Current controversies over metaphase chromosome architecture arise from the extreme chromatin packing density in the chromatid which precludes observation of individual chromatin fibers over significant distances. Also, little is known concerning cellular mechanisms controlling chromosome condensation at mitosis. I will approach these problems by analysing the controlled condensation of minute chromosomes in vivo. Double minutes are tiny chromosomes found only in tumor-derived cell lines. They apparently contain amplifications of cellular DNA sequences. Normal chicken cells also contain minute chromosomes. Chromosome condensation will be induced by fusing interphase cells containing minute chromosomes with metaphase-arrested HeLa cells. This induces premature chromosome condensation in the interphase nuclei. the process resembles normal prophase, but the final degree of condensation varies greatly as a function of the interphase cell cycle position. I will produce samples for electron microscope analysis covering a wide range of condensation. This range, coupled with the tiny size of the minute chromosomes should permit great improvement in the visualization of individual chromain fiber paths. Also, since minute and normal chromosomes are readily separable, the approach should permit identification of protein factors involved in the premature condensation. In both microscope and biochemical analysis, particular attention will be given to components of the non-histone protein scaffold, a substructure thought to be involved in maintaining the compact structure of mitotic chromosomes. The electron microscope techniques I will use feature modified Miller spreading procedures, involving sedimentation of chromosomes onto carbon-coated microscope grids. These procedures have already permitted me to improve visualization of substructural detail in normal chromosomes and residual scaffolds. I will also exploit a new ultra-quick-freeze: deep-etch method which virtually eliminates two major problems of chromosome microscopy; structural distortion during sample preparation, and superposition of image detail from thick specimens.
Showing the most recent 10 out of 26 publications
