CAA: Characterization of the Molting Cycle of a Freshwater Crustacean

点击翻译按钮获取中文摘要

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.