I propose to investigate the phenomenon of nuclear localization using yeast H2B as a prototype. Preliminary data suggest that a H2B-lacZ fusion which contains approximately one half of the amino terminal end of the H2B gene is localized in the nucleus as determined by indirect immuno-fluorescence (IIF) of whole cells using anti-beta-galactosidase primary antibody. The proposed research can be divided along two separate lines. The first will involve determination of the H2B domain(s) required for localization and the site(s) to which the fusion proteins are localized. This will involve construction of a series of H2B-lacZ fusion containing variable portions of the H2B gene. These fusions will then be assayed for localization by IIF. Precise localization will be determined by immuno-electron microscopy using gold labelled antibodies. The second line of experimentration is directed towards understanding the mechanisms of localization. Specifically we will determine whether histones are nuclear limited by asking whether dikaryone, only one of whose nuclei carries a H2B-lacZ gene, exhibit fluorescence over both nuclei. We will also determine whether localization is coupled to the cell division cycle. H2B-lacZ fusions containing the Gal 10 regulatory sequence will be constructed, thereby allowing induction, via galactose, at any point in the cell division cycle. Induction will be performed in asynchronous and synchronous cells as well as in cell division cycle (CDC) mutants.
| Moreland, R B; Langevin, G L; Singer, R H et al. (1987) Amino acid sequences that determine the nuclear localization of yeast histone 2B. Mol Cell Biol 7:4048-57 |
| Moreland, R B; Nam, H G; Hereford, L M et al. (1985) Identification of a nuclear localization signal of a yeast ribosomal protein. Proc Natl Acad Sci U S A 82:6561-5 |
