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At least 1,153 records · Page 64Linked to original sources
PubMed2005

A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1.

We describe the design and detailed characterization of 6-hydroxy-nicotine (6HNic)-adjustable transgene expression (NICE) systems engineered for lentiviral transduction and in vivo modulation of angiogenic responses. Arthrobacter nicotinovorans pAO1 encodes a unique catabolic machinery on its plasmid pAO1, which enables this Gram-positive soil bacterium to use the tobacco alkaloid nicotine as the exclusive carbon source. The 6HNic-responsive repressor-operator (HdnoR-O(NIC)) interaction, controlling 6HNic oxidase production in A.nicotinovorans pAO1, was engineered for generic 6HNic-adjustable transgene expression in mammalian cells. HdnoR fused to different transactivation domains retained its O(NIC)-binding capacity in mammalian cells and reversibly adjusted transgene transcription from chimeric O(NIC)-containing promoters (P(NIC); O(NIC) fused to a minimal eukaryotic promoter [P(min)]) in a 6HNic-responsive manner. The combination of transactivators containing various transactivation domains with promoters differing in the number of operator modules as well as in their relative inter-O(NIC) and/or O(NIC)-P(min) spacing revealed steric constraints influencing overall NICE regulation performance in mammalian cells. Mice implanted with microencapsulated cells engineered for NICE-controlled expression of the human glycoprotein secreted placental alkaline phosphatase (SEAP) showed high SEAP serum levels in the absence of regulating 6HNic. 6HNic was unable to modulate SEAP expression, suggesting that this nicotine derivative exhibits control-incompatible pharmacokinetics in mice. However, chicken embryos transduced with HIV-1-derived self-inactivating lentiviral particles transgenic for NICE-adjustable expression of the human vascular endothelial growth factor 121 (VEGF121) showed graded 6HNic response following administration of different 6HNic concentrations. Owing to the clinically inert and highly water-soluble compound 6HNic, NICE-adjustable transgene control systems may become a welcome alternative to available drug-responsive homologs in basic research, therapeutic cell engineering and biopharmaceutical manufacturing.

Animals↗
PubMed1988

A new recombinant procoagulant protein derived from the cDNA encoding human factor VIII.

We have constructed new B domain deletion derivatives of human factor VIII (FVIII) by manipulating the cDNA using recombinant DNA techniques. One of these new derivatives, FVIII delta II, in which amino acids 771(pro)-1666(asp) have been deleted, no longer contains the protease cleavage site at amino acid position 1648(arg)-1649(glu) known to be involved in the initial step of FVIII processing. We have expressed this molecule in both baby hamster kidney (BHK) 21 cells using the vaccinia virus (VV) expression system and have established Chinese hamster ovary (CHO) derived permanent cell lines expressing either recombinant (r)FVIII or FVIII delta II. The characteristics of FVIII delta II have been compared to those of rFVIII and/or plasma derived (pd) FVIII. FVIII delta II has the following properties: (i) it exhibits FVIII procoagulant activity; (ii) it is expressed at 5-fold higher levels than is the complete molecule in comparable systems; (iii) it migrates for the most part as a single major band on SDS-PAGE, in contrast to the complete molecule; (iv) it is activated to a greater extent by thrombin than is either rFVIII or pdFVIII; and (v) it retains the ability to bind von Willebrand factor (vWf).

Antibodies, Monoclonal↗
PubMed1991

Temperature-sensitive production of rabbit muscle glycogen phosphorylase in Escherichia coli.

In order to understand how allosteric switches regulate both the catalytic activity and molecular interactions of glycogen phosphorylase, it is necessary to design and analyze variant proteins that test hypotheses about the structural details of the allosteric mechanism. Essential to such an investigation is the ability to obtain large amounts of variant proteins. We developed a system for obtaining milligram amounts (greater than 20 mg/l) of rabbit muscle phosphorylase from bacteria. Phosphorylase aggregates as inactive protein when a strong bacterial promoter is used under full inducing conditions and normal growth conditions. However, when the growth temperature of bacteria expressing phosphorylase is reduced to 22 degrees C we obtain active muscle phosphorylase. The degree to which the induced expression of phosphorylase protein is temperature sensitive depends on the strain of bacteria used. New assay and purification methods were developed to allow rapid purification of engineered phosphorylase proteins from bacterial cultures. The rabbit muscle phosphorylase obtained from the bacterial expression system is enzymatically identical to the enzyme purified from rabbit muscle. The expressed protein crystallizes in the same conditions used for growing crystals of protein from rabbit muscle and the crystal form is isomorphous. Rabbit muscle phosphorylase is one of the largest oligomeric mammalian enzymes successfully expressed in Escherichia coli. Our results indicate that optimization of a combination of growth and induction conditions will be important in the expression of other heterologous proteins in bacteria.

Animals↗
PubMedDate not supplied

The persistence of engineered Agrobacterium tumefaciens in agroinfected plants.

Several plant species, including tomato (Lycopersicon esculentum), Gynura aurantiaca, avocado (Persea americana), and grapefruit (Citrus paradisi) grafted on Troyer citrange (Poncirus trifoliata x C. sinensis) were "agro-infected" with Agrobacterium tumefaciens strain LBA-4404, carrying a mini-Ti plasmid with a dimeric cDNA of citrus exocortis viroid (CEVd). Extracts prepared from tissues of the agroinfected plants 38-90 days after inoculation were plated on selective media and found to contain large amounts of the engineered bacteria. These observations suggest the need for more stringent quarantine measures when handling A. tumefaciens cells harboring constructs for "agroinoculation" with plant viruses or viroids.

Agrobacterium tumefaciens↗
PubMed1997

Characterization of acquired resistance in lesion-mimic transgenic potato expressing bacterio-opsin.

The lesion-mimic mutants of certain plants display necrotic lesions resembling those of the hypersensitive response and activate local and systemic defense responses in the absence of pathogens. We have engineered a lesion-mimic phenotype in transgenic Russet Burbank potato plants through constitutive expression of a bacterio-opsin (bO) proton pump derived from Halobacterium halobium. Transgenic potato plants exhibiting a lesion-mimic phenotype had increased levels of salicylic acid and overexpressed several pathogenesis-related messenger RNAs, all hallmarks of systemic acquired resistance (SAR). The lesion-mimic plants also displayed enhanced resistance to the US1 isolate (A1 mating type) of a fungal pathogen, Phytophthora infestans, a causal agent of late blight disease. In contrast, little resistance was observed against the US8 isolate (A2 mating type) of this pathogen. Furthermore, a majority of the transgenic plants displaying the lesion-mimic phenotype had increased susceptibility to potato virus X. The tubers of these plants were not resistant to the bacterial pathogen Erwinia carotovora. These results indicate that expression of bO can result in the activation of defense responses in transgenic potato plants and show for the first time that bO expression can confer resistance to a pathogenic fungus. However, our results also demonstrate that like SAR, this "engineered" resistance is likely to be limited to certain pathogens and particular cultivars.

Bacteriorhodopsins↗
PubMed1980

Genetic engineering: possibilities and prospects for its application in industrial microbiology.

A wide range of techniques is now available for the construction of hybrid DNA molecules comprising components from disparate species. Transfer of segments of DNA from other organisms, and especially eukaryotes, to Escherichia coli permits their preparation in quantities sufficient for detailed analysis of their structure and mechanism of expression. This information could be exploited to enhance the quantity or quality of polypeptide products from bacterial cells. Greatly increased yields of bacterial enzymes have been obtained in this way in several instances. The approaches that have been pioneered with bacteria are currently being applied to higher organisms. Much work is in progress with yeasts, in which transformation has been successfully demonstrated, with animal viruses and cells in culture and with some plant systems and offers the promise of wider application of genetic engineering in the not too distant future.

Bacteria↗
PubMed1992

Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha.

Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.

DNA Repair↗
PubMed1999

Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria.

Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptII (encoding kanamycin resistance) or aacCI (encoding gentamicin resistance) genes were equipped with the tac promoter (Ptac) and the trpA terminator (TtrpA) and then cloned between NotI sites to construct the CAS-Nm (Ptac-nptII-TtrpA) and CAS-Gm (Ptac/PaacCI-aacCI-TtrpA) cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (gusA-Ptac-nptII-TtrpA) or CAS-GGm (gusA-Ptac/PaacCI-aacCI-TtrpA) cassettes. Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional activity. The promoterless gusA gene has also been cloned into a broad-host-range IncP1 plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette was cloned as a NotI fragment into the NotI site of a pUT derivative to construct four minitransposons. The mTn5-Nm (containing Ptac-nptII-TtrpA) and mTn5-Gm (containing Ptac/PaacCI-aacCI-TtrpA) minitransposons have been constructed specifically for insertional inactivation studies. The minitransposons mTn5-GNm (containing gusA-Ptac-nptII-TtrpA) and mTn5-GGm (containing gusA-Ptac/PaacCI-aacCI-TtrpA) can be used for transcription signal localization or insertional inactivation. The TAC-31R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, mTn5-GNm and mTn5-GGm. The specific application of these constructs to generate acid- or nodule-inducible fusions is presented. The new constructs provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other gram-negative bacteria.

Base Sequence↗
PubMed2004

Many paths to many clones: a comparative look at high-throughput cloning methods.

The creation of genome-scale clone resources is a difficult and costly process, making it essential to maximize the efficiency of each step of clone creation. In this review, we compare the available commercial and open-source recombinational cloning methods with regard to their use in creating comprehensive open reading frame (ORF) clone collections with an emphasis on the properties requisite to use in a high-throughput setting. The most efficient strategy to the creation of ORF clone resources is to build a master clone collection that serves as a quality validated source for producing collections of expression clones. We examine the methods for recombinational cloning available for both the creation of master clones and their conversion into expression clones. Alternative approaches to creating clones involving mixing of cloning methods, including gap-repair cloning, are also explored.

Animals↗