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R F Margolskee

Publications and source records attributed to R F Margolskee.

13 recordsLinked to original sources
PubMed1992

Gustducin is a taste-cell-specific G protein closely related to the transducins.

A novel G protein alpha-subunit (alpha-gustducin) has been identified and cloned from taste tissue. alpha-Gustducin messenger RNA is expressed in taste buds of all taste papillae (circumvallate, foliate and fungiform); it is not expressed in non-sensory portions of the tongue, nor is it expressed in the other tissues examined. alpha-Gustducin most closely resembles the transducins (the rod and cone photoreceptor G proteins), suggesting that gustducin's role in taste transduction is analogous to that of transducin in light transduction.

Amino Acid Sequence↗
PubMed1992

Isolation of a clone which induces expression of the gene encoding the human tumor necrosis factor receptor.

Tumor necrosis factor (TNF) is a cytokine with pleiotropic effects upon cell growth, inflammation and immunologic responsiveness. High-affinity TNF receptors (TNFRs) of 55 and 75 kDa are found in many cell types. Using an Epstein-Barr virus (EBV)-based mammalian expression library, we have isolated a clone from human lymphoblastoid transfectants that induces overexpression of the TNFR-encoding gene (TNFR). Transfectants overproducing the TNFR were isolated by multiple rounds of sorting on a fluorescence-activated cell sorter using fluorescent TNF ligand binding as the selection procedure. Among the sorted transfectants were cells producing approx. 150,000 receptors per cell (Kd of approx. 1 nM). These cells have multiple copies of the TNFR gene present as extrachromosomal plasmids. These cells also overproduced the mRNA for TNFR. Low-Mr EBV episomes were isolated from these overproducing cells and used to transform Escherichia coli. One of the colonies isolated contained a plasmid encoding a portion of the noncoding region of the TNFR gene. Transfection of human lymphoblastoid cells with this DNA gave rise to high-level production of TNFR. Fluorescent TNF bound to these transfectants is fully and specifically displaced by an excess of TNF. The rescued clone contains approx. 10 kb of human genomic DNA including the 3'-untranslated region of TNFR and several Alu sequences; apparently during the selection procedure in human cells, recombination occurred to rescue a portion of the TNFR gene. Transient transfection was used to narrow down the region responsible for TNFR induction to 5.2 kb. The mechanism by which this clone induces TNFR expression has not been determined.

Base Sequence↗
PubMed1992

Cloning muscle isoforms of neural cell adhesion molecule using an episomal shuttle vector.

Three distinct transcripts encoding two phosphatidylinositol (PI) linked isoforms of the neural cell adhesion molecule (NCAM) are induced during the differentiation of C2C12 myoblasts into myotubes. Corresponding NCAM clones were isolated from a mouse muscle cDNA library made in an Epstein-Barr virus shuttle vector that replicates extrachromosomally in human cells. Following transfection with the library, human cells expressing mouse NCAM were enriched using the fluorescence-activated cell sorter. Episomal NCAM clones recovered from sorted cells contain an 18-bp insert between exons 12 and 13. Two other NCAM cDNAs encode identical polypeptides containing a 108-bp insert homologous to the complete MSD1 domain, but differ in their 3' untranslated regions. Induction of MSD1-containing transcripts in advance of myotube formation suggests that muscle-specific NCAMs contribute to myogenesis from the earliest stages of differentiation. Moreover, our studies demonstrate the feasibility of cloning tissue-specific molecules by transfection and expression of cDNA libraries in episomal vectors.

Amino Acid Sequence↗
PubMed1992

Hepatitis B virus (HBV)-specific cytotoxic T-cell (CTL) response in humans: characterization of HLA class II-restricted CTLs that recognize endogenously synthesized HBV envelope antigens.

In this study, we show that CD4+, hepatitis B virus (HBV) envelope-specific T-cell clones produced by stimulation with a particulate antigen preparation are able to recognize and kill not only autologous antigen-presenting cells incubated with exogenous HBV envelope antigens but also autologous HLA class II-positive cells expressing endogenously synthesized HBV envelope antigens following infection with recombinant vaccinia viruses or transfection with recombinant Epstein-Barr virus expression vectors. Experiments with lysosomotropic agents and brefeldin A suggest that the endosomal compartment is likely involved in the processing of endogenously synthesized viral proteins for recognition by CD4+ T cells. Our study indicates that HBV envelope-specific, HLA class II-restricted CD4+ cytotoxic T lymphocytes can potentially participate in the immune clearance of HBV-infected cells and the pathogenesis of hepatocellular injury in hepatitis B.

Ammonium Chloride↗
PubMed1992

Hepatitis B virus (HBV)-specific cytotoxic T-cell response in humans: production of target cells by stable expression of HBV-encoded proteins in immortalized human B-cell lines.

To analyze the hepatitis B virus (HBV)-specific cytotoxic T-cell (CTL) response during acute and chronic viral hepatitis, target cells that express HBV-encoded antigens in the context of the appropriate HLA restriction element must be available for each subject studied. Since HBV is not infectious for human cells in vitro, such target cells must be produced by DNA-mediated gene transfer into cultured human primary cells or cell lines. For this purpose, we have developed a panel of Epstein-Barr virus-based episomal expression vectors containing each of the HBV open reading frames under the transcriptional control of the simian virus 40 early promoter. Transfection of Epstein-Barr virus-immortalized B-cell lines with this panel of recombinants consistently leads to stable expression of the HBV envelope, nucleocapsid, and polymerase proteins. The HBV X gene product is transiently expressed following transfection, but stable expression of this protein cannot be maintained on a long-term basis. To assess the suitability of this system for the identification of HBV-specific CTL in humans, a panel of EBO-HBV transfectants of defined HLA haplotype was used to monitor the HBV-specific CTL response in a patient with acute viral hepatitis type B. Transfectants that stably express the HBV nucleocapsid (core) antigen were found to serve as excellent targets for the detection of HLA class I-restricted CTL that recognize endogenously synthesized HBV core antigen in this patient; they were also successfully used to stimulate the specific expansion of these CTL in vitro.

B-Lymphocytes↗
PubMed1991

A highly efficient directional cDNA cloning method utilizing an asymmetrically tailed linker-primer plasmid.

A new procedure using an asymmetrically tailed linker-primer plasmid has been developed to prepare extremely high complexity cDNA libraries. This procedure yields plasmid primed libraries with a final form equivalent to those made by the procedure of Okayama and Berg. However, the number of steps involved in library preparation is decreased. The form of the vector is such that one end of the linearized linker-primer plasmid has a 3' terminal extension of 40 deoxythymidylate residues (the dT end). The other end has a 3' terminal extension of 10 deoxycytidylate residues (the dC end). The dC end of the plasmid is blocked to further 3' extension by a 3' phosphate group. This configuration enables one to prime first strand cDNA synthesis at the dT end, tail the 3' end of the cDNA with deoxyguanylate residues without tailing the dC end (due to the 3' phosphate block). The plasmid primed cDNA can then be self-annealed and the 3' phosphate blocking group removed during the synthesis of double stranded cDNA. The efficiency of this procedure is significantly higher than other methods (including phage based libraries): linker-primer libraries have 15 to 900-fold higher complexity than libraries prepared by other methods. A cloning efficiency of 9 x 10(8) colonies per microgram of linker-primer DNA was achieved. This method should be useful for the cloning of cDNAs corresponding to extremely rare mRNAs.

Base Sequence↗
PubMed1990

Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector.

Site-directed mutagenesis was used to identify residues responsible for the greater than 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase alpha 1 and alpha 2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat alpha 2 subunit with the corresponding residues from the rat alpha 1 subunit. These cDNAs were cloned into a mammalian episomal expression vector (EBOpLPP) and expressed in ouabain-sensitive primate cells. Either of two single substitutions introduced into the rat alpha 2 subunit cDNA (Leu-111----Arg or Asn-122----Asp) conferred partial resistance (approximately 10 microM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat alpha 1 cDNA (approximately 500 microM ouabain) and the rat alpha 2 cDNA (approximately 0.2 microM ouabain). A double substitution of the rat alpha 2 cDNA (Leu-111----Arg and Asn-122----Asp) conferred a resistance level equivalent to that obtained with rat alpha 1. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the end of the H1-H2 extracellular domain. The combination of site-directed mutagenesis and episomal expression provides a useful system for the selection and analysis of mutants.

Amino Acid Sequence↗
PubMed1988

Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells.

Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1979

Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin.

The circular genome of simian virus 40 is a model mammalian replicon, containing a unique origin of replication (ori) and coding for a protein (SV40 T antigen) known to be involved in initiation of viral DNA replication and to bind in vitro to the origin region. Mutations within the ori sequence lead to defective viral DNA replication and the formation of small viral plaques after infection of a cell monolayer. Second-site revertants (pseudorevertants) of ori mutants were isolated by random local mutagenesis of mutant DNA followed by transfection of cultured cells and the selection of large plaques. In each case, reversion of the plaque phenotype was associated with an increased rate of viral DNA replication. The second-site mutations that suppressed the replication defects were localized by in vitro recombination or marker rescue experiments to the gene for T antigen. Their map positions differ from those of previously described T antigen mutants, possibly reflecting a specific ori-binding domain of T antigen. From these results we infer that T antigen interacts with the ori signal during virus development as it does in vitro and that this interaction regulates the rate of viral DNA replication.

Antigens, Neoplasm↗
PubMed1979

Glutamine synthetase mutations which affect expression of nitrogen fixation genes in Klebsiella pneumoniae.

Previous studies have implicated glutamine synthetase (L-glutamate:ammonia ligase [adenosine diphosphate for-ing], EC 6.6.1.2) as a major controlling element of the nitrogen fixation (nif) genes in Klebsiella pneumoniae. We report here the isolation of a new class of K. pneumoniae mutants which exhibit altered patterns of nif and hut (histidine utlization) regulation. The expression of nif in these mutants, which were isolated as Gln+ (glutamine nonrequiring) revertants of a particular glnA mutation, is extremely sensitive to ammonia repression. These mutants have a Nif- Hut- phenotype at external ammonia concentrations at which wild-type strains are Nif+ Hut+. On the other hand, these mutants can be fully derepressed for nif at very low ammonia concentrations. We adopted the nomenclature "GlnR- (Nif- Hut-)" to facilitate discussion of the phenotype of these mutant strains. The mutations in these strains which confer the GlnR- phenotype map at or near glnA, the structural gene for glutamine synthetase.

Ammonia↗