The long term objectives of this work are to understand the relationship of the nuclear lamina to nuclear envelope and chromosome structure, and to comprehend the mechanisms of nucleocytoplasmic transport of macromolecules by the pore complex. Lamina-related investigations will focus on defining the molecular interactions of the three polypeptides that comprise the lamina (the lamins), and the biochemical mechanisms for modulation of lamina structure. The organization of the lamins will be examined with ultrastructural and biochemical techniques, and by lamina subunit reconstitution. Furthermore, the interaction of the lamina with the inner nuclear membrane will be examined in membrane reconstitution studies, and the lamina polypeptides and DNA sequences mediating the lamina-chromatin association will be studied by molecular crosslinking and subnuclear fractionation. Finally, the disassembly of the lamina during cell division by phosphorylation will be analyzed in mitotic cell-free systems. Pore complex-related investigations will identify and characterize pore complex polypeptides involved in structure and transport functions. A pore complex-enriched fraction of the nuclear envelope will be used to prepare monoclonal antibodies, to identify a nunber of polypeptides occurring in different structural domains of the pore complex with electron microscopic immunocytochemistry. Gp190, a pore complex-associated intrinsic membrane protein, will be studied in vitro translation and protein crosslinking to define its membrane topology and polypeptide interactions. Finally, a putative pore complex-associated protein kinase and its substrates will be studied by in vivo and in vitro phosphate labelling, and monoclonal antibodies will be prepared to the kinase and selected substrates for immunocytochemical localization and for use as probes in investigations of interactions between macromolecules and the pore complex in vitro.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Modified Research Career Development Award (K04)
Project #
5K04HD000573-03
Application #
3073138
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1984-05-01
Project End
1989-04-30
Budget Start
1986-05-01
Budget End
1987-04-30
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218