Core A: VENOM COLLECTION AND DISCOVERY SUPPORT CORE

核心A。抽象的。 核心A的目标是保存大量来自不同芋螺物种的毒液和组织 以及纯化的毒液肽、合成肽及其类似物的集合。生物材料将 主要来自菲律宾的各个地方。虽然毒液将被用作 纯化的肽，组织将用作分子生物学的mRNA和基因组DNA的来源。 鉴别毒素。将通过钙成像试验（项目I）鉴定肽， 异源表达的通道和受体（项目II和III），并通过分子生物学鉴定 编码肽的核酸序列。毒液肽将通过生物化学方法纯化。从 我们将系统地分析芋螺物种之间的进化关系， 特定配体的毒液。用于分支分析的遗传标记将通过PCR扩增 使用特异性引物检测基因组DNA。完整毒素（前原成熟毒素）序列将通过以下方法测定： 从由毒管mRNA构建的cDNA文库中鉴定相应的cDNA。PCR 用家族特异性引物扩增cDNA文库将用于确定 表达毒素。我们将使用下一代测序方法来获得毒素的完整列表 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 其他锥形蜗牛中的相关毒素。我们还将实施芋螺毒素发现管道， 蛋白质组学和下一代测序，通过直接分析毒液和测序毒液导管 转录组分子生物学方法提供了一种鉴定潜在配体（肽）的方法 非常有限的生物组织。具有丰富分子生物学经验的核心将协助 检测和定量的受体和离子通道表达的解离神经元最初使用 实时PCR方法。由核心分离的肽被所有项目使用。设备 所要求的是用于肽的生产和分离。真实的时间PCR仪器将由 不同的项目来量化受体/通道表达（详见设备预算理由）。的 该中心将继续向科学界提供肽和毒液样本。

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.