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Biomedical subjects

H Busch

Publications and source records attributed to H Busch.

At least 127 records · Page 7Linked to original sources
PubMed1985

Purification and partial characterization of a nucleolar scleroderma antigen (Mr = 34,000; pI, 8.5) rich in NG,NG-dimethylarginine.

A new scleroderma antigen of Mr = 34,000; pI, 8.5 has been identified. This 34-kDa protein is a nucleolar protein as determined by immunostaining procedures with affinity-purified antibodies. The 34-kDa protein was shown to localize to the fibrillar regions of the nucleolus by immunoelectron microscopy. Antibodies against the 34-kDa protein precipitate U3 RNA-containing particles. The 34-kDa protein has been isolated from Novikoff hepatoma cell nucleoli by ion exchange and reverse-phase column chromatography. The protein contains 4.1 mol % NG,NG-dimethylarginine (DMA) and 22.8 mol % glycine. It is the most highly arginine-methylated protein thus far detected in higher eukaryotes. This nucleolar 34-kDa protein resembles several nucleoplasmic proteins that are associated with heterogeneous nuclear RNA with respect to isoelectric point, Mr, presence of NG,NG-dimethylarginine, and its high glycine content. The amino-terminal sequence of the first 31 residues of the 34-kDa protein is: Met-Lys-Pro-Gly-Phe-Ser-Pro-DMA-Gly-Gly-Gly-Phe-Gly-Gly-DMA-Gly-Gly- Phe-Gly-Asp-DMA-Gly-Gly-DMA-Gly-Gly-Gly-DMA-Gly-Gly-DMA. In the first 31 residues, there are 16 glycine, 6 DMA, and 3 phenylalanine residues. This is a novel demonstration of clusters of glycine and DMA in a protein.

Amino Acid Sequence↗
PubMed1985

Clustering of glycine and NG,NG-dimethylarginine in nucleolar protein C23.

Protein C23 (Mr 110 000, pI = 5.5), a major phosphoprotein in the nucleolus of mammalian cells, has been shown to contain 1.3 mol% of NG,NG-dimethylarginine (DMA) [Lischwe, M.A., Roberts, K.D., Yeoman, L.C., & Busch, H. (1982) J. Biol. Chem. 257, 14600-14602]. A tryptic peptide from protein C23 that contains DMA has been isolated and sequenced. Its sequence is Gly-Glu-Gly-Gly-Phe-Gly-Gly-DMA-Gly-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA- Gly-Gly- Gly-DMA-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA-Gly-DMA-Gly-Gly-Phe-Gly-Gly- DMA-Gly-Gly-Phe-DMA-Gly-Gly-DMA-Gly-Gly-Gly-Gly-Asp-Phe-Lys. This peptide contains 34 glycine, 10 DMA, and 6 phenylalanine residues and has clusters of glycine and NG,NG-dimethylarginine interspersed with phenylalanine residues. A similar domain has been found at the amino terminus of a nucleolar protein of Mr 34,000, pI = 8.5. This sequence array may represent a conserved domain characteristic of a certain class of nuclear proteins. All of the methylated arginine residues in protein C23, the 34-kilodalton protein, and myelin basic protein [Carnegie, P.R. (1971) Biochem. J. 123, 57-67] have at least one adjacent glycine. Access of certain arginine methylases to arginine residues may be sterically possible because of the lack of a side chain on the adjacent glycine residue(s).

Amino Acid Sequence↗
PubMed1985

Primary and secondary structure of U8 small nuclear RNA.

U8 small nuclear RNA is a new, capped, 140 nucleotides long RNA species found in Novikoff hepatoma cells. Its sequence is: m3GpppAmUmCGUCAGGA GGUUAAUCCU UACCUGUCCC UCCUUUCGGA GGGCAGAUAG AAAAUGAUGA UUGGAGCUUG CAUGAUCUGC UGAUUAUAGC AUUUCCGUGU AAUCAGGACC UGACAACAUC CUGAUUGCUU CUAUCUGAUUOH. This RNA is present in approximately 25,000 copies/cell, and it is enriched in nucleolar preparations. Like U1, U2, U4/U6, and U5 RNAs, U8 RNA was also present as a ribonucleoprotein associated with the Sm antigen. The rat U8 RNA was highly homologous (greater than 90%) to a recently characterized 5.4 S RNA from mouse cells infected with spleen focus-forming virus (Kato, N., and Harada, F. (1984) Biochim. Biophys. Acta, 782, 127-131). In addition to the U8 RNA, three other U small nuclear RNAs were found in anti-Sm antibody immunoprecipitates from labeled rat and HeLa cells. Each of these contained a m3GpppAm cap structure; their apparent chain lengths were 60, 130, and 65 nucleotides. These U small nuclear RNAs are designated U7, U9, and U10 RNAs, respectively.

Animals↗
PubMed1985

U4 small nuclear RNA pseudogenes from rat genome have common truncated 3'-ends.

Four U4 RNA pseudogenes were isolated and characterized from a rat genomic bank. The four pseudogenes contained sequences completely homologous to U4 RNA from nucleotides 1 to 67 and had common truncated 3'-ends. Three of the four pseudogenes were flanked by 14 to 18 nucleotide-long direct repeats. The structural features of these four U4 RNA pseudogenes are consistent with the hypothesis that these pseudogenes arose by RNA self-primed complementary DNA synthesis and integration into the genome (Van Arsdell et al., Cell 26:11-17, 1981).

Animals↗
PubMed1985

Purification of an 86-70 kDa nuclear DNA-associated protein complex.

In the course of studies on nucleolar antigens, monoclonal antibodies were developed, one of which recognized an 86 kDa antigen as shown by analysis of nuclear extracts from HeLa or Namalwa cells. Immunofluorescence studies on HeLa cells showed a nucleoplasmic and phase-dependent nucleolar localization of the monoclonal antibody was decreased after digestion with DNAase I but not with RNAase A. For purification, the antigen was released from nuclei by digestion with DNAase I and then purified by chromatography on DEAE cellulose, phosphocellulose and antibody-Sepharose affinity chromatography. Interestingly, the immunoaffinity purified product contained two polypeptide chains; the immunoreactive polypeptide had an Mr of 86 000 and a pI of 6.0. The complex also contained a 70 kDa, pI 6.5 nonantigenic polypeptide in a 1:1 ratio. The overall purification of the complex was 5700-fold. Both polypeptides contained approx. 15 mol% glutamic acid and the 70 kDa polypeptide contained approx. 15 mol% serine.

Amino Acids↗
PubMed1985

Isolation and characterization of three rat U3 RNA pseudogenes colinear with U3 RNA.

Three different 15-kilobase rat genomic clones that contained sequences colinear with U3 RNA were isolated. These inserts hybridized only to U3 RNA in a mixture of total cellular 4-8 S RNA labeled in vivo which showed that genes or pseudogenes for most other small RNAs were absent in these U3 DNA clones. DNA sequence analysis showed that the three subcloned genes contained full-length U3-coding sequences but each had sequence variations, insertions, and/or deletions when compared to rat U3A or U3B RNA. Two of these pseudogenes contained poly(A) sequences on the 3'-end and were flanked by 6-15-nucleotide long direct repeats. None of the three clones was transcribed when injected into Xenopus oocyte nuclei. One clone was a template for a small RNA slightly larger than U3 RNA, but this transcript was not related to the U3 RNA sequences. The structural features of two of these three U3 DNAs are supportive of the hypothesis that some pseudogenes arose from RNA-mediated DNA synthesis and insertion into the genome at random sites (Van Arsdell, S. W., Denison, R.A., Bernstein, L.B., Weiner, A.M., Manser, T., and Gesteland, R.F. (1981) Cell 26, 11-20). This is the first instance where full-length, colinear, U3 RNA pseudogenes have been isolated and characterized.

Animals↗
PubMed1985

Discrimination of epitopes identified by monoclonal antibodies by competitive binding to nitrocellulose bound antigens.

A new method is described for determining the distribution of epitopes identified by monoclonal antibodies. The method utilizes nitrocellulose membranes as a solid support for antigens which are rapidly adsorbed to nitrocellulose by vacuum-blotting and then used in competitive antibody binding assays. The distribution of epitopes is established by the reciprocal cross-blocking of radiolabeled antibody by increasing concentrations of unlabeled antibody. When unlabeled antibody does not block the binding of labeled antibody to antigen, the 2 antibodies recognize distinct epitopes. When unlabeled antibody blocks the binding of labeled antibody to antigen, the 2 antibodies recognize the same epitope. The method is rapid, sensitive and should be applicable to screening monoclonal antibodies to any epitope.

Antibodies, Monoclonal↗
PubMed1985

Identification and characterization of a polyadenylated small RNA (s-poly A+ RNA) in dinoflagellates.

A 104 nucleotide-long small RNA, referred to as s-poly A+ RNA, containing 30 adenosine residues on its 3' -end was found in dinoflagellates, purified and its nucleotide sequence was determined. The sequence is: (sequence text) The polyadenylation signal AAUAAA was not found in this RNA; this result indicates that the 30 nucleotide-long poly A on the 3' -end is either coded for by this gene, or the poly A chain is added on this small RNA by a mechanism different from that for polyadenylation of messenger RNAs. Two polyadenylated small RNAs identified previously were implicated in differentiation of chicken heart muscle cells (Deshpande, A. K., Jakowlew, S. B., Arnold, H., Crawford, P. A. and Siddiqui, M. A. Q. (1977) J. Biol. Chem. 252, 6521-6527), and in brain specific mRNA transcription (Sutcliffe, J. G., Milner, R. J., Gottesfeld, J. M. and Lerner, R. A. (1984) Nature 309, 237-241). This RNA is the first polyadenylated small RNA to be sequenced.

Animals↗
PubMed1985

A new moderately repetitive rat DNA sequence detected by a cloned 4.5 SI DNA.

4.5 SI RNA is an abundant, noncapped, small nuclear RNA found in rodent cells. The 4.5 SI RNA is 98 or 99 nucleotides long and contains no modified nucleotides; it is synthesized by RNA polymerase III, is partly hydrogen-bonded to poly(A+) hnRNA, and was the first small nuclear RNA to be purified and sequenced (Busch, H., Reddy, R., Ruthblum, L., and Choi, Y. C. (1982) Annu. Rev. Biochem. 51, 617-654). In studies on the structure and organization of genes coding for this abundant RNA, it was found that this RNA is homologous to an apparently novel family of repetitive sequences. Two clones were characterized; one clone showed that its sequence is identical to the RNA in the first 92 residues and differed only in the last six nucleotides. In addition, the 3'-end of the sequence contained an A,T-rich region, and the sequence was flanked by a 15-nucleotide long direct repeat of AAAATATAGACACTG. The second clone characterized contained nucleotide sequences 1-57 corresponding to the RNA and was flanked by a 15-nucleotide long direct repeat. The structural features of these two DNAs are consistent with RNA-mediated DNA synthesis and integration of this DNA into the genome at random sites. It is estimated that there are about 10,000 copies of this family of sequences in the haploid rat genome.

Animals↗
PubMed1985

Topoisomerase I phosphorylation in vitro and in rapidly growing Novikoff hepatoma cells.

Changes in phosphorylation modulate the activity of topoisomerase I in vitro. Specifically, enzymatic activity is stimulated by phosphorylation with a purified protein kinase (casein kinase type II). The purpose of this study was to compare the sites that are phosphorylated in vitro by casein kinase type II with the site(s) phosphorylated in vivo in rapidly growing Novikoff hepatoma cells. Topoisomerase I labeled in vitro was characterized by three major tryptic phosphopeptides (I-III). Separation of these peptides by a C18-reverse phase h.p.l.c. column resulted in their elution at fractions 18 (I), 27 (II) and 44 (III) with 17%, 22.5% and 33% acetonitrile, respectively. In contrast, only one major phosphopeptide was identified by h.p.l.c. in topoisomerase I labeled in vivo. This phosphopeptide eluted at fraction 18 corresponding to the elution properties of phosphopeptide I labeled in vitro. It also co-migrated with tryptic phosphopeptide I when subjected to high-voltage electrophoresis on thin-layer cellulose plates. Preliminary experiments suggest that phosphorylation occurs at a serine residue six amino acids from the N-terminus of the peptide. These data indicate that topoisomerase I is phosphorylated in vivo and in vitro within the same tryptic peptide and suggest that topoisomerase I is phosphorylated in vivo by casein kinase II.

Amino Acid Sequence↗
PubMed1985

Epitope distribution and immunochemical characterization of nucleolar phosphoprotein C23 using ten monoclonal antibodies.

Extractable nucleolar proteins from HeLa cells were used as a source of antigen to immunize mice for monoclonal antibody (MAb) production. Ten of the resulting MAbs shown to identify nucleolar phosphoprotein (110 kD/pI 5.5) were purified and used in immunochemical studies to further characterize protein C23. All ten MAbs showed nucleolar localization by indirect immunofluorescence; one antibody (FR2) also showed some nucleoplasmic localization that was attributed to a shared epitope between protein C23 and a 72 kD nuclear/nucleolar antigen. Reciprocal antibody cross blocking studies indicated that the ten MAbs identified nine distinct epitopes on protein C23. Interestingly, seven of the nine epitopes were shown by immunofluorescence and competitive ELISA studies to be species related. Immunostained patterns of exponentially growing HeLa cells suggest that protein C23 exists in vivo solely as a 110 kD peptide. However, protein C23 was subject to rapid degradation into a number of proteolytic fragments upon extraction or storage of isolated nucleoli. The failure to find protein C23 related peptides with molecular sizes less than 110 kD in exponentially growing cells and the lack of cytoplasmic localization of any of the ten MAbs suggests that protein C23 is not a prepro-protein processed in vivo to form ribosomal proteins as previously suggested (1).

Animals↗
PubMed1985

Nucleologenesis: composition and fate of prenucleolar bodies.

A time course study was conducted on nucleologenesis after release from a mitotic block in the presence and absence of actinomycin D to determine the composition and fate of prenucleolar bodies (PNBs). Prenucleolar bodies, whether naturally occurring or induced by actinomycin D treatment, stain with silver and contain phosphoproteins B23 and C23, two of the major proteins of the interphase nucleolus as determined by double label immunofluorescence with specific antibodies. The nucleolus is formed by fusion of PNBs, which subsequently "reorganize" and form internal fibrillar and peripheral granular regions. Actinomycin D prevents fusion of PNBs, which are then randomly dispersed throughout the nucleus but they still contain proteins B23 and C23. These results demonstrate that the nucleolus is formed by fusion of prenucleolar structures whose biochemical composition resembles the mature nucleolus, since PNBs contain at least two of the major nucleolar proteins.

Animals↗
PubMed1985

Alterations in immunolocalization of the phosphoprotein B23 in HeLa cells during serum starvation.

Bright nucleolar immunofluorescence was observed in HeLa S3 cells by immunostaining with a monoclonal antibody to the nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1). After 48 h of incubation in a serum-free medium, the nucleolar fluorescence was diminished and a general nuclear immunofluorescence was observed. This change in localization of fluorescence indicated that protein B23 had migrated out of the nucleoli. No gross morphological change in nucleoli was observed by light microscopy and the immunolocalization of another nucleolar phosphoprotein, C23, was unaffected by serum deprivation. Relocation of protein B23 in nucleoli was observed after refeeding with serum-containing medium. This re-entry process was not observed after treatment with actinomycin D (50 ng/ml-5 micrograms/ml), but the process was unaffected by cycloheximide (0.2 mM). Quantitation of protein B23 in the nucleoli of the control (fed) or starved HeLa cells was done by ELISA immunoassay. A marked decrease in the amount of protein B23 occurred in the nucleoli of the starved cells (11.8 micrograms B23/mgDNA) as compared with the control nucleoli (20.8 micrograms B23/mgDNA). The amount of protein B23 in the nucleoplasm (excluding nucleoli) was 70% higher in the starved cells. Protein B23 was analysed by one- and two-dimensional PAGE. Three components of protein B23 with slightly different molecular weights and pIs (37 kD/5.1, 35 kD/5.1 and 35 kD/5.3) were observed in nucleoli. The lower molecular weight components were predominantly found in the nucleoplasm.

Blood↗
PubMed1985

Detection of a 140 KDA nucleolar protein with an anti-PCNA autoantibody.

To assess the numbers and types of PCNA (proliferating cell nuclear antigen) species, immunoprecipitation studies on HeLa cell, nuclear and nucleolar extracts were performed. A 140 KDa protein from HeLa nucleoli was immunoprecipitated by an autoantibody (E.B.) previously used to detect the proliferating cell nuclear antigens (PCNA). The 140 KDa protein was also detected in the nuclear extract of colon carcinoma cells (omega) labeled in vitro with 125I-Bolton Hunter reagent. When the growth of the colon carcinoma cells was inhibited by 1% N,N-dimethylformamide for two weeks, the 140 KDa protein was not detected which suggests this protein is associated with cell growth.

Autoantibodies↗
PubMed1985

Fibrillarin: a new protein of the nucleolus identified by autoimmune sera.

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.

Animals↗
PubMed1985

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↗