundant and 72,281 distinct peptide identifications. This unprecedented high proteome coverage for a complex eukaryote was achieved by combining sample diversity, multidimensional biochemical fractionation and analysis-driven experimentation feedback loops, whereby data collection is guided by statistical analysis of prior data. We show that high-quality proteomics data provide crucial information to amend genome annotation and to confirm many predicted gene models. We also present experimentally identified proteotypic peptides matching approximately 50% of D. melanogaster gene models. This library of proteotypic peptides should enable fast, targeted and quantitative proteomic studies to elucidate the systems biology of this model organism.