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At least 1,117 records · Page 62Linked to original sources
PubMed1991

Rescue of a foreign gene by Sendai virus.

A simple protocol for the rescue of a synthetic genome into a paramyxovirus has been developed. First, a synthetic Sendai virus-like RNA, containing the antisense coding region of the chloramphenicol acetyltransferase gene replacing the coding region of the Sendai virus genome, was transcribed from a cDNA. When introduced into cells that are infected with Sendai virus, this RNA construct was transcribed, replicated, and packaged into infectious virions. The addition of infected cell extract to the RNA prior to transfection markedly enhanced levels of chloramphenicol acetyltransferase expression and rescue. However, this enhancement is not due to encapsidation of the RNA into nucleocapsids as the RNA remains nuclease-sensitive. Uninfected cell extract also enhances expression and rescue efficiency, implying involvement of a cellular factor(s) with the synthetic viral-like RNA construct that allows for enhanced polymerase recognition. This system should allow for the dissection of the various cis-acting RNA signals within the paramyxovirus genome.

Animals↗
PubMed1993

Copper-controllable gene expression system for whole plants.

We describe a system for gene expression in plants based on the regulation mechanism of the yeast metallothionein (MT) gene. The system consists of two elements: (i) the yeast ace1 (activating copper-MT expression) gene encoding a transcription factor under control of the cauliflower mosaic virus (CaMV) 35S RNA promoter, and (ii) a gene of interest under control of a chimeric promoter consisting of the 90-base-pair domain A of the CaMV 35S RNA promoter linked to the ACE1 transcription factor-binding site. At elevated copper ion concentrations, the ACE1 protein changes conformation, binds to, and activates transcription from the chimeric promoter. To test the functioning of the system in plants, a construct containing the beta-glucuronidase (GUS) reporter gene under control of the chimeric promoter was prepared, and transgenic tobacco plants were produced. It was shown that GUS activity in the leaves of transgenic plants increased up to 50-fold, either after addition of 50 microM CuSO4 to the nutrient solution or after application of 0.5 microM CuSO4 to the plants in a foliar spray. This GUS expression was repressed after the removal of copper ions. The results show that the activity of the described chimeric promoter directly depends on copper ion concentration and that this system can be used in experiments that demand precise timing of expression.

Base Sequence↗
PubMed1997

A yeast genetic system for selecting small molecule inhibitors of protein-protein interactions in nanodroplets.

Cellular processes are mediated by complex networks of molecular interactions. Dissection of their role most commonly is achieved by using genetic mutations that alter, for example, protein-protein interactions. Small molecules that accomplish the same result would provide a powerful complement to the genetic approach, but it generally is believed that such molecules are rare. There are several natural products, however, that illustrate the feasibility of this approach. Split-pool synthesis now provides a simple mechanical means to prepare vast numbers of complex, even natural product-like, molecules individually attached to cell-sized polymer beads. Here, we describe a genetic system compatible with split-pool synthesis that allows the detection of cell-permeable, small molecule inhibitors of protein-protein interactions in 100- to 200-nl cell culture droplets, prepared by a recently described technique that arrays large numbers of such droplets. These "nanodroplets" contain defined media, cells, and one or more beads containing approximately 100 pmol of a photoreleasable small molecule and a controlled number of cells. The engineered Saccharomyces cerevisiae cells used in this study express two interacting proteins after induction with galactose whose interaction results in cell death in the presence of 5-fluoroorotic acid (inducible reverse two-hybrid assay). Disruption of the interaction by a small molecule allows growth, and the small molecule can be introduced into the system hours before induction of the toxic interaction. We demonstrate that the interaction between the activin receptor R1 and the immunophilin protein FKBP12 can be disrupted by the small molecule FK506 at nanomolar concentrations in nanodroplets. This system should provide a general method for selecting cell-permeable ligands that can be used to study the relevance of protein-protein interactions in living cells or organisms.

Activin Receptors, Type I↗
PubMed1998

Characterization of hematopoietic progenitor cells that express the transcription factor SCL, using a lacZ "knock-in" strategy.

Gene targeting experiments have demonstrated that the transcription factor SCL is essential for primitive and definitive hematopoiesis in the mouse. To study the functional properties of hematopoietic cells expressing SCL, we have generated mutant mice (SCLlacZ/w) in which the Escherichia coli lacZ reporter gene has been "knocked in" to the SCL locus, thereby linking beta-galactosidase expression to transcription from the SCL promoter. Bone marrow cells from heterozygous SCLlacZ/w mice were sorted into fractions expressing high, intermediate and low levels of beta-galactosidase (designated lacZhigh, lacZint, and lacZneg). Cells that were lacZhigh or lacZint were enriched for day 12 spleen colony-forming units and myeloid and erythroid colony-forming cells (CFCs). These fractions included >99% of the erythroid and >90% of the myeloid CFCs. Culture of sorted bone marrow populations on stromal cells secreting interleukin-7 or in fetal thymic organ cultures showed that B and T lymphoid progenitors were also present in the lacZhigh and lacZint fractions. These data provide a functional correlation between SCL expression and colony-forming ability in immature hematopoietic cells. Our data also suggested that expression of SCL was transient and confined to hematopoietic stem and/or progenitor cells, because the differentiated progeny of most lineages (except the erythroid) were beta-galactosidase-negative.

Animals↗
PubMed1998

Charge-dependent translocation of Bordetella pertussis adenylate cyclase toxin into eukaryotic cells: implication for the in vivo delivery of CD8(+) T cell epitopes into antigen-presenting cells.

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400-aa residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver T cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted CD8(+) cytotoxic T cell responses in vivo. Here, we constructed a series of recombinant toxins harboring at the same insertion site various peptide sequences of 11-25 amino acids, corresponding to defined CD8(+) T cell epitopes and differing in the charge of the inserted sequence. We show that inserted peptide sequences containing net negative charges (-1 or -2) decreased or completely blocked (charge of -4) the internalization of the toxin into target cells in vitro and abolished the induction of cytotoxic T cell responses in vivo. The blocking of translocation due to the inserted acidic sequences can be relieved by appropriate mutations in the flanking region of CyaA that counterbalance the inserted charges. Our data indicate that (i) the electrostatic charge of the peptides inserted within the catalytic domain of CyaA is critical for its translocation into eukaryotic cells and (ii) the delivery of T cell epitopes into the cytosol of antigen-presenting cells by recombinant CyaA toxins is essential for the in vivo stimulation of specific cytotoxic T cells. These findings will help to engineer improved recombinant CyaA vectors able to stimulate more efficiently cellular immunity.

Adenylate Cyclase Toxin↗
PubMed1996

Selection of linkers for a catalytic single-chain antibody using phage display technology.

Phage display has been evaluated as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. Preliminary experiments with a conventional linker failed to yield a functional single-chain version of a catalytic antibody with chorismate mutase activity. A random linker library was therefore constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire ( approximately 5 x 10(6) different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VH C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers. There are apparently many viable solutions to the problem of linking individual VH and VL domains, but subtle differences in sequence dramatically influence the production, stability, and recognition properties of the scFv. The success of these experiments suggests that phage display will be generally useful for identifying peptide sequences for covalently linking any two protein domains.

Amino Acid Sequence↗
PubMed1996

Human coproporphyrinogen oxidase is not a metalloprotein.

Coproporphyrinogen oxidase (CPO) (EC 1.3.3.3), the antepenultimate enzyme in the heme biosynthetic pathway, catalyzes the conversion of coproporphyrinogen III to protoporphyrinogen IX. Previously, based upon metal analysis and site-directed mutagenesis of purified recombinant enzyme, it has been suggested that CPO contains and requires copper for activity (Kohno, H., Furukawa, T., Tokunaga, R., Taketani, S., and Yoshinaga, T. (1996) Biochim. Biophys. Acta 1292, 156-162). To examine this putative metal site in human CPO, the cDNA encoding human CPO was engineered into an expression vector with a His6 tag at its amino terminus, and the protein was expressed in Escherichia coli and purified to apparent homogeneity using nickel-nitroliotriacetic acid resin. Activity of the purified protein was monitored by a coupled fluorometric assay that employed purified protoporphyrinogen oxidase to convert protoporphyrinogen to protoporphyrin, thereby allowing the direct fluorescent determination of protoporphyrin IX produced. CPO has an apparent Km of 0.6 microM and an apparent Kcat of 16 min-1 with coproporphyrinogen III as substrate. Metal analysis of the enzyme was carried out via ultraviolet and visible spectroscopy, inductively coupled plasma atomic emission spectroscopy metal analysis, and electron paramagnetic resonance spectroscopy. The data presented demonstrate that human CPO contains no metal center, that it is not stimulated in vitro by iron or copper, and that addition of these metals to cultures expressing the protein has no effect.

Amino Acid Sequence↗
PubMed1998

A biochemical and genetic model for parasite resistance to antifolates. Toxoplasma gondii provides insights into pyrimethamine and cycloguanil resistance in Plasmodium falciparum.

We have exploited the experimental accessibility of the protozoan parasite Toxoplasma gondii and its similarity to Plasmodium falciparum to investigate the influence of specific dihydrofolate reductase polymorphisms known from field isolates of drug-resistant malaria. By engineering appropriate recombinant shuttle vectors, it is feasible to examine mutations by transient or stable transformation of T. gondii parasites, in bacterial and yeast complementation assays, and through biochemical analysis of purified enzyme. A series of mutant alleles that mirror P. falciparum variants reveals that the key mutation Asn-108 (Asn-83 in T. gondii) probably confers resistance to pyrimethamine by affecting critical interactions in the ternary complex. Mutations such as Arg-59 (T. gondii 36) have limited effect in isolation, but in combination with other mutations they enhance the competitive ability of folate by increasing the speed of product turnover. Val-16 (T. gondii 10) confers low level resistance to cycloguanil but hypersensitivity to pyrimethamine. This mutation precludes Asn-108, probably because compression of the folate binding pocket introduced by this combination is incompatible with enzyme function. These studies permit detailed biochemical, kinetic, and structural analysis of drug resistance mutations and reconstruction of the probable phylogeny of antifolate resistance in malaria.

Alleles↗
PubMed2003

Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes.

Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

Animals↗
PubMed1983

Expression of Semliki Forest virus proteins from cloned complementary DNA. II. The membrane-spanning glycoprotein E2 is transported to the cell surface without its normal cytoplasmic domain.

The E2 protein (422 amino acid residues long) of Semliki Forest virus is a spanning membrane protein which is made in the rough endoplasmic reticulum of the infected cell and transported to the cell surface. The cytoplasmic domain of this protein comprises 31 amino acid residues. We introduced deletions of various sizes into the gene region encoding this part of the protein molecule and analyzed the transport behavior of the mutant proteins. The deletions were made using exonuclease digestions of cloned cDNA encoding the E2 protein. When the mutated DNA molecules, engineered into an expression vector, were introduced into nuclei of baby hamster kidney 21 cells, membrane proteins with cytoplasmic deletions were expressed and routed to the cell surface in the same way as the wild-type protein. This suggests that the cytoplasmic domain of the E2 protein does not carry information that is needed for its transport from the rough endoplasmic reticulum to the cell surface.

Amino Acid Sequence↗
PubMed1999

Retrovirus-mediated correction of the metabolic defect in cultured Farber disease cells.

Farber disease is a rare severe lysosomal storage disorder due to a deficient activity of the enzyme acid ceramidase (AC). Patients have granulomas along with lipid-laden macrophages that accumulate in a number of tissues, leading to multiple diverse clinical symptoms. There is no therapy for the disorder and most patients succumb to the disease in early childhood. The severity of the disease progression seems to correlate with the amount of the accumulated ceramide substrate. Since the cDNA for human AC has been elucidated we sought to establish if genetic transfer of this sequence would lead to enzymatic and, especially, functional correction of the catabolic defect in Farber patient cells. To do this, a novel amphotropic recombinant retrovirus was constructed that engineers transfer of the human AC cDNA. On infection of patient fibroblasts, AC enzyme activity in cell extracts was completely restored. Further, substrate-loading assays of intact living cells showed a fully normalized catabolism of lysosomal ceramide. Lastly, as reported for some other corrected enzymatic defects of lysosomes, metabolic cooperativity was seen, in that functionally corrected patient fibroblasts secreted AC that was taken up through the mannose 6-phosphate receptor and used by uncorrected fibroblasts as well as recipient Farber lymphoblastoid cells. This overall transduction and uptake scenario for Farber disease allows future treatment of this severe disorder to be envisioned using gene transfer approaches.

Acid Ceramidase↗