Plasmid Amplification Research Articles

Oscillations by Minimal Bacterial Suicide Circuits Reveal Hidden Facets of Host-Circuit Physiology

Synthetic biology seeks to enable programmed control of cellular behavior though engineered biological systems. These systems typically consist of synthetic circuits that function inside, and interact with, complex host cells possessing pre-existing metabolic and regulatory networks. Nevertheless, while designing systems, a simple well-defined interface between the synthetic gene circuit and the host is frequently assumed. We describe the generation of robust but unexpected oscillations in the densities of bacterium Escherichia coli populations by simple synthetic suicide circuits containing quorum components and a lysis gene. Contrary to design expectations, oscillations required neither the quorum sensing genes (luxR and luxI) nor known regulatory elements in the PluxI promoter. Instead, oscillations were likely due to density-dependent plasmid amplification that established a population-level negative feedback. A mathematical model based on this mechanism captures the key characteristics of oscillations, and model predictions regarding perturbations to plasmid amplification were experimentally validated. Our results underscore the importance of plasmid copy number and potential impact of “hidden interactions” on the behavior of engineered gene circuits - a major challenge for standardizing biological parts. As synthetic biology grows as a discipline, increasing value may be derived from tools that enable the assessment of parts in their final context.

Suppression of lysyl oxidase gene expression by methylation in pelvic organ prolapse

We measured promoter methylation in the LOX gene in women with pelvic organ prolapse and in women without prolapse. Genomic DNA was isolated from the uterosacral ligaments of eight women with prolapse and eight women without prolapse as controls. Genomic DNA was digested with BamHI and underwent sodium bisulfite modification. The LOX gene promoter region of -246 to +74 was then amplified by PCR, cloned into PCR2.1-TOPO and transformed into an Escherichia coli DH5alpha strain. Amplified plasmid DNA samples containing the LOX gene promoter region from each woman were sequenced and methylated CpG islands were identified by sequence comparison. A total of 66 methylated CpG sites were found in the group of patients with prolapse, while only one methylated CpG site was found in the non-prolapse control group. Methylation in the promoter region may suppress LOX gene expression in women with pelvic organ prolapse.

Upstream Regulatory Region Alterations Found in Human Papillomavirus Type 16 (HPV-16) Isolates from Cervical Carcinomas Increase Transcription, ori Function, and HPV Immortalization Capacity in Culture

Human papillomavirus (HPV) DNAs isolated from cervical and head and neck carcinomas frequently contain nucleotide sequence alterations in the viral upstream regulatory region (URR). Our study has addressed the role such sequence changes may play in the efficiency of establishing HPV persistence and altered keratinocyte growth. Genomic mapping of integrated HPV type 16 (HPV-16) genomes from 32 cervical cancers revealed that the viral E6 and E7 oncogenes, as well as the L1 region/URR, were intact in all of them. The URR sequences from integrated and unintegrated viral DNA were found to harbor distinct sets of nucleotide substitutions. A subset of the altered URRs increased the potential of HPV-16 to establish persistent, cell growth-altering viral-genome replication in the cell. This aggressive phenotype in culture was not solely due to increased viral early gene transcription, but also to augmented initial amplification of the viral genome. As revealed in a novel ori-dependent HPV-16 plasmid amplification assay, the altered motifs that led to increased viral transcription from the intact genome also greatly augmented HPV-16 ori function. The nucleotide sequence changes correlate with those previously described in the distinct geographical North American type 1 and Asian-American variants that are associated with more aggressive disease in epidemiologic studies and encompass, but are not limited to, alterations in previously characterized sites for the negative regulatory protein YY1. Our results thus provide evidence that nucleotide alterations in HPV regulatory sequences could serve as potential prognostic markers of HPV-associated carcinogenesis.

Simultaneous one-tube full-length amplification of the NA, NP, M, and NS genes of influenza A viruses for reverse genetics

Reverse genetics of influenza A viruses has expedited increasingly basic research and vaccine development. Target-primed plasmid amplification using full-length PCR amplicons as inserts was established previously for strain-independent and rapid cloning of all eight influenza A virus genes. This method involves separate amplification of each viral gene using segment-specific primers. Four different primer pairs are required for PCR amplification of the neuraminidase gene depending on the subtype. In order to reduce the number of necessary PCRs, a pair of primers with truncated 3′ ends was designed in the present study. This primer pair permitted reliable amplification of the NA, NP, M, and NS genes in one tube whose products can be separated subsequently by their sizes. Full-length amplicons can be generated with this one primer pair from the NA genes of all nine subtypes. By avoiding separate assays for several viral genes, this parallel PCR steps up rapid universal cloning of influenza A virus genes further.

Cellular factors are required to activate bovine papillomavirus-1 early gene transcription and to establish viral plasmid persistence but are not required for cellular transformation

Transcription from the major upstream early gene promoter, P89, of bovine papillomavirus (BPV)-1 is detectable in transfected cells lacking viral gene products yet also responds to viral E2 proteins. In contrast to human papillomaviruses (HPVs), the BPV upstream regulatory region (URR) functions as a transcriptional enhancer in epithelial cells and fibroblasts of bovine, murine or human origin. Mutations of Sp1 and/or two novel transcriptional enhancer factor (TEF)-1 sites within the 5' URR of the intact BPV-1 genome dramatically reduced P89-initiated mRNA levels, leading to decreased BPV-1 plasmid amplification and inefficient formation of transformed cell foci. However, cell lines transformed with wt or mutant BPV-1 genomes contained similar levels of unintegrated BPV-1 DNA, P89 mRNA and E2-dependent transactivation. We conclude that cellular factors necessary for activating viral early gene transcription, establishment of viral plasmid replication and cell immortalization are not required during the maintenance phase of BPV-1 infection.

Influence of Growth Conditions on Plasmid DNA Production

The obtention of high yields of purified plasmid DNA is viewed as an essential issue to be considered towards efficient production of DNA vaccines and therapeutic plasmids. In this work, Escherichia coli DH5alpha bearing the pVAX1-LacZ plasmid was grown in a developed semi-defined medium at different temperatures and tryptone concentrations. Analysis of pDNA yields and E. coli morphology revealed that at higher temperatures (37 and 40 degrees C), higher specific yields and E. coli filamentation were obtained. However, the best results were achieved when a lower tryptone concentration was used. This approach was shown to be a powerful tool to promote plasmid amplification, keeping the desirable plasmid structure, and favoring the attainment of quality. Our results suggest that by using tryptone alone as an amino acid source, pDNA amplification was improved and a specific yield of 20.43 mg pDNA/g dcw was achieved, proving that this strategy can improve pDNA yield even at a small scale.

100 ASSESSMENT OF LOCUS-SPECIFIC DNA METHYLATION: OPTIMIZATION FOR BOVINE EMBRYOS

Assessment of chromatin remodeling in early embryos is a major focus of studies today, and evaluation of DNA methylation at specific loci is one approach to study these epigenetic modifications. Our objective was to optimize the bisulfite sequencing methodology for use with very small cell numbers originating from pre-implantation embryos, making the process more time- and cost-efficient. The optimized steps include bisulfite conversion of small samples, bisulfite primer design, high-throughput plasmid DNA amplification, and preparation for sequencing. Methylation at 2 loci, Satellite I and Oct4, was investigated in bovine in vitro-produced (IVP) embryos collected at the 2-cell, 8-cell, and blastocyst stages. Bovine skin fibroblasts were first used to optimize the particular steps of the process. All reactions were run in duplicate and no-template negative and somatic cell-positive controls were treated alongside samples. Incorporating the use of Methyl Primer Express (Applied Biosystems, Foster City, CA), MacVector (Oxford Molecular Ltd., Campbell, CA), and Mfold software (Mathews DH et al. 1999 J. Mol. Biol. 288, 911–940; Zuker M 2003 Nucleic Acids Res. 31, 3406–3415) improved the specificity of bisulfite primers by exclusion of secondary or tertiary structures. The DNA from bisulfite treatment for 15 to 16 h was of better quality than DNA treated for 18 h. After initial PCR optimization, different cell concentrations were used to establish that detectable PCR products and subsequent methylation data could be obtained from DNA isolated from as few as 8 cells. Treating single blastocysts and pools of ten 8-cell and forty 2-cell embryos was sufficient for the entire scope of the experiment, allowing use of the same samples across all loci. After molecular cloning, plasmid DNA was amplified by 3 different methods and evaluated for efficiency: miniprep, TempliPhi (GE Healthcare, Piscataway, NJ), or 96-well glycerol stocks and automated TempliPhi format. Although TempliPhi alone was better than miniprep for small-scale experiments, it was the 96-well format that saved weeks of time and was most cost-effective. Sequencing was performed on a minimum of 8 clones/sample using ABI Prism sequencers (Applied Biosystems), and results were analyzed using Chromas Pro software (Technelysium Pty. Ltd., Helensvale, Australia). Percentage methylation of bovine IVP 2-cell, 8-cell, and blastocyst stage embryos for Satellite I was 25, 10, and 22%, respectively, and for Oct4 was 88, 88, and 79%, respectively. However, somatic cell methylation was 74% for Satellite I and 88% for Oct4, implying that Satellite I is demethylated during early embryo development, whereas Oct4 remains hypermethylated. In conclusion, these improved methods will benefit further studies of chromatin remodeling in early bovine pre-implantation embryos. This project was supported by National Research Initiative Competitive Grant no. 2006-35203-16620 from the USDA Cooperative State Research, Education, and Extension Service.

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

Expressional Control of a Cadmium-transporting P1B-type ATPase by a Metal Sensing Degradation Signal

Cadmium is a highly toxic environmental contaminant implicated in various diseases. Our previous data demonstrated that Pca1, a P1B-type ATPase, plays a critical role in cadmium resistance in yeast S. cerevisiae by extruding intracellular cadmium. This illustrates the first cadmium-specific efflux pump in eukaryotes. In response to cadmium, yeast cells rapidly enhance expression of Pca1 by a post-transcriptional mechanism. To gain mechanistic insights into the cadmium-dependent control of Pca1 expression, we have characterized the pathway for Pca1 turnover and the mechanism of cadmium sensing that leads to up-regulation of Pca1. Pca1 is a short-lived protein (t1/2 < 5 min) and is subject to ubiquitination when cells are growing in media lacking cadmium. Distinct from many plasma membrane transporters targeted to the vacuole for degradation via endocytosis, cells defective in this pathway did not stabilize Pca1. Rather, Pca1 turnover was dependent on the proteasome. These data suggest that, in the absence of cadmium, Pca1 is targeted for degradation before reaching the plasma membrane. Mapping of the N terminus of Pca1 identified a metal-responding degradation signal encompassing amino acids 250-350. Fusion of this domain to a stable protein demonstrated that it functions autonomously in a metal-responsive manner. Cadmium sensing by cysteine residues within this domain circumvents ubiquitination and degradation of Pca1. These data reveal a new mechanism for substrate-mediated control of P1B-type ATPase expression. Cells have likely evolved this mode of regulation for a rapid and specific cellular response to cadmium.

Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification

Reverse genetics has become pivotal in influenza virus research relying on rapid generation of tailored recombinant influenza viruses. They are rescued from transfected plasmids encoding the eight influenza virus gene segments, which have been cloned using restriction endonucleases and DNA ligation. However, suitable restriction cleavage sites often are not available. Here, we describe a cloning method universal for any influenza A virus strain which is independent of restriction sites. It is based on target-primed plasmid amplification in which the insert provides two megaprimers and contains termini homologous to plasmid regions adjacent to the insertion site. For improved efficiency, a cloning vector was designed containing the negative selection marker ccdB flanked by the highly conserved influenza A virus gene termini. Using this method, we generated complete sets of functional gene segments from seven influenza A strains and three haemagglutinin genes from different serotypes amounting to 59 cloned influenza genes. These results demonstrate that this approach allows rapid and reliable cloning of any segment from any influenza A strain without any information about restriction sites. In case the PCR amplicon ends are homologous to the plasmid annealing sites only, this method is suitable for cloning of any insert with conserved termini.

The E8 ∧ E2 Gene Product of Human Papillomavirus Type 16 Represses Early Transcription and Replication but Is Dispensable for Viral Plasmid Persistence in Keratinocytes

A conserved E8(wedge)E2 spliced mRNA is detected in keratinocytes transfected with human papillomavirus type 16 (HPV-16) plasmid DNA. Expression of HPV-16 E8--E2 (16-E8--E2) is independent of the major early promoter, P97, and is modulated by both specific splicing events and conserved cis elements in the upstream regulatory region in a manner that differs from transcriptional regulation of other early viral genes. Mutations that disrupt the predicted 16-E8--E2 message also increase initial HPV-16 plasmid amplification 8- to 15-fold and major early gene (P97) transcription 4- to 5-fold over those of the wild type (wt). Expressing the 16-E8--E2 gene product from the cytomegalovirus (CMV) promoter represses HPV-16 early gene transcription from P97 in a dose-dependent manner, as detected by RNase protection assays. When expressed from the CMV promoter, 16-E8--E2 also inhibits the amplification of an HPV-16 plasmid and a heterologous simian virus 40 (SV40) ori plasmid that contains E2 binding sites in cis. In contrast, cotransfections with HPV-16 wt genomes that express physiologic levels of 16-E8--E2 are sufficient to repress HPV-16 plasmid amplification but are limiting and insufficient for the repression of SV40 amplification. 16-E8--E2-dependent repression of HPV-16 E1 expression is sufficient to account for this observed inhibition of initial HPV-16 plasmid amplification. Unlike with other papillomaviruses, primary human keratinocytes immortalized by the HPV-16 E8 mutant genome contain more than eightfold-higher levels of unintegrated plasmid than the wt, demonstrating that 16-E8(wedge)E2 limits the viral copy number but is not required for plasmid persistence and maintenance.

Improving live attenuated bacterial carriers for vaccination and therapy

Live attenuated bacteria are well established as vaccines. Thus, their use as carriers for prophylactic and therapeutic macromolecules is a logical consequence. Here we describe several experimental applications of bacteria to carry heterologous macromolecules into the murine host. First, Listeria monocytogenes are described that are able to transfer eukaryotic expression plasmids into host cells for gene therapy. High multiplicities of infection are still required for efficient gene transfer and we point out some of the bottlenecks that counteract a more efficient transfer and application in vivo. Then, we describe Salmonella enterica serovar Typhimurium ( S. typhimurium) as an expression plasmid transfer vehicle for oral DNA vaccination of mice. We demonstrate that the stabilization of the plasmid transformants results in an improved immune response. Stabilization was achieved by replacing the origin of replication of the original high-copy-number plasmid by a low-copy-number origin. Finally, we describe Salmonella carriers for the improved expression of heterologous proteins. We introduce a system in which the plasmid is carried as a single copy during cultivation but is amplified several fold upon infection of the host. Using the same in vivo inducible promoter for both protein expression and plasmid amplification, a substantial increase in antigen expression in vivo can be achieved. A modification of this approach is the introduction of inducible gene expression in vivo with a low-molecular-weight compound. Using P BAD promoter and l-arabinose as inducer we were able to deliberately activate genes in the bacterial carrier. No background activity could be observed with P BAD such that an inducible suicide gene could be introduced. This is adding an important safety feature to such live attenuated carrier bacteria.

A New Helicobacter pylori Vacuolating Cytotoxin Determinant, the Intermediate Region, Is Associated With Gastric Cancer

Helicobacter pylori is the main cause of peptic ulceration and gastric adenocarcinoma. The vacuolating cytotoxin gene, vacA, is a major determinant of virulence. Two naturally polymorphic sites in vacA, the signal region and midregion, are well-characterized determinants of toxicity and markers of pathogenesis. The aim of this study was to characterize a new vacA polymorphic site, the intermediate (i) region. The vacA i-region was identified and characterized by constructing isogenic vacA exchange mutants and determining their vacuolating activity on HeLa, AGS, and RK13 cell lines. The vacA i-region types of H pylori isolates from patients undergoing routine endoscopy were determined by nucleotide sequencing and allele-specific polymerase chain reaction. Two i-region types were identified, i1 and i2, and both were common among 42 Western clinical isolates. Interestingly, only naturally occurring s1/m2 strains varied in i-type; s1/m1 and s2/m2 strains were exclusively i1 and i2, respectively. Vacuolation assays showed that i-type determined vacuolating activity among these s1/m2 strains, and exchange mutagenesis confirmed that the i-region itself was directly responsible. Using a simple i-region polymerase chain reaction-based typing system, it was shown for 73 Iranian patients that i1-type strains were strongly associated with gastric adenocarcinoma (P < 10(-3)). Finally, logistic regression analysis showed this association to be independent of, and larger than, associations of vacA s- or m-type or cag status with gastric adenocarcinoma. Together these data show that the vacA i-region is an important determinant of H pylori toxicity and the best independent marker of VacA-associated pathogenicity.

Interconversion of intra‐ and extra‐chromosomal sites of gene amplification by modulation of gene expression and DNA methylation

We previously showed that plasmids containing a mammalian replication initiation region and a matrix attachment region were efficiently amplified to few thousand copies per cell, and that they formed extrachromosomal double minutes (DMs) or chromosomal homogeneously staining regions (HSRs). In these structures, the plasmid sequence was arranged as a tandem repeats, and we suggested a mechanism of plasmid amplification. Since amplification was very efficient, easy, and convenient, it might be adapted to a novel method for protein production. In the current study, we found that gene expression from the tandem plasmid repeat was suppressed. We identified several strategies to overcome this suppression, including: (1) use of higher concentrations of antibiotic during cell selection; (2) treatment of cells with agents that influence DNA methylation (5-azacytidine) or histone acetylation (butyrate); (3) co-amplification of an insulator sequence; and (4) co-amplification of sequences that encode a transcriptional activator. Expression from the plasmid repeat was always higher at DMs compared to HSRs. We found that continuous activation of a plasmid-encoded inducible promoter prevented the generation of long HSRs, and favored amplification at DMs. Consistent with this finding, there was a synergistic effect of transcriptional activation and inhibition of DNA methylation on the fragmentation of long HSRs and the generation of DMs and short HSRs. Our results indicate that both transcriptional activation and DNA methylation regulate the interconversion between extra- and intra-chromosomal gene amplification. These results have important implications for both protein production technology, and the generation of chromosomal abnormalities found in human cancer cells.

A cryptic promoter in potato virus X vector interrupted plasmid construction

BackgroundPotato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV-2) VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation).ResultsA cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR.ConclusionIt is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but in E. coli) can interrupt the downstream work.

Dissection of mammalian replicators by a novel plasmid stability assay

A plasmid, bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR) was previously shown to be efficiently amplified to high copy number in mammalian cells and to generate chromosomal homogeneously staining regions (HSRs). The amplification mechanism was suggested to entail a head-on collision at the MAR between the transcription machinery and the hypothetical replication fork arriving from the IR, leading to double strand breakage (DSB) that triggered HSR formation. The experiments described here show that such plasmids are stabilized if collisions involving not only promoter-driven transcription but also promoter-independent transcription are avoided, and stable plasmids appeared to persist as submicroscopic episomes. These findings suggest that the IR sequence that promotes HSR generation may correspond to the sequence that supports replication initiation (replicator). Thus, we developed a "plasmid stability assay" that sensitively detects the activity of HSR generation in a test sequence. The assay was used to dissect two replicator regions, derived from the c-myc and DHFR ori-beta loci. Consequently, minimum sequences that efficiently promoted HSR generation were identified. They included several sequence elements, most of which coincided with reported replicator elements. These data and this assay will benefit studies of replication initiation and applications that depend on plasmid amplification.

Amplification of plasmid DNA bound on soil colloidal particles and clay minerals by the polymerase chain reaction

Polymerase chain reaction (PCR) was used to amplify a 600-base pair (bp) sequence of plasmid pGEX-2T DNA bound on soil colloidal particles from Brown soil (Alfisol) and Red soil (Ultisol), and three different minerals (goethite, kaolinite, montmorillonite). DNA bound on soil colloids, kaolinite, and montmorillonite was not amplified when the complexes were used directly but amplification occurred when the soil colloid or kaolinite-DNA complex was diluted, 10- and 20-fold. The montmorillonite-DNA complex required at least 100-fold dilution before amplification could be detected. DNA bound on goethite was amplified irrespective of whether the complex was used directly, or diluted 10- and 20-fold. The amplification of mineral-bound plasmid DNA by PCR is, therefore, markedly influenced by the type and concentration of minerals used. This information is of fundamental importance to soil molecular microbial ecology with particular reference to monitoring the fate of genetically engineered microorganisms and their recombinant DNA in soil environments.

Identification and characterization of IS1 transposition in plasmid amplification mutants of E. coli clones producing DNA vaccines

Merck Research Laboratories has developed a highly productive Escherichia coli fermentation process to produce plasmid DNA for use as vaccines. The process consists of a fed-batch fermentation in a chemically defined medium. Initiation of the feed stream precedes a growth-limited phase in which plasmid DNA is amplified. The fermentation is only maximally productive for a small fraction of E. coli transformants designated as high-producers, while the predominant low-producer population does not amplify plasmid DNA. In experiments undertaken to probe this phenomenon, transposition of the 768-bp E. coli insertion sequence IS1 into an HIV DNA vaccine vector was observed in several low-producer clones. IS1 was found to insert in or near the neomycin resistance gene in nearly a dozen unique sites from within a single population of plasmid molecules. The fraction of IS1-containing plasmids within several clones was determined by quantitative polymerase chain reaction and was found to increase with increasing cultivation time in the chemically defined medium. Because transposition into an antibiotic-resistance gene is unlikely to affect plasmid amplification, the genomes of high- and low-producers of three different HIV DNA vaccine vectors were subsequently profiled by restriction fragment length polymorphism analysis. In all three cases, IS1 insertional mutations were found in the genomes of the predominant low-producers, while the genomes of the high-producers were indistinguishable from untransformed cells. The insertions reside on similarly sized fragments for two of the low-producer clones, and the fragment size is smaller for the third clone. The third clone also produces much less plasmid DNA than a typical low-producer. The results suggest the presence of an IS1 insertional mutation that affects plasmid replication and amplification, possibly in a position-dependent manner.

Adenosine monophosphate-induced amplification of ColE1 plasmid DNA in Escherichia coli

ColE1 plasmid copy number was analyzed in relaxed ( relA) and stringent ( relA +) Escherichia coli cells after supplementation of culture media with adenosine monophosphate (AMP). When a relaxed E. coli strain bearing ColE1 plasmid was cultured in LB medium for 18 h and induced with AMP for 4 h, the plasmid DNA yield was significantly increased, from 2.6 to 16.4 mg l −1. However no AMP-induced amplification of ColE1 plasmid DNA was observed in the stringent host. Some plasmid amplification was observed in relA mutant cultures in the presence of adenosine, while adenine, ADP, ATP, ribose, potassium pyrophosphate and sodium phosphate caused a minor, if any, increase in ColE1 copy number. A mechanism for amplification of ColE1 plasmid DNA with AMP in relA mutant bacteria is suggested, in which AMP interferes with the aminoacylation of tRNAs, increases the abundance of uncharged tRNAs, and uncharged tRNAs promote plasmid DNA replication. According to this proposal, in relA + cells, the AMP induction could not increase ColE1 plasmid copy number because of lower abundance of uncharged tRNAs. Our results suggest that the induction with AMP can be used as an effective method of amplification of ColE1 plasmid DNA in relaxed strains of E. coli.

Development of a Neuroscience-oriented “Methods” Course for Graduate Students of Pharmacology and Toxicology

To provide graduate students in pharmacology/toxicology exposure to, and cross-training in, a variety of relevant laboratory skills, the Duquesne University School of Pharmacy developed a "methods" course as part of the core curriculum. Because some of the participating departmental faculty are neuroscientists, this course often applied cutting-edge techniques to neuroscience-based systems, including experiments with brain G protein-coupled receptors. Techniques covered by the course include animal handling and behavioral testing, bacterial and mammalian cell culture, enzyme-linked immunosorbent assay, western blotting, receptor binding of radioligands, plasmid DNA amplification and purification, reverse transcriptase-polymerase chain reaction, gel electrophoresis, and UV-visible and fluorescence spectroscopy. The course also encompasses research aspects such as experimental design and record keeping, statistical analysis, and scientific writing. Students were evaluated via laboratory reports and examinations, and students in turn evaluated the course using a detailed exit survey. This course introduces the graduate student to many more techniques and approaches than can be provided by the traditional graduate "rotation" format alone and should serve as a template for graduate programs in many basic research disciplines.