CAA: Characterization of the Molting Cycle of a Freshwater Crustacean

这是一个职业晋升奖，使PI能够通过休假获得所需的现代技术，以研究小龙虾钙离子atp酶的分子生物学和在换壳周期中调节基因表达的因素。初步工作采用异源哺乳动物cDNA探针鉴定小龙虾钙离子atp酶mRNA。本研究的目的是利用基于聚合酶链反应和cDNA文库筛选的策略，生成组织特异性的小龙虾钙离子atp酶的cDNA克隆。将对全长克隆进行测序，并与已发表的哺乳动物序列进行比较。部分克隆将用于研究脱毛周期中相关组织中钙离子atp酶的表达，使用定量Northern blotting和Western分析，并检查类固醇蜕皮激素对体外基因表达的可能调控。这些发现将进一步加深我们对离子动机atp酶进化的理解，并可能有助于理解它们的形式和功能。最后，类固醇调节钙离子转移对所有形成矿化组织的动物都有重要意义。在生物体中，矿物质钙起着许多重要的生物学作用。在动物中，它对骨骼的形成至关重要。这个过程很容易在甲壳类动物（螃蟹、龙虾、小龙虾）中进行研究。首先，它们的骨骼是外部的，因此比脊椎动物更容易接近。更重要的是，为了生长，它们经历了蜕皮周期。在准备蜕皮时，钙从旧骨架中被重新吸收并有效地回收；与此同时，一个新的柔软的骨架在下面铺设。在从旧骨架中“钻出来”后，新骨架迅速矿化以提供保护。因此，在蜕皮周期的不同阶段，科学家们可以研究钙进出骨骼的运动。这种运动是由特化细胞表面的蛋白质“泵”影响的。蛋白质的制造受细胞核内遗传物质的控制。在这项拨款中，钙泵编码的适当遗传信息将被提取出来，以评估在蜕皮周期的不同阶段是什么调节了钙泵的丰度和活性。

This is a career advancement award to enable the PI to acquire, through a sabbatical leave, modern techniques required to examine the molecular biology of the crayfish calcium ion ATPase and factors that regulate expression of the gene during the molting cycle. Preliminary work has employed a heterologous mammalian cDNA probe to identify mRNA for the crayfish calcium ion ATPase. The objective of the present study is to generate tissue specific authentic cDNA clones of the crayfish calcium ion ATPase using strategies based on the polymerase chain reaction and on screening a cDNA library. The full length clone will be sequenced and compared to published mammalian sequences. Partial clones will be used to study expression of calcium ion ATPase in tissues of interest during the molting cycle using quantitative Northern blotting and Western analysis, and to examine possible regulation of gene expression in vitro by the steroid ecdysone. Findings will further our understanding of the evolution of ion motive ATPases and perhaps assist in understanding their form and function. Finally the steroidal regulation of calcium ion transfer is of significance for all animals that form mineralized tissues. In living organisms the mineral calcium plays many important biological roles. In animals it is crucial for the formation of the skeleton. This process is easily studied in crustaceans (crabs, lobsters, crayfish). First their skeleton is external and therefore far more accessible than that of vertebrates. More importantly, in order to grow, they undergo the molting cycle. In preparation for molting, calcium is reabsorbed from the old skeleton and effectively recycled; at the same time a new soft skeleton is laid down underneath. After "busting out" of the old skeleton, the new skeleton is rapidly mineralized to provide protection. Therefore at different stages in the molting cycle scientists can study the movement of calcium either into or out of the skeleton. This movement is effected by protein "pumps" on the surface of specialized cells. Manufacture of protein is under the control of genetic material in the nucleus of the cell. In this grant appropriate genetic information that codes for the calcium pump will be extracted to assess what regulates the abundance and activity of the calcium pumps at different stages in the molting cycle.