CAA: Characterization of the Molting Cycle of a Freshwater Crustacean

这是一个职业发展奖，使PI能够通过休假获得现代技术，以检查小龙虾钙离子ATPase的分子生物学和调节蜕皮周期中基因表达的因素。初步的工作是利用异源哺乳动物的cDNADNA探针来鉴定小龙虾钙离子ATPase的mRNA。本研究的目的是利用基于聚合酶链式反应和筛选文库的策略，获得小龙虾钙离子ATPase的组织特异性真核表达克隆。全长克隆将被测序，并与已公布的哺乳动物序列进行比较。部分克隆将用于利用定量Northern blotting和Western分析研究蜕皮周期中感兴趣组织中钙离子ATPase的表达，并研究类固醇蜕皮激素对体外基因表达的可能调节。这些发现将进一步加深我们对离子动力ATPase进化的理解，并可能有助于理解它们的形式和功能。最后，类固醇对钙离子转移的调节对所有形成矿化组织的动物都具有重要意义。在生物体中，矿物质钙起着许多重要的生物学作用。在动物身上，它对骨骼的形成至关重要。这一过程在甲壳类动物(螃蟹、龙虾、小龙虾)中很容易研究。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。在为蜕皮做准备的过程中，钙被从旧骨骼中重新吸收并有效地回收利用，同时在下面铺设了一个新的柔软骨骼。从旧骨架中“冲出来”后，新骨架被迅速矿化，以提供保护。因此，在蜕皮周期的不同阶段，科学家可以研究钙进入或离开骨骼的运动。这种运动是通过特殊细胞表面的蛋白质“泵”来实现的。蛋白质的制造受细胞核中遗传物质的控制。在这笔赠款中，将提取钙泵编码的适当遗传信息，以评估是什么在蜕皮周期的不同阶段调节钙泵的丰度和活性。

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.