BIOCHEMISTRY OF GENE EXPRESSION IN HIGHER EUKARYOTES

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We are using the giant single cell of the early Drosophila embryo (500 um x 150 um) as a model system in which to examine the biochemical basis for selected aspects of cytoplasmic organization. An understanding of the highly organized cytoplasm in this unusual cell will be required to understand the spatially patterned cell determination processes that underlie its development into a complex multicellular organism. Our approach centers on a study of the cytoskeletal proteins that organize the syncytial cytoplasm. The cytoskeleton is very complicated, and our major goal is to analyze the function of a few of the major multiprotein complexes involved in molecular detail. We also wish to develop methods for identifying and isolating more minor cytoskeletal proteins that are suspected to localize selected mRNAs (such as bicoid mRNA) or proteins to specific regions of the egg cortex. Most of our research plans begin by exploiting two affinity chromatography procedures that have taken us several years to develop to a satisfactory state: actin filament-affinity chromatography and microtubule-affinity chromatography. These procedures have been validated by experiments with known proteins that bind to actin filaments and microtubules. When used to fractionate extracts of early (pre-gastrula) Drosophila embryos, they enable more than 40 (actin filament columns) and 60 (micro- tubule columns) different proteins to be selected and purified. The antibodies that have thus far been produced to these proteins and used to stain embryos suggest that most of the proteins isolated by affinity chromatography are associated with their respective cytoskeletal network (actin filament or microtubule) inside the cell. The main problem now is to make a wise selection of individual proteins to purify to homogeneity and study in detail. This selection is being made after considering the results of cytological localizations, antibody injections into embryos to block function, and biochemical fractionation that identify protein complexes and activities. We have thus far selected for detailed study a 205 kilodalton microtubule-binding protein that is released from microtubules with ATP and localizes to centrosomes, and a 230 kilodalton actin-binding protein whose monoclonal antibody interferes with nuclear spacing when injected into Drosophila embryos. In addition, we have been purifying to homogeneity several intermediate filament-type proteins that are present in large amounts in the syncytial stage embryo.