scholarly journals hnRNA and its attachment to a nuclear protein matrix.

1981 ◽  
Vol 88 (3) ◽  
pp. 554-563 ◽  
Author(s):  
C A van Eekelen ◽  
W J van Venrooij
Keyword(s):  
Nuclear Protein ◽  
Protein Complexes ◽  
Intact Cell ◽  
Published Data ◽  
Intact Cells ◽  
Nuclear Protein Matrix ◽  
The Matrix ◽  
Covalently Linked ◽  
Protein Matrices

In this study, DNA-depleted nuclear protein matrices are isolated from HeLa S3 cells. These nuclear matrices consist of peripheral laminae, residual nucleoli, and internal fibrillar structures. High molecular weight, heterogeneous nuclear RNA (hnRNA) is quantitatively associated with these structures and can be released intact only by affecting the integrity of the matrices. It is, therefore, concluded that hnRNA is part of a highly organized nuclear structure. By irradiation of intact cells or isolated nuclear matrices with ultraviolet light, proteins tightly associated with hnRNA can be induced to cross-link with the RNA. Performing the cross-linking in vivo is an extra guarantee that only hnRNA-protein (hnRNP) complexes existing in the intact cell are covalently linked. Such hnRNP complexes were isolated and purified under conditions that completely dissociate nonspecific RNA-protein complexes. By comparison of the hnRNP found in nuclear matrices and the published data on the composition of hnRNP particles, it was found that the so-called hnRNP "packaging" proteins (32,000-38,000 mol wt) were not efficiently cross-linked to hnRNA by UV irradiation. They were, however, present in the matrix preparations, bound to hnRNA, because they were released from nuclear matrices after ribonuclease treatment of these structures. On the other hand, two major hnRNPs (41,500 and 43,000 mol wt) were efficiently cross-linked to hnRNA. These proteins were not released by ribonuclease treatment, which suggests that they are involved in the binding of hnRNA to the nuclear matrix.

scholarly journals Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.

1984 ◽  
Vol 4 (6) ◽  
pp. 1104-1114 ◽  
Author(s):  
G Dreyfuss ◽  
Y D Choi ◽  
S A Adam
Keyword(s):  
Monoclonal Antibodies ◽  
Uv Light ◽  
Protein Complexes ◽  
Cross Linking ◽  
Intact Cells ◽  
Molecular Weights ◽  
Nuclear Rna ◽  
Nonionic Detergent ◽  
Sufficient Intensity

Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.

Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies

1984 ◽  
Vol 4 (6) ◽  
pp. 1104-1114
Author(s):  
G Dreyfuss ◽  
Y D Choi ◽  
S A Adam
Keyword(s):  
Monoclonal Antibodies ◽  
Uv Light ◽  
Protein Complexes ◽  
Cross Linking ◽  
Intact Cells ◽  
Molecular Weights ◽  
Nuclear Rna ◽  
Nonionic Detergent ◽  
Sufficient Intensity

Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.

scholarly journals COPII-coated membranes function as transport carriers of intracellular procollagen-1

2017 ◽  
Author(s):  
Amita Gorur ◽  
Lin Yuan ◽  
Samuel J Kenny ◽  
Satoshi Baba ◽  
Ke Xu ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Imaging Techniques ◽  
Coated Vesicles ◽  
Intact Cells ◽  
Vesicle Budding ◽  
Coated Membranes ◽  
Copii Vesicles ◽  
Optical Reconstruction

AbstractThe coat protein complex II (COPII) is essential for the secretion of large cargo, such as the 300 nm precursor fibrils of procollagen I (PC1). Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles to over 300 nm in diameter and accelerates the secretion of PC1; however, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy (STORM), correlated light electron microscopy (CLEM), and live cell imaging we report the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We have also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude from in vivo and in vitro evidence that large COPII vesicles are bona fide carriers of PC1.SummaryCOPII may play a direct or indirect role in the traffic of large protein complexes such as procollagen. Using high resolution imaging techniques in intact cells and in vitro reconstituted vesicles, Gorur et al. show that COPII coated vesicles carry procollagen1.

scholarly journals Removal of Spo11 from meiotic DNA breaksin vitrobut notin vivoby Tyrosyl DNA Phosphodiesterase 2

2019 ◽  
Author(s):  
Dominic Johnson ◽  
Rachal M Allison ◽  
Elda Cannavo ◽  
Petr Cejka ◽  
Matthew J Neale
Keyword(s):  
Meiotic Recombination ◽  
Dna Cleavage ◽  
Protein Complexes ◽  
Dna Lesions ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Alternative Processing ◽  
Covalently Linked

ABSTRACTMeiotic recombination events are initiated by DNA double-strand breaks (DSBs) created by the topoisomerase-like protein, Spo11. Similar to type-II topoisomerases, Spo11 becomes covalently linked to the 5′ ends generated on each side of the DSB. Whilst Spo11-oligos—the product of nucleolytic removal by Mre11—have been detected in a number of biological systems, the lifetime of the covalent Spo11-DSB precursor has not been systematically determined and may be subject to alternative processing reactions. Here we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2, on Spo11-DSBs isolated fromS. cerevisiaecells. We demonstrate that TDP2 can remove Spo11 from natural ssDNA-oligos, and dsDNA ends even when in the presence of excess competitor genomic DNA. Interestingly, TDP2-processed Spo11-DSBs are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essentialin vivoto facilitate resection-dependent HR at Spo11-DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptidesin vitro, TDP2 was unable to remove Spo11in vivo—unlike during the repair of topoisomerase-induced DNA lesions. These results suggest that Spo11-DNA, but not topoisomerase-DNA cleavage complexes, are inaccessible to the TDP2 enzyme, perhaps due to occlusion by higher order protein complexes resident at sites of meiotic recombination.

scholarly journals In vitro DNA synthesis in the macronuclear replication band of Euplotes eurystomus.

1987 ◽  
Vol 104 (5) ◽  
pp. 1125-1132 ◽  
Author(s):  
D E Olins ◽  
A L Olins
Keyword(s):  
Microscopic Analysis ◽  
Published Data ◽  
Ciliated Protozoan ◽  
Intact Cells ◽  
Vitro Assay ◽  
Rabbit Igg ◽  
Replication Band ◽  
Prolonged Heat

Isolated macronuclei from the hypotrichous ciliated protozoan Euplotes eurystomus incorporate biotinylated dUTP specifically into the replication band (RB) as detected with immunofluorescence, using rabbit anti-biotin antibodies followed by fluorescein-conjugated goat anti-rabbit IgG. When gold-conjugated goat anti-rabbit IgG was used in a preembedded reaction, subsequent immunoelectron microscopic analysis demonstrated that the biotinylated nucleotide appeared more concentrated in the rear zone of the RB, with almost no labeling in the forward zone. It was possible to use the immunofluorescent assay to establish that incorporation of biotinylated dUTP is inhibited by simultaneous addition of N-ethyl maleimide or aphidicolin, and by omission of any one of the other unlabeled dNTPs. In addition, prolonged heat shock of the intact cells, before lysis and in vitro assay, yielded markedly reduced incorporation. Comparison with published data on the in vivo incorporation of [3H]thymidine into Euplotes eurystomus RBs indicates the fidelity of the in vitro reaction.

Radiolabeled r-Hirudin as a Measure of Thrombin Activity at, or within, the Rabbit Aorta Wall In Vitro and In Vivo

1994 ◽  
Vol 71 (04) ◽  
pp. 499-506 ◽  
Author(s):  
Mark W C Hatton ◽  
Bonnie Ross-Ouellet
Keyword(s):  
Rabbit Aorta ◽  
Balloon Injury ◽  
Recombinant Hirudin ◽  
Aorta Wall ◽  
Thrombin Activity ◽  
Before And After ◽  
The Matrix

SummaryThe behavior of 125I-labeled recombinant hirudin towards the uninjured and de-endothelialized rabbit aorta wall has been studied in vitro and in vivo to determine its usefulness as an indicator of thrombin activity associated with the aorta wall. Thrombin adsorbed to either sulfopropyl-Sephadex or heparin-Sepharose bound >95% of 125I-r-hirudin and the complex remained bound to the matrix. Binding of 125I-r-hirudin to the exposed aorta subendothelium (intima-media) in vitro was increased substantially if the tissue was pre-treated with thrombin; the quantity of l25I-r-hirudin bound to the de-endothelialized intima-media (i.e. balloon-injured in vitro) correlated positively with the quantity of bound 131I-thrombin (p <0.01). Aortas balloon-injured in vivo were measured for thrombin release from, and binding of 125I-r-hirudin to, the de-endothelialized intimal surface in vitro; 125I-r-hirudin binding correlated with the amount of active thrombin released (p <0.001). Uptake of 125I-r-hirudin by the aorta wall in vivo was proportional to the uptake of 131I-fibrinogen (as an indicator of thrombin activity) before and after balloon injury. After 30 min in the circulation, specific 125I-r-hirudin binding to the uninjured and de-endo- thelialized (at 1.5 h after injury) aorta wall was equivalent to 3.4 (± 2.5) and 25.6 (±18.1) fmol of thrombin/cm2 of intima-media, respectively. Possibly, only hirudin-accessible, glycosaminoglycan-bound thrombin is measured in this way.

Photoinhibition in vivo and in vitro Involves Weakly Coupled Chlorophyll–Protein Complexes‡¶

2002 ◽  
Vol 75 (6) ◽  
pp. 613 ◽  
Author(s):  
Stefano Santabarbara ◽  
Ilaria Cazzalini ◽  
Andrea Rivadossi ◽  
Flavio M. Garlaschi ◽  
Giuseppe Zucchelli ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Weakly Coupled ◽  
Chlorophyll Protein Complexes ◽  
Chlorophyll Protein

Conjugates of Classical DNA/RNA Binder with Nucleobase: Chemical, Biochemical and Biomedical Applications

2019 ◽  
Vol 26 (30) ◽  
pp. 5609-5624
Author(s):  
Dijana Saftić ◽  
Željka Ban ◽  
Josipa Matić ◽  
Lidija-Marija Tumirv ◽  
Ivo Piantanida
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Biomedical Applications ◽  
Protein Targets ◽  
New Class ◽  
Rna Targets ◽  
Covalently Linked ◽  
Structural Aspects

: Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class is nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder – nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets are involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules.

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