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At least 397 records · Page 22Linked to original sources
PubMed1990

Optimization of the hygromycin B resistance-conferring gene as a dominant selectable marker in mammalian cells.

The HyR gene, conferring resistance to hygromycin B (Hy), has been modified for optimal expression in mammalian cells. Modifications to the HyR gene and its expression cassette include: (1) removal of all upstream start codons, (2) conversion of the region around the start codon to the consensus sequence associated with efficient translation initiation, and (3) removal of downstream splice donor and acceptor sequences. The resulting HyR gene is an efficient dominant selectable marker that is useful for studies requiring resistance from a low-copy-number gene driven by a promoter of moderate strength. The HyR gene was also tested for its compatibility with BPV vectors. Mouse C127 cells harboring pHyR-BPV plasmids exhibited properties of BPV-transformed cells and were resistant to toxic levels of Hy. The vectors were stable as episomes and present in high copy. The HyR gene thus joins the NmR (neo) gene as the only dominant selectable markers that are known to be compatible with BPV replication.

Animals↗
PubMed1990

Construction and hormone regulation of a novel retroviral vector.

We report the analysis of a self-inactivating retroviral vector, constructed to allow inducible gene expression of inserted sequences from the mouse mammary tumour virus hormonal response element. The cloning strategy has been designed to allow for ease of insertion of the genes of interest. The vector contains the aph gene, allowing geneticin-resistance selection in mammalian cells. We have characterised dexamethasone (Dex)-induced increase in gene expression using the reporter gene encoding chloramphenicol acetyltransferase (CAT) inserted into the retroviral vector. We observe low basal levels of CAT activity in infected cells which is increased up to 50-fold by induction with Dex. The induction of pooled clones is 13.3-fold. Variation in Dex-induced CAT activity is observed in independently infected clones, which is not explained by proviral copy number.

Animals↗
PubMed2005

Cyanide detoxification by recombinant bacterial rhodanese.

Cyanide is a major environmental pollutant of the chemical and metallurgical industries. Although extremely toxic, cyanide can enzymatically be converted to the less toxic thiocyanate by rhodaneses (thiosulfate:cyanide sulfurtransferases, EC 2.8.1.1). We engineered a genetic system to express high levels of recombinant Pseudomonas aeruginosa rhodanese (r-RhdA) in Escherichia coli, and used this organism to test the role of r-RhdA in cyanide detoxification. Inducible expression of the rhdA gene under the control of the hybrid T7-lacO promoter yielded active r-RhdA over a 4-h period, though r-RhdA-expressing E. coli showed decreased viability starting from 1 h post-induction. At this time, Western blot analysis and enzymatic assay showed r-RhdA partition between the cytoplasm (95%) and the periplasm (5%). The accessibility of thiosulfate to r-RhdA was a limiting step for the sulfur transfer reaction in the cellular system, but cyanide conversion to thiocyanate could be increased upon permeabilization of the bacterial membrane. Specific r-RhdA activity was higher in the whole-cell assay than in the in vitro assay with pure enzyme (2154 vs. 816 micromol min-1 mg-1 r-RhdA, respectively), likely reflecting enzyme stability. The r-RhdA-dependent cyanide detoxification resulted in increased resistance of r-RhdA overexpressing E. coli to 5 mM cyanide. Bacterial survival was paralleled by release of thiocyanate into the medium. Our results indicate that cyanide detoxification by engineered E. coli cells is feasible under laboratory conditions, and suggest that microbial rhodaneses may contribute to cyanide transformation in natural environments.

Biodegradation, Environmental↗
PubMed2006

Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin.

Artemisinin, a sesquiterpene lactone endoperoxide derived from the plant Artemisia annua, forms the basis of the most important treatments of malaria in use today. In an effort to elucidate the biosynthesis of artemisinin, an expressed sequence tag approach to identifying the relevant biosynthetic genes was undertaken using isolated glandular trichomes as a source of mRNA. A cDNA clone encoding a cytochrome P450 designated CYP71AV1 was characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of the proposed biosynthetic intermediates amorpha-4,11-diene, artemisinic alcohol and artemisinic aldehyde. The identification of the CYP71AV1 gene should allow for the engineering of semi-synthetic production of artemisinin in appropriate plant or microbial hosts.

Antimalarials↗
PubMed2006

Expression of recombinant puroindolines for the treatment of staphylococcal skin infections (acne vulgaris).

Puroindolines are antimicrobial peptides that occur in wheat seed, and are characterized by broad antimicrobial activity. We describe the heterologous expression of puroindoline A and B in the Origami strain of Escherichia coli. The recombinant puroindolines showed the same antimicrobial activity on Staphylococcus epidermidis as compared to the native peptides (MIC(90)=30microgml(-1)). The bactericidal activity was 125microgml(-1) for recombinant puroindoline A and 42microgml(-1) for recombinant puroindoline B. Neither protein shows in vitro haemolytic activity or toxicity towards the murine macrophage cell line J774, but they are able to kill intracellular staphylococci. Our preliminary results suggest that recombinant puroindolines deserve further attention as alternatives to the conventional antibiotics in the control of S. epidermidis skin infections.

Acne Vulgaris↗
PubMed2003

Lignins and lignocellulosics: a better control of synthesis for new and improved uses.

The composition and structure of lignified walls has a dramatic impact on the technological value of raw materials. The chemical flexibility of the secondary cell wall has been demonstrated and it is now possible to develop strategies to optimize its composition through genetic engineering. Thanks to functional genomics, new target genes of both plant and microbial origin are rapidly becoming available for this purpose and their use will open new avenues for producing tailor-made plant products with improved properties. Moreover, the major proportion of terrestrial plant biomass comprises lignified cell walls and this reservoir of carbon should be increasingly exploited for the production of chemicals and energy within the context of sustainable development. For example, the design of plants suitable for downstream conversion processes, such as the production of bioethanol, and the exploitation of microorganisms and microbial enzymes for biomass pretreatments or for the production of novel chemicals.

Biomass↗
PubMed2001

Construction of desosamine containing polyketide libraries using a glycosyltransferase with broad substrate specificity.

BACKGROUND: Combinatorial biosynthesis techniques using polyketide synthases (PKSs) in heterologous host organisms have enabled the production of macrolide aglycone libraries in which many positions of the macrolactone ring have been manipulated. However, the deoxysugar moieties of macrolides, absent in previous libraries, play a critical role in contributing to the antimicrobial properties exhibited by compounds such as erythromycin. Since the glycosidic components of polyketides dramatically alter their molecular binding properties, it would be useful to develop general expression hosts and vectors for synthesis and attachment of deoxysugars to expand the nature and size of such polyketide libraries. RESULTS: A set of nine deoxysugar biosynthetic and auxiliary genes from the picromycin/methymycin (pik) cluster was integrated in the chromosome of Streptomyces lividans to create a host which synthesizes TDP-D-desosamine. The pik desosaminyl transferase was also included so that when the strain was transformed with a previously constructed library of expression plasmids encoding genetically modified PKSs that produce different macrolactones, the resulting strains produced desosaminylated derivatives. Although conversion of the macrolactones was generally low, bioassays revealed that, unlike their aglycone precursors, these novel macrolides possessed antibiotic activity. CONCLUSIONS: Based on the structural differences among the compounds that were glycosylated it appears that the desosaminyl transferase from the pik gene cluster is quite tolerant of changes in the macrolactone substrate. Since others have demonstrated tolerance towards modifications in the sugar substituent, one can imagine employing this approach to alter both polyketide and deoxysugar pathways to produce 'unnatural' natural product libraries.

Amino Sugars↗
PubMed1997

Therapeutic potential of antiidiotypic single chain antibodies with yeast killer toxin activity.

Single chain fragment (ScFv) antiidiotypic antibodies (antilds) of a killer toxin (KT) from the yeast Pichia anomala have been produced by recombinant DNA methodology from the splenic lymphocytes of mice immunized by idiotypic vaccination with a KT-neutralizing monoclonal antibody (Mab KT4). ScFv KT-like antilds (KTIdAb) react with specific Candida albicans KT cell wall receptors (KTR) exerting a candidacidal activity in vitro could be neutralized by adsorption with Mab KT4. ScFv KTIdAb displayed an effective therapeutic activity in an experimental model of rat candidal vaginitis.

Animals↗
PubMed1967

Microbiological burden on the surfaces of Explorer 33 spacecraft.

The Explorer XXXIII Spacecraft (Anchored Interplanetary Monitoring Platform, or AIMP) was decontaminated to prevent gross contamination of the moon with terrestrial microorganisms. Assay of the total spacecraft surface before and after decontamination showed that the decontamination procedure reduced the viable microbiological burden from 1.40 x 10(6) to 3.60 x 10(4). However, assembly of parts which were not decontaminated for engineering reasons or were not assembled under cleanroom conditions increased the viable microbial burden at the time of launch to 2.62 x 10(5).

Humans↗
PubMed1996

Apramycin resistance as a selective marker for gene transfer in mycobacteria.

We have explored the potential of using the apramycin resistance gene as a marker in mycobacterial gene transfer studies. Shuttle plasmids available for both electroporation and conjugation studies have been constructed, and we have successfully validated the use of the apramycin resistance gene as a component of cloning vectors for Mycobacterium smegmatis, M. bovis BCG, and M. tuberculosis.

Anti-Bacterial Agents↗
PubMed2003

aadA confers streptomycin resistance in Borrelia burgdorferi.

To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.

Anti-Bacterial Agents↗
PubMed1992

Purine biosynthetic genes are required for cadmium tolerance in Schizosaccharomyces pombe.

Phytochelatins (PCs) are metal-chelating peptides produced in plants and some fungi in response to heavy metal exposure. A Cd-sensitive mutant of the fission yeast Schizosaccharomyces pombe, defective in production of a PC-Cd-sulfide complex essential for metal tolerance, was found to harbor mutations in specific genes of the purine biosynthetic pathway. Genetic analysis of the link between metal complex accumulation and purine biosynthesis enzymes revealed that genetic lesions blocking two segments of the pathway, before and after the IMP branchpoint, are required to produce the Cd-sensitive phenotype. The biochemical functions of these two segments of the pathway are similar, and a model based on the alternate use of a sulfur analog substrate is presented. The novel participation of purine biosynthesis enzymes in the conversion of the PC-Cd complex to the PC-Cd-sulfide complex in the fission yeast raises an intriguing possibility that these same enzymes might have a role in sulfur metabolism in the fission yeast S. pombe, and perhaps in other biological systems.

Adenylosuccinate Synthase↗
PubMedDate not supplied

Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes.

The number of medical applications using autologous fibroblasts is increasing rapidly. We investigated thoroughly the procedure to isolate cells from skin using the enzymatic tissue dissociation procedure. Tissue digestion efficiency, cell viability, and yield were investigated in relation to size of tissue fragments, digestion volume to tissue ratio, digestion time, and importance of other protease activities present in Clostridium histolyticum collagenase (CHC) (neutral protease, clostripain, and trypsin). The results showed that digestion was optimal with small tissue fragments (2-3 mm3) and with volumes tissue ratios > or =2 ml/g tissue. For incubations < or =10 h, the digestion efficiency and cell isolation yields were significantly improved by increasing the collagenase, neutral protease, or clostripain activity, whereas trypsin activity had no effects. However, a too high proteolytic activity of one of the proteases present in CHC digestion solution or long exposure times interfered with cell viability and cell culture yields. The optimal range of CHC proteases activities per milliliter digestion solutions was determined for digestions < or =10 h (collagenase 2700-3900 Mandl U/ml, neutral protease 5100-10,000 caseinase U/ml, and clostripain 35-48 BAEE U/ml) and for longer digestions (>14 h) (collagenase 1350- 3000 U/ml, neutral protease 2550-7700 U/ml, and clostripain 18-36 U/ml). Using these conditions, a maximum fibroblast expansion was achieved when isolated cells were seeded at 1 x 10(4) cells/cm2. These results did not only allow selection of optimal CHC batches able to digest dermal tissue with an high cell viability but also significantly increased the fibroblast yields, enabling us to produce autologous dermal tissue in a clinically acceptable time frame of 3 wk.

Fibroblasts↗