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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM028349-07A1
Application #
3275635
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1980-07-01
Project End
1990-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
7
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Mississippi Medical Center
Department
Type
Schools of Medicine
DUNS #
928824473
City
Jackson
State
MS
Country
United States
Zip Code
39216
Szebeni, A; Olson, M O (1999) Nucleolar protein B23 has molecular chaperone activities. Protein Sci 8:905-12
Dundr, M; Olson, M O (1998) Partially processed pre-rRNA is preserved in association with processing components in nucleolus-derived foci during mitosis. Mol Biol Cell 9:2407-22
Savkur, R S; Olson, M O (1998) Preferential cleavage in pre-ribosomal RNA byprotein B23 endoribonuclease. Nucleic Acids Res 26:4508-15
Xie, J; Briggs, J A; Morris, S W et al. (1997) MNDA binds NPM/B23 and the NPM-MLF1 chimera generated by the t(3;5) associated with myelodysplastic syndrome and acute myeloid leukemia. Exp Hematol 25:1111-7
Szebeni, A; Mehrotra, B; Baumann, A et al. (1997) Nucleolar protein B23 stimulates nuclear import of the HIV-1 Rev protein and NLS-conjugated albumin. Biochemistry 36:3941-9
Dundr, M; Meier, U T; Lewis, N et al. (1997) A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase. Chromosoma 105:407-17
Herrera, J E; Correia, J J; Jones, A E et al. (1996) Sedimentation analyses of the salt- and divalent metal ion-induced oligomerization of nucleolar protein B23. Biochemistry 35:2668-73
Bharti, A K; Olson, M O; Kufe, D W et al. (1996) Identification of a nucleolin binding site in human topoisomerase I. J Biol Chem 271:1993-7
Xie, J; Briggs, J A; Olson, M O et al. (1995) Human myeloid cell nuclear differentiation antigen binds specifically to nucleolin. J Cell Biochem 59:529-36
Szebeni, A; Herrera, J E; Olson, M O (1995) Interaction of nucleolar protein B23 with peptides related to nuclear localization signals. Biochemistry 34:8037-42

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