Microsatellite mutation directed by an external stimulus

Microsatellites are regions of DNA containing tandem repeats of a core 2-6 bp nucleotide sequence. To test the hypothesis that microsatellite mutation can be directed by exposure to specific external cues, control and treatment groups of resistant and susceptible wheat varieties were grown under controlled conditions and genotyped at a number of microsatellite loci that map to chromosomes known to contain Fusarium head blight (FHB) resistance/susceptibility loci. Genotyping was undertaken both prior to and following exposure to Fusarium graminearum, the FHB pathogen. Within a month of inoculation of inflorescences, 58% of experimental plants, and no control plants, had acquired a novel allele at the locus Xgwm112.1. This allele was detected only in head blight affected tissue. Uninoculated control plants, and leaf samples from inoculated plants, showed no mutation. Cloning and sequencing of PCR products indicates that the new allele was generated by contraction of the (CT)(n) repeat motif. Observation of the same deletion-based mutation in all varieties, its absence in control plants not exposed to the head blight pathogen, and the detection of no similar mutational events in a control panel of loci not expected to show mutation, indicates that this example of microsatellite mutation is induced and/or caused by FHB infection.     

Mathematical issues arising from the directed mutation controversy

During the past 14 years or so a large body of new evidence that supposedly supports the directed mutation hypothesis has accumulated. Interpretation of some of the evidence depends on mathematical reasoning, which can be subtler than it appears at first sight. This article attempts to clarify some of the mathematical issues arising from the directed mutation controversy, thereby offering alternative interpretations of some of the evidence.     

Modeling DNA mutation and recombination for directed evolution experiments

Directed evolution experiments rely on the cyclical application of mutagenesis, screening and amplification in a test tube. They have led to the creation of novel proteins for a wide range of applications. However, directed evolution currently requires an uncertain, typically large, number of labor intensive and expensive experimental cycles before proteins with improved function are identified. This paper introduces predictive models for quantifying the outcome of the experiments aiding in the setup of directed evolution for maximizing the chances of obtaining DNA sequences encoding enzymes with improved activities. Two methods of DNA manipulation are analysed: error-prone PCR and DNA recombination. Error-prone PCR is a DNA replication process that intentionally introduces copying errors by imposing mutagenic reaction conditions. The proposed model calculates the probability of producing a specific nucleotide sequence after a number of PCR cycles. DNA recombination methods rely on the mixing and concatenation of genetic material from a number of parent sequences. This paper focuses on modeling a specific DNA recombination protocol, DNA shuffling. Three aspects of the DNA shuffling procedure are modeled: the fragment size distribution after random fragmentation by DNase I, the assembly of DNA fragments, and the probability of assembling specific sequences or combinations of mutations. Results obtained with the proposed models compare favorably with experimental data.     

Fluorescence-based directed termination PCR: direct mutation characterization without sequencing

We describe a fluorescence-based directed termination PCR (fluorescent DT–PCR) that allows accurate determination of actual sequence changes without dideoxy DNA sequencing. This is achieved using near infrared dye-labeled primers and performing two PCR reactions under low and unbalanced dNTP concentrations. Visualization of resulting termination fragments is accomplished with a dual dye Li-cor DNA sequencer. As each DT–PCR reaction generates two sets of terminating fragments, a pair of complementary reactions with limiting dATP and dCTP collectively provide information on the entire sequence of a target DNA, allowing an accurate determination of any base change. Blind analysis of 78 mutants of the supF reporter gene using fluorescent DT–PCR not only correctly determined the nature and position of all types of substitution mutations in the supF gene, but also allowed rapid scanning of the signature sequences among identical mutations. The method provides simplicity in the generation of terminating fragments and 100% accuracy in mutation characterization. Fluorescent DT–PCR was successfully used to generate a UV-induced spectrum of mutations in the supF gene following replication on a single plate of human DNA repair-deficient cells. We anticipate that the automated DT–PCR method will serve as a cost-effective alternative to dideoxy sequencing in studies involving large-scale analysis for nucleotide sequence changes.     

A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique

Background

Gene knockdown analyses using the in utero electroporation method have helped reveal functional aspects of genes of interest in cortical development. However, the application of this method to analyses in later stages of brain development or in the adult brain is still difficult because the amount of injected plasmids in a cell decreases along with development due to dilution by cell proliferation and the degradation of the plasmids. Furthermore, it is difficult to exclude the influence of earlier knockdown effects.

Methodology/Principal Findings

We developed a tightly controlled conditional knockdown system using a newly constructed vector, pT2K-TBI-shRNAmir, based on a Tol2 transposon-mediated gene transfer methodology with the tetracycline-inducible gene expression technique, which allows us to maintain a transgene for a long period of time and induce the knockdown of the gene of interest. We showed that expression of the endogenous amyloid precursor protein (APP) was sharply decreased by our inducible, stably integrated knockdown system in PC12 cells. Moreover, we induced an acute insufficiency of Dab1 with our system and observed that radial migration was impaired in the developing cerebral cortex. Such inhibitory effects on radial migration were not observed without induction, indicating that our system tightly controlled the knockdown, without any expression leakage in vivo.

Conclusions/Significance

Our system enables us to investigate the brain at any of the later stages of development or in the adult by utilizing a knockdown technique with the aid of the in utero electroporation gene transfer methodology. Furthermore, we can perform knockdown analyses free from the influence of undesired earlier knockdown effects.     

Functional genomics of Plasmodium falciparum through transposon-mediated mutagenesis

Plasmodium falciparum is the causative agent for the most lethal form of human malaria, killing millions annually. Genetic analyses of P. falciparum have been relatively limited due to the lack of robust techniques to manipulate this parasite. Development of transfection technologies and whole genome analyses have helped in understanding the complex biology of this parasite. Even with this wealth of information functional genomics approaches are still very limited in P. falciparum due to the cumbersome and inefficient methods of genetic manipulation. This review focuses on a recently developed, highly efficient method for transposon-based mutagenesis and transgene expression in P. falciparum that will allow functional genomics studies to be performed proficiently on this deadly malaria parasite. By using a piggyBac-based transposition system, multiple random integrations have been obtained into the genome of the parasite. This technique could hence be employed to set up several biological screens in this lethal protozoan parasite that may lead to identification of novel drug targets and vaccine candidates.     

Tol2 transposon-mediated transgenesis in Xenopus tropicalis

The diploid frog Xenopus tropicalis is becoming a powerful developmental genetic model system. Sequencing of the X. tropicalis genome is nearing completion and several labs are embarking on mutagenesis screens. We are interested in developing insertional mutagenesis strategies in X. tropicalis. Transposon-mediated insertional mutagenesis, once used exclusively in plants and invertebrate systems, is now more widely applicable to vertebrates. The first step in developing transposons as tools for mutagenesis is to demonstrate that these mobile elements function efficiently in the target organism. Here, we show that the Medaka fish transposon, Tol2, is able to stably integrate into the X. tropicalis genome and will serve as a powerful tool for insertional mutagenesis strategies in the frog.     

Potential of transposon-mediated cellular reprogramming towards cell-based therapies

Induced pluripotent stem(iPS) cells present a seminal discovery in cell biology and promise to support innovative treatments of so far incurable diseases. To translate i PS technology into clinical trials, the safety and stability of these reprogrammed cells needs to be shown. In recent years, different non-viral transposon systems have been developed for the induction of cellular pluripotency, and for the directed differentiation into desired cell types. In this review, we summarize the current state of the art of different transposon systems in i PS-based cell therapies.     

Single base pair mutation analysis by PNA directed PCR clamping.

A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and bind to their complementary nucleic acid sequences with higher thermal stability and specificity than the corresponding deoxyribooligonucleotides and that they cannot function as primers for DNA polymerases. We show that a PNA/DNA complex can effectively block the formation of a PCR product when the PNA is targeted against one of the PCR primer sites. Furthermore, we demonstrate that this blockage allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers.     

Active site directed inhibitors and mechanism of action of glycosidases

Summary A review is given on the inactivation of glycosidases by active site directed inhibitors, their use to identify functional groups at the active site, and deductions with respect to the mechanism of catalysis. The following enzymes are discussed:-glucosidases from various sources,-glucosidase from S. cerevisiae,-galactosidases from E. coli and Helix pomatia, cellulase and lysozyme. Epoxide type inhibitors appear especially suitable because their reactivity is enhanced selectively by acidic groups at the active site.In those cases where definite conclusions regarding the mechanism can be drawn, catalysis seems to occur by a carboxylate ion as base and an acid that has been identified as a carboxyl group with-glucosidase from Asp. wentii. The hydrolysis probably proceeds via a glycosyl-enzyme intermediate, which may be a stabilized carbonium ion or a covalent intermediate.an invited article     

A soluble acid invertase is directed to the vacuole by a signal anchor mechanism

Enzyme activities in the vacuole have an important impact on the net concentration of sucrose. In sugarcane (Saccharum hybrid), immunolabelling demonstrated that a soluble acid invertase (β-fructofuranosidase; EC 3.2.1.26) is present in the vacuole of storage parenchyma cells during sucrose accumulation. Examination of sequences from sugarcane, barley and rice showed that the N-terminus of the invertase sequence contains a signal anchor and a tyrosine motif, characteristic of single-pass membrane proteins destined for lysosomal compartments. The N-terminal peptide from the barley invertase was shown to be capable of directing the green fluorescent protein to the vacuole in sugarcane cells. The results suggest that soluble acid invertase is sorted to the vacuole in a membrane-bound form.     

In vivo packaging of cosmids in transposon-mediated mutagenesis.

A technique was developed that permits the analysis of large regions of DNA by transposition mutagenesis. Large fragments of the pTiA6NC plasmid were cloned into the broad host range cosmid pHK17 and subjected to transposition mutagenesis by Tn3. Cosmids containing Tn3 insertions were selected by in vivo packaging by lambda cI857 and transduction to a new host. The insertions were localized by DNA restriction endonuclease analysis and transferred to the Ti-plasmid by marker exchange.     

The impact of dissociation on transposon-mediated disease control strategies

Vector-borne diseases such as malaria and dengue fever continue to be a major health concern through much of the world. The emergence of chloroquine-resistant strains of malaria and insecticide-resistant mosquitoes emphasize the need for novel methods of disease control. Recently, there has been much interest in the use of transposable elements to drive resistance genes into vector populations as a means of disease control. One concern that must be addressed before a release is performed is the potential loss of linkage between a transposable element and a resistance gene. Transposable elements such as P and hobo have been shown to produce internal deletion derivatives at a significant rate, and there is concern that a similar process could lead to loss of the resistance gene from the drive system following a transgenic release. Additionally, transposable elements such as Himar1 have been shown to transpose significantly more frequently when free of exogenous DNA. Here, we show that any transposon-mediated gene drive strategy must have an exceptionally low rate of dissociation if it is to be effective. Additionally, the resistance gene must confer a large selective advantage to the vector to surmount the effects of a moderate dissociation rate and transpositional handicap.     

Hysteresis-based mechanism for the directed motility of the Ncd motor

Ncd is a Kinesin-14 family protein that walks to the microtubule's minus end. Although available structures show its α-helical neck in either pre- or post-stroke orientations, little is known about the transition between these two states. Using a combination of molecular dynamics simulations and structural analyses, we find that the neck sequentially makes intermediate contacts with the motor head along its mostly longitudinal path, and it develops a 24° twist in the post-stroke orientation. The forward (pre-stroke to post-stroke) motion has an ∼4.5 k_BT (where k_B is the Boltzmann constant, and T = 300 K) free-energy barrier and is a diffusion guided by the intermediate contacts. The post-stroke free-energy minimum is higher and is formed ∼10° before reaching the orientation in the post-stroke crystal structure, consistent with previous structural data. The importance of intermediate contacts correlates with the existing motility data, including those for mutant Ncds. Unlike the forward motion, the recovery stroke goes nearly downhill in free energy, powered in part by torsional relaxation of the neck. The hysteresis in the energetics of the neck motion arises from the mechanical compliance of the protein, and together with guided diffusion, it may be key to the directed motility of Ncd.     

A role for p53 in the frequency and mechanism of mutation

The tumor suppressor protein, p53, is often referred to as the guardian of the genome. When p53 function is impaired, its ability to preserve genomic integrity is compromised. This may result in an increase in mutation on both a molecular and chromosomal level and contribute to the progression to a malignant phenotype. In order to study the effect of p53 function on the acquisition of mutation, in vitro and in vivo models have been developed in which both the frequency and mechanism of mutation can be analyzed. In human lymphoblastoid cells in which p53 function was impaired, both the spontaneous and induced mutant frequency increased at the autosomal thymidine kinase (TK) locus. The mutant frequency increased to a greater extent in cell lines in which p53 harbored a point mutation than in those lines in which a "null" mutation had been introduced by molecular targeting or by viral degradation indicating a possible "gain-of-function" associated with the mutant protein. Further, molecular analysis revealed that the loss of p53 function was associated with a greater tendency towards loss-of-heterozygosity (LOH) within the TK gene that was due to non-homologous recombination than that found in wild-type cells. Most data obtained from the in vivo models uses the LacI reporter gene that does not efficiently detect mutation that results in LOH. However, studies that have examined the effect of p53 status on mutation in the adenine phosphoribosyl transferase (APRT) gene in transgenic mice also suggest that loss of p53 function results in an increase in mutation resulting from non-homologous recombination. The results of these studies provide clear and convincing evidence that p53 plays a role in modulating the mutant frequency and the mechanism of mutation. In addition, the types of mutation that occur within the p53 gene are also of importance in determining the mutant frequency and the pathways leading to mutation.     

Suitability of Vader for transposon-mediated mutagenesis in Aspergillus niger

The filamentous fungus Aspergillus niger is widely used in biotechnological applications. Strain CBS513.88 is known to harbor 21 copies of the nonautonomous transposon Vader. Upon selection of chlorate-resistant A. niger colonies, one Vader copy was found integrated in the nirA gene. This copy was used for vector construction and development of a transposon-tagging method. Vader showed an excision frequency of about 1 in 2.2 × 10(5) conidiospores. A total of 95 of 97 colonies analyzed exhibited an excision event at the DNA level, and Vader footprints were found. By employing thermal asymmetric interlaced (TAIL)-PCR, the reintegration sites of 21 independent excision events were determined. All reintegration events occurred within or very close to genes. Therefore, this method can be used for transposon mutagenesis in A. niger.     

piggyBac transposon-mediated transgenesis in the apicomplexan parasite Eimeria tenella

piggyBac, a type II transposon that is useful for efficient transgenesis and insertional mutagenesis, has been used for effective and stable transfection in a wide variety of organisms. In this study we investigate the potential use of the piggyBac transposon system for forward genetics studies in the apicomplexan parasite Eimeria tenella. Using the restriction enzyme-mediated integration (REMI) method, E. tenella sporozoites were electroporated with a donor plasmid containing the enhanced yellow fluorescent protein (EYFP) gene flanked by piggyBac inverted terminal repeats (ITRs), an Asc I-linearized helper plasmid containing the transposase gene and the restriction enzyme Asc I. Subsequently, electroporated sporozoites were inoculated into chickens via the cloacal route and transfected progeny oocysts expressing EYFP were sorted by flow cytometry. A transgenic E. tenella population was selected by successive in vivo passage. Southern-blotting analysis showed that exogenous DNA containing the EYFP gene was integrated into the parasite genome at a limited number of integration sites and that the inserted part of the donor plasmid was the fragment located between the 5' and 3' ITRs as indicated by primer-specific PCR screening. Genome walking revealed that the insertion sites were TTAA-specific, which is consistent with the transposition characteristics of piggyBac.