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At least 19 recordsLinked to original sources

PubMed1993

Reactivity of workshop antibodies on L cell and COS cell transfectants expressing bovine CD antigens.

Mouse L cells and COS cells were transfected with either genomic DNA or cDNAs encoding leukocyte differentiation antigens. Positive transfectants were isolated by FACS and cloned by limiting dilution. Transfectants expressing CD4, CD5, CD8, CD25, CD44, two different WC1 gene products and a transfectant expressing an unknown gene product were isolated using these techniques. Antibodies from the various preliminary clusters were analysed for reactivity on these transfectants. The results confirm the fine specificity of mAbs for two alleles of CD4 and CD5; they also subdivide mAbs recognizing CD8 into two groups based on their specificity for the CD8 alpha chain or a combination of both alpha and beta chains. The data also provides support for the clustering of mAbs recognizing CD25 and CD44 based on transfection of cDNAs encoding these specificities. These results reflect the ability of transfection technology to elucidate the fine specificity of mAbs recognizing bovine CD antigens.

Animals↗

PubMed1990

Beta-galactosidase-deficient human fibroblasts: uptake and processing of the exogenous precursor enzyme expressed by stable transformant COS cells.

COS-1 cells were transfected by electroporation with a cDNA for human acid beta-galactosidase cloned in our laboratory and stable transformants expressing the enzyme activity were selected. The precursor form of the enzyme was secreted in large quantities into the culture medium. The fibroblasts from patients with GM1-gangliosidosis or Morquio B disease showed a remarkable increase of enzyme activity, up to the normal level, after culture in this medium for 2 days; the amount of uptake was essentially the same as that for the precursor form in human fibroblasts. After endocytosis, the precursor molecules were processed normally to the mature form and remained as stable as those produced by human fibroblasts. On the other hand, cells from galactosialidosis patients did not show any increase of enzyme activity in a similar experiment. It was concluded that the transformants are useful as the source of precursor proteins for the study of intracellular turnover of enzyme molecules in mutant cells.

Animals↗

PubMed1992

The binding specificity of normal and variant rat Kupffer cell (lectin) receptors expressed in COS cells.

A receptor uniquely found on the surface of rat Kupffer cells was shown previously to bind oligosaccharides terminating in galactose, N-acetylgalactosamine, and fucose. To analyze further the binding specificity of the receptor, receptor-mediated adhesion of transfected COS cells to immobilized glycolipids of known structure was measured. The glycolipid Gb4Cer (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1Cer) was the best ligand. Gb5Cer (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1Cer) and LacCer (Gal beta 1-4Glc beta 1Cer) bound more weakly (five times less than Gb4Cer) and Gb3Cer (Gal alpha 1-4Gal beta 1-4Glc beta 1Cer), and g3Cer(GalNAc beta 1-4Gal beta 1-4Glc beta 1Cer) bound even more weakly (60 times less than Gb4Cer). Gangliosides did not support adhesion of transfected cells. The adhesion of COS cells transfected with plasmids encoding variants of the receptor was also examined. In each variant, either tryptophan 498 or 523, which are conserved in most C-type lectins, was replaced by one of several amino acids. Variants that retained binding activity had the same specificity as the normal receptor. Differences between variants were noted, however, in maximal levels of adhesion and these differences correlated with altered expression of the receptor variants in COS cells.

Acetylgalactosamine↗

PubMed1993

Overexpression of deoxyribonuclease I (DNase I) transfected into COS-cells: its distribution during apoptotic cell death.

COS-cells were transiently transfected with the pSG5 plasmid containing the cDNA of rat parotid deoxyribonuclease I (DNase I) either in right or inverse orientation. Expression of DNase I in transfected cells was only observed when the plasmid contained the cDNA in the right orientation. Expression of DNase I was monitored by measuring the DNase I specific DNA-degrading activity present in the conditioned cell culture medium and in cell homogenates. The expressed DNase I activity could be inhibited by monospecific polyclonal antibodies and by G-actin. Immunofluorescence indicated that approximately 20% of the COS-cells transfected with the DNase I-cDNA in right orientation expressed DNase I. These transfected cells contained large amounts of DNase I, which was found to be localized within the rough endoplasmic reticulum, the Golgi-complex and finally concentrated in a perinuclear location. Occasionally cells were observed which contained the DNase I in small apparently secretory transport vesicles. Transfected cells with perinuclear concentration of DNase I exhibited progressive nuclear destruction, i.e., pyknosis and cytoplasmic shrinkage. Solely the DNA extracted from isolated nuclei of cells transfected with the DNase I-cDNA in correct orientation revealed an internucleosomal DNA-degradation (ladder formation) typical for apoptosis after incubation in the presence of CaCl2 and MgCl2. Only the conditioned medium of COS-cells transfected with the right-oriented DNase I-cDNA contained the nucleolytic activity able to internucleosomally degrade the chromatin of substrate nuclei. Thus, these results indicate that overexpression of DNase I alone is sufficient to induce the morphological and biochemical changes observed during apoptosis.

Animals↗

PubMed1995

An assay for the identification of antigens recognized by cytotoxic T cells, based on transient transfection of COS cells.

The studies reported here describe methodology permitting the direct identification of antigens recognized by cytotoxic T lymphocytes. We demonstrated that bovine alloreactive CTL can detect a bovine MHC molecule transiently expressed in a COS cell population in a standard microcytotoxicity assay. We then showed that alloreactive CTL can detect cells expressing the bovine class I MHC molecule in a population of cells transfected with the plasmid containing the corresponding gene plus 100-fold as many plasmids containing an irrelevant gene. In addition, the transiently transfected COS cells can specifically restimulate CTL as detected by a standard microcytotoxicity assay using the target cell line. Overall, the results suggest that COS cells could be employed for the direct screening of an antigen or antigen gene library by immune CTL.

Animals↗

PubMed1987

Permanent cell lines established from ts-COS cells that regulate by temperature the amplification and expression of cloned genes.

Temperature-sensitive COS cells have been transformed at restrictive temperature with SV40 replicons containing the neo or pac markers. Puromycin-resistant cell clones maintained at the restrictive temperature contain the pac gene integrated into the cell DNA. However, when the cells are shifted to the permissive temperature the pac gene is amplified in episomal forms up to 2-4 X 10(4) copies per cell. Concomitant with this, an induction of 35-300 fold in the levels of puromycin acetyl transferase activity is observed, leading to the accumulation of the enzyme up to 10-60 mU/mg of total cell protein. A band of apparent molecular weight 26,500 daltons is observed by polyacrylamide gel electrophoresis of induced culture extracts, that accounts for approximately 3% of the newly synthesized protein. The expression of non-selectable genes can also be regulated, as shown by the induction of influenza virus nucleoprotein synthesis in transformed cells. These results indicate that the ts-COS cells can be used as a highly efficient, regulable mammalian expression system.

Acetyltransferases↗

PubMed1997

Characterization and evaluation of NGF antisense oligonucleotides: inhibition of NGF synthesis in transfected COS cells.

We present a system for the assessment of the inhibiting capacity of antisense oligonucleotides. The aim of this study was to identify an oligonucleotide that can inhibit chicken nerve growth factor (NGF) synthesis. Five antisense chicken NGF phosphorothioate oligonucleotides, AS1-5, were designed and were tested for their capacity to inhibit NGF expression in COS cells. COS cells that transiently expressed chicken NGF were treated with the oligonucleotides, and NGF expression was analyzed using a bioassay and Western blotting for NGF protein. Two oligonucleotides, AS 1 and AS 5, were more capable than the others of inhibiting expression compared with nonsense oligonucleotide, and they targeted the translational initiation and stop sites. The chicken NGF is expressed at a high level from an adenovirus major late promoter, and AS 1 was capable of inhibiting more than 80% of the NGF expression as determined using the bioassay and Western blotting. Expression of another member of the NGF gene family, neurotrophin-4, was not affected by treatment of the antisense oligonucleotides. A 10-fold lower concentration of the AS 1 oligonucleotide could be used to inhibit NGF synthesis if the cellular uptake was facilitated using lipofectin compared with addition of oligonucleotide directly to the culture medium. The amount of oligonucleotide taken up by the cells was similar in the lipofectin-treated cells as in the cells treated by a 10-fold higher concentration of medium-supplemented nucleotide. This system based on COS cells can facilitate evaluation of the capacity of inhibiting antisense oligonucleotides, particularly targeting those genes in which endogenous products are present in low levels and are difficult to analyze.

Animals↗

PubMed1995

Effect of nicotine on dopamine uptake in COS cells possessing the rat dopamine transporter and in PC12 cells.

The effect of nicotine on the uptake of dopamine (DA) is not completely understood. We studied its effect on PC12 cells and on COS cells transfected with the rat DA transporter cDNA (pcDNADAT1). DA uptake by PC12 cells was inhibited by nicotine in a concentration-related fashion. Treatment of PC12 cells with nerve growth factor (NGF) increased such inhibition. This inhibitory effect was abolished by hexamethonium and mecamylamine, indicating that nicotine acted via the nicotinic acetylcholine (nACh) receptors in PC12 cells. This view is also supported by evidence that acetylcholine (ACh) reduced the uptake of DA in a hexamethonium-, but not atropine-, sensitive fashion. However, nicotine failed to inhibit DA uptake by COS cells possessing the DA transporter. These results suggest that the inhibitory effect of nicotine on DA uptake, when coupled with an nACh receptor leading to an indirect action on the transporter, may play a role in regulating extracellular concentrations of DA.

Acetylcholine↗

PubMed1996

The same sequence mediates activation of the human urokinase promoter by cAMP in mouse Sertoli cells and by SV40 large T antigen in COS cells.

Cell-specific activation by follicle-stimulating hormone and its intracellular mediator, cAMP, of the human urokinase promoter in mouse Sertoli cells requires overlapping purine-rich and GC-rich sequences between -54 and -42 from the transcriptional start site. We have previously shown that binding of unidentified nuclear factors to these sequences is induced by cAMP stimulation, and that sequences from the enhancerless SV40 replication origin can interfere with the binding, whereas consensus Sp1 binding sites are ineffective. We now show that sequences within the SV40 origin able to compete for the formation of cAMP-induced DNA-protein complexes in Sertoli cell nuclear extracts are binding sites for the SV40 large T antigen. Large T antigen expressed in COS cells binds the cAMP-responsive sequences of the human urokinase gene and transactivates the proximal promoter, thus mimicking the effect of nuclear factors induced by cAMP in Sertoli cells. We show that Egr-1 is one of the factors present in cAMP-induced DNA-protein complexes formed between the human urokinase promoter and Sertoli cell nuclear extracts. However, Egr-1 levels are similar in unstimulated and cAMP-treated Sertoli cells, suggesting that this factor interacts with a different GC-box binding factor, that we have previously shown to be strongly induced by cAMP treatment of Sertoli cells. We propose that SV40 large T antigen in COS cells can mimick the action of heterodimers formed in cAMP stimulated Sertoli cells between Egr-1 and a cell specific cAMP-induced GC-box binding factor.

Animals↗

PubMed1995

Reconstitution of the B cell antigen receptor signaling components in COS cells.

To elucidate interactions occurring between B cell protein tyrosine kinases and the signaling components of the B cell antigen receptor, we have co-transfected into COS cells individual tyrosine kinases together with chimeric cell surface receptors containing the cytoplasmic domains of Ig alpha or Ig beta. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with cotransfected Ig alpha and Ig beta chimeras in vivo. Association between Blk and the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Ig alpha and Ig beta chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Ig alpha chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Ig alpha. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation.

Animals↗

PubMed1987

Molecular cloning of two CD7 (T-cell leukemia antigen) cDNAs by a COS cell expression system.

The human CD7 antigen (gp40) is a cell surface glycoprotein found on thymocytes and mature T-cells. It is one of the earliest antigens to appear on cells of the T-lymphocyte lineage, and the most reliable clinical marker of T-cell acute lymphocytic leukemia. This report describes the isolation and nucleotide sequence of a full length CD7 cDNA, and of a cDNA for an unusual intron-bearing precursor. The DNA sequence of the clone predicts a highly glycosylated membrane protein with homology to members of the immunoglobulin superfamily, and no relationship to known oncogenes. Over-expression of CD7 RNA was observed in only one T-cell tumor line, and genomic DNA rearrangement was not observed in any lines. Prompted by a recent suggestion that CD7 plays a role in IgM binding, COS cells expressing CD7 were tested and found not to bind IgM or IgM immune complexes.

Amino Acid Sequence↗

PubMed1997

Three different endogenous alpha-L-fucosyltransferases expressed in COS cells.

The monkey kidney COS cell line is frequently used for the transient expression of cloned human fucosyltransferase cDNAs in the belief that negligible endogenous expression of fucosyltransferase genes occurs in these cells. In the course of transfection experiments we observed weak cell surface expression of sialyl-Lex and weak fucosyltransferase activity in extracts of control untransfected cells. Since these activities could complicate interpretation of the results with the transfected genes, a more detailed examination was undertaken that has now revealed expression of three different fucosyltransferases in the cells. One enzyme, which utilises N-acetyllactosamine as substrate, has a pH optimum of 7.0, is resistant to heat inactivation, and has been tentatively identified as an alpha1,3-fucosyltransferase. A second enzyme which acts on asialo-fetuin has a pH optimum of 5.5 and is rapidly inactivated by heat; the acceptor sugar and positional linkage of the transferred fucose are not yet established. A third enzyme that utilises asialo-agalacto-fetuin as acceptor is provisionally identified as an alpha1,6-fucosyltransferase.

Animals↗

PubMed1999

Expression of human alpha-l-fucosyltransferase gene homologs in monkey kidney COS cells and modification of potential fucosyltransferase acceptor substrates by an endogenous glycosidase.

Previous investigations on the monkey kidney COS cell line demonstrated the weak expression of fucosylated cell surface antigens and presence of endogenous fucosyltransferase activities in cell extracts. RT-PCR analyses have now revealed expression of five homologs of human fucosyltransferase genes, FUT1, FUT4, FUT5, FUT7, and FUT8, in COS cell mRNA. The enzyme in COS cell extracts acting on unsialylated Type 2 structures is closely similar in its properties to the alpha1,3-fucosyltransferase encoded by human FUT4 gene and does not resemble the product of the FUT5 gene. Although FUT1 is expressed in the COS cell mRNA, it has not been possible to demonstrate alpha1,2-fucosyltransferase activity in cell extracts but the presence of Le(y) and blood-group A antigenic determinants on the cell surface imply the formation of H-precursor structures at some stage. The most strongly expressed fucosyltransferase in the COS cells is the alpha1,6-enzyme transferring fucose to the innermost N -acetylglucosamine unit in N -glycan chains; this enzyme is similar in its properties to the product of the human FUT8 gene. The enzymes resembling the human FUT4 and FUT8 gene products both had pH optima of 7.0 and were resistant to 10 mM NEM. The incorporation of fucose into asialo-fetuin was optimal at 5.5 and was inhibited by 10 mM NEM. This result initially suggested the presence of a third fucosyltransferase expressed in the COS cells but we have now shown that triantennary N- glycans with terminal nonreducing galactose units, similar to those present in asialo-fetuin, are modified by a weak endogenous beta-galactosidase in the COS cell extracts and thereby rendered suitable substrates for the alpha1,6-fucosyltransferase.

ABO Blood-Group System↗

PubMed1990

Expression of the phospholipid-dependent Escherichia coli sn-1,2-diacylglycerol kinase in COS cells perturbs cellular lipid composition.

The Escherichia coli sn-1,2-diacylglycerol (DAG) kinase has been successfully expressed in COS cells. The E. coli dgkA locus which contains the coding sequences for DAG kinase was subcloned into an eukaryotic expression vector, pMT2. COS cells transfected with the vector pMT2dgk expressed the DAG kinase as shown by Western analysis. Immunofluorescence studies revealed that the E. coli DAG kinase was prominently but not exclusively located in the endoplasmic reticulum. In addition, mixed micellar assays in beta-octyl glucoside revealed that membranes prepared from pMT2dgk-transfected COS cells contained over a 1500-fold increase in DAG kinase activity: 107 nmol/min/mg compared with only 0.067 nmol/min/mg for controls. DAG kinase activity from the E. coli enzyme was distinguished from endogenous COS cell activity based on differences in thermolability and the ability of the E. coli enzyme to use ceramide as a substrate. No ceramide kinase activity was detected in control COS cells, so the activity detected in pMT2dgk transfectants must have resulted from the expressed E. coli DAG kinase. The Km values for DAG kinase derived from E. coli and COS cells were nearly identical. Finally, transfected COS cells were labeled with [32P]Pi to investigate possible perturbations in lipid composition induced by the action of the E. coli DAG kinase. Ceramide (generated by the action of sphingomyelinase) was also used to clearly implicate the E. coli enzyme. Levels of ceramide phosphate increased more than 150-fold in pMT2dgk-transfected cells relative to controls. The results of these studies show that the E. coli enzyme expressed in COS cells is active and perturbs lipid composition in the intact cell system; the absolute lipid cofactor requirement of E. coli DAG kinase can be satisfied in COS cells.

Animals↗

PubMed1988

Transfection of genes encoding the T cell receptor-associated CD3 complex into COS cells results in assembly of the macromolecular structure.

The T cell antigen receptor (TCR) consists of a disulfide-linked TCR-alpha/beta heterodimer that is both structurally and functionally associated with a set of four non-covalently linked membrane proteins termed CD3-gamma, -delta, -epsilon, and -zeta. An additional protein described recently, CD3-omega, has been suggested to play a role in assembly of the CD3 complex on the basis of its transient association with the CD3 proteins early during biosynthesis. Association of all the proteins seems to be a prerequisite for intracellular transport, since mutants lacking either the TCR-alpha or -beta protein do not express the CD3 complex on the cell surface. CD3-cDNAs were transfected into COS cells in order to study the protein-protein interactions ruling the assembly of the CD3 macromolecular structure. CD3-delta-epsilon, CD3-gamma-epsilon, and CD3-gamma-delta-epsilon intermediates could be detected. These data indicated that a CD3 core structure could be formed in the absence of the other members of the complex (CD3-zeta, -omega, TCR-alpha, and -beta). Both the individual CD3 chains and the assembled CD3.gamma.delta.epsilon complexes could not be detected on the cellular surface but in an intracellular compartment, probably the endoplasmic reticulum or the cis Golgi. The transfection experiments allowed us to identify the 25-kDa member of the murine CD3 complex as CD3-epsilon m. Furthermore, a 23-kDa glycoprotein seen upon metabolic labeling of human T cells was shown to be an immature form of the CD3-gamma h protein.

Animals↗

PubMed1994

Expression of human aspartyl-tRNA synthetase in COS cells.

Mammalian aspartyl-tRNA synthetase (DRS) occurs in a multi-enzyme complex of aminoacyl-tRNA synthetases, while DRS exists as free soluble enzymes in bacteria and yeast. The properties of human DRS transient expressed in COS cells were examined. After transfection of COS cells with the recombinant plasmids pSVL-63 that contained hDRS cDNA coding and non-coding sequences, and pSV-hDRS where the non-coding sequences were deleted, DRS in the transfected COS cells significantly increased compared to mock transfected cells. COS cells transfected with pSV-hDRS delta 32 that contained N-terminal 32 residue-coding sequence deleted hDRS cDNA showed no increase in DRS activity. Northern blot analysis showed that concentrations of corresponding mRNAs of hDRS and hDRS delta 32 were greatly enhanced in transfected cells. The increases in the level of the transcripts were much higher than those of the corresponding proteins. Gel filtration analysis showed that hDRS in pSV-hDRS transfected cells expressed as a low molecular weight form of hDRS and pSV-hDRS delta 32 transfected cells did not. Epitope tagging and indirect immunofluorescence microscopy was used to localize hDRS. Both hDRSmyc and hDRS delta 32myc were localized in the cytoplasm and showed diffused patterns. These results showed that hDRS has little tendency to aggregate in vivo and suggested that the N-terminal extension in hDRS was not involved in the expression and sub-cellular localization of hDRS, but may play a role in the maintenance of enzymatic activity of hDRS in COS cells.

Animals↗

PubMed1996

Detection and isolation of lectin-transfected COS cells based on cell adhesion to immobilized glycosphingolipids.

Two methods are described, one for detection and one for isolation of COS cells transiently expressing vertebrate lectins. The methods are based on specific cell adhesion to polystyrene microwells or magnetic beads adsorbed with glycosphingolipids. In the first method, glycolipids were adsorbed to wells of 96-well polystyrene plates. A suspension of lectin-transfected COS cells was added and the plate was incubated to allow cell adhesion to occur. The plate was then immersed in buffer, inverted (while immersed), and placed in a fluid-filled Plexiglas centrifugation chamber which was sealed to avoid introducing an air-liquid interface. The chamber, with the inverted plate enclosed, was centrifuged to remove nonadherent cells. The plate was then removed from the carrier (while immersed) and righted, and adherent cells were quantitated enzymatically or immunochemically using a 96-well plate reader. COS cells transfected with an expression plasmid carrying the gene for the rat Kupffer cell lectin (fucose and N-acetylgalactosamine specific) adhered specifically to globotetraosylceramide. Glycolipid- and lectin-specific cell adhesion was readily detected even when COS cells were transfected with a plasmid mixture containing 0.5% lectin-carrying plasmid and 99.5% irrelevant plasmid. This sensitivity will facilitate screening of plasmid pools to detect and isolate plasmids expressing mammalian lectin genes. To isolate COS cells transiently expressing lectin, glycosphingolipids were adsorbed to carboxylated magnetic polystyrene microspheres, which were mixed with the lectin-transfected COS cells. Adherent cells were collected on a fixed magnet and plasmid recovered for subsequent amplification.

Adsorption↗

PubMed1994

Effects of transient expression of spermidine/spermine N1-acetyltransferase in COS cells.

Mammalian spermidine/spermine N1-acetyltransferase (SSAT) was transiently expressed in COS cells. As compared to COS cells transfected with control vector alone, cells transfected with the expression vector containing SSAT cDNA contained lower concentrations of spermidine and spermine. The putrescine content, on the other hand, was markedly increased in the COS cells expressing large amounts of SSAT. These changes in polyamine content were most likely caused by an interconversion of spermine and spermidine into putrescine. The SSAT-induced changes in cellular polyamine content resulted in a compensatory increase in the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, i.e. the enzymes catalyzing the rate-limiting steps in polyamine biosynthesis. This is the first demonstration that a primary increase in SSAT activity will induce an interconversion-like change in the polyamine levels and the physiological role of SSAT is most likely to protect cells against too high concentrations of spermidine and spermine.

Acetyltransferases↗