We propose to microinject purified nuclear proteins into cultured mammalian cells to study dynamic aspects of chromosome structure. Purified radiolabeled core histones and histone H1 will be injected and the recipient cells will be fused to 3T3 cells to permit hybrid cell formation. We will then determine using autoradiography whether core histones and H1 remain permanently associated with recipient cell chromosomes or exchange onto 3T3 chromosomes. Microinjection of radiolabeled HMG proteins will be used to study the processes underlying internuclear transfer of these proteins. Radiolabeled ubiquitin will be injected and acrylamide gel analyses will be used to determine the number of different nuclear and cytoplasmic proteins modified by covalent attachment to this unusual protein. We will also microinject small DNA fragments into cultured mammalian cells. It is hoped that these small fragments will be integrated and/or interact with endogenous DNA-binding proteins. if this should occur, we will have a system to study integration reactions within living cells, and we should be able to test for genetic interference by these small fragments. Our long-term goal is to contribute to a better understanding of eucaryotic chromosome structure and gene regulation. This should provide useful information about, and perhaps eventual therapy for, a number of human genetic diseases.
