scholarly journals Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

1993 ◽  
Vol 13 (2) ◽  
pp. 869-876 ◽  
Author(s):  
D F Smith ◽  
W P Sullivan ◽  
T N Marion ◽  
K Zaitsu ◽  
B Madden ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Simian Virus 40 ◽  
Immunoblot Analysis ◽  
Simian Virus ◽  
Rabbit Reticulocyte Lysate ◽  
Striking Similarity ◽  
Peptide Fragments ◽  
Receptor Complexes ◽  
Reticulocyte Lysate ◽  
A Cell

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.

scholarly journals Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70

1993 ◽  
Vol 13 (2) ◽  
pp. 869-876
Author(s):  
D F Smith ◽  
W P Sullivan ◽  
T N Marion ◽  
K Zaitsu ◽  
B Madden ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Simian Virus 40 ◽  
Immunoblot Analysis ◽  
Simian Virus ◽  
Rabbit Reticulocyte Lysate ◽  
Striking Similarity ◽  
Peptide Fragments ◽  
Receptor Complexes ◽  
Reticulocyte Lysate ◽  
A Cell

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.

Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes

1994 ◽  
Vol 14 (3) ◽  
pp. 1956-1963
Author(s):  
J L Johnson ◽  
T G Beito ◽  
C J Krco ◽  
D O Toft
Keyword(s):  
Progesterone Receptor ◽  
Cdna Clone ◽  
Peptide Fragments ◽  
Receptor Complexes ◽  
Human Cdna ◽  
Partial Clone ◽  
Chicken Oviduct ◽  
Brain Cdna Library ◽  
Carboxy Terminal ◽  
And Function

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.

scholarly journals Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific.

1986 ◽  
Vol 6 (12) ◽  
pp. 4697-4708 ◽  
Author(s):  
R H Costa ◽  
E Lai ◽  
J E Darnell
Keyword(s):  
Hela Cells ◽  
Hepg2 Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Proximal Promoter ◽  
Start Site ◽  
Beta Globin ◽  
Human Hepatoma Cells ◽  
Enhancer Element ◽  
A Cell

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.

Role of HCO3- in regulation of cytoplasmic pH in ciliary epithelial cells

1989 ◽  
Vol 257 (4) ◽  
pp. C696-C705 ◽  
Author(s):  
H. Helbig ◽  
C. Korbmacher ◽  
F. Stumpff ◽  
M. Coca-Prados ◽  
M. Wiederholt
Keyword(s):  
Cell Clone ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Disulfonic Acid ◽  
Ciliary Epithelium ◽  
Cytoplasmic Ph ◽  
Acid Load ◽  
A Cell ◽  
Free Media ◽  
Phi Regulation

Cytoplasmic pH (pHi) was monitored using the pH-sensitive absorbance of 5(6)carboxy-4',5'-dimethylfluorescein in monolayers of a cell clone derived from bovine pigmented ciliary epithelium (PE) transformed with the simian virus 40. 1) Changing extracellular media from a nominally HCO3(-)-free solution to a solution containing 28 mM HCO3(-)-5% CO2 at constant extracellular pH (7.4) resulted in a delayed alkalinization of pHi, which was 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) sensitive and was inhibited in Na+-free medium and in Cl(-)-depleted cells. 2) DIDS pretreatment acidified pHi in HCO3(-)-containing media. 3) Replacing extracellular Cl- resulted in a DIDS-sensitive, HCO3(-)-dependent, and Na+-independent alkalinization. 4) Replacing extracellular Na+ in HCO3(-)-containing media led to a partly DIDS-sensitive intracellular acidification. 5) Recovery of pHi after an alkali load (acetate prepulse) had a HCO3(-)-dependent and DIDS-sensitive component. 6) Two Na+-dependent components participated in pHi regulation after an acid load (NH4+ prepulse) in HCO3(-)-containing solution. One was amiloride sensitive, the other was DIDS sensitive and was inhibited in HCO3(-)-free media and after Cl- depletion. We conclude that in cultured PE, in addition to Na+-H+ exchange, two HCO3-transporters participate in pHi regulation. Cl(-)-dependent Na+-HCO3-symport regulates pHi during steady state and after an acid load, and Na+-independent Cl(-)-HCO3-exchange is involved in pHi recovery after an alkali load.

Hyperexpression of recombinant CFTR in heterologous cells alters its physiological properties

1998 ◽  
Vol 274 (2) ◽  
pp. C310-C318 ◽  
Author(s):  
Raha Mohammad-Panah ◽  
Sophie Demolombe ◽  
David Riochet ◽  
Veronique Leblais ◽  
Gildas Loussouarn ◽  
...  
Keyword(s):  
Cell Metabolism ◽  
A549 Cells ◽  
Simian Virus 40 ◽  
Ionic Channels ◽  
Simian Virus ◽  
Chloride Current ◽  
Patch Clamp Technique ◽  
A Cell ◽  
High Level ◽  
Camp Stimulation

We investigated whether high levels of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) would alter the functional properties of newly synthesized recombinant proteins. COS-7, CFPAC-1, and A549 cells were intranuclearly injected with a Simian virus 40-driven pECE-CFTR plasmid and assayed for halide permeability using the 6-methoxy- N-(3-sulfopropyl)quinolinium fluorescent probe. With increasing numbers of microinjected pECE-CFTR copies, the baseline permeability to halide dose dependently increased, and the response to adenosine 3′,5′-cyclic monophosphate (cAMP) stimulation decreased. In cells hyperexpressing CFTR, the high level of halide permeability was reduced when a cell metabolism poisoning cocktail was applied to decrease intracellular ATP and, inversely, was increased by orthovanadate. In CFPAC-1 cells investigated with the patch-clamp technique, CFTR hyperexpression led to a time-independent nonrectifying chloride current that was not sensitive to cAMP stimulation. CFPAC-1 cells hyperexpressing CFTR exhibited no outward rectifying chloride current nor inward rectifying potassium current either spontaneously or under cAMP stimulation. We conclude that hyperexpression of recombinant CFTR proteins modifies their properties inasmuch as 1) CFTR channels are permanently activated and not susceptible to cAMP regulation and 2) they lose their capacity to regulate heterologous ionic channels.

scholarly journals Novel Activation Step Required for Transcriptional Competence of Progesterone Receptor on Chromatin Templates

2003 ◽  
Vol 17 (12) ◽  
pp. 2543-2553 ◽  
Author(s):  
Varykina G. Thackray ◽  
David O. Toft ◽  
Steven K. Nordeen
Keyword(s):  
Progesterone Receptor ◽  
Transcriptional Activity ◽  
Specific Binding ◽  
Specific Inhibitor ◽  
Transcription System ◽  
Naked Dna ◽  
Activation Of Transcription ◽  
Chromatin Template ◽  
Reticulocyte Lysate ◽  
A Cell

Abstract To elucidate the earliest molecular steps in the activation of transcription by the progesterone receptor (PR), we investigated its activity in a cell-free transcription system utilizing chromatin templates. PR prepared as a ligand-free, recombinant protein failed to induce transcription on chromatin templates. However, transcriptional competence could be restored by coincubation with rabbit reticulocyte lysate (RRL). The interaction of PR with chaperones results in a receptor conformation competent to bind ligand and RRL contains abundant chaperone-mediated protein folding activity. Blocking this activity with the specific inhibitor geldanamycin inhibited receptor-dependent transcriptional activity. However, recombinant chaperones could not replace RRL in the restoration of transcriptional activity on chromatin templates, suggesting the presence of an additional activity in the lysate. Under chromatin assembly conditions, PR could bind naked DNA and RRL did not increase that binding. In contrast, PR bound to a chromatin template only poorly. Interestingly, RRL stimulated sequence-specific binding by PR to target sites in chromatin and the concomitant recruitment of the steroid receptor coactivator 1 to the promoter. Thus, our results indicate that a novel protein-mediated activity in RRL is involved in an additional, heretofore unrecognized, activation step required for PR to become transcriptionally competent on chromatin templates.

Murine leukemia virus long terminal repeat sequences can enhance gene activity in a cell-type-specific manner

1985 ◽  
Vol 5 (10) ◽  
pp. 2832-2835 ◽  
Author(s):  
F K Yoshimura ◽  
B Davison ◽  
K Chaffin
Keyword(s):  
Long Terminal Repeat ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Manner ◽  
A Cell ◽  
Cell Type Specific ◽  
Murine Leukemia

We tested the ability of sequences in the long terminal repeat (LTR) of a mink cell focus-forming (MCF) murine leukemia virus to function as an enhancer in a cell-type-specific manner. In a stable transformation assay, the MCF or Akv LTR and the simian virus 40 enhancer had similar activities in murine fibroblasts. In contrast, the MCF LTR had a significantly greater activity in murine T lymphoid cells than did either the simian virus 40 enhancer or the Akv LTR.

scholarly journals The expression of Escherichia coli threonine synthase and the production of threonine from homoserine in mouse 3T3 cells

1993 ◽  
Vol 291 (1) ◽  
pp. 315-322 ◽  
Author(s):  
W D Rees ◽  
S M Hay
Keyword(s):  
Escherichia Coli ◽  
Protein A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Eukaryotic Expression ◽  
Cell Protein ◽  
3T3 Cells ◽  
Threonine Synthase ◽  
Homoserine Kinase ◽  
A Cell

We have subcloned the coding sequence for the Escherichia coli threonine synthase gene into a eukaryotic expression vector based on the simian-virus-40 early promoter. When mouse 3T3 cells which already expressed homoserine kinase were transfected with the new plasmid, the cells were able to incorporate radioactivity from [14C]homoserine into their cell proteins. Stable cell lines were established by co-transfecting 3T3 cells with the plasmid coding for threonine synthase and another coding for homoserine kinase and G-418 (Geneticin) resistance. Cells were selected for G-418 resistance and then screened for an ability to synthesize threonine from homoserine and incorporate it into the cell protein. A cell line which expressed both the homoserine kinase and threonine synthase genes was capable of growth in a threonine-deficient medium containing homoserine.

Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis

1980 ◽  
Vol 70 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Glen B. Zamansky ◽  
Lawrence F. Kleinman ◽  
Paul H. Black ◽  
Joan C. Kaplan
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Line ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
A Cell ◽  
Simplex Virus
Sign in / Sign up

Export Citation Format

Share Document