The dramatic cell differentiation undergone by Naegleria gruberi, from amoebae to flagellates, is unique as a model system in the rapidity, synchrony, reproducibility, homogeneity, and Accessibility of a major phenotypic change. Differentiation-specific molecules synthesized during this change include two calmodulins, one that becomes localized in the flagella and the other that remains in the cell body. Cloning and sequencing of the single flagellar calmodulin gene revealed provocative structural features that may be implicated in regulation of its programmed transcription, in the stability of its mRNA, and in the localization of its protein. In order to test these possibilities, and to make this system advantageous for other studies, this laboratory is working vigorously to obtain DNA-mediated transformation of Naegleria. In addition, initial searches for DNA clones to cell body calmodulin have led to the discovery of two additional calcium-binding protein genes expressed during differentiation. One encodes a calcineurin B-like protein; the other and undefined calcium-binding protein whose gene sequence is related to flagellar calmodulin DNA but which is expressed earlier in differentiation. The goals of this research are to find the calmodulin-2 gene and to dissect these multiple calmodulin-like proteins in Naegleria, while continuing to seek DNA-mediated transformation in this remarkable organism. The unicellular eukaryote, Naegleria gruberi reproduces as amoebae. If conditions are suitable, the amoebae undergo a rapid, dramatic differentiation into lively, swimming flagellates with a fixed, streamlined body contour. The flagellates are temporary, do not reproduce, and eventually revert back to amoebae. The long-term goal of this research is to elucidate the sequence of events (from environmental trigger, through programmed changes in gene expression, to localization of differentiation-specific gene products) that underlie these differentiation events. The current goals of this project are to identify and characterize genes for proteins involved in the regulation of cellular functions by calcium. Since the genes for these regulatory molecules are expressed differently at different stages of the differentiation process in different parts of the organism, it is likely that they represent an important link in this chain of events.
