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PubMedDate not supplied

Development of auxotrophic agrobacterium tumefaciens for gene transfer in plant tissue culture.

Auxotrophic strains of Agrobacterium tumefaciens were generated for use in liquid co-culture with plant tissue for transient gene expression. Twenty-one auxotrophs were recovered from 1,900 tetracycline-resistant insertional mutants generated with a suicide vector transposon mutagenesis system. Twelve of these auxotrophs were characterized on a nutrient matrix. Isolates were screened for growth in plant cell and root culture, and three auxotrophs were identified that had limited growth: adenine (ade-24), leucine (leu-27), and cysteine (cys-32). Ade-24 displayed poor T-DNA delivery in a transient expression test delivering GUS from a binary vector, while cys-32 displayed the best ability to deliver DNA of these three auxotrophs. The growth yield of cys-32 on cysteine was assessed to provide a quantitative basis for co-culture nutrient supplementation. The utility of cys-32 for delivering T-DNA to plant tissues is demonstrated, where an 85-fold enhancement in GUS expression over wild-type A. tumefaciens was achieved.

Agrobacterium tumefaciens↗
PubMed2003

Stability of plasmid pA387 derivatives in Amycolatopsis mediterranei producing rifamycin.

Genetic studies on the biosynthesis of rifamycins in producer strains such as Amylcolaptopsis mediterranei U-32 are severely hampered by the availability of efficient transformation procedures and stable plasmid vectors. Using an efficient electroporation procedure we have studied the replication and stability of a pA387 derivative, pDXM32. This plasmid confers enhanced plasmid stability and copy number compared to pA387 derivatives commonly used as cloning vectors in A. mediterranei. Deletion derivatives in the region previously identified as being a minimal replication origin were also examined with respect to their ability to transform A. mediterranei and at least one locus was essential for replication. A 5.4 kbp DNA fragment was sequenced and annotated encoding the replication and plasmid stability functions. A parA homologue was identified which is likely to confer plasmid stability.

Actinomycetaceae↗
PubMed1995

Two-phase approach for the expression of high-affinity human anti-human immunodeficiency virus immunoglobulin Fab domains in Escherichia coli.

We describe here a two-phase approach for the development of high-affinity human anti-HIV immunoglobulin Fab domains in a bacterial expression system. The first phase of this technique involves the generation of human hybridoma cell lines producing high-affinity antibodies (MAbs). Anti-HIV-1 human MAbs from peripheral blood lymphocytes (PBLs) were prepared from an HIV-1-seropositive patient and from an HIV-1-seronegative volunteer immunized with HIV-1 rgp160. One MAb (T15G1), derived from the blood of the seropositive donor, was specific for HIV-1 gp41, recognized gp41 on the surface of HIV-1-infected cells and bound this antigen with an apparent dissociation constant of 4 x 10(-10) M. A second MAb (M7B5), developed from the immunized volunteer, was specific for HIV-1 gp120 with a dissociation constant on the order of 8 x 10(-10) M, but was unable to recognize cell surface antigen. In the second phase of this technique the Fab domains of these two MAbs were molecularly cloned into a bacterial expression vector. mRNA was isolated from the M7B5 and T15G1 hybridoma cell lines and used as a template for the production of cDNA. The cDNA was amplified using the polymerase chain reaction (PCR) technique, and then fused, in frame, into a bacterial expression vector. The recombinant Fabs (rFabM7B5 and rFabT15G1) were expressed as dicistronic messages in bacteria using the IPTG-inducible lactose promoter (LacZ). DNA sequencing was used to define the gamma chain isotypes and the VH and VL chain gene usage. The binding specificities of rFabM7B5 and rFabT15G1 were indistinguishable from their respective intact MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2003

Ultrasound enhances the transfection of plasmid DNA by non-viral vectors.

Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: material improvement of non-viral vectors and their combination with various external physical stimuli. This paper reviews the latter research trials, with special attention paid to the enhancement of gene expression by ultrasound (US). The expression level of plasmid DNA by various cationized polymers and liposomes is promoted by US irradiation in vitro as well as in vivo. This US-enhanced expression of plasmid DNA will be discussed to emphasize the technical feasibility of US in gene therapy and biotechnology.

Animals↗
PubMed1996

Stable expression of human inducible nitric oxide synthase in V79 Chinese hamster cells.

A recombinant expression vector containing the full-length cDNA for human inducible nitric oxide (NO) synthase was constructed for constitutive expression in V79 Chinese hamster cells. Expression was followed by Western analyses using three different NO synthase antisera. Activity remained stable during 4 months of continued cultivation. Activities were 25 pmol min-1 mg-1 cytosolic protein with L-arginine and 47 pmol min-1 mg-1 cytosolic protein with NG-hydroxy-L-arginine as substrates. Activity was concentration-dependently inhibited by inhibitors such as NG-methyl-L-arginine, NG-nitro-L-arginine, NG-nitro-L-arginine methyl ester, aminoguanidine, and S-methyl-isothiourea. The rank order of inhibitor potencies was different from published results obtained with rodent inducible NOS. Parental V79 cells do not express and cannot be induced for NO synthase activity. Therefore, the genetically engineered V79 cell line is defined for the cDNA-encoded human inducible NO synthase. The new cell line may serve as a useful tool to study human inducible NO synthase.

Animals↗
PubMed2005

A versatile system for the expression of nonmodified bacteriocins in Escherichia coli.

AIMS: To develop a method and plasmid vectors suitable for expression of class II bacteriocins from Escherichia coli. METHODS AND RESULTS: The expression vector pSuV1 was constructed by inserting the PelB secretion signal coding sequence and a number of restriction endonuclease sites for cloning, into pTYB1. Codon optimized genes encoding the active mature region of each bacteriocin were constructed and inserted into pSuV1. Transfer of these constructs to a host expressing T7 RNA polymerase allowed for expression of secreted mature or fusion forms of the bacteriocins. Generation of the fusion, to the adjacent intein-chitin-binding domain gene, was achieved by removal of a small intervening BseRI fragment. The bacteriocins BacR1, divercin V41, enterocin P, pediocin PA-1 and piscicolin 126 were expressed from this system. For piscicolin 126, expression levels of 200 microg l(-1) in the mature form and 1100 microg l(-1) when cleaved from the fusion partner were achieved. All expressed bacteriocins displayed antimicrobial activity. CONCLUSIONS: Several class II bacteriocins have been expressed in E. coli using purpose designed plasmid vectors described here. SIGNIFICANCE AND IMPACT OF THE STUDY: This method provides a common expression system capable of producing a range of different class II bacteriocins. It allows researchers to study class II bacteriocins without access to the original producer strain, the native bacteriocin gene, or highly specific heterologous producing strains. Resulting expression levels are as high or higher than those previously reported for related bacteriocins.

Bacteriocins↗
PubMed1995

Isolation of promoters from Brevibacterium flavum strain MJ233C and comparison of their gene expression levels in B. flavum and Escherichia coli.

A promoter probe shuttle vector suitable for the isolation of promoter elements from coryneform bacteria was constructed. This vector carried the neomycin phosphotransferase (NPTII) gene from transposon Tn5 as a reporter gene, and was capable of replication in both Escherichia coli and Brevibacterium flavum. The vector was used in the construction of a B. flavum library of 899 independently isolated promoter clones. Promoters with a wide range of activities in B. flavum, including some very strong promoter elements, were isolated. Comparative analysis suggests that significant differences between B. flavum and E. coli may exist in the determinants of promoter strength.

Base Sequence↗
PubMed1997

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus.

Amino Acid Sequence↗
PubMed2002

Development of multiple cloning site cis-vectors for recombinant adeno-associated virus production.

Recombinant adeno-associated virus (rAAV) has become a very popular gene therapy vector in the past several years. A cis-plasmid is used to generate the rAAV stocks. In this plasmid, the entire expression cassette is incorporated between two AAV inverted terminal repeats. The construction of cis-plasmid has been problematic because of the high-frequency recombination of the viral inverted terminal repeats. Here we describe the design and construction of several multiple cloning site cis-plasmids that are driven by five different promoters, including the ubiquitous cytomegalovirus enhancer/chicken beta-actin (CAG), cytomegalovirus (CMV), rous sarcoma virus (RSV), simian virus 40 (SV40), and a muscle-specific promoter (CK6). The application of these multiple cloning site cis-plasmids improves the cloning efficiency. As an example of the utilization of these multiple cloning site vectors, the prokaryotic beta-galactosidase cDNA was cloned in the multiple cloning site cis-plasmids. High-level rAAV-mediated beta-galactosidase expression was achieved in HeLa cells from CAG, CMV, RSV and SV40 promoters, respectively, but notfrom the CK6 promoter. In vivo application in the adult mdx mouse (mouse model for Duchenne muscular dystrophy) muscle revealed efficient transgene expression from CMV and CK6 promoters, followed by CAG and RSV promoters. The SV40 promoter was the least efficient.

Animals↗
PubMed1997

An autocatalytic expression system for regulated production of recombinant protein in mammalian cells.

An autocatalytic inducible mammalian expression system was established. This system is composed of two sets of vectors: one carries a selectable marker gene and the other carries a bicistronic expression unit consisting of a target gene, an internal ribosomal entry site, and a tetracycline-controlled transactivator gene. Both the selectable marker and the bicistronic unit are controlled by the tetracycline-responsive promoter (PhCMV*-1). When the two vectors are cotransfected into host cells, only a single selection round in the absence of tetracycline is necessary to generate clones expressing the target gene. The expression level is high and easily regulated by tetracycline. Combination with a gene amplification system may allow further enhance the foreign gene expression. Because the PhCMV*-1 promoter is relatively independent of cellular regulation signals, this system is expected to work in a wide variety of cell types.

Animals↗
PubMed2003

[Construction of plant expressive vector of human beta-defensin-2 gene].

OBJECTIVE: Several evidences suggested that transgenic plants would be a facile and economic bioreactor for large-scale production of industrial and pharmaceutical recombinant proteins. This study is made in an attempt to establish plant bioreactor for expression of recombinant hBD-2. METHODS: Recombinant hBD-2 gene with C terminal of bi-tags of myc and 6xHis was inserted into a plant expressive vector-pCAMBIA1304, which closely located the down-stream of CaMV35S promoter. Agrobacterium tumefaciens LBA4404 was transformed with the recombinant plant expressive vector: rpCAMBIA1304/hBD-2/His. The positive clones of LBA4404 transformed by rpCAMBIA1304/hBD-2/His were selected on a culture plate containing kanamycin. The callus tissues were transfected by positive clones of LBA4404, and positive callus were examined by using the resistant selection of hygromycin gene. RESULTS: The evidences of enzyme digestion, PCR and sequence analysis confirmed that recombinant hBD-2 gene with C terminal of bi-tags of myc and 6xHis was correctly inserted into pCAMBIA1304 and was located between CaMV35S promoter and Nos terminal cordon to construct recombinant plant expressive vector: rpCAMBIA1304/hBD-2/His, thus indicating that rpCAMBIA1304/hBD-2/His has successfully transformed Agrobacterium tumefaciens LBA4404 and positive clones have been isolated. The results from resistant selection of hygromycin gene showed that rpCAMBIA1304/hBD-2/His has been transferred into the callus of wheat, and the differentiation of callus tissue under selective pressure of hygromycin is carried out continually. CONCLUSION: The above data suggest that the technique of transgenic plant is workable for the production of recombinant hBD-2.

Base Sequence↗
PubMed1990

A phasmid optimised for protein design projects: pMAMPF.

Protein design requires the rapid production of recombinant genes and active recombinant proteins, the latter in sufficient amounts for functional and physical studies. We present here the construction and application of a new phasmid vector system, using the fd phage origin, lambda pL promoter, ompA-leader sequence and pMB1 origin, which allows the preparation of secretable proteins in active form, mutagenesis and gene sequencing, without subcloning steps. The vector can be used in plasmid form in a stably transformed culture to induce product formation, or as a packaged single-stranded phasmid, which, via batch transduction in a growing culture, leads directly to recombinant protein formation. This latter method has the advantage that, during the short period required for phasmid amplification, little counterselection against clones with high rDNA-protein synthesis potential occurs. The total sequence of pMAMPF-1 and pMAMPF-3 can be assembled from known sequences of constituent fragments. Mutated regions were directly sequenced.

Bacteriophage lambda↗
PubMed1998

Cold-sensitive E-lysis systems.

The release of recombinant bacteria into the environment is undesirable because of possible risks associated with the genetically modified organisms. The aim of this study was to establish a cold-sensitive killing system with a lethal gene, activated when bacteria encounter lower environmental temperatures. To obtain cold-sensitive lysis vectors, the lambdacI857 repressor/pR promoter expression system was combined with either the lacI/lacZpo or the phage 434 cI/pR system that control the expression of the lysis gene E of bacteriophage phiX174. Escherichia coli strains harbouring such suicide vectors are able to grow at 37 degrees C, but cell lysis takes place at temperatures below 30 degrees C. By replacing gene E with a beta-galactosidase reporter gene we also showed that the onset of beta-galactosidase activity corresponds with the onset of lysis at 28 degrees C. Results indicate that these newly combined promoter/repressor systems can also be used to confer cold-sensitive expression to any gene of interest.

Bacterial Proteins↗
PubMed1992

Chemical synthesis, molecular cloning, and expression of the gene coding for the Trichosanthes trypsin inhibitor--a squash family inhibitor.

The gene coding for a Trichosanthes trypsin inhibitor analog (Ala-6-TTI) in which methionine at position 6 was replaced by alanine was synthesized chemically. The synthetic gene was cloned into plasmid pWR590-1 and expressed in Escherichia coli as a fusion protein composed of beta-galactosidase fragment of 590 amino acid residues and (Ala-6)-TTI, with methionine as a connecting residue. After cyanogen bromide cleavage and reduction of the fusion protein, followed by refolding with trypsin-Sepharose 4B as a matrix and affinity chromatography on the immobilized enzyme, the fully active (Ala-6)-TTI was obtained. The trypsin inhibitory activity and amino acid composition of the recombinant (Ala-6)-TTI were consistent with those of the natural one. The (Ala-6)-TTI gene was also cloned into the secretion expression vector, pVT102U/alpha, in Saccharomyces cerevisiae. In order to make the reading frame of the gene compatible with the vector, a nucleotide was inserted into the (Ala-6)-TTI gene via site-directed mutagenesis. The secreted (Ala-6)-TTI was purified and found to be correctly processed at the junction between the alpha-factor leader peptide and (Ala-6)-TTI downstream. Of the two expression systems, the latter is more advantageous in the high yield (greater than 2 mg/liter), easy purification and needlessness of disulfide refolding.

Amino Acid Sequence↗
PubMed1984

Prospects for human gene therapy.

Procedures have now been developed for inserting functional genes into the bone marrow of mice. The most effective delivery system at present uses retroviral-based vectors to transfer a gene into murine bone marrow cells in culture. The genetically altered bone marrow is then implanted into recipient animals. These somatic cell gene therapy techniques are becoming increasingly efficient. Their future application in humans should result in at least partial correction of a number of genetic disorders. However, the safety of the procedures must still be established by further animal studies before human clinical trials would be ethical.

Animal Experimentation↗
PubMed1990

Gene replacement and expression of foreign DNA in mycobacteria.

A system that permits molecular genetic manipulation of mycobacteria was developed on the basis of the yeast paradigm of gene replacement by homologous recombination. A shuttle vector that can replicate autonomously at a high copy number in Escherichia coli but must integrate into homologous DNA for survival in Mycobacterium smegmatis was constructed. The vector contains a ColE1 origin of replication, antibiotic resistance markers for ampicillin and kanamycin, a nutritional marker (pyrF) that allows both positive and negative selection in E. coli and M. smegmatis, and unique restriction sites that permit insertion of foreign DNA. Transformation of mycobacteria with this vector results in integration of its DNA into the genomic pyrF locus by either a single or a double homologous recombination event. With this system, the 65-kilodalton Mycobacterium leprae stress protein antigen was inserted into the M. smegmatis genome and expressed. This gene replacement technology, together with a uniquely useful pyrF marker, should be valuable for investigating mycobacterial pathobiology, for the development of candidate mycobacterial vaccine vehicles, and as a model for the development of molecular genetic systems in other pathogenic microorganisms.

Blotting, Southern↗
PubMed2003

Chimeric and pseudotyped parvoviruses minimize the contamination of recombinant stocks with replication-competent viruses and identify a DNA sequence that restricts parvovirus H-1 in mouse cells.

Recent studies demonstrated the ability of the recombinant autonomous parvoviruses MVMp (fibrotropic variant of the minute virus of mice) and H-1 to transduce therapeutic genes in tumor cells. However, recombinant vector stocks are contaminated by replication-competent viruses (RCVs) generated during the production procedure. To reduce the levels of RCVs, chimeric recombinant vector genomes were designed by replacing the right-hand region of H-1 virus DNA with that of the closely related MVMp virus DNA and conversely. Recombinant H-1 and MVMp virus pseudotypes were also produced with this aim. In both cases, the levels of RCVs contaminating the virus stocks were considerably reduced (virus was not detected in pseudotyped virus stocks, even after two amplification steps), while the yields of vector viruses produced were not affected. H-1 virus could be distinguished from MVMp virus by its restriction in mouse cells at an early stage of infection prior to detectable viral DNA replication and gene expression. The analysis of the composite viruses showed that this restriction could be assigned to a specific genomic determinant(s). Unlike MVMp virus, H-1 virus capsids were found to be a major determinant of the greater permissiveness of various human cell lines for this virus.

Animals↗
PubMed1987

Human globin gene expression after gene transfer.

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.

Anemia, Sickle Cell↗