Plasmids

The MspI restriction-modification system, which recognizes the sequence 5'-CCGG-3', has been previously cloned and sequenced (1). We subcloned the methyltransferase gene (M.MspI) downstream of the ptac promoter in the multicopy vector pUC119 and overexpressed it in E. coli. Upon induction with IPTG, M.MspI constitutes more than 10% of cellular protein. A scheme has been devised to purify large amounts of biologically active M.MspI to apparent homogeneity from these overexpressing E. coli cells. Approximately 0.8 mg of pure M.MspI per gram of cells (wet weight) can be obtained. The apparent molecular weight of M.MspI is 49 kD, by SDS gel electrophoresis and 48-54 kD by gel filtration. At low concentrations (less than 0.4 mg/ml), the methyltransferase is a monomer in solution but at higher concentrations (greater than 3.0 mg/ml) it exists predominantly as a dimer. Polyclonal antibodies raised against M.MspI cross-react with the DNA-methyltransferases of several other restricti...

Mucosal (and in minor extent transcutanous) stimulation can induce local or distant mucosa secretory IgA. Liposomes and other vesicles as mucosal and transcutaneous adjuvants are attractive alternatives to parenteral vaccination. Liposomes can be massively produced under good manufacturing practices and stored for long periods, at high antigen/vesicle mass ratios. However, their uptake by antigen-presenting cells (APC) at the inductive sites remains as a major challenge. As neurotoxicity is a major concern in intranasal delivery, complexes between archaeosomes and calcium as well as cationic liposomes complexed with plasmids encoding for antigenic proteins could safely elicit secretory and systemic antigen-specific immune responses. Oral bilosomes generate intense immune responses that remain to be tested against challenge, but the admixing with toxins or derivatives is mandatory to reduce the amount of antigen. Most of the current experimental designs, however, underestimate the mu...

We have established stably transformed human embryonic kidney cell lines (HEK293) containing bicistronic constructs to study regulation of viral internal ribosome entry site (IRES)-mediated translation in vivo. These cells produce Renilla luciferase (Rluc) in a cap-dependent manner, while Firefly luciferase (Luc) synthesis is mediated by IRES elements. Using these cell lines, we demonstrate here that IRES-mediated translation directed by both hepatitis C (HCV) and encephalomyocarditis (EMCV) virus varies with the cell cycle. Experiments involving arrest of the cell lines at different phases of the cell cycle, release of synchronized cells from cell cycle arrest, as well as direct sorting of the cells based on position in the cell cycle have shown that the activity of the HCV and EMCV IRES elements is lowest during the G2/M phase in HEK293 cells. These results suggest that cellular transacting factors either stimulate viral IRES-mediated translation during G1 and S phases or repress translation during the G2/M phase in HEK293 cells.

A modified gel electrophoresis technique provided a reproducible way of detecting and isolating plasmids with molecular weights ranging from 12 X 10(6) to 370 X 10(6) for Azospirillum species. Analysis with the nifHD region of Rhizobium trifolii showed that the Azospirillum nif genes were chromosomally located in all eight strains investigated and not on endogenous plasmids.

As an approach to the study of mammalian gene expression, the promoters and translation initiation regions of the rat preproinsulin II and the simian virus 40 early genes were fused to the structural gene of Escherichia coli 3-galactosidase, a sensitive probe for gene expression. These fusions were introduced into COS-7 cells, a simian virus 40 large tumor-antigen-producing monkey kidney cell line, where they directed the synthesis of enzymatically active hybrid 3-galactosidase proteins. Conditions for transfection were varied to optimize the expression of 13-galactosidase activity in the transfected cells. The pH optimum of this activity was found to be 7.0, the same as that of native E. coli fgalactosidase and distinct from the major lysosomal "acid" fl-galactosidase. The fused preproinsulin-fl-galactosidase was further characterized by gel electrophoresis of nondenatured cell extracts stained by a fluorogenic substrate and by immunoprecipitation and gel electrophoresis of 3H-labeled cell proteins. These results all indicate that fully active tetrameric P-galactosidase hybrids can be produced in mammalian cells. The expression of preproinsulin-,B-galactosidase activity was measured in the presence of high glucose, insulin, dexamethasone, or epidermal growth factor but no regulatory changes were observed. Gene fusions utilizing the Escherichia coli lacZ structural gene provide an especially suitable method for studying gene expression and regulation (1). Its product, f3-galactosidase, can be as

Aging is associated to a decline in immune functions that are in part related to a defective protein kinase C dependent signal transduction machinery. RACK-1 (Receptor for Activated C Kinase 1) is a scaffold protein for different kinases and membrane receptors. We have previously demonstrated, in the elderly, a defective PCKβII (Protein Kinase C βII) translocation related to a decrease in RACK-1 protein expression, which is correlated to the age-associated decline in DHEA (dehydroepiandrosterone) levels. As a consequence of this signal transduction impairment, a significant decrease in immune cells functionality was observed. Furthermore, we could demonstrate that in vivo and in vitro DHEA administration restored RACK-1 level and immune functions, indicating that this hormone behaved as a positive RACK-1 regulator. We have most recently characterized the human GNB2L1 promoter region, coding for RACK-1 protein. Although no direct DHEA responsive elements were found, a glucocorticoid responsive element (GRE) was identified. The purpose of this work was to investigate, in the human pro-myelocytic cell line THP-1, whether physiological cortisol concentrations were able to modulate GNB2L1 promoter activity, RACK-1 transcription as well as cytokine production. As DHEA is endowed of anti-glucocorticoid properties in several cellular systems, and as cortisol:DHEA ratio imbalance is relevant in aging, we also investigated their possible interaction at the RACK-1 expression level. We could demonstrate that cortisol acted in a dose-related manner as a GNB2L1 promoter repressor, reducing RACK-1 mRNA expression and protein level. Probably by interfering with glucocorticoid receptor binding to GRE sequence, prolonged DHEA exposure counteracted cortisol effects, restoring RACK-1 levels and cytokine production, as assessed by LPS-induced TNF-α release.

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30-45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications.

antigen expression and potentiate CD8+ T cell immune responses CD4+ T lymphocytes mediate in vivo clearance of plasmid DNA vaccine (5019 articles) Immunobiology Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests

Stannous chloride (SnCl 2 ) is widely used in daily human life, for example, to conserve soft drinks, in food manufacturing and biocidal preparations. In nuclear medicine, stannous chloride is used as a reducing agent of Technetium-99m, a radionuclide used to label different cells and molecules. In spite of this, stannous chloride is able to generate reactive oxygen species (ROS) which can damage DNA. In this work, plasmid DNA (pUC 9.1) was incubated with SnCl 2 under different conditions and the results analyzed through DNA migration in agarose gel electrophoresis. Our data reinforce the powerful damaging effect induced by stannous ion and suggest that this salt can play a direct role in inducing DNA lesions.

We report the successful introduction of heterologous DNA sequences into embryos of the mosquito Aedes aegypti (L.) by microinjection. The injected DNA carrried P transposable element sequences, derived from and known to facilitate transformation in Drosophilu rnelanogaster. Two plasmids, one of which carried a dominant selectable marker, were introduced into the posterior of embryos prior to pole cell formation and subsequently taken up into the germ line of transformed individuals. Stable transfer of the selectable marker (G418 resistance) was demonstrated over two generations. The precise nature of these putative P mediated integration events is currently being investigated. However, the results presented here establish the technique of DNA transformation for the genetic manipulation of Aedes uegypti.

Background Interferons (IFNs) play an important role in host antiviral responses, but viruses, including vaccinia viruses (VV), employ mechanisms to disrupt IFN activities, and these viral mechanisms are often associated with their virulence. Here, we explore an attenuation strategy with a vaccine strain of VV lacking a virus-encoded IFN-γ receptor homolog (viroceptor).

The two opponents, toxin (CcdB, LetB or LetD, protein G, LynB) and antidote (CcdA, LetA, protein H, LynA), in the plasmid addiction system ccd of the F plasmid were studied by different biophysical methods. The thermodynamic stability was measured at different temperatures combining denaturant and thermally induced unfolding. It was found that both proteins denature in a two-state equilibrium (native dimer versus unfolded monomer) and that CcdA has a signi®cantly lower thermodynamic stability. Using a numerical model, which was developed earlier by us, and on the basis of the determined thermodynamic parameters the concentration dependence of the denaturation transition temperature was obtained for both proteins. This concentration dependence may be of physiological signi®cance, as the concentration of both ccd addiction proteins cannot exceed a certain limit because their expression is controlled by autoregulation. The in¯uence of DNA on the thermal stability of the two proteins was probed. It was found that cognate DNA increases the melting temperature of CcdA. In the presence of non-speci®c DNA the thermal stability was not changed. The melting temperature of CcdB was not in¯uenced by the applied double-stranded oligonucleotides, neither cognate nor unspeci®c.

Nitric oxide (NO) and reactive oxygen species (ROS) are emerging as important regulators of angiogenesis. NO enhances VEGF synthesis in several cell types and is required for execution of VEGF angiogenic effect in endothelial cells. Similarly, hydrogen peroxide induces VEGF synthesis and recent studies indicate the involvement of ROS in signaling downstream of VEGF stimulation. VEGF synthesis can not only be enhanced by gene transfer of VEGF but also by overexpression of NO synthase genes. Here, we examined the possibility of augmentation of VEGF production by gene transfer of copper/zinc superoxide dismutase (CuZnSOD, SOD1). Overexpression of human SOD1 in mouse NIH 3T3 fibroblasts increased SOD activity, enhanced intracellular generation of H 2 O 2 and significantly stimulated VEGF production as determined by increase in VEGF promoter activity, VEGF mRNA expression and VEGF protein synthesis. The stimulatory effect on VEGF synthesis induced by SOD1 gene transfer was reverted by overexpression of human catalase. The effect of H 2 O 2 produced by engineered cells is mediated by activation of hypoxia-inducible factor response element (HRE) as well as Sp1 recognition site of VEGF promoter. This data suggest the feasibility of stimulation of angiogenesis by overexpression of SOD1. (Mol Cell Biochem 264: [169][170][171][172][173][174][175][176][177][178][179][180][181] 2004)

Autofluorescent protein tags represent one of the major and, perhaps, most powerful tools in modern cell biology for visualization of various cellular processes in vivo. In addition, advances in confocal microscopy and the development of autofluorescent proteins with different excitation and emission spectra allowed their simultaneous use for detection of multiple events in the same cell. Nevertheless, while autofluorescent tags are widely used in plant research, the need for a versatile and comprehensive set of vectors specifically designed for fluorescent tagging and transient and stable expression of multiple proteins in plant cells from a single plasmid has not been met by either the industrial or the academic communities. Here, we describe a new modular satellite (SAT) vector system that supports N-and C-terminal fusions to five different autofluorescent tags, EGFP, EYFP, Citrine-YFP, ECFP, and DsRed2. These vectors carry an expanded multiple cloning site that allows easy exchange of the target genes between different autofluorescence tags, and expression of the tagged proteins is controlled by constitutive promoters, which can be easily replaced with virtually any other promoter of interest. In addition, a series of SAT vectors has been adapted for high throughput Gateway recombination cloning. Furthermore, individual expression cassettes can be assembled into Agrobacterium binary plasmids, allowing efficient transient and stable expression of multiple autofluorescently tagged proteins from a single vector following its biolistic delivery or Agrobacteriummediated genetic transformation.

One limiting factor in studies of tuberculosis and leprosy is the difficulty of genetic analysis and manipulation of mycobacteria. Two approaches were adopted for the construction of vectors, based on different Escherichia coli plasmids and using Mycobacterium smegmatis as the host. In both cases we found that the original E. coli plasmid is capable of being replicated in M. smegmatis, yielding chloramphenicol-resistant colonies. One such plasmid has been recovered from a M. smegmatis transformant and used to re-transform both M. smegmatis and E. coli to chloramphenicol resistance. This plasmid is indistinguishable from the original plasmid by restriction analysis, and can be used as a shuttle vector for the genetic manipulation of mycobacterial species.

Purpose: To develop an optimal nonviral aerosol formulation for locoregional treatment of early lung cancer. Experimental Design: The formulation was made of polylysine/protamine combination (AND) as the carrier and p53 gene (p53sm) as therapeutic agent. To estimate the aerosol deposition, the aerodynamic size of the AND-p53sm was measured with extrusion-precipitation method. To accurately determine the dose, the aerosol efficiency in mice was measured with a fluorescent dye. The transfection efficiency and DNA protection function of the aerosolized formulation in cultured cells and mouse lungs were detected with reporter gene assays and/or reverse transcription-PCR.The preclinical safety and efficacy of AND-p53sm were studied in healthy mice and mice bearing orthotopic human non^small-cell lung cancer (NSCLC) xenograft. Results: After aerosolization, AND is 3-to 17-fold more effective than commonly used PEI or cationic lipid formulations in transfecting the NSCLC cells (relative light units, 1,494 versus 534 and 86; P < 0.003). Aerodynamic size of AND-p53sm ranged 0.2 to 3 Am is the optimal aerosol droplets for deposition in the entire human respiratory tract. Significant gene expression was detected in the lungs of mice given aerosolized AND-p53sm and AND-luciferase. Aerosolized AND-p53sm significantly prolonged the life of mice bearing orthotopic human NSCLC xenografts, and it was more effective than an optimal i.v. cisplatin chemotherapy (increased life span, 93% versus 25%; P = 0.014). Inhalation of AND produced low and reversible pulmonary toxicity and no systemic toxicity. Conclusions: This optimal formulation is suitable for delivering biological materials to human lung with aerosol administration. This therapeutic strategy is an option for patients with early lung cancer and bronchoalveolar carcinoma.

An episomal RNAi silencing construct containing the inducible cbhB promoter and a hairpin structure has been made to downregulate the alb1 gene in the human pathogen Aspergillus fumigatus. Transformation of fungal protoplasts resulted in a high number of transformants with an inducible silenced phenotype (white spores). Efficient downregulation of the alb1 gene using this system suggests that this approach may overcome the variable downregulation observed with integrative constructs.

Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were dfrA1, dfrA15, dfrA5, and dfrA12 for trimethoprim; aac(6)-Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance. To our knowledge, this is the first report of the presence of dfrA5, dfrA12, aac(6)-Ib, and ereA2 cassettes in class I integrons of V. cholerae. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance.

A molecular PCR-based assay was developed and applied to macrophyte and seawater samples containing mixed microphytobenthic and phytoplanktonic assemblages, respectively, in order to detect toxic Ostreopsis species in Mediterranean Sea. The specificity and sensitivity of the molecular PCR assay were assessed with both plasmidic and genomic DNA of the target genus or species using taxon-specific primers in the presence of background macrophyte DNA. The PCR molecular technique allowed rapid detection of the Ostreopsis cells, even at abundances undetectable within the resolution limit of the microscopy technique. Species-specific identification of Ostreopsis was determined only by PCR-based assay, due to the inherent difficulty of morphological identification in field samples. In the monitoring of the toxic Ostreopsis blooms PCR-based methods proved to be effective tools complementary to microscopy for rapid and specific detection of Ostreopsis and other toxic dinoflagellates in marine coastal environments.

To determine the genetic structures at the origin of the mobilization of the extended-spectrum b-lactamase (ESBL) bla GES-1 gene in an Escherichia coli clinical isolate. Methods: ESBL-encoding genes and class 1 or class 3 integron-specific motifs were screened. Conjugation experiments were performed to determine whether the plasmid-carrying bla GES-1 gene was self-transferable. Plasmid sequencing was achieved by a primer-walking approach. Results: The bla GES-1 gene was located in a class 3 integron. That unusual genetic structure was itself located on an 9 kb plasmid, pQ7, which was not self-transferable. Sequence analysis revealed that plasmid pQ7 belonged to a novel subtype of the IncQ group. Conclusions: This study identified for the first time the bla GES-1 gene in E. coli and in Switzerland. It describes a novel IncQ-type plasmid subgroup that possesses original features, in particular iteron sequences that constitute a hot spot for integration of foreign DNA.

The HSV gene encoding ICP4 is negatively regulated and the HSV gene encoding thymidine kinase is positively regulated by ICP4 in vivo. We report that ICP4 is a component of a stable complex that contains protein and a sequence of approximately 28 nucleotides that span the ICP4 gene transcription initiation site. The association of ICP4 with DNA sequences between positions-103 and +32 relative to the ICP4 mRNA start site was demonstrated by DNA binding immunoassays. DNase footprinting revealed that nucleotides between positions-8 and +20 are protected by ICP4. In contrast, binding of ICP4 to sequences flanking the mRNA start site in the thymidine kinase gene was not observed. Models for ICP4-mediated positive or negative regulation of HSV gene transcription are discussed.

Rust fungi are devastating plant pathogens and several Puccinia species have a large economic impact on wheat production worldwide. Disease protection, mostly offered by introgressed host-resistance genes, is often racespecific and rapidly overcome by newly-emerging virulent strains. Extensive new genomic resources have identified vital pathogenicity genes but their study is hampered because of the biotrophic life styles of rust fungi. In cereals, Barley stripe mosaic virus (BSMV)-induced RNAi has emerged as a useful tool to study loss-of-function phenotypes of candidate genes. Expression of pathogenderived gene fragments in this system can be used to obtain in planta-generated silencing of corresponding genes inside biotrophic pathogens, a technique termed host-induced gene silencing (HIGS). Here we test the effectiveness of BSMV-mediated HIGS in the wheat leaf rust fungus Puccinia triticina (Pt) by targeting three predicted pathogenicity genes, a MAPK, a cyclophilin, and a calcineurin regulatory subunit. Inoculation of BSMV RNAi constructs generated fungal gene-specific siRNA molecules in systemic leaves of wheat plant. Subsequent Pt inoculation resulted in a suppressed disease phenotype and a reduction in endogenous transcript levels of the targeted fungal genes indicating translocation of siRNA molecules from host to fungal cells. Efficiency of this host-generated trans-specific RNAi was enhanced by using BSMV silencing vectors defective in coat protein coupled with introducing fungal gene sequences simultaneously in sense and antisense orientation. The disease suppression indicated the likely involvement of these fungal genes in pathogenicity. This study demonstrates that BSMV-mediated in planta-generated RNAi is an effective strategy for functional genomics in rust fungi.

PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Q␤. Here we show that PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase. Its RNA binding activity is specific since it represses the translational operator of PP7, but does not repress the operators of the MS2 or Q␤ phages. Conditions for the purification of coat protein and for the reconstitution of its RNA binding activity from disaggregated virus-like particles were established. Its dissociation constant for PP7 operator RNA in vitro was determined to be about 1 nM. Using a genetic system in which coat protein represses translation of a replicase-␤-galactosidase fusion protein, amino acid residues important for binding of PP7 RNA were identified.

Polyethylenimine is a popular DNA transfection reagent, and many approaches have been explored to further enhance its transfection efficiency. Substitution of branched polyethylenimine's primary amine groups is an attractive approach because it is amenable to a variety of chemistries and is also implicated as a primary factor in its cytotoxicity. The purpose of this work was to serially substitute saturated hydrocarbons to branched polyethylenimine and determine what structure/function relationships exist between the hydrocarbon length and its degree of substitution, relative to transfection efficiency in multiple cell lines. Specifically, acetate, butanoate and hexanoate were conjugated to branched polyethylenimine (M w = 25,000) using an aqueous condensation protocol. Transfections were performed in culture using HeLa, NIH/3T3 and Clone 9 cell lines. Biophysical characteristics of the polyelectrolyte complexes were also measured (hydrodynamic diameter, relative binding affinity) and correlated to transfection efficiency. The results show that substitution of the primary amines generally increases transfection efficiency relative to unconjugated polyethylenimine, but increasing the degree of substitution beyond ∼ 25 mol% generally decreases transfection efficiency from the optimum. Additionally, increasing hydrocarbon length generally decreased transfection efficiency. There was little correlation between particle size and binding efficiency to transfection efficiency.

The inducible acetamidase of Mycobacterium smegmatis NCTC 81 59 is expressed at high levels in the presence of a suitable inducer, such as acetamide. The gene and 1.5 kb of upstream sequence had previously been sequenced. A further 14 kb of upstream sequence has now been determined, containing an additional ORF on the opposite strand to the acetamidase gene. This ORF has significant homologies to genes encoding regulatory proteins involved in amidase expression in other organisms. Restriction fragments from the 4 kb region were subcloned into a promoter-probe shuttle vector to locate the approximate region of the acetamidase promoter and investigate the mechanism of regulation. An inducible promoter was found to lie in the 1.4 kb region situated 1-5 kb upstream from the acetamidase coding region. Expression of the acetamidase was studied at the protein and mRNA levels. Using immunoblotting, induction of the enzyme was demonstrated in minimal medium containing succinate plus acetamide, but not in a richer medium (Lemco broth) plus acetamide, confirming that regulation of acetamidase expression is mediated by both positive and negative control elements. Afier induction by acetamide, an increase above basal level could be detected after 1 h for both protein levels (using ELISA) and mRNA levels (using Northern blot analysis), indicating that control of expression is at the mRNA level. The size of the mRNA transcript detected was approximately 1.2 kb, the size of the acetamidase coding region. Since no promoter was identified immediately upstream of the coding region, this raises the possibility that a larger, primary transcript (possibly polycistronic) is cleaved to produce a stable form encoding the acetamidase protein.

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4 + gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C-or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C-or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was d50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.

An ~se~richiu coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein (Recombinant DNA; malE; factor X, protease; paramyosin; crosslinked amylose; /$galactosidase; Dirofilaria immi~; affinity purification; over expression; phage rl vectors)

Gene electrotransfer is an established method for plasmid delivery into different tissues. Contrary to extensive in vitro studies demonstrating increased gene electrotransfer by changing the electric field direction during the pulse delivery, little is known about the efficiency of both polarities pulses in vivo. Therefore the aim of our study was to evaluate the effect of pulse polarity and orientation on the efficacy of gene electrotransfer in the murine fibrosarcoma tumor model by using the luciferase and GFP reporter gene expression plasmids. Our results demonstrated no significant difference in luciferase activity, GFP transfected area or fluorescence intensity between different sets of electric pulses. Inversion of the pulse polarity did not result in the increase of gene transfer, but non-significant enhancement up to 7-fold was detected by changing the electric field orientation in perpendicular direction. Also, transfection of surrounding skin tissue was observed, meaning t...

The genes encoding staphylococcal protein A and bacterial luciferase (Vibrio harveyi) were fused in-frame in order to obtain a general marker enzyme for bioluminescent immunoassays. Two constructs were made where protein A was ligated to the first and the 12th amino acid residue, respectively, of the N terminus of the fl subunit of luciferase. Only the first fusion protein encoding the entire/3 subunit was able to form an enzymatically active luciferase complex when expressed together with the a subunit. The fusion of protein A to luciferase did not notably alter the emitted wavelength spectrum or its stability to urea treatment. The fusion protein was found to retain at least 50% of the specific bioluminescent activity compared to native luciferase. In preliminary tests, this hybrid protein was shown to be useful in bioluminescent immunoassays.

The use of natural microorganisms in biotransformations is frequently constrained by their limited tolerance to the high concentrations of metabolites and solvents required for effective industrial production. In many cases, more robust strains have to be generated by random mutagenesis and selection. This process of directed evolution can be accelerated in mutator strains, which carry defects in one or more of their DNA repair genes. However, in order to use mutator strains, it is essential to restore the normal low mutation rate of the selected organisms immediately after selection to prevent the accumulation of undesirable spontaneous mutations. To enable this process, we constructed temperature-sensitive plasmids that temporarily increase the mutation frequency of their hosts by 20-to 4,000-fold. Under appropriate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. By using this approach, we were able to increase the tolerance of three bacterial strains to dimethylformamide by 10 to 20 g/liter during only two subsequent transfers. Subsequently, the evolved strains were returned to their normal low mutation rate by curing the cells of the mutator plasmids. Our results demonstrate a new and efficient method for rapid strain improvement based on in vivo mutagenesis.

The present study was designed to analyze the regulation of the levels of the polyamines and their biosynthetic enzymes during embryonic development of Xenopus laet:is. The activity of omithine decarboxylase (ODC), a rate-controlling enzyme in polyamine biosynthesis, is elevated until, during gastrulation, there is a precipitous drop in activity. This is not attributable to a decrease in ODC mRNA content and polysome profiles reveal no apparent decrease in ODC message associated with polysomes. ODC synthesis seems to be maintained at a low, relatively constant rate until neurulation whereupon ribosome loading of ODC mRNA increases. During gastrulation the rate of ODC degradation increases dramatically, which can account for the decrease in ODC. S-Adenosylmethionine decarboxylase (AdoMetDC), another rate-controlling enzyme in polyamine biosynthesis, shows a low and constant activity from cleavage to neurulation. Subsequently, the AdoMetDC activity increases dramatically. The changes in AdoMetDC activity parallel the changes in AdoMetDC mRNA levels, suggesting a transcriptional control of AdoMetDC expression during this developmental period. The activities of ODC and AdoMetDC produce a steady increase in putrescine and spermidine content of the embryo. The spermine content also increases until gastrulation, but then decreases until the tailbud stage. . Fax: + 46 90 16669 I. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 01 67-4781 (95)001 36-0

One major barrier limiting the transfection efficiency of polyplexes is poor endosomal release, especially when small particles are applied. In an approach to overcome this barrier, covalent attachment of the membrane-active peptide all-(L)-melittin to polyethylenimine (PEI) polyplexes was found to enhance gene transfer efficiency.

The transcriptional complex hypoxia-inducible factor-1 (HIF-1) has emerged as an important mediator of gene expression patterns in tumors, although the range of responding genes is still incompletely defined. Here we show that the tumor-associated carbonic anhydrases (CAs) are tightly regulated by this system. Both CA9 and CA12 were strongly induced by hypoxia in a range of tumor cell lines. In renal carcinoma cells that are defective for the von Hippel-Lindau (VHL) tumor suppressor, up-regulation of these CAs is associated with loss of regulation by hypoxia, consistent with the critical function of pVHL in the regulation of HIF-1. Further studies of CA9 defined a HIF-1-dependent hypoxia response element in the minimal promoter and demonstrated that tight regulation by the HIF/ pVHL system was reflected in the pattern of CA IX expression within tumors. Generalized up-regulation of CA IX in VHL-associated renal cell carcinoma contrasted with focal perinecrotic expression in a variety of non-VHL-associated tumors. In comparison with vascular endothelial growth factor mRNA, expression of CA IX demonstrated a similar, although more tightly circumscribed, pattern of expression around regions of necrosis and showed substantial although incomplete overlap with activation of the hypoxia marker pimonidazole. These studies define a new class of HIF-1-responsive gene, the activation of which has implications for the understanding of hypoxic tumor metabolism and which may provide endogenous markers for tumor hypoxia.

Nearly precise excision of a transposon related to Tn1O from an Escherichia coli plasmid was used as a model to study illegitimate DNA recombination between short direct repeats. The excision was stinulated 100-1000 times by induction of plasmid single-stranded DNA synthesis and did not involve transfer of DNA from the parental to the progeny molecule. We conclude that it occurred by copy-choice DNA recombination, and propose that other events of recombination between short direct repeats might be a result of the same process.

We report the construction of a Pichia pastoris integrating vector which contains the inducible CUP1 promoter from Saccharomyces cerevisiae. We show that the promoter is indeed inducible by copper when used in P. pastoris and that the level of induction is dependent on the amount of copper in the medium.

Bradyrhizobium sp. DOA9, a non-photosynthetic bacterial strain originally isolated from the root nodules of the legume Aeschynomene americana, is a divergent nod-containing strain. It exhibits a broad host range, being able to colonize and efficiently nodulate the roots of most plants from the Dalbergioid, Millettioid, and Robinioid tribes (7 species of Papilionoideae). In all cases, nodulation was determinate. The morphology and size of DOA9 bacteroids isolated from the nodules of various species of Papilionoideae were indistinguishable from the free-living form. However, they were spherical in Arachis hypogaea nodules. GusA-tagged DOA9 also colonized rice roots as endophytes. Since broad-host-range legume symbionts often carry multiple replicons in their genome, we analyzed the replicons for symbiosis genes by electrophoresis. DOA9 carried two replicons, a chromosome (cDOA9) and single megaplasmid (pDOA9) larger than 352 kb. The genes for nodulation (nodA, B, C) and nitrogen fixation (nifH) were localized on the megaplasmid. Southern blot hybridization revealed two copies of nodA on the megaplasmid, single copies of nodB and C on the megaplasmid, and one copy each of nifH on the chromosome and megaplasmid. These results suggested that Bradyrhizobium sp. DOA9 may have the unusual combination of a broad host range, bacteroid differentiation, and symbiosis-mediating replicons.

KATHLEEN M. HESS*, MATTHEW W. DUDLEY*, DAVID G. LYNN*t, ROLF D. JOERGERf, AND ANDREW N. BINNSt *Searle Chemistry Laboratory, 5735 Ellis Avenue, The University of Chicago, Chicago IL 60637; and tPlant Science Institute, Department of Biology, ...