Nuclear antigenic structures.
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Antibodies, Neoplasm↗
Biomedical subjects
H Busch
Publications and source records attributed to H Busch.
Electrophoretic analysis of HeLa cell and human liver nucleolar proteins and antigens.
The major antigens in HeLa nucleolar extracts recognized by immunoblots with rabbit antisera had molecular weights of 145, 110, and 34 kd. The major antigens in the HeLa nucleolar residues had molecular weights of 145, 110, 86, 68, 55, 48, and 34 kd. In the liver, the major antigens in the nucleolar extracts had molecular weights of 86 and 76 kd. In the liver residues, the major antigens had molecular weights of 110, 86, 76, 65, and 55 kd. On two-dimensional gels stained with Coomassie blue, the HeLa nucleolar extract contained large amounts of protein B23 and lesser amounts of protein C23. In the liver nucleolar samples separated on two-dimensional (2-D) gels, protein 55/7.6 (Mr/pI) was the major protein in the extract. Lesser amounts of protein B23 were identified. Addition of protease inhibitors markedly improved the quality of the protein samples as shown in one-dimensional gel patterns for liver nucleolar proteins and to a lesser extent for HeLa nucleolar proteins. In the 2-D immunoblots of the HeLa extract and HeLa residues, the major stained band was protein C23 (110/5.2-5.6). In the liver extract, the major bands were 70/5.8-7 and 60/5.8-7. With a monoclonal antibody (MS-3) to protein C23 a 76/5.8 band was more notable in the residue which supported the results of the Coomassie blue and immunostains with rabbit antinucleolar antibodies. Some degradation products of protein C23 were observed in both the liver extract and the liver residue despite the use of protease inhibitors. Protein C23 along with other proteins are major antigens in HeLa nucleoli. In human liver nucleoli, a major protein 55/7.6 was identified which was not observed in the HeLa extracts. These studies show that a combination of protease inhibitors markedly reduced degradation of proteins in liver samples and provided a more satisfactory sample for comparison with HeLa cells. Qualitative and quantitative differences were found in the nucleolar proteins by Coomassie blue and immunostaining.
Antigens↗
Masking of nontumorous antigens for development of human tumor nucleolar antibodies with improved specificity.
Nucleoli purified from HeLa cells were spread by homogenization in low ionic strength buffer and by chelation of divalent cations. The antigenic determinants of these tumor nucleoli that are shared by normal liver nucleoli were masked by addition of rabbit anti-liver nucleolar antisera to form immune complexes. New Zealand White rabbits were immunized with these antibody-nucleolar complexes. The resulting antisera produced nucleolar fluorescence in HeLa cells but not in normal human liver or human kidney cells. One- and two-dimensional immunoblots identified a major Mr 58,000/pl 5.8 antigen in HeLa cells, which was not detected on corresponding immunoblots of normal human liver nucleoli. This method offers an improved approach to selection of tumor-associated antigens.
Animals↗
Multiple states of U3 RNA in Novikoff hepatoma nucleoli.
U3 RNA, a capped small nuclear RNA found thus far only in the nucleolus, has been implicated in the processing and/or transport of preribosomal RNA [Busch, H., Reddy, R., Rothblum, L., & Choi, Y. C. (1982) Annu. Rev. Biochem. 51, 617-654]. Tris(hydroxymethyl)aminomethane (Tris) (10 mM, pH 7.0) extracts of Novikoff hepatoma nucleoli, which contained about 80% of total nucleolar U3 RNA, were analyzed by sucrose density gradient centrifugation. Approximately 65% of the U3 RNA was bound to greater than 60S preribosomal ribonucleoprotein (RNP) particles, and about 15% sedimented at less than 20 S. The association between the 65% of U3 RNA that was bound to the preribosomal RNP particles was stable up to 55 degrees C. About 10% of U3 RNA was base paired to preribosomal RNA after deproteinization at 22 degrees C. The base-paired fraction of U3 RNA was released from the preribosomal RNA by heating to 45 degrees C or treating with 4 M urea. These results show that of the total nucleolar U3 RNP, (a) about 55% is bound to preribosomal RNP particles primarily by protein interactions, (b) about 10% is base paired to preribosomal RNA, (c) approximately 15% sedimented slowly and consisted presumably of free U3 RNP particles, and (d) the remaining 20% of U3 RNP was not extractable using 10 mM Tris buffer. On the basis of the different association states of U3 RNP particles, a model is proposed for the binding and dissociation events which take place between U3 RNP and preribosomal RNP particles.
Animals↗
Actinomycin D inhibition of monoclonal antibody binding to nucleolar phosphoprotein 37/5.2 (B23).
Actinomycin D was found to block binding of a monoclonal antibody to nucleolar phosphoprotein 37/5.2 (B23) when incubated either simultaneously with or prior to addition of the antibody. Daunorubicin had no significant blocking activity of this antigen-antibody reaction. In comparative studies with a monoclonal antibody to nucleolar phosphoprotein 110/5.2 (C23), actinomycin D exhibited little blocking activity. When a 42-mer peptide containing the epitope of protein 37/5.2 (B23) was tested as the antigen, similar inhibition by actinomycin D of binding of the monoclonal antibody was found. These results provide evidence for binding of actinomycin D to a specific epitope of protein 37/5.2 (B23).
Amino Acid Sequence↗
Primary and secondary structure of 7-3 (K) RNA of Novikoff hepatoma.
7-3 RNA (also known as K-RNA and 7SK-RNA) is a distinct small RNA found in insect to mammalian cells. Previous studies showed that this RNA is not capped, contains no modified nucleotides, is conserved through evolution, is synthesized by RNA polymerase III, and, in part, is associated by polyribosomes. In this study, the complete nucleotide sequence of 7-3 RNA was determined by RNA-sequencing methods, and the sequence is compared with several small RNAs and repetitive DNA sequences for homology. This 330-nucleotide-long RNA contained pppGp as its 5' terminus and exhibited heterogeneity with respect to the 3'-terminal AoH. The nucleotide sequence is: (sequence in text) The RNA is G-C rich, and evidence is presented that 7-3 RNA is in a ribonucleoprotein particle in the cytoplasm.
Animals↗
Purification and partial characterization of a 19kD/pI 4.5 nucleolar phosphoprotein.
Two-dimensional PAGE analysis of proteins associated with the slowly sedimenting "fibrillar" structures of HeLa nucleoli revealed a protein with a M of 19,000 and a pI of 4.5 which was highly labeled both with 32P-orthophosphate and 35S-methionine. The protein was isolated from Novikoff hepatoma nucleoli by extraction in 0.35 M NaCl and 5 mM DTT followed by chromatography in EDTA on DEAE-cellulose and Sephadex G-100. The protein was homogeneous with respect to two-dimensional PAGE, number of tryptic peptides and carboxyl terminal analysis. The protein contained an acidic/basic amino acid ratio of 2.1, 7 residues of methionine, 2 residues of cysteine, a blocked amino terminus and a carboxyl terminal lysylleucine.
Amino Acids↗
Characterization of rat ribosomal DNA. The highly repetitive sequences that flank the ribosomal RNA transcription unit are homologous and contain RNA polymerase III transcription initiation sites.
The non-transcribed spacers (NTS) of the ribosomal genes of a number of organisms have been studied and were found to contain repetitive sequences. In these studies with plasmid subclones of NTS, designated p3.4, p2.6 and p1.7, which come from both 5' and 3' flanking regions of the rat ribosomal genes, respectively, it has been determined that these sequences are found elsewhere within the genome. Southern hybridization analysis has demonstrated that the 5' and 3' NTS subclones cross-hybridize, and that the cross-hybridizing regions are synonymous with the highly repetitive regions. Sequences homologous to the rat NTS were specifically localized to both 5' and 3' flanking regions as well as to a number of the introns of cloned genes including rat serum albumin, rat alpha-fetoprotein, rat casein and human serum albumin. No hybridization was detected of the 5' NTS subclone to the human Alu sequence clone, Blur 8, or to the rodent equivalent, a clone containing Chinese hamster ovary type I and II Alu sequences. However, as reported for type II Alu sequences, the subcloned rat NTS sequences contain RNA polymerase III initiation sites and also hybridize to a number of small RNAs, but not 4.5 S or 7 S RNA. Sequence analysis of two distinct repetitive regions in p1.7 has revealed a region of alternating purine-pyrimidine nucleotides, potentially of Z DNA, and stretches of repetitive sequences. The possible roles for these repetitive sequences in recombination and in maintaining a hierarchical structure for the ribosomal genes are discussed.
Animals↗
Identification of a tumor inhibitory factor in rat ascites fluid.
A polypeptide which inhibits the growth of human carcinoma cells has been characterized from Novikoff rat ascites fluid. This tumor inhibitory factor co-purified with transforming growth factor activity through acid/ethanol extraction and Bio-Gel chromatography. The two activities were completely separated by reverse phase HPLC. The tumor inhibitory factor is heat stable and requires disulfide bonds for bioactivity. This factor inhibited the anchorage independent growth of the more differentiated human colon carcinoma cell lines but did not affect the less differentiated carcinoma cells. The presence of stimulatory and inhibitory activities in the same extracts suggests that the relative concentrations of these factors may be important in the control of cell growth.
Animals↗
Primary and secondary structure of dinoflagellate U5 small nuclear RNA.
U5 RNA is one of the six capped small nuclear RNAs present in most eukaryotic cells. Like U1, U2, U4 and U6 RNAs, U5 RNA is associated with hnRNP particles and is thus probably involved in some, as yet undefined, aspects of pre-messenger RNA processing. In this study, the complete nucleotide sequence of U5 RNA of a dinoflagellate, Crypthecodinium cohnii was determined. The analysis of this dinoflagellate U5 RNA sequence showed that a) the sequence homology between human, rat and chicken U5 RNA sequences and dinoflagellate U5 RNA sequence is 64%; b) the extent and the position of post-transcriptional modifications are similar to those found in U5 RNA of higher eukaryotes; c) although the dinoflagellate U5 RNA is shorter in length (108 nucleotides long vs 117 long in human, rat and chicken cells), the RNA fits well into the same secondary structure proposed for U5 RNA of higher eukaryotes (Krol et al. (1981) Nucl. Acids Res. 9, 769); and d) the AUn nucleotide sequence protected by the Sm-antigen and the tight secondary structure found near the 3'-end of other U-RNAs was also found in dinoflagellate U5 RNA. The high order of homology observed between dinoflagellate U5 RNA and U5 RNA of higher eukaryotes indicates that dinoflagellates are more closely related to metazoans than to early eukaryotes.
Animals↗
Molecular lesions in cancer.
Newer methods of identifying biochemical events associated with cancer include recombinant DNA technology, monoclonal antibodies and improved analysis of nuclear and other cell functions to determine specific events which occur commonly in cancer cells. 'Onc-gene' products offer potential opportunities for new approaches to cancer treatment and the hope of inducing differentiation of cancer cells toward their normal counterparts. Studies on antigens which react with monoclonal antibodies offer the opportunity for 'epitope attack' which may be effected by improved drugs or by design of totally new drugs to bind to specific reactive sites. The complexity and pleiomorphism of cancer do not permit predictions as to whether these approaches will be more effective than the empirical approach to cancer treatment.
Animals↗
Interactions of U1 RNP with heterogeneous nuclear RNA in rat Novikoff hepatoma nuclei.
The nature of the association of U1 RNA with rapidly sedimenting RNP structures in rat hepatoma nuclei was investigated. The effects of salt and proteinase K treatment on the stability of this 'bound' form of U1 RNA were studied using sucrose density gradient analyses. Quantitation of the amount of U1 RNA remaining associated with large structures after treatment was used to assess the relative contribution of protein-protein (and protein-RNA) versus RNA-RNA interactions. Forty-eight percent of the total nuclear U1 RNA released by sonication was found in a 'bound' form when the sonicate was centrifuged through gradients containing 50 mM NaCl. Fifty percent of this 'bound' U1 RNA remained associated with rapidly sedimenting RNPs when the NaCl concentration was raised to 500 mM. To assess the contribution of protein independent interactions, large RNPs were completely deproteinized and their RNA moieties were then recentrifuged on gradients. By this analysis, 27% of the U1 RNA originally 'bound' to hnRNPs was associated with rapidly sedimenting (greater than 30 S) RNA (at 50 mM NaCl) suggesting their association by RNA-RNA hydrogen bonds. When the concentration of NaCl was 500 mM, 31% of the U1 RNA was associated with large RNA. Hence, approximately 30% of the U1 RNA molecules originally 'bound' (or about 15% of the total nuclear U1 RNA) were found to be associated by RNA-RNA hydrogen bonds while the remainder of the binding of U1 RNP to hnRNP was by protein-protein and/or protein-RNA interactions.
Animals↗
Purification of p53/55 kinase from nuclear ribonucleoproteins of Namalwa cells.
A nuclear p53/55 protein kinase has been isolated from nuclear ribonucleoprotein particles from human tumor cells. The enzyme was purified approximately 2200-fold cell nuclei by sequential ribonuclease digestion of the RNP particles, DEAE cellulose and phosphocellulose chromatography. The kinase which was cAMP independent, catalyzed the phosphorylation of rabbit muscle glycogen synthase in the amino terminal domain, and conversion of the I to D form. The D synthase had a phosphorylation stoichiometry of 8 moles 32P per mole of synthase subunit with maximal specificity for ATP as phosphate donor; its Km was 30 microM. An antinucleolar antibody inhibited enzyme activity by 80%. Substrates for most other kinases were inactive. The kinase was essentially unaffected by the Walsh inhibitor, EGTA, regulatory subunits of protein kinase, calmodulin, trifluoperazine or heparin. Its activity was lost at 1 mM polyamine, but was enhanced 3-fold by MnCl2 and 4- to 9-fold by deoxymononucleotides. The nuclei of HeLa cells contained 64% of the total kinase of which 64% of the total kinase of which 11% were in nucleoli; the specific activity of the nucleolar kinase was twice that of the nuclear supernatant and four times that of the cytoplasmic kinase. These results indicate that nucleolar ribonucleoprotein particles of human tumor cells contain a cAMP-independent protein kinase which is similar to glycogen synthase kinase.
Burkitt Lymphoma↗
Silver staining, immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23.
Nucleolar organizer region (NOR)-specific silver staining and immunolocalization of nucleolar phosphoproteins B23 and C23 were compared in Novikoff hepatoma ascites cells. Silver staining and protein C23 immunostaining were both localized in the fibrillar shell surrounding the fibrillar center and in the fibrillar center. During mitosis, silver staining and protein C23 were localized at the NORs. Therefore, protein C23 and the silver-staining protein both seem to be associated with rDNA-containing structures (Mirre and Stahl 1981). A comparison of toluidine blue staining specific for RNA and B23 immunostaining demonstrated that protein B23 was associated with RNA-containing regions of the nucleolus and was absent from the fibrillar centers. Localization of these proteins and their functions are discussed in relation to the organization of the nucleolus.
Animals↗
Onc genes and other new targets for cancer chemotherapy.
Recent advances in molecular biology have raised the hope that understanding of human cancer might progress rapidly and that improvements in therapy might result (Bishop 1983a, b; Busch 1962; Busch 1976; Duesberg 1983). With the development of gene cloning, DNA sequence analysis and improved hybridization methods, it became possible to evaluate whether cancer results from alteration in gene dosage, point or multiple mutation of genes, translocations, deletions, insertions, inversions, cis or trans altered promoters, amplification, and a variety of other genetic factors, including enhancer elements that alter rates of readouts of particular mRNA species. "Onc genes" are under intensive study because they offer manageable probes for evaluation of these various possibilities and also because the study of their cellular analogs may further understanding of the molecular biology of normal fetal and malignant cells. Despite the excessive enthusiasm of some proponents of this field and the negativism of its critics (Bishop 1983 a, b; Duesberg 1983), it is clear that analytical tools and new information will be of value in further studies on experimental cancer, regardless of whether cellular oncogenes (c-onc genes) have anything to do with human cancer or not. In the meantime, studies on enzymes, proteins and epitopes involved in growth processes, have opened new avenues for inhibition of human cancer by quantitative reduction of biosynthetic reactions.
Animals↗
Immunofluorescence studies on proteins B23 and C23 in nucleoli of human lymphocytes.
Nucleoli of normal and leukemic lymphocytes were studied by cytochemical and immunofluorescence methods to provide more information on the nucleolar presence and distribution of proteins B23 and C23. Annular nucleoli of human lymphocytes represent a very convenient subject for such studies, since they consist of one centrally located large fibrillar center surrounded by RNP components. In such nucleoli, protein C23 was present mainly in the central nucleolar region and protein B23 was found mostly in the periphery. The nucleolar area immunostained for protein B23 was usually larger than that stained for protein C23. The distribution of protein C23 appeared to be similar to that of intensely stained nucleolar argyrophilic components. No substantial differences were found between the distribution of proteins B23 and C23 in nucleoli of normal and leukemic lymphocytes. In lymphocytes of patients treated with chemotherapy, the immunofluorescence was diminished for protein B23 and particularly so for protein C23.
Cell Nucleolus↗
Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein.
Previously we demonstrated a similar distribution between nucleolar organizing region-(NOR)-specific silver staining and localization of nucleolar phosphoprotein C23 (MW 110 kD/pI 5.1) [1, 2]. We now report that under fixation conditions which allow for antibody binding and subsequent silver staining, monoclonal antibody against protein C23 blocks NOR silver staining as well as silver staining in interphase nucleoli. Monoclonal antibody against nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1) did not block silver staining in either NORs or interphase nucleoli. These, along with earlier observations, provide evidence that nucleolar phosphoprotein C23 is the major silver staining protein of the nucleolus and that it is directly or indirectly associated with rDNA.
Animals↗
Further studies on satellite nucleoli in rat and mouse hepatocytes.
To provide more information on satellite nucleoli, these nuclear structures were studied by means of cytochemical and immunofluorescence procedures in rat and mouse hepatocytes without and following experimental inhibition of the RNA synthesis. The immuno-staining specific for nucleoli or B23 as well as C23 proteins demonstrated that satellite nucleoli and characteristic nucleoli exhibit the same fluorescence. The number of satellite nucleoli decreased after inhibition of nucleolar RNA synthesis in a similar way to the number of silver-stained granules (SSGs) of characteristic nucleoli. Inhibition of RNA synthesis also reduced the number of hepatocytes containing satellite nucleoli. Thus, satellite nucleoli seem to be real nucleoli from single NORs which did not fuse in the formation of a characteristic nucleolus.
Animals↗