The aim of the proposed research is to analyze the structures and elucidate the functions of two major nucleolar nonribosomal, nonhistone proteins, B23 (Mr 38,000) and C23 (Mr 110,000). The two proteins are believed to organize the various components of the nucleolus. They contain highly acidic, phosphorylated regions, they are found in nucleolar preribosomal particles and they have a high affinity for silver. Protein C23 is located at the nucleolus organizer regions of chromosomes and binds DNA with an apparent preference for sequences upstream from the 18 S expressed region. Approximately 15% of the sequence of protein C23 and the location of major fragments have been determined. The major goal of this study is to complete the sequence of protein C23. This will be achieved by using known sequence information to synthesize oligonucleotide primers for production of complementary DNA from protein C23 messenger RNA. The cDNA will be cloned in appropriate vectors, the DNA will be sequenced and the amino acid sequence will be deduced from the DNA sequence. The location of the approximately 35 phosphoryl groups will be determined by automated protein sequencing. A similar approach will be applied to protein B23; i.e., partial protein sequencing, cDNA cloning and DNA sequencing. Additional studies related to the structure and function of these two proteins will be conducted: (1) the DNA binding domains of protein C23 will be determined by assaying large fragments of the protein for DNA binding activity and by proteolysis of the protein C23-DNA complex; DNA binding domains will be identified by automated protein sequencing; (2) the native molecular weight will be determined by sedimentation studies and chemical crosslinking; (3) the secondary structure of protein C23 and its fragments will be studied by circular dichroism; and (4) the interactions of these proteins with other macromolecules will be determined by use of cleavable chemical crosslinkers and photo crosslinking. These studies will contribute to the long range goal of understanding the structure and function of the nucleolus. Because the nucleolus is highly susceptible to biochemical and morphological alterations in neoplastic processes and in drug treatment, these studies should aid in elucidation of molecular mechanism of disease and chemotherapy.
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