An exciting worldwide commitment to elucidate the molecular events of the eukaryotic cell cycle has resulted in a unified view of the basic events of the cell cycle that are consistent throughout all taxa. These studies have greatly contributed to our understanding of developmental processes and proliferative diseases. The goal of the proposed research is to describe, for the first time, the molecular and biochemical regulation of the cell cycle in the teleost embryonic model system, Fundulus heteroclitus. Teleosts offer many of the same advantages of other vertebrates and in addition offer a greater potential for variation in cell cycle regulatory mechanisms due to their wide taxonomic, developmental nd adaptive diversification. Further, a distinct advantage of a teleost is the ability to produce thousands of synchronously cleaving embryos via the simple act of fertilization. Our initial strategy has been to use the affinity of the yeast p13suc1 protein for the p34cdc2 cyclin heterodimer (MPF - M phase Promoting Factor) to partially purify and characterize MPF and its activity by immunoblotting and the histone H1 kinase assay. Evidence from Western blots indicates 4 p34cdc2 like proteins are present in cleaving F. heteroclitus egg extracts, two of which appear to be novel. p34cdc2 kinase activity appears to peak 4 minutes prior to completion of anaphase in cleaving eggs. The unusual degree of cleavage synchrony and sharp peak of p34cdc2 kinase activity (~4 min./35 min. cell cycle) should make this model an excellent choice to study the temporal aspects of cell cycle regulation during development. Our research plan is to first assess the degree of synchrony in embryos from fertilization through the midblastula transition with the histone H1 kinase assay and flow cytometry. We will then conduct studies to describe the biochemical and temporal aspects of p34cdc2 kinase activity in cleaving embryos using p13suc1 affinity beads to isolate the fish homolog(s) of p34cdc2 for further purification and analysis by SDS, native and 2D gel electrophoresis; the histone H1 kinase assay; and Western blots. Cyclin synthesis and degradation will be followed by labelling protein precursor pools in vivo with 35S methionine by microinjection. Incorporated label will be isolated by p13suc1 immunoprecipitation and then visualized by autoradiographic analysis of SDS polyacrylamide gels. The results of these experiments will form a preliminary picture of the embryonic cell cycle in fish that will be directly comparable to other popular biomedical models such as the frog and form the basis for more intensive research.
