Centromere plasmid: a new genetic tool for the study of Plasmodium falciparum

The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite.     

Virtual profiling: a new way to analyse phenotypes

Simulation models can be used to perform virtual profiling in order to analyse eco‐physiological processes controlling plant phenotype. To illustrate this, an eco‐physiological model has been used to compare and contrast the status of a virtual fruit system under two situations of carbon supply. The model simulates fruit growth, accumulation of sugar, citric acid and water, transpiration, respiration and ethylene emission, and was successfully tested on peach (Prunus persica L. Batsch) for two leaf‐to‐fruit ratios (6 and 18 leaves per fruit). The development stage and the variation in leaf number had large effects of the fruit model variables dealing with growth, metabolism and fruit quality. A sensitivity analysis showed that changing a single parameter value, which could correspond to a genotypic change induced by a mutation, either strongly affects most of the processes, or affects a specific process or none. Correlation analysis showed that, in a complex system such as fruit, the intensity of many physiological processes and quality traits co‐varies. It also showed unexpected co‐variations resulting from emergent properties of the system. This virtual profiling approach opens a new route to explore the impact of mutations, or naturally occurring genetic variations, under differing environmental conditions.     

A novel neisserial shuttle plasmid: a useful new tool for meningococcal research

We report the identification and nucleotide sequence analysis of a cryptic plasmid pMIDG2830 from the Gram-negative bacterium Neisseria flavescens. The largest open reading frame encodes a protein similar to the replication protein, RepA, found in pAB49 from Acinetobacter baumannii and pNI10 from Pseudomonas. Modified by the incorporation of a kanamycin resistance cassette, the plasmid can be stably maintained in Escherichia coli and Neisseria meningitidis, and can be used as a shuttle plasmid in meningococcal research.     

incR: a new R package to analyse incubation behaviour

Incubation represents a life stage of crucial importance for the optimal development of avian embryos. For most birds, incubation poses a trade‐off between investing in self‐maintenance and offspring care. Furthermore, incubation is affected by environmental temperatures and, therefore, will be likely impacted by climate change. Despite its relevance and readily available temperature logging methods, avian incubation research is hindered by recognised limitations in available software. In this paper, a new quantitative approach to analyse incubation behaviour is presented. This new approach is embedded in a free R package, incR. The flexibility of the R environment eases the analysis, validation and visualisation of incubation temperature data. The core algorithm in incR is validated here and it is shown that the method extracts accurate metrics of incubation behaviour (e.g. number and duration of incubation bouts). This paper also presents a suggested workflow along with detailed R code to aid the practical implementation of incR.     

Chromosome mobilization by the R plasmid R68.45: a tool in Pseudomonas genetics.

Summary The conjugative plasmid R68.45 mobilizes the chromosome of Pseudomonas aeruginosa strain PAO from multiple sites located in different chromosome regions. In interrupted matings on the plate, selection for any single marker tested resulted in entry times of 3–5 min. When selection was imposed for two markers linked in R68.45-mediated conjugation, double recombinants appeared after a delay which corresponded approximately to the map distance between the two markers as measured by the sex factor FP2. Thus, R68.45 and FP2 appear to promote chromosome transfer at similar rates, but R68.45, unlike FP2, seems to give non-polarized transfer. R68.45 may be used to estimate map distances between linked markers located in those chromosome regions where other sex factors do not produce enough recombinants to permit accurate measurement of entry times.In R68.45 matings on the plate, most recombinants inherited short donor chromosome fragments (usually less than 10 min long) and lost the R plasmid during purification. Used like a large generalized transducing phage, R68.45 has proved valuable in construction of PAO strains with desired genotypes.     

Data analysis of plasmid maintenance in a CSTR

A simple procedure is developed to process experimental data from plasmid maintenance studies of recombinant cells in a chemostat with nonselective medium. This procedure, based on the model proposed by Imanaka and Aiba, provides quantitative information on the rate of plasmid loss and the difference in the specific growth rate between the plasmid-carrying and plasmid-free cells. The performance of the proposed method is evaluated through simulation studies. In addition, the method is applied to a set of previously reported experimental data. The two-parameter model, together with the estimated parameter values, provides an excellent fit to the experimental data.     

The protelomerase of the phage-plasmid N15 is responsible for its maintenance in linear form

The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularises via cohesive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). We demonstrate that this enzyme acts in vivo on specific substrates, and show that it is necessary for replication of the linear prophage. We show that protelomerase is an end-resolving enzyme responsible for processing of replicative intermediates. Removal of protelomerase activity resulted in accumulation of replicative intermediates that were found to be circular head-to-head dimers. N15 protelomerase and its target site constitute a functional unit acting on other replicons independently of other phage genes; a mini-F or mini-P1 plasmid carrying this unit replicates as a linear plasmid with covalently closed ends. Our results suggest the following model of N15 prophage DNA replication. Replication is initiated at an internal ori site located close to the left end of plasmid DNA and proceeds bidirectionally. After replication of the left telomere, protelomerase cuts this sequence and forms two hairpin loops telL. After duplication of the right telomere (telR) the same enzyme resolves this sequence producing two linear plasmids. Alternatively, full replication of the linear prophage to form a circular head-to-head dimer may precede protelomerase-mediated formation of hairpin ends.     

Construction of a tRNA-embedded-ribozyme trimming plasmid.

We have combined Cotten and Birnstiel's tRNA-embedded ribozymes and our 5'- and 3'-trimming system. Although the activity of the tRNA-embedded ribozyme was ca. 30% lower than those of naked ribozymes, since the stability of the former in bovine serum was higher than those of naked ribozymes, the tRNA-embedded ribozymes appear useful especially when the 5'- and 3'-trimming units are concatenated in tandem.     

Polyene-lipids: a new tool to image lipids

Microscopy of lipids in living cells is currently hampered by a lack of adequate fluorescent tags. The most frequently used tags, NBD and BODIPY, strongly influence the properties of lipids, yielding analogs with quite different characteristics. Here, we introduce polyene-lipids containing five conjugated double bonds as a new type of lipid tag. Polyene-lipids exhibit a unique structural similarity to natural lipids, which results in minimal effects on the lipid properties. Analyzing membrane phase partitioning, an important biophysical and biological property of lipids, we demonstrated the superiority of polyene-lipids to both NBD- and BODIPY-tagged lipids. Cells readily take up various polyene-lipid precursors and generate the expected end products with no apparent disturbance by the tag. Applying two-photon excitation microscopy, we imaged the distribution of polyene-lipids in living mammalian cells. For the first time, ether lipids, important for the function of the brain, were successfully visualized.     

Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid.

A composite plasmid has been constructed in vitro from colicin E1 factor (mass of 4.2 megadaltons [Md]) and nontransmissible resistance factor RSF 1010 (mass, 5.5. Md) deoxyribonucleic acids (DNAs) by the sequential action of Escherichia coli endonuclease (RI (Eco RI) and T4 phage DNA ligase on the covalently closed circular forms of the constituents. The composite plasmid was selected and amplified in vivo by sequential transformation of E. coli C600 with the ligated mixture and selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-cesium chloride solution. Treatment of the composite plasmid with Eco RI yielded two fragments with mobilities corresponding to the linear forms of the parental plasmids, whereas Serratia marscesens endonuclease R (SmaR), which introduces a single scission in the colicin E1 factor but not in RSF 1010, convErted the composite plasmid to a single linear molecule (mass, 9.7 Md). Sequential degradation of colicin E1 factor with Sma R and Eco RI produced two fragments with masses of 3.5 and 0.7 Md; sequential degradation of RSF 1010 produced only one fragment (due to the cleavage with Eco RI), and sequential degradation of the composite plasmid produced the expected three fragments--an RSF 1010 Eco RI linear and the two expected products from the colicin E1 factor moiety. The composite plasmid conferred on the host cell resistance to streptomycin, sulfonamides, and colicin E1, but colicin E1 itself was not synthesized. In contrast, colicin E1 was synthesized by cells containing simultaneously both colicin E1 factor and RSF 1010 as separate entities. In the presence of chloramphenicol, the composite plasmid continued to replicate for 6 h. whereas replication of RSF 1010 and chromosomal DNA stopped within 2 h. Continued replication in the presence of chloramphenicol suggests that the replicator of the colicin E1 factor is functional in the composite plasmid.     

Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.

We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.     

Effect of medium composition on the maintenance of a recombinant plasmid in Bacillus subtilis.

Recombinant plasmid pCED3 [confers beta-galactosidase production (LacZ+) and kanamycin resistance (Kmr)] in Bacillus subtilis was found to be both segregationally and structurally unstable. Since many solutions to segregational instability are already available, the problem of structural instability was specifically addressed by inclusion of kanamycin in the growth media. Culture instability was found to be highest in complex and defined media supporting high growth rates. Stabilization over the duration of the experiment (40 generations) was achieved by use of a recently developed chemically defined medium supporting a lower growth rate. Slowing down growth by decreasing temperature was much less effective. A major effect of the growth medium appears to be that of decreasing the growth rate advantage held by cells with plasmid deletions over parental cells containing the intact plasmid.     

PreP+07: improvements of a user friendly tool to preprocess and analyse microarray data

Background  

Nowadays, microarray gene expression analysis is a widely used technology that scientists handle but whose final interpretation usually requires the participation of a specialist. The need for this participation is due to the requirement of some background in statistics that most users lack or have a very vague notion of. Moreover, programming skills could also be essential to analyse these data. An interactive, easy to use application seems therefore necessary to help researchers to extract full information from data and analyse them in a simple, powerful and confident way.     

Electroporation as a tool to transfer the plasmid pRL489 in Oscillatoria MKU 277

To establish the gene transfer system in the cyanobacteria, we successfully introduced the shuttle plasmid pRL489 into Oscillatoria MKU 277 by electroporation with transformation frequencies of up to 102 cfu/mug of plasmid DNA. These findings suggest that Oscillatoria MKU 277 can be used as an experimental tool for genetic maneuvering.     

Restriction endonuclease BglI as a tool for in vitro reconstruction and recombination of plasmid and bacteriophage genomes

The restriction endonuclease BglI produces different individual fragment ends from different cut sites. This property has allowed us to reconstruct efficiently several commonly used plasmid and bacteriophage genomes and a number of recombinant plasmids containing up to seven BglI restriction sites from their constituent BglI fragments. It is demonstrated that in vitro reconstitution from BglI fragments can be used to create, in a simple way, recombinant DNA molecules by recombining in vitro BglI fragments from different mutated or otherwise related genomes. Further applications of the method are discussed.     

Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomplete repression of the lytic replicon by the c1 immunity repressor. Incomplete repression was particularly evident in lysogens of the thermoinducible P1 c1.100 prophage, whose replication at 32 degrees C remained almost unaffected when use of the plasmid replicon was prevented. Moreover, the average plasmid copy number of P1 in a P1 c1.100 lysogen was elevated with respect to the copy number of P1 c1+. The capacity of the lytic replicon to act as an auxiliary in plasmid maintenance may contribute to the extraordinary stability of P1 plasmid prophage.