Plasmid Amplification Research Articles

Propagation of an amplifiable recombinant plasmid in Saccharomyces cerevisiae: flow cytometry studies and segregated modeling

Efficient expression of a foreign protein product by the yeast Saccharomyces cerevisiae requires a stable recombinant vector present at a high number of copies per cell. A conditional centromere yeast plasmid was constructed which can be amplified to high copy number by a process of unequal partitioning at cell division, followed by selection for increased copy number. However, in the absence of selection pressure for plasmid amplification, copy number rapidly drops from 25 plasmids/cell to 6 plasmids/cell in less than 10 generations of growth. Copy number subsequently decreases from 6 plasmids/cell to 2 plasmids/cell over a span of 50 generations. A combination of flow cytometric measurement of copy number distributions and segregated mathematical modeling were applied to test the predictions of a conceptual model of conditional centromere plasmid propagation. Measured distributions of plasmid content displayed a significant subpopulation of cells with a copy number of 4-6, even in a population whose mean copy number was 13.5. This type of copy number distribution was reproduced by a mathematical model which assumes that a maximum of 4-6 centromere plasmids per cell can be stably partitioned at cell division. The model also reproduces the observed biphasic kinetics of plasmid number instability. The agreement between simulation and experimental results provides support for the proposed model and demonstrates the utility of the flow cytometry/segregated modeling approach for the study of multicopy recombinant vector propagation.

RelA mutation and pBR322 plasmid amplification in amino acid-starved cells of Escherichia coli.

Plasmid pBR322 is amplified following amino-acid limitation in Escherichia coli relA hosts. In relA+ hosts there was no significant amplification or a much smaller one. Plasmid amplification is due to the relA mutation; when the relA+ allele is transferred into the relA mutant CP79 this strain no longer amplifies plasmid DNA during amino acid starvation. It is concluded that ppGpp is a negative effector of plasmid replication. Amplification is temperature dependent, being maximal at 32 degrees C and negligible at 37 degrees C.

Antigenic properties and cellular localization of herpes simplex virus glycoprotein H synthesized in a mammalian cell expression system

Herpes simplex virus type 1 glycoprotein H (HSV-1 gH) was synthesized in an inducible mammalian cell expression system, and its properties were examined. The gH coding sequence, together with the stable 5' untranslated leader sequence from xenopus beta-globin, was placed under control of the strong promoter from the human cytomegalovirus major immediate-early gene in an amplifiable plasmid which contains the simian virus 40 (SV40) virus origin for replication (ori). This expression vector was transfected into ts COS cells constitutively expressing a temperature-sensitive SV40 T antigen which allows utilization of the SV40 ori at permissive temperatures. The results of transient expression assays at the permissive temperature showed that HSV-1 gH could be synthesized in greater amounts than those produced by a high-multiplicity virus infection. The proteins produced were detected in Western blots (immunoblots) with a HSV-1 gH-specific polyclonal serum raised against a TrpE-gH fusion protein. The transfected gH had an apparent molecular weight of approximately 105,000, intermediate in size to those of the precursor (100,000) and fully processed forms (110,000) of HSV-1 gH from infections. Antigenicity was investigated by reactions with three virus-neutralizing monoclonal antibodies specific for conformational epitopes on gH. Only one of these monoclonal antibodies could immunoprecipitate the synthesized gH. However, equal recognition of the transfected gH was achieved by superinfection with virus. In addition, detectable amounts of gH were not expressed on the cell surface unless the cells were superinfected with virus. Studies with a temperature-sensitive mutant, ts1201, defective in encapsidation showed that the changes in antigenic structure and cell surface expression caused by superinfection with virus were not due simply to incorporation of gH into virions. These results suggest that gH requires additional virus gene products for cell surface localization and formation of an antigenic structure important for its function in mediating infectivity.

Effects of plasmid amplification and recombinant gene expression on the growth kinetics of recombinant E. coli

An experimental study was undertaken to identify and quantitate the effects of plasmid amplification and recombinant gene expression on Escherichia coli growth kinetics. Identification of these effects was possible because recombinant gene expression and plasmid copy number were controlled by different mechanisms on plasmid pVH106/172. Recombinant gene expression of the lactose operon structural genes was under the control of the lac promoter and was activated by the addition of the chemicals, IPTG and cyclic AMP, to the fermentation medium. Plasmid content was amplified in a separate fermentation by increasing culture temperature since the plasmid replicon was temperature-sensitive. A final fermentation was performed in which both plasmid content and recombinant gene expression were induced simultaneously by adding chemicals and raising the culture temperature. Recombinant growth rates were found to be reduced by the expression of high levels of recombinant lac proteins in the chemical induction experiments and by the amplification of plasmid levels in the temperature induction experiment. High expression of recombinant lac proteins following chemical induction was accompanied by a loss in recombinant cell viability. In the plasmid amplification experiment, the recombinant cells did not lose viability but the recombinant product yields were much lower than those achieved in the chemical induction experiments. Combining temperature and chemical induction increased the recombinant product yield by a factor of 4400 but also lowered cellular growth rates by 70%.

Escherichia coli plasmid production in fermenter

Escherichia coli harboring a recombinant plasmid was grown in a fermenter to study the effects of selected process parameters on the growth of the microbe and on plasmid amplification with chloramphenicol treatment. Eighteen fermentations were carried out according to a statistical experimental design in which the fermentation temperature, pH, and turbidity of culture at the onset of plasmid amplification were the selected independent process variables. Static regression models describing the process were derived from the experimental results. It turned out that recombinant plasmid copy numbers could be influenced by controlling fermentation temperature and pH. The maximal copy number during bacterial growth phase and the optimal plasmid production were found to require fermentation conditions different from those needed for optimal bacterial growth and cell division. The conditions also differed significantly from those routinely used in research laboratories for plasmid preparation. The chloramphenicol treatment increased the plasmid copy number compared with chromosome numbers up to fivefold. Some of the data suggest that under certain conditions the number of chromosome molecules in E. coli cells may rise during the plasmid amplification stage. Statistical experimental design, a nucleic acid sandwich hybridization technique for plasmid quantification, and regression models proved to be useful in this study.

Identification of an amplification promoting DNA sequence from the hypotrichous cilateStylonychia lemnae

The macronucleus of the hypotrichous ciliate Stylonychia lemnae contains a 1218 bp long DNA molecule which becomes highly amplified during vegetative growth due to a continuous overreplication over a long time range. The region which is located upstream the open reading frame of the overamplified 1.2kbp Stylonychia DNA molecule enabled plasmids containing an inefficiently transcribed thymidine kinase gene to persist and amplify upon transfection into mouse L fibroblasts under selective conditions. This region contains long AT-rich stretches. The AT-rich sequences interact with a previously characterized HMG-I like protein from mouse Ehrlich ascites tumour cells. A binding activity for AT-rich stretches could also be identified in macronuclear extracts from Stylonychia lemnae. We suggest a common mechanism for overamplification in Stylonychia macronuclei during vegetative growth and amplification of plasmid DNA in heterologous mouse cells under the influence of a common element.

Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support.

Approaches to direct solid phase sequencing of genomic and plasmid DNA have been developed using magnetic beads, coated with streptavidin, as solid support. The DNA is immobilized through selective incorporation of biotin into one of the strands. A single stranded template, suitable for sequencing, is obtained through strand-specific elution. Using this concept, in vitro amplified plasmid DNA and chromosomal DNA were sequenced directly from single colonies. The solid phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The system was found to be suitable for sequencing using both isotope- and fluorescent-labelled primers.

Cis-acting sequences from mouse rDNA promote plasmid DNA amplification and persistence in mouse cells: implication of HMG-I in their function

Searching for amplification promoting sequences within the murine rDNA cistrons, we isolated two elements from the nontranscribed spacer region. These 370 bp and 423 bp long cis-acting elements, referred to as muNTS1 and muNTS2, are localized 4.1 kb and 4.6 kb upstream the RNA polymerase I transcriptional start site. They contain ca. 50 bp long AT-rich sequences that strongly interact with a protein from nuclear extracts. The protein could be purified and identified as HMG-I. A synthetic oligonucleotide encompassing the AT-rich stretch from muNTS1 is able to substitute for the muNTS elements. A similar sequence from the nontranscribed spacer of rat has previously been reported to be important for the function of the RNA polymerase I enhancer (1). Therefore the interaction of HMG I with the muNTS elements may play a role both in the stimulation of DNA amplification and transcription.

Expression of a cloned beta-glucanase gene from Bacillus amyloliquefaciens in an Escherichia coli relA strain after plasmid amplification.

Amino acid starvation of cells of the Escherichia coli relA strain, CP79, which cannot accumulate guanosine tetraphosphate (ppGpp) in response to amino acid limitation, increased the pEG1 plasmid content about 5- to 7-fold in comparison with exponentially growing cells (pEG1:pBR322 with an insertion of Bacillus amyloliquefaciens DNA coding for beta-glucanase). In contrast, no pEG1 amplification occurred in E. coli CP78, the stringently controlled counterpart, after amino acid starvation. In order to verify these results, the plasmid DNA content was monitored by measuring the expression of pEG1-encoded beta-glucanase from B. amyloliquefaciens both before and after plasmid amplification. When amino acid starved CP79 cells were given an additional dose of amino acids, a more than 10-fold increase in pEG1-encoded beta-glucanase activity (per cell mass) was measured. This increase in enzyme activity correlates with pEG1 amplification during amino acid limitation. Under comparable conditions the activity of beta-glucanase was not increased in strain CP78, which did not amplify the plasmid. We suggest that the replication of pEG1 in amino acid starved E. coli cells is somehow under negative control by ppGpp. Moreover, we found the Bacillus beta-glucanase in E. coli relA cells to be excreted into the growth medium after starvation and overexpression.

Conditional high copy number ColE1 mutants: resistance to RNA1 inhibition in vivo and in vitro.

We describe three independently isolated copy number mutants of a plasmid ColE1 derivative which undergo temperature- and growth-phase-dependent DNA amplification in Escherichia coli. These mutants have single base-pair alterations in a highly localized region of the plasmid genome encoding the replication primer RNA. The mutations map immediately upstream of the RNA1 transcript, altering the sequence between conserved elements of the RNA1 promoter. These mutants have 2- to 4-fold increased copy number relative to wild-type plasmids in exponential growth at 37 degrees C but undergo 20-fold amplification of copy number relative to wild-type when cells enter stationary phase. Cells containing these plasmids grow with normal kinetics at 37 degrees C but grow poorly at 42 degrees C. The poor growth is associated with high-level plasmid amplification. Both the temperature and growth phase plasmid DNA amplification are suppressed if the ColE1 rop gene product is provided in trans from a compatible plasmid. Analysis of steady-state RNA1 levels indicates that DNA amplification occurs in the presence of RNA1 made by the mutant plasmid. Thus, the DNA amplification of the copy mutant is not due to an inability to synthesize RNA1. Using an in vitro transcription system containing RNase H, we show that mutant primer processing by RNase H is resistant to levels of the replication initiation inhibitor RNA1 that inhibit wild-type primer processing. The defect in inhibition appears not to be at the level of association of RNA1 with nascent primer. These results indicate that mutant plasmid amplification is due to the ability of its primer precursor transcripts to serve as substrates for RNase H despite the presence of RNA1.

Transient response simulations of recombinant microbial populations

An asynchronous bacterial population has been approximated using a finite number of "computer" cells, each based on a complex single-cell model for Escherichia coli. This formulation correctly simulates the transient responses of protein and total cell mass synthesis rate to the sudden increase in the concentration of limiting energy source in the growth medium. Experimentally observed responses of rRNA and mRNA synthesis rates to growth rate shifts are qualitatively mirrored by the model. Simulation trends following those of a rel(-) mutant suggest that model modifications are needed to describe the dynamics of the stringent response. Simulations of the responses of recombinant populations to plasmid amplification or plasmid promoter induction also result in behavior similar to that determined experimentally. The calculated responses for recombinant populations subjected to constant promoter induction or cyclic induction-noninduction lead to the conclusion that inducible systems give greater productivity than those with fixed promoter strength. This formulation may be utilized as a basis for exploring other aspects of recombinant population dynamics.

Construction of a runaway vector and its use for a high-level expression of a cloned human superoxide dismutase gene

A runaway cloning vector pR3 from plasmid pBEU17 has been constructed by introducing three deletions (ΔI, ΔH and ΔEHi), multiple cloning sites and transcription terminator sequences. Introduction of the deletion ΔH restored a drastic plasmid amplification at high temperature (37° C). This suggested the presence of unknown sequences around the ΔH region which repressed runaway replication. To examine the usefulness of a pR3 runaway vector on the high-level expression of a cloned gene, a pRT8 plasmid containing a cloned human superoxide dismutase (SOD) gene in the pR3 was constructed. A host with tolerance to high copy numbers of pRT8 was selected, and the cultural conditions required for high-level expression were investigated. It was shown that the copy number of pRT8 increased in the stationary phase even at 30° C, and that cells carrying an excess number of plasmid did not support the runaway replication, yielding less SOD protein. SOD synthesis in the production culture lasted for about 24 h after the thermal shift, though viability was soon lost. The maximum yield was determined as approximately 20% of total cellular protein. This was 12-fold higher than that of a vector having the ColE1 plasmid replicon.

A mathematical model of recombinational amplification of the 2μ plasmid in the yeast Saccharomyces cerevisiae

A mathematical model of 2 mu plasmid recombinational amplification in Saccharomyces cerevisiae has been developed, based on mechanisms of 2 mu recombination and replication presented in the literature. A probabilistic description reveals the limits inherent in the recombinational mode of plasmid amplification. These limits correspond well with values calculated from reported results. In the model, copy number control is effected by the constitutive expression of a repressor of recombinase expression. Estimation of the model parameters is accomplished via a set of heuristic rules which restrict the feasible parameter space considerably. It is demonstrated that many parameter sets arbitrarily chosen from the feasible parameter space reproduce the observed characteristics of 2 mu plasmid amplification: rapid correction of downward copy number deviations, with a lack of strict control of steady-state copy number.

Reconstruction of the yeast 2 micron plasmid partitioning mechanism.

The effect of the yeast 2 micron circle encoded REP1 and REP2 gene products on plasmid partitioning and copy number control was analyzed by removing the open reading frames from their normal sequence context and transcriptional control regions and directing their expression using heterologous promoters in [cir0] host strains. Both the REP1 and REP2 gene products are directly required at appropriate levels of expression to reconstitute the 2 microns circle partitioning system in conjunction with REP3 and the origin of replication. The level of expression of REP2 appears to be critical to re-establishing proper partitioning and may also play a role in monitoring and thereby regulating the plasmid copy number. Constitutive expression of the REP1 and REP2 open reading frames using heterologous expression signals can be used to reconstruct efficient plasmid partitioning even in the absence of FLP-mediated plasmid amplification and a functional D open reading frame.

Site-specific recombination promotes plasmid amplification in yeast

All stable, naturally occurring circular yeast DNA plasmids contain a pair of long, nontandem inverted repeats that undergo frequent reciprocal recombination. This yields two plasmid inversion isomers that exist in the cell in equal numbers. In the 2μ circle plasmid of S. cerevisiae such inversion is catalyzed by a plasmid-encoded site-specific recombinase, FLP. We show that the site-specific recombination system of 2μ circle enables the plasmid to increase its mean intracellular copy number in yeast cells growing under nonselective conditions. This apparently occurs by a FLP-induced transient shift in the mode of replication from theta to double rolling circle as initially proposed by Futcher. This capability may ensure stable maintenance of the plasmid by enabling it to correct downward deviations in copy number that result from imprecision of the plasmid-encoded partitioning system.

Deregulated expression of c-myc by murine erythroleukaemia cells prevents differentiation.

Friend murine erythroleukaemia (F-MEL) cells are a permanent line of primitive erythroid precursors originally derived from the spleens of mice infected with the Friend strain of murine leukaemia virus. F-MEL cells differentiate in vitro in response to various chemical inducers. Concomitantly with induction, a biphasic regulation of c-myc oncogene transcripts is observed. Within one hour of the addition of dimethyl sulphoxide (DMSO) or hypoxanthine (Hyp), the levels of c-myc transcripts fall dramatically and remain virtually undetectable for the next few hours. Between 8 and 24 hours after induction, c-myc transcripts return to pre-induction levels and then decline again between 3 and 5 days as most of the cells undergo terminal differentiation. To explore the potential relationship between c-myc expression and F-MEL terminal differentiation, we have investigated here whether reversing the early fluctuations in c-myc transcript levels affects the ability of F-MEL cells to differentiate. We therefore constructed an amplifiable plasmid vector containing a full-length mouse c-myc complementary DNA and introduced it stably into recipient F-MEL cells. The exogenous c-myc sequences are transcribed in F-MEL cells and the transcript levels do not change significantly in response to inducing agents. The net result is continued c-myc expression following DMSO or Hyp induction and a complete or partial inhibition of F-MEL differentiation.

Physiological studies on pBR 322 DNA amplification in an Escherichia coli relA strain.

Amino acid limitation leads in E. coli relA cells which cannot synthesize guanosine tetraphosphate (ppGpp) under these conditions to an amplification of pBR 322 DNA. We previously proposed that ppGpp produced in E. coli relA+ cells subjected to amino acid limitation inhibits pBR 322 DNA replication (Hecker et al. 1983). In further experiments it was established that an E. coli relA strain shows plasmid amplification during amino acid limitation (arginine, threonine, leucine or histidine) only in the presence of sufficient concentrations of phosphate, ammonia and glucose. Plasmid amplification does not occur if ammonia or phosphate is depleted. We suggest that glucose, ammonia and phosphate are needed for nucleotide and deoxynucleotide synthesis as the essential prerequisite for plasmid amplification. The activity of beta-lactamase was determined as an indicator for the expression of plasmid-encoded genes. The enzyme activity remains on a low level during plasmid amplification because of arginine exhaustion. A remarkable increase in the activity of beta-lactamase was observed after resumption of growth of relA cells containing amplified plasmid DNA. The plasmid content decreased as the cells continued to grow. We found that plasmid amplification and expression of plasmid-localized genes are opposite reactions which do not occur at the same time.

An inducible eukaryotic host-vector expression system: amplification of genes under the control of the polyoma late promoter in a cell line producing a thermolabile large T antigen

We have taken advantage of the inherent instability of integrated polyoma (Py) DNA sequences in the presence of a functional viral large T antigen (LT) to develop a eukaryotic host-vector system where copy number is controlled by temperature. A mouse cell line WOP32-4, that constitutively expresses a temperature sensitive (ts) LT, was transfected with plasmids containing the Py origin of DNA replication ( ori) and either a neomycin-resistance gene ( neo) or chloramphenicol acetyl transferase gene ( cat) linked to the Py late promoter. Stable transformants were selected at 39°C, the non-permissive temperature for the ts LT function. Upon shift to 33 °C, the resident Py sequences present in the WOP32-4 cells cannot excise due to an ori deletion. However, excision of the transfected plasmid molecules and subsequent extrachromosomal replication occur at high rates leading in some cases to the production of 1000–2000 copies per cell (average) of the plasmid. Proportional increases in either neo-specific mRNA or CAT activity were also observed. In situ hybridization for one cell line indicated that about 20% of temperature-shifted cells contained amplified plasmid DNA.

An economical large scale procedure to purifyE. coli amplifiable plasmids for DNA sequencing, in vitro transcription and in vitro mutagenesis

A reproducible and economical procedure for obtaining a large and quantitative yield of highly purified covalently closed circular plasmid DNA is described. The procedure departs in several ways from more commonly used methods. These are a) avoidance of the use of CsCl, ethidium bromide and ultracentrifuge, b) enrichment of the plasmid DNA by selective denaturation of chromosomal DNA with an alkaline-SDS solution, c) enrichment of covalently closed circular plasmid DNA by extraction with acid-phenol, and d) removal of small degraded RNA fragments by molecular sieve chromatography after digestion with RNase A. The plasmid DNA prepared by this new procedure is free of contaminants and has been used for DNA sequencing, in vitro transcription, transformation and in vitro mutagenesis.

Flow Cytometry Analysis of Plasmid Amplification in Escherichia coli

Large quantities of cloned DNAs for use in characterization, or in subsequent recombinant plasmid construction, may be obtained by plasmid amplification, which is the increase of cellular plasmid DNA content relative to the amount of host cell chromosomal DNA. Plasmid amplification has been investigated with flow cytometry in chloramphenicol (Cm) treated cell populations of Escherichia coli HB101 carrying plasmids with pMBI replication origins at different copy number levels ranging from 12 to 122. The flow cytometry method is based on the determination of the total cellular DNA content after staining with mithramycin, a DNA-specific fluororescent dye. This method permits direct measurement of the rate of plasmid amplification and classification of cell populations according to chromosomal replication forks at the time of Cm treatment. Both initial rates of plasmid amplification and final contents of amplified plasmids were found to be proportional to the average initial plasmid copy numbers. The maximum degree of amplification, defined as the ratio of plasmid content after eighteen hours incubation to the preamplification plasmid content at steady-state growth, was approximately equal for the strains studied.Larger cells, with four active chromosomal replication forks at Cm addition, exhibit faster plasmid replication rates than cells initially at earlier cell cycle states. A kinetic model was proposed that agrees well with the present and previous experimental observations. This work clearly demonstrates the advantage of flow cytometry for experimental analysis of plasmid propagation in recombinant organisms.