Nuclear RNA-associated proteins and their relationship to the nuclear matrix and related structures in HeLa cells

1985 ◽  
Vol 63 (6) ◽  
pp. 631-643 ◽  
Author(s):  
Dominique Bouvier ◽  
Jean Hubert ◽  
Annie-Pierre Sève ◽  
Michel Bouteille
Keyword(s):  
Nuclear Matrix ◽  
Dnase I ◽  
High Salt ◽  
Relative Mass ◽  
Two Dimensional Gel Electrophoresis ◽  
Basic Proteins ◽  
Salt Extraction ◽  
Nuclear Rna ◽  
Associated Proteins ◽  
Rnase Digestion

The ultrastructure and the polypeptide composition of residual nuclear substructures including nuclear matrices, nuclear ghosts, and residual envelopes were investigated by means of electron microscopy and two-dimensional gel electrophoresis. Nuclear matrices were prepared by digesting isolated nuclei with DNase I alone, followed by high-salt extraction in 2 M NaCl. Nuclear ghosts were obtained by high-salt extraction of nuclei previously digested with DNase and RNase in MgCl2-containing buffers. To prepare residual envelopes, nuclei were first digested with RNase in the presence of EDTA, then digested with Mg2+-activated DNase I, and extracted in 2 M NaCl. The results of this comparative study support the conclusion that the intranuclear matrix is made of two distinct RNA-containing elements. One of these elements appears on ultrathin sections as a thin fibrillar network. It disappears from RNase-digested nuclei, together with numerous basic proteins, whatever the conditions of digestion. Although this element is present in extranucleolar territories, it is a major component of residual nucleoli. The second element appears as coarse-beaded fibers absent from the nucleolar areas. Its preservation in residual nuclear substructures depends on the presence of Mg2+ ions during RNase digestion of nuclei. It is enriched in two minor basic proteins of relative mass 49 000 and 70 000. The involvement of this fibrogranular element in heterogeneous nuclear RNA attachment to the nuclear matrix is discussed.

Protein composition of nuclear matrix preparations from HeLa cells: an immunochemical approach

1986 ◽  
Vol 80 (1) ◽  
pp. 103-122
Author(s):  
R. Verheijen ◽  
H. Kuijpers ◽  
P. Vooijs ◽  
W. van Venrooij ◽  
F. Ramaekers
Keyword(s):  
Nuclear Matrix ◽  
Connective Tissue Diseases ◽  
Protein Composition ◽  
Cytoskeletal Proteins ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Core Proteins ◽  
Nuclear Rna ◽  
Hela S3 ◽  
Protein Components

Procedures for the isolation of HeLa S3 nuclear matrices were re-examined with special emphasis on the use of various nucleases and detergents as well as on the ionic strength of the final salt extraction. The protein composition of the resulting nuclear matrix preparations was analysed by one- and two-dimensional gel electrophoresis and found to be extremely reproducible. By means of co-electrophoresis several typical cytoskeletal proteins (actin, vimentin and cytokeratins) and heterogeneous nuclear RNA (hnRNA)-associated core proteins (hnRNP) were shown to be present in such nuclear matrix preparations. The nature of some other protein components was elucidated using two-dimensional immunoblotting and immunofluorescence. For this purpose mouse monoclonal antibodies to cytoskeletal components (vimentin, cytokeratins), small nuclear RNP (70 X 10(3) Mr protein of U1-RNP), hnRNP (C1/C2) and the pore-complex lamina (lamins A, B and C) were used next to human autoimmune sera obtained from patients with connective tissue diseases and directed against the residual nucleoli and the internal fibrillar mass. These antibodies enabled us to identify a number of proteins present specifically in the nuclear matrix and to show that part of the cytoskeletal proteins are still present in the isolated structures.

Adenoviral heterogeneous nuclear RNA is associated with the host nuclear matrix during splicing

1982 ◽  
Vol 154 (1) ◽  
pp. 103-119 ◽  
Author(s):  
Edwin C.M. Mariman ◽  
Chris A.G. van Eekelen ◽  
Rita J. Reinders ◽  
Anton J.M. Berns ◽  
Walther J. van Venrooij
Keyword(s):  
Nuclear Matrix ◽  
Nuclear Rna

The analysis of 40 kDa nuclear protein, p40, in interphase cells and mitotic cells

1993 ◽  
Vol 106 (3) ◽  
pp. 741-748 ◽  
Author(s):  
Y. Kaneda ◽  
K. Kinoshita ◽  
M. Sato ◽  
K. Tanaka ◽  
Y. Kaneda
Keyword(s):  
Monoclonal Antibody ◽  
Nuclear Envelope ◽  
Nuclear Protein ◽  
Dnase I ◽  
High Salt ◽  
The Other ◽  
Interphase Nuclei ◽  
Interphase Cells ◽  
Detergent Treatment ◽  
Sequential Treatments

We previously reported that the monoclonal antibody M108 recognized a 40 kDa protein both in the nucleus and the cytoplasm. This nuclear 40 kDa antigen was located in the nuclear envelope in interphase cells and in the perichromosomal region during mitosis. Now, we have analyzed this nuclear 40 kDa protein (p40) further, through morphological and biochemical approaches. At the beginning of mitosis, the perinuclear p40 detached from the nuclear envelope and moved to surround the condensing chromatin, while in the late stage of mitosis, the perichromosomal p40 moved back to the reassembled nuclear envelope. Most of the perichromosomal p40 on the metaphase chromosome was solubilized only by DNase I treatment, not by either high salt or detergent treatment. On the other hand, the perinuclear p40 was not solubilized by DNase1 alone, or high salt detergent alone. Sequential treatments with DNase I and high salt detergent were required to extract p40 in interphase nuclei. These results suggest that p40 was associated both with the nuclear envelope and chromatin DNA in interphase nuclei, while it bound only to chromatin DNA in mitosis.

Identification of secreted and surface proteins from Enterococcus faecalis

2009 ◽  
Vol 55 (8) ◽  
pp. 967-974 ◽  
Author(s):  
Abdellah Benachour ◽  
Thierry Morin ◽  
Laurent Hébert ◽  
Aurélie Budin-Verneuil ◽  
André Le Jeune ◽  
...  
Keyword(s):  
Cell Surface ◽  
Enterococcus Faecalis ◽  
Virulence Factors ◽  
Gel Electrophoresis ◽  
Ion Trap ◽  
Surface Proteins ◽  
Two Dimensional Gel Electrophoresis ◽  
Proteomic Approach ◽  
Associated Proteins

Secreted and surface proteins of bacteria are key molecules that interface the cell with the environment. Some of them, corresponding to virulence factors, have already been described for Enterococcus faecalis , the predominant species involved in enterococcal nosocomial infections. In a global proteomic approach, the identification of the most abundant secreted and surface-associated proteins of E. faecalis JH2-2 strain was carried out. These proteins were separated by gel electrophoresis or directly subjected to in vivo trypsinolysis and then analyzed by liquid chromatography – electrospray ion trap tandem mass spectrometry. Putative functions were assigned by homology to the translated genomic database of E. faecalis. A total of 44 proteins were identified, eight secreted proteins from the supernatant culture and 38 cell surface proteins from two-dimensional gel electrophoresis and in vivo trypsinolysis among which two are common to the two groups. Their sequences analysis revealed that 35 of the 44 proteins harbour characteristic features (signal peptide or transmembrane domains) consistent with an extracellular localization. This study may be considered as an important step to encourage proteomic-based investigations of E. faecalis cell surface associated proteins that could lead to the discovery of virulence factors and to the development of new therapeutic tools.

scholarly journals Structural analyses of the 7SK ribonucleoprotein (RNP), the most abundant human small RNP of unknown function.

1991 ◽  
Vol 11 (7) ◽  
pp. 3432-3445 ◽  
Author(s):  
D A Wassarman ◽  
J A Steitz
Keyword(s):  
Secondary Structure ◽  
Rnase H ◽  
Small Nuclear Rna ◽  
Base Pairing ◽  
Structural Determinants ◽  
Nuclear Rna ◽  
Sequence Complementarity ◽  
Associated Proteins ◽  
Stem Structure

The human 7SK ribonucleoprotein (RNP) has been analyzed to determine its RNA secondary structure and protein constituents. HeLa cell 7SK RNA alone and within its RNP have been probed by chemical modification and enzymatic cleavage, and sites of modification or cleavage have been mapped by primer extension. The resulting secondary structure suggests that structural determinants necessary for capping (a 5' stem followed by the sequence AUPuUPuC) and nuclear migration (the sequence AUPuUPuC) of 7SK RNA may be similar to those for U6 small nuclear RNA (snRNA). It also supports existence of a 3' stem structure which could serve to self-prime cDNA synthesis during pseudogene formation. Oligonucleotide-directed RNase H digestion indicated regions of 7SK RNA capable of base pairing with other nucleic acids. Antisense 2'-O-methyl RNA oligonucleotides were used to affinity select the 7SK RNP from an in vivo 35S-labeled cell sonic extract and identify eight associated proteins of 83, 48, 45, 43, 42, 21, 18, and 13 kDa. 7SK RNA has extensive sequence complementarity to U4 snRNA, within the U4/U6 base pairing domain, and also to U11 snRNA. The possibility that the 7SK RNP is an unrecognized component of the pre-mRNA processing machinery is discussed.

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue

PROTEOMICS ◽  
2004 ◽  
Vol 4 (1) ◽  
pp. 27-30 ◽  
Author(s):  
Kyla Pennington ◽  
Emma McGregor ◽  
Clare L. Beasley ◽  
Ian Everall ◽  
David Cotter ◽  
...  
Keyword(s):  
Human Brain ◽  
Brain Tissue ◽  
Gel Electrophoresis ◽  
Two Dimensional ◽  
Human Brain Tissue ◽  
Two Dimensional Gel Electrophoresis ◽  
Basic Proteins

scholarly journals A possible skeletal substructure of the macronucleus of tetrahymena

1980 ◽  
Vol 84 (1) ◽  
pp. 160-171 ◽  
Author(s):  
J Wolfe
Keyword(s):  
Citric Acid ◽  
Inner Core ◽  
Protein Composition ◽  
High Salt ◽  
Skeletal Structure ◽  
Digestion Procedure ◽  
Salt Extraction ◽  
Nonhistone Protein ◽  
Fibrillar Component ◽  
Triton X

Upon removal of chromatin from isolated macronuclei of tetrahymena, residual structures are obtained, the organization of which faithfully reflects the distinctive architecture of the macronucleus. Macronuclei are isolated by a new procedure in which cells are lysed by immersion in citric acid and Triton X-100. This method is rapid and efficient and leaves the nuclear structures stripped of nuclear envelope and nucleoli. The remaining interconnected chromatin bodies are structurally differentiated into a dense outer shell and a fibrillar inner core. The fibrillar component is identified as chromatin because it is removed upon digestion with DNase and extraction with 2 M NaCl. The dense shell of the chromatin body is unaffected by the digestion procedure, which leaves a skeletal structure comprised of hollow spherical bodies. Analysis of the protein composition by SDS acrylamide gel electrophoresis before and after digestion with DNase and RNase and high-salt extraction shows that histones are diminished, whereas the nonhistone protein composition remains unchanged. It was found the DNase not only extracts chromatin but also protects the nonchromatin structure from the otherwise disruptive effects of high-salt extraction. The method used for isolating the nuclei also affects the structure remaining after the digestion procedure the citric acid/Triton X-100 method enhances the stability of the interconnected spherical bodies. The results indicate that the method for isolating nuclei and the procedure by which chromatin is extracted are both major factors contributing to the detection of a possible nonchromatin nuclear skeleton.

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