Core A: VENOM COLLECTION AND DISCOVERY SUPPORT CORE

核心A。摘要。 核心A的目标是保持来自不同圆锥体物种的大量毒液和组织的收集 以及精制的蛇毒多肽、合成肽及其类似物的集合。生物材料将是 主要从菲律宾的不同地点获得。而毒液将被用作直接来源 纯化的多肽、组织将作为mRNA和基因组DNA的来源进行分子生物学研究 毒素的鉴定。多肽将通过钙成像分析(项目I)进行鉴定，测试 异源表达的通道和受体(项目II和项目III)，并通过分子生物学鉴定 编码多肽的核酸序列。蛇毒多肽将通过生化方法进行提纯。从… 了解圆锥体物种之间的进化关系，我们将系统地分析 针对特定配体的毒液。分支分析的遗传标记将通过PCR扩增获得 基因组DNA的特异性引物扩增。完整的毒素(早熟毒素)序列将通过以下方式确定 从蛇毒导管基因文库中鉴定相应的cDNA。聚合酶链反应 用家族特异性引物扩增cDNA库将被用来确定 表达的毒素。我们将使用下一代测序方法来获得完整的毒素清单 序列和其他蛋白质，在毒液导管中表达。这将使我们能够设计引物来识别 其他锥形蜗牛体内的相关毒素。我们还将实施芋螺毒素发现管道，将 直接分析毒液和测序毒液管道的蛋白质组学和下一代测序 转录组。分子生物学方法提供了鉴定潜在配体(肽)的手段。 生物组织的数量非常有限。具有丰富分子生物学经验的核心将有助于 分离神经元中受体和离子通道表达的初步检测和定量 实时荧光聚合酶链式反应方法。所有项目都使用由核心分离出来的多肽。这些设备 要求，是为了生产和分离多肽。实时聚合酶链式反应仪器将由 量化受体/通道表达的不同项目(详情见设备预算理由)。这个 CORE将继续向科学界提供多肽和毒液样本。

Core A. Abstract. The objective of Core A is to maintain a large collection of venoms and tissues from different Conus species and a collection of purified venom peptides, synthetic peptides and their analogs. Biological material will be obtained mainly from various locations in the Philippines. While the venom will be used as a direct source of purified peptides, tissues will be used as a source of mRNA and genomic DNA for the molecular biological identification of toxins. Peptides will be identified by calcium imaging assay (Project I), testing on heterologously expressed channels and receptors (Projects II and III), and by molecular biological identification of peptide-encoding nucleic acid sequences. Venom peptides will be purified by biochemical methods. From knowledge of the evolutionary relationship among the Conus species we will systematically analyze the venoms for specific ligands. Genetic markers for the cladistic analysis will be obtained by PCR amplification of genomic DNA using specific primers. Complete toxin (pre-pro-mature toxin) sequences will be determined by identification of corresponding cDNAs from cDNA libraries constructed from venom-duct mRNA. PCR amplification of cDNA libraries with family-specific primers will be used to determine the repertoire of expressed toxins. We will use Next Generation sequencing methods to obtain the complete list of toxin sequences and other proteins, expressed in the venom duct. This will enable us to design primers to identify related toxins in other cone snails. We will also implement a conotoxin discovery pipeline combining proteomics and next-gen sequencing by direct analysis of venoms and sequencing the venom-duct transcriptome. Molecular biological methods offer a means of identification of potential ligands (peptides) from very limited amount of biological tissue. The core with extensive molecular biological experience will assist in the detection and quantification of receptors and ion channels expressed in dissociated neurons initially using real-time PCR methods. The peptides isolated by the core are used by all the projects. The equipment requested, is for the production and isolation of the peptides. The real time PCR instrument will be used by the different projects to quantify receptor/channel expression (see equipment budget justification for details). The core will continue to provide peptide and venom samples to the scientific community.