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At least 127 records · Page 7Linked to original sources
PubMed1987

Expression of human atrial natriuretic polypeptide gene in Cos 7 cells.

Cos 7 cells transfected with human atrial natriuretic polypeptide (hANP) gene with SV40 enhancer and replication origin sequences expressed hANP gene. The expressed RNA was indistinguishable from native hANP mRNA and the transcribed protein seemed to be properly processed to alpha-hANP and beta-hANP. This system provides a useful approach to investigate the processing of hANPs and the structure-function relationship of amino acid sequences of hANPs.

Animals↗
PubMed1995

Protein kinase C inhibitor potentiates the agonist-induced GTPase activity in COS cell membranes expressing delta-opioid receptor.

In COS-7 cell membranes expressing cloned delta-opioid receptor, [D-Ser2, Leu5]enkephalin-Thr6, an opioid delta-agonist, showed no significant stimulation of high-affinity GTPase, while this agonist binding showed a guanine nucleotide sensitivity. Significant stimulation of GTPase activity by this agonist was observed only when the cells were pretreated with 0.1 microM calphostin C, a protein kinase C inhibitor, and when this inhibitor was further added to the reaction mixture at 1 microM. These findings suggest that protein kinase C is involved in the heterologous desensitization of delta-opioid receptor in the cells.

Analgesics↗
PubMed1998

Transient overexpression of human H- and L-ferritin chains in COS cells.

The understanding of the in vitro mechanisms of ferritin iron incorporation has greatly increased in recent years with the studies of recombinant and mutant ferritins. However, little is known about how this protein functions in vivo, mainly because of the lack of cellular models in which ferritin expression can be modulated independently from iron. To this aim, primate fibroblastoid COS-7 cells were transiently transfected with cDNAs for human ferritin H- and L-chains under simian virus 40 promoter and analysed within 66 h. Ferritin accumulation reached levels 300-500-fold higher than background, with about 40% of the cells being transfected. Thus ferritin concentration in individual cells was increased up to 1000-fold over controls with no evident signs of toxicity. The exogenous ferritin subunits were correctly assembled into homopolymers, but did not affect either the size or the subunit composition of the endogenous heteropolymeric fraction of ferritin, which remained essentially unchanged in the transfected and non-transfected cells. After 18 h of incubation with [59Fe]ferric-nitrilotriacetate, cellular iron incorporation was similar in the transfected and non-transfected cells and most of the protein-bound radioactivity was associated with ferritin heteropolymers, while H- and L-homopolymers remained iron-free. Cell co-transfection with cDNAs for H- and L-chains produced ferritin heteropolymers that also did not increase cellular iron incorporation. It is concluded that transient transfection of COS cells induces a high level of expression of ferritin subunits that do not co-assemble with the endogenous ferritins and have no evident activity in iron incorporation/metabolism.

Animals↗
PubMed1994

Rabbit skeletal muscle glycogen synthase expressed in COS cells. Identification of regulatory phosphorylation sites.

Rabbit skeletal muscle glycogen synthase contains multiple sites for phosphorylation. To investigate the relative importance of these sites, the enzyme was overexpressed in COS M9 cells, and Ser-->Ala mutations were introduced singly, or in combinations, at nine known phosphorylation sites. Overexpressed wild-type enzyme had a very low +/- glucose-6-P activity ratio of approximately 0.01, indicative that the glycogen synthase is in a highly phosphorylated state. No single Ser-->Ala mutation was able to cause a substantial increase in activity ratio; rather, simultaneous mutation at both NH2- and COOH-terminal sites was needed. The most effective combinations were mutations at site 3a (Ser-640) or site 3b (Ser-644) together with site 2 (Ser-7). The results were consistent with site 2 phosphorylation being a prerequisite for phosphorylation of site 2a (Ser-10). Mutation of site 5 (Ser-656) perturbed COOH-terminal phosphorylation but did not prevent inactivation. Expression of the most active mutants correlated with increased glycogen accumulation in the COS M9 cells. In summary, we conclude that (i) the sites most important for activating the enzyme are sites 2, 2a, 3a, and 3b; (ii) removal of phosphate from both NH2- and COOH-terminal sites is required for activation; and (iii) sites 3a and/or 3b can be phosphorylated in COS cells by mechanisms that do not depend on phosphorylation of site 5.

Amino Acid Sequence↗
PubMed1991

Identification of two amino acid residues in the epsilon subunit that promote mammalian muscle acetylcholine receptor assembly in COS cells.

We have used a species difference in epsilon subunits of the acetylcholine receptor (AChR) to investigate regions of the subunit protein that are important in receptor assembly. Upon transient transfection of COS cells, mouse epsilon subunit cDNA is approximately 10 times more effective than that of the rat in supporting expression of surface AChRs when the other subunits are from either mouse or rat. In cells transfected with only alpha and epsilon subunit cDNAs, the formation of an alpha epsilon heterodimer, a presumed assembly intermediate, is also less efficient with rat than with mouse epsilon subunit. By site-directed mutagenesis, we have found that these differences can be accounted for by 2 amino acid differences in the N-terminal domain at positions 106 and 115 of the rat and mouse epsilon subunits, suggesting that the region near these 2 amino acid residues is important for AChR assembly.

Amino Acid Sequence↗
PubMed1995

Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli.

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

Animals↗
PubMed1988

The production of normal and variant human glucose-6-phosphate dehydrogenase in cos cells.

Full-length cDNA coding for human glucose-6-phosphate dehydrogenase (G6PD) was inserted into a eukaryotic expression vector containing the immediate early promoter of cytomegalovirus. When this plasmid was introduced into cos cells by transfection it led to the production of high levels of human G6PD. cDNAs containing mutations found in G6PD-deficient individuals were constructed by in vitro mutagenesis and expressed in the same system. Characterization of the G6PD proteins obtained in this way confirmed the primary structure inferred for the variant enzymes. An enzyme in which lysine-205 had been mutated to threonine was produced and found to have no G6PD activity, proving that this lysine residue is essential for enzyme activity in human G6PD.

Cell Line↗
PubMed1991

Analysis of rubella virus E1 glycosylation mutants expressed in COS cells.

cDNA clones encoding the envelope glycoprotein E1 of rubella virus (RV) were altered by site-directed mutagenesis at consensus sites for addition of N-linked glycans. The resulting plasmids were introduced into COS cells and the mutant E1 proteins were analyzed by indirect immunofluorescence, radioimmunoprecipitation, and immunoblotting. We found that RV E1 contains three N-linked oligosaccharides, each approximately 2 kDa in size. Although lack of glycosylation did not appear to affect targeting of E1 to the Golgi region, mutants lacking N-linked glycans at Asn 177 and Asn 209 failed to bind anti-E1 antibodies under nonreducing conditions. Our results suggest that glycosylation may be important for expression of important immunologic epitopes on RV E1.

Animals↗
PubMed1994

Expression of a novel human myotonin protein kinase (MtPK) cDNA clone which encodes a protein with a thymopoietin-like domain in COS cells.

A full-length cDNA of human myotonin protein kinase (MtPK) was cloned and expressed in COS-1 cells. MtPK is recovered from the cytosolic fraction of the COS extract as a 70 kDa protein, which coincides with the size deduced from the predicted amino acid sequence. The sequence has a significant homology to thymopoietin, a peptide hormone of the thymus. Biochemical characteristics of MtPK expressed in COS-1 cells and its expression in rat tissues are investigated.

Amino Acid Sequence↗
PubMed1988

Expression of four types of human tyrosine hydroxylase in COS cells.

Alternative splicing from a single gene produces four kinds of human tyrosine hydroxylase (types 1-4), which have structural diversity only in the N-terminal region. We attempted expression of the type 1-4 enzymes in COS cells and performed kinetic analyses. All had enzymatic activities. The Km values of the four types for L-tyrosine and 6-methyl-5,6,7,8-tetrahydropteridine were similar, although their relative homospecific activities were clearly different. The type 1 enzyme displayed the highest activity.

Blotting, Western↗
PubMed1996

Formation of crystalloid endoplasmic reticulum in COS cells upon overexpression of microsomal aldehyde dehydrogenase by cDNA transfection.

When rat liver microsomal aldehyde dehydrogenase (msALDH) was overexpressed in COS-1 cells by cDNA transfection, large granular structures containing both msALDH and endogenous protein disulfide isomerase appeared (Masaki et al. (1994) J. Cell Biol. 126, 1407-1420). Confocal laser microscopy revealed that these granular structures are dispersed throughout the cytoplasm. Electron microscopy showed that the structures are composed of regularly arranged crystalloid smooth endoplasmic reticulum (ER). The formation of the crystalloid ER was accompanied by a remarkable proliferation of smooth ER, which appeared occasionally continuous to the rough ER. We suggest that the smooth ER, proliferated from the rough ER, is transformed and assembled into the crystalloid ER by head-to-head association of the msALDH molecules on the apposed smooth ER membranes. In order to understand the molecular mechanism of the crystalloid ER formation, we asked which portions of the msALDH molecules are needed for the crystalloid ER formation by expressing deletion mutants or chimera protein of msALDH in COS-1 cells. The overexpression of msALDH molecules lacking the stem region preceding the membrane spanning region, although they were exclusively localized in the ER, did not induce the formation of crystalloid ER. More detailed analysis showed that the amino acid sequence FFLL, located in the stem region, is necessary to form the crystalloid ER. The chimera protein containing the last 35 amino acids of msALDH at the carboxyl terminus of chloramphenicol acetyltransferase was localized to the ER, but did not induce the formation of the crystalloid ER. These results suggest that at least two regions, the bulky amino-terminal region and the FFLL sequence in the stem region of msALDH molecules are required for the formation of the crystalloid ER.

Aldehyde Dehydrogenase↗
PubMed1985

Polyadenylylation of sea urchin histone RNA sequences in transfected COS cells.

The region of pSV2neo that encompasses the simian virus 40 early polyadenylylation signal was replaced with a DNA fragment that spans the 3' end of a sea urchin (Psammechinus miliaris) histone H2A gene. This clone, pMK2.H2A(3'), was used to transfect COS cells. RNA analysis revealed that transcripts from pMK2.H2A(3') were polyadenylylated at a site 85 nucleotides downstream from the expected 3' end of mature H2A mRNA. Nucleotide sequencing showed that the site of poly(A) addition was located 10 nucleotides downstream from a cluster of four A-A-U-A-A-A sequences. The lower accumulation of MK2.H2A(3') mRNA, which was 5-10% that of SV2neo mRNA, suggests that the H2A polyadenylylation signal is relatively inefficient. The relationship of the above findings to the 3' end processing of other histone mRNAs is discussed.

Animals↗
PubMed1997

Overexpression in COS cells of p50, the major core protein associated with mRNA, results in translation inhibition.

p50, the major core protein of messenger ribonucleoprotein particles (mRNPs) in the cytoplasm of somatic mammalian cells, has been characterized previously as a member of the Y-box binding transcription factor family of proteins (YB-protein) by both high structural homology and ability to bind specifically the Y-box sequence in double-stranded DNA. YB proteins are present in a whole range of cell types and some have been identified as germ-specific cytoplasmic proteins masking stored mRNA from translation. Western blot analysis of the distribution of p50 in subcellular fractions of COS-1 cells shows that p50 is a cytoplasmic protein quantitatively associated with mRNA, both in polyribosomes and in free mRNPs. The level of p50 in COS-1 cells determined by Western immunoblotting is 0.10% of total protein, which is nearly equimolar to that of ribosomes and is approximately 5-10-fold higher than the mRNA level. Transient transfection of COS-1 cells with a p50-expressing vector results in a dramatic inhibition of protein synthesis. A control transfection with a vector expressing a frameshift mutant of p50 does not cause translation inhibition. Therefore the increase in p50 protein level is responsible for the inhibitory effect in these cells.

Animals↗
PubMed1984

In monkey COS cells only the TATA box and the cap site region are required for faithful and efficient initiation of the fibroin gene transcription.

The DNA sequences necessary for faithful and efficient initiation of transcription of Bombyx mori fibroin gene have been studied in vivo using monkey COS cells and a SV40 origin vector. Transcriptional analysis of 5' deletion genes and of exact substitution genes including a series of single base substitution mutants indicates that the TATA box and the cap region are required for faithful and efficient initiation of the fibroin gene transcription in vivo, whereas far upstream sequences and the CAAT-like sequences are not required. In addition, single base substitution genes of the cap site region have clearly shown that there exists a strict sequence requirement of the cap region of the fibroin gene. These results define the basic promoter of the fibroin gene to the region spanning the TATA box and the cap site region.

Animals↗
PubMed1989

Stimulation of chloramphenicol acetyltransferase mRNA translation by reovirus capsid polypeptide sigma 3 in cotransfected COS cells.

The mammalian reovirus S4 gene has been implicated in the serotype-dependent inhibition of host cell protein synthesis during viral replication in mouse L cells. To examine the effect(s) of this gene on transcription or translation or both, a DNA copy of the serotype 3 S4 gene was inserted into a eucaryotic expression vector. Cotransfection of COS cells with plasmids containing S4 and the reporter gene, chloramphenicol acetyltransferase (CAT), resulted in a marked stimulation of CAT expression, predominantly at the level of translation. The significance of these findings is discussed in relation to the double-stranded-RNA-binding activity of the S4 gene product, polypeptide sigma 3.

Animals↗
PubMed1994

Amino acids 327-350 of the human C5a-receptor are not essential for [125I]C5a binding in COS cells and signal transduction in Xenopus oocytes.

The anaphylatoxic peptide C5a is an important inflammatory mediator of the complement system. We have generated human C5a-receptor (hC5aR) mutants with truncation of its cytosolic carboxyl-terminus (C-terminus). Both mutants were analysed for C5a-binding in transiently expressing COS cells, and one mutant additionally for GTP-binding regulatory protein (G-protein) coupling in cRNA-injected Xenopus oocytes. Our data suggest that (a) amino acids (aa) 314 to 326 as part of the C-terminus are necessary for proper receptor folding or expression and (b) the receptor C-terminus distal from position 327 is not critical for receptor expression, folding, binding and G-protein coupling.

Animals↗
PubMed1992

HPV-1 L1 protein expressed in cos cells displays conformational epitopes found on intact virions.

Seven polyclonal and monoclonal antibodies were characterized for their ability to react specifically with either conformational or nonconformational epitopes of the HPV-1 virion. Using these antibodies, it was shown that the HPV-1 L1 protein (when expressed by an SV40 vector in cos cells) displayed conformational epitopes characteristic of intact viral particles. In addition, the L1 capsid protein was translocated normally into cell nuclei, was of appropriate size (57 kDa), and could be isolated in native form by immunoprecipitation techniques. Most importantly, the screening of expressed papillomavirus capsid proteins for reactivity with conformation-dependent antibodies represents a new, general methodology for ensuring that such proteins will be suitable for use in vaccine development or in the serologic detection/typing of human papillomavirus infections.

Animals↗