The objective of Dr. Walsh's work is to understand how synthesis of the more than 200 different proteins which make up the flagellar apparatus is regulated during the differentiation of Naegleria gruberi amebae into flagellates. He has been investigating this question using four classes of cDNA clones specific to differentiating cells, the DS clones, which represent proteins of the flagellar apparatus. The current proposal attempts to answer the question: how is transcription of the DS genes regulated? The approach involves a comparison of the DS genes and genes for mRNAs that are present in both amebae and flagellates (NS genes). This analysis will begin with sequencing recently isolated genomic clones for the four DS genes and 2 NS genes. The functional significance of the resulting sequence information will then be evaluated using DNase hypersensitivity to define regions of the DS chromatin which change during changes in their transcription, followed by a nucleotide level analysis of selected regions of the DS and NS genes using "photofootprinting". Dr. Walsh also plans to develop a transfection system for Naegleria in order to test hypotheses about gene regulation developed from the structural analysis. The differentiation of the amebae of Naegleria gruberi into swimming flagellates is a dramatic example of the structural changes that can occur in organisms in response to environmental stimulae. As such it represents an excellent model system in which to study the regulation of differentiation.
