The primary object of the proposed studies is to determine quantitative and qualitative differences in nucleolar proteins of cancer cells, normal growing cells and normal resting cells normal as related to mechanisms involved in the 15-100 fold greater pre- rRNA synthesis in cancer cells compared to normal cells. With improved techniques for removal of immunodominant antigens, we developed a """"""""library"""""""" of monoclonal antibodies to nucleolar proteins. We demonstrated that tumor cells contain nucleolar proteins not found in normal resting cells, e.g., antigens P145, P120, P86/70, and P40. We will evaluate the use of these monoclonal antibodies for isolation and analysis of the functions of these antigens, immunodetection of cancer and for isolation and sequence analysis of these antigens. We will study the roles of these antigens in RNA and DNA synthesis, gene control, processing, transport, topoisomerase, and oncogene related activities in nucleoli during the mitogenic cascade. We are utilizing these antibodies in studies on inhibition or acceleration of in vitro RNA and DNA synthesis in isolated nuclei and nucleoli and in vivo using microinjection techniques. We are analyzing cDNA clones that express antigens P120, P86/70, P40 and topoisomerase I and other nucleolar antigens. We will use these gene clones to determine the amino acid sequences of these antigens and for further studies on gene control mechanisms involved in the activation and timing of the readout of these genes. We will evaluate the effects of these antibodies on the synthesis and gene controls for rRNA, U3 snRNA, and U8 snRNA, and the functions of their RNPs. Studies are being made on activity on promoters of rRNA genes (-170 to +15), particularly the holopromoter which has a core promoter element (CPE) and an upstream promoter element (UPE). Deletion mutants are being used to map the upstream promoter element of rat rDNA between nucleotides -114 and -167 (+1 being the transcription initiation site). U3 and U8 small nuclear RNAs (snRNAs) are localized to nucleoli of eukaryotic cells and are synthesized 10-15 times faster in tumor cells than in normal cells. This investigation is designed to understand the factors involved in the increased synthesis of nucleolar pre-ribosomal ribonucleoprotein particles (pre-rRNPs) in cancer cells and particularly the role of U3 snRNPs, U8 snRNPs and other nucleolar small RNP particles, in the hyperactivity of cancer cell nucleoli.
| Rosenberg, N L (1986) Isolation of an MSP1-derived transcriptionally active nucleolar particle. Exp Cell Res 165:41-52 |
