Foreign DNA Molecules Research Articles

GAS-STING: a classical DNA recognition pathways to tumor therapy.

Cyclic GMP-AMP synthetase (cGAS), recognized as the primary DNA sensor within cells, possesses the capability to identify foreign DNA molecules along with free DNA fragments. This identification process facilitates the production of type I IFNs through the activator of the interferon gene (STING) which induces the phosphorylation of downstream transcription factors. This action characterizes the most archetypal biological functionality of the cGAS-STING pathway. When treated with anti-tumor agents, cells experience DNA damage that triggers activation of the cGAS-STING pathway, culminating in the expression of type I IFNs and associated downstream interferon-stimulated genes. cGAS-STING is one of the important innate immune pathways,the role of type I IFNs in the articulation between innate immunity and T-cell antitumour immunity.type I IFNs promote the recruitment and activation of inflammatory cells (including NK cells) at the tumor site.Type I IFNs also can promote the activation and maturation of dendritic cel(DC), improve the antigen presentation of CD4+T lymphocytes, and enhance the cross-presentation of CD8+T lymphocytes to upregulating anti-tumor responses. This review discussed the cGAS-STING signaling and its mechanism and biological function in traditional tumor therapy and immunotherapy.

Historical Aspects of Restriction Endonucleases as Intelligent Scissors for Genetic Engineering

Restriction endonucleases are a component of restriction–modification systems, where the main biological function is to protect bacterial cells from incoming foreign DNA molecules. There are four main types of restriction enzymes (types I, II, III, and IV), which differ in protein composition, cofactor requirements, and mode of action. The most studied are representatives of type II, which specifically recognize DNA sequences of 4–8 bp and catalyze DNA cleavage within these sequences or not far from them. The exceptional precision of type II enzymes has made them indispensable for DNA manipulations. Although hundreds of DNA restriction enzymes are currently known, there is still a need for enzymes that recognize new DNA targets. For this reason, the discovery of new natural restriction endonucleases and rational design of their properties (to obtain enzymes with high specificity for a unique nucleotide sequence at a restriction site and without nonspecific activity) will expand the list of enzymes for use in biotechnology and genetic engineering. This review briefly touches upon the main types of restriction endonucleases, their classification, nomenclature, and typical properties, and it concisely describes approaches to the construction of enzymes with altered properties.

Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts.

Genetic engineering of economically important traits in plants is an effective way to improve global welfare. However, introducing foreign DNA molecules into plant genomes to create genetically engineered plants not only requires a lengthy testing period and high developmental costs but also is not well-accepted by the public due to safety concerns about its effects on human and animal health and the environment. Here, we present a high-throughput nucleic acids delivery platform for plants using peptide nanocarriers applied to the leaf surface by spraying. The translocation of sub-micrometer-scale nucleic acid/peptide complexes upon spraying varied depending on the physicochemical characteristics of the peptides and was controlled by a stomata-dependent-uptake mechanism in plant cells. We observed efficient delivery of DNA molecules into plants using cell-penetrating peptide (CPP)-based foliar spraying. Moreover, using foliar spraying, we successfully performed gene silencing by introducing small interfering RNA molecules in plant nuclei via siRNA-CPP complexes and, more importantly, in chloroplasts via our CPP/chloroplast-targeting peptide-mediated delivery system. This technology enables effective nontransgenic engineering of economically important plant traits in agricultural systems.

High-efficiency transformation of archaea by direct PCR products with its application to directed evolution of a thermostable enzyme.

SummaryHyperthermophilic archaea with unique biochemical and physiological characteristics are important organisms for fundamental research of life science and have great potential for biotechnological applications. However, low transformation efficiency of foreign DNA molecules impedes developments in genetic modification tools and industrial applications. In this study, we applied prolonged overlap extension PCR (POE‐PCR) to generate multimeric DNA molecules and then transformed them into two hyperthermophilic archaea, Thermococcus kodakarensis KOD1 and Pyrococcus yayanosii A1. This study was the first example to demonstrate the enhanced transformation efficiencies of POE‐PCR products by a factor of approximately 100 for T. kodakarensis KOD1 and 8 for P. yayanosii A1, respectively, relative to circular shuttle plasmids. Furthermore, directed evolution of a modestly thermophilic enzyme, Methanothermococcus okinawensis 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR), was conducted to obtain more stable ones due to high transformation efficiency of T. kodakarensis (i.e. ~3 × 104 CFU per μg DNA). T. kodakarensis harbouring the most thermostable MoHMGR mutant can grow in the presence of a thermostable antibiotic simvastatin at 85°C and even higher temperatures. This high transformation efficiency technique could not only help develop more hyperthermophilic enzyme mutants via directed evolution but also simplify genetical modification of archaea, which could be novel hosts for industrial biotechnology.

Integer Programming Problem Based on Plasmid DNA Computing Model

A DNA algorithm by operating on plasmids was presented to solve a special integer programming, a typical hard computing problem. The DNA algorithm employed double-stranded molecules to encode variables of 0-1 programming problem, the encoded DNA molecules were inserted into circular plasmids as foreign DNA molecules. Followed by, a series of enzymatic treatments to plasmids were performed in order to find feasible solutions to the given problem. The final optimum was obtained by applying founded feasible solutions to object function. Compared with other DNA algorithms of integer programming problem, the proposed algorithm is simple, error-resistant, above all, feasible. Our work clearly showed the distinct advantages of plasmid DNA computing model when solving integer related programming problem.

Diatom centromeres suggest a mechanism for nuclear DNA acquisition

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequence features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.

Soma to germline inheritance of extrachromosomal genetic information via a LINE-1 reverse transcriptase-based mechanism.

Mature spermatozoa are permeable to foreign DNA and RNA molecules. Here I propose a model, whereby extrachromosomal genetic information, mostly encoded in the form of RNA in somatic cells, can cross the Weismann barrier and reach epididymal spermatozoa. LINE-1 retrotransposon-derived reverse transcriptase (RT) can play key roles in the process by expanding the RNA-encoded information. Retrotransposon-encoded RT is stored in mature gametes, is highly expressed in early embryos and undifferentiated cells, and becomes downregulated in differentiated cells. In turn, RT plays a role in developmental control, as its inhibition arrests developmental progression of early embryos with globally altered transcriptomic profiles. Thus, sperm cells act as recipients, and transgenerational vectors of somatically derived genetic information which they pass to the next generation with the potential to modify the fate of the developing embryos.

Asymmetric partitioning of transfected DNA during mammalian cell division

Foreign DNA molecules and chromosomal fragments are generally eliminated from proliferating cells, but we know little about how mammalian cells prevent their propagation. Here, we show that dividing human and canine cells partition transfected plasmid DNA asymmetrically, preferentially into the daughter cell harboring the young centrosome. Independently of how they entered the cell, most plasmids clustered in the cytoplasm. Unlike polystyrene beads of similar size, these clusters remained relatively immobile and physically associated to endoplasmic reticulum-derived membranes, as revealed by live cell and electron microscopy imaging. At entry of mitosis, most clusters localized near the centrosomes. As the two centrosomes split to assemble the bipolar spindle, predominantly the old centrosome migrated away, biasing the partition of the plasmid cluster toward the young centrosome. Down-regulation of the centrosomal proteins Ninein and adenomatous polyposis coli abolished this bias. Thus, we suggest that DNA clustering, cluster immobilization through association to the endoplasmic reticulum membrane, initial proximity between the cluster and centrosomes, and subsequent differential behavior of the two centrosomes together bias the partition of plasmid DNA during mitosis. This process leads to their progressive elimination from the proliferating population and might apply to any kind of foreign DNA molecule in mammalian cells. Furthermore, the functional difference of the centrosomes might also promote the asymmetric partitioning of other cellular components in other mammalian and possibly stem cells.

Multifunctional Inorganic Nanocontainers for DNA and Drug Delivery into Living Cells

AbstractInvited for the cover of this issue is the group of Luisa De Cola at the University of Strasbourg, ISIS, (France) and her collaborator, Roberto Corradini from Università degli Studi di Parma (Italy). Their study demonstrates the use of multifunctional zeolite L nanoparticles for the simultaneous delivery of foreign DNA and organic molecules into living cells. The fate and the kinetics of the different labeled components have been followed by confocal microscopy. Read the full text of the article at 10.1002/chem.201403232.

Nonhomologous end joining-mediated gene replacement in plant cells.

Stimulation of the homologous recombination DNA-repair pathway via the induction of genomic double-strand breaks (DSBs) by zinc finger nucleases (ZFNs) has been deployed for gene replacement in plant cells. Nonhomologous end joining (NHEJ)-mediated repair of DSBs, on the other hand, has been utilized for the induction of site-specific mutagenesis in plants. Since NHEJ is the dominant DSB repair pathway and can also lead to the capture of foreign DNA molecules, we suggest that it can also be deployed for gene replacement. An acceptor DNA molecule in which a green fluorescent protein (GFP) coding sequence (gfp) was flanked by ZFN recognition sequences was used to produce transgenic target plants. A donor DNA molecule in which a promoterless hygromycin B phosphotransferase-encoding gene (hpt) was flanked by ZFN recognition sequences was constructed. The donor DNA molecule and ZFN expression cassette were delivered into target plants. ZFN-mediated site-specific mutagenesis and complete removal of the GFP coding sequence resulted in the recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was unlinked to the acceptor DNA. More importantly, ZFN-mediated digestion of both donor and acceptor DNA molecules resulted in NHEJ-mediated replacement of the gfp with hpt and recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was physically linked to the acceptor DNA. Sequence and phenotypical analyses, and transmission of the replacement events to the next generation, confirmed the stability of the NHEJ-induced gene exchange, suggesting its use as a novel method for transgene replacement and gene stacking in plants.

Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system.

A widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules consists of the clustered regularly interspaced short palindromic repeats (CRISPR) coupled with cas (CRISPR-associated) genes. Similar to RNA interference in eukaryotes, these CRISPR/Cas systems use small RNAs for sequence-specific detection and neutralization of invading genomes. Here we describe the first examples of genes that mediate the inhibition of a CRISPR/Cas system. Five distinct 'anti-CRISPR' genes were found in the genomes of bacteriophages infecting Pseudomonas aeruginosa. Mutation of the anti-CRISPR gene of a phage rendered it unable to infect bacteria with a functional CRISPR/Cas system, and the addition of the same gene to the genome of a CRISPR/Cas-targeted phage allowed it to evade the CRISPR/Cas system. Phage-encoded anti-CRISPR genes may represent a widespread mechanism for phages to overcome the highly prevalent CRISPR/Cas systems. The existence of anti-CRISPR genes presents new avenues for the elucidation of CRISPR/Cas functional mechanisms and provides new insight into the co-evolution of phages and bacteria.

Homologous illegitimate random integration of foreign DNA into the X chromosome of a transgenic mouse line

BackgroundIt is not clear how foreign DNA molecules insert into the host genome. Recently, we have produced transgenic mice to investigate the role of the fad2 gene in the conversion of oleic acid to linoleic acid. Here we describe an integration mechanism of fad2 transgene by homologous illegitimate random integration.ResultsWe confirmed that one fad2 line had a sole integration site on the X chromosome according to the inheritance patterns. Mapping of insertion sequences with thermal asymmetric interlaced and conventional PCR revealed that the foreign DNA was inserted into the XC1 region of the X chromosome by a homologous illegitimate replacement of an entire 45,556-bp endogenous genomic region, including the ovarian granulosa cell tumourigenesis-4 allele. For 5' and 3' junction sequences, there were very short (3-7 bp) common sequences in the AT-rich domains, which may mediate the recognition of the homologous arms between the transgene and the host genome. In addition, analysis of gene transcription indicated that the transgene was expressed in all tested fad2 tissues and that its transcription level in homozygous female tissues was about twice as high as in the heterozygous female (p < 0.05).ConclusionsTaken together, the results indicated that the foreign fad2 behaved like an X-linked gene and that foreign DNA molecules were inserted into the eukaryotic genome through a homologous illegitimate random integration.

Exploring The Spatiotemporaloral Dynamics of DNA Binding and Cleavage by Restriction Endonucleaes

Using restriction endonucleases to catalyze the double-stranded DNA (dsDNA) breakage at certain recognition sequences is an important molecular biology technique. The restriction endonucleases constitute an important defense mechanism of bacteria against viral attacks; this mechanism is to destroy invading foreign DNA molecules via cleaving a specific site (phosphodiester bond) of a dsDNA. By cleaving recognition sites on dsDNA with extraordinary specificity can lead to the DNA double strand breaks (dsb). We presented a novel single-molecule approach to investigate the interaction between DNA and restriction endonucleases, including DNA recognition and cleavage. To elucidate how fast restriction endonucleases recognize and cleavage DNA sequence, we constructed a high resolution dual-beam laser tweezers system to manipulate single DNA molecule, together with the site-specific restriction enzymes, namely, EcoRI (one-site endonuclease) and Cfr9I (two-site endonuclease), conjugated to nanometer-sized fluorescence particle. Because most endonucleases work in the presence of magnesium ions, we will apply optically based reaction mechanism to control and synchronize the restriction endonuclease activity in this study. Furthermore, both laser tweezers and fluorescence particle imaging will be used to probe whether the DNA double strand breaks occurred due to the molecular cutting. Hence, this single-molecule approach allows us to directly observe and visualize the spatiotemporal dynamics of DNA binding and cleavage by restriction endonucleases, and can be further applied to determine the DNA cleavage rate due to the presence of EcoRI and Cfr9I. Finally, we extend this approach, together with the light-induced molecular cutting, to investigate the DNA binding and cleavage by restriction endonucleases under tension at different temperatures.

An improved protocol for electroporation ofOenococcus oeniATCC BAA-1163 using ethanol as immediate membrane fluidizing agent

To finalize an effective and reproducible electroporation procedure to transform Oenococcus oeni ATCC BAA-1163 strain. The vector pGID052 was selected to optimize the electroporation procedure. Transformation efficiency was 5.8 x 10(3) per microg of DNA. Transformation was improved when competent cells were prepared with exponential phase cultures; optimum electroporation parameters were an electric pulse of 125 kV cm(-1), under a resistance of 200 omega and the presence of 10% (v/v) ethanol in the electroporation buffer (EPB). An effective protocol to transform O. oeni ATCC BAA-1163 strain by electroporation has been obtained by addition of ethanol to the EPB. A heterologous expression was obtained in O. oeni ATCC BAA-1163 by introducing a recombinant vector encoding a truncated form of ClpL2 protein. This is the first report of a successful electroporation of O. oeni ATCC BAA-1163. The major improvement was the addition of ethanol to the EPB, which has never been reported before as means of enhancing the incorporation of foreign DNA molecules into prokaryote cells by electroporation. This method constitutes a useful tool for the genetic study of this lactic bacterium.

Comments on Usage of the Term “Restriction Enzyme”

The term “restriction enzyme” is derived from the phenomenon of host-controlled restriction and modification in viral infection of bacteria, not from the ability of enzymes to recognize the specific target nucleotide sequence in the DNA and break the DNA chain. That is, the term “restriction enzyme” is not derived from the fact that the cleavage site is restricted. Bacteria initially resist infections by new bacteriophages, and this “restriction” of viral growth stems from endonucleases within the cells that destroy foreign DNA molecules from bacteriophages. Restriction enzymes are a kind of nuclease that catalyze hydrolysis of DNA strands and were discovered during investigations of the phenomenon of host-controlled restriction and modification of bacteriophages. They are not peptidases (proteases). However, it is a common mistake to use the term “restriction enzyme” for amino acid residue-specific endopeptidase such as trypsin.

Generation of biologically active retro‐genes upon interaction of mouse spermatozoa with exogenous DNA

Mature spermatozoa of most animal species can spontaneously take up foreign DNA molecules which can be delivered to embryos upon fertilization. Following this procedure, transgenic animals of various species have been generated. We recently discovered a reverse transcriptase (RT) activity in mouse spermatozoa that can reverse-transcribe exogenous RNA molecules into cDNA copies. These cDNA copies are transferred to embryos at fertilization, mosaic propagated as non-integrated structures in tissues of founder individuals and further transmitted to F1 progeny. Reverse-transcribed sequences behave as functional genes, being correctly expressed in tissues of F0 and F1 animals. To learn more about this mechanism and further characterize the reverse transcription step, we have now incubated spermatozoa with a plasmid harboring a green fluorescent protein (EGFP) retrotransposition cassette interrupted by an intron in the opposite orientation to the EGFP gene. We found that reverse-transcribed spliced EGFP DNA sequences are generated in sperm cells and transmitted to embryos in IVF assays. After implantation in foster mothers, embryos developed into mice that expressed EGFP in the blood vessel endothelia of a variety of organs. The EGFP-encoding cDNA sequences were detected in positive tissues as extrachromosomal mosaic-propagated structures, maintained in low-copy number (<1 copy/genome), and mosaic transmitted from founders to the F1 progeny. These results indicate that an efficient machinery is present in mature spermatozoa, which can transcribe, splice, and reverse-transcribe exogenous DNA molecules. This mechanism is implicated in the genesis and non-Mendelian propagation of new genetic information besides that contained in chromosomes.

344ICSI-MEDIATED GENE TRANSFER SKEWS SEX RATIO AGAINST FEMALE BIRTHS IN MICE

ICSI-mediated gene transfer has been used as an alternative method to pronuclear microinjection for the genomic modification of many species. With this method, transgenic embryos are produced by the microinjection of metaphase II oocytes with spermatozoa previously incubated with foreign DNA. Recently, it was shown in mice that the low percentage of transgenic animals produced from injected oocytes, results from the fact that the expression of foreign DNA is associated with paternal chromosome degradation (Szczygiel M.A. et al., 2003 Biol. Reprod. 68, 1903–1910). It is also known that sex chromosomes localize preferentially, at least in humans, on the periphery of the sperm nucleus on sub-acrosomal regions (Sbracia M. et al., 2002 Hum. Reprod. 17, 320–324), suggesting a high level of interaction with foreign DNA molecules with possible impact on the sex ratio of the offspring. In order to test this hypothesis we have compared ICSI (no DNA), and with ICSI-mediated EGFP (5Kb plasmid DNA from Clonetech, Spain) transfer, with ICSI-mediated YRT3 (a mouse tyrosinase gene derivative YAC-DNA with 100Kb; Montoliu L. et al., 1996 EMBO) transfer. Gametes were from 6–8 weeks old CD1 mice. ICSI-mediated gene transfer with post-thawed immotile spermatozoa, extended in M2 medium in the absence of ion chelators (EDTA and EGTA), was done as previously described (Szczygiel M.A. et al., 2003 Biol. Reprod. 68, 1903–1910). Table 1 below summarizes the data collected. Relative to our control, sex ratio deviation was a consequence of the coinjection of DNA. Forty-three percent of males were obtained with regular ICSI, whereas 64% and 65% were the respective percentages when EGFP or YRT3 DNA was coinjected with spermatozoa. This statistically significant (P&amp;lt;0.05, z-test, Sigma Stat, Jandel Scientific, USA) sex ratio deviation, favoring male ICSI offspring when foreign DNA is coinjected, may result from a higher female embryo susceptibility to parental sex chromosome fragmentation induced by the interaction with foreign DNA molecules. Possible impairment of X chromosome inactivation and dosage compensation resulting from the fragmentation of the sex chromosome on X-carrying spermatozoa could explain this female embryo degeneration. Supporting this view, it was recently shown in mice that sex ratio can be skewed against female births by a mutation in a single gene of the X chromosome (Tsix) involved in such mechanisms (Lee J.T., 2002 Nat. Genet.). In conclusion, mouse ICSI-mediated gene transfer induces sex ratio deviation favoring male offspring. Table 1 Sex ratio of the offspring obtained with ICSI, ICSI-mediated EGFP transfer, and ICSI-mediated YRT3 transfer

Sperm endogenous reverse transcriptase as mediator of new genetic information

Mature spermatozoa spontaneously take up foreign DNA molecules which can be delivered to embryos at fertilization. Recently we discovered an endogenous reverse transcriptase (RT) activity in mouse spermatozoa which can reverse-transcribe exogenous RNA molecules into cDNA copies. Here we have sought to establish whether foreign RNA is a suitable substrate for the sperm RT to generate new functional genes. In vitro fertilization (IVF) experiments were carried out with spermatozoa that were preincubated with RNA from hybrid murine leukemia virus/virus-like 30S (MLV/VL30) β-galactosidase (β-gal) gene-containing vector. The RNA was taken up by sperm cells, reverse-transcribed, delivered to embryos upon IVF, and propagated in a mosaic pattern in founders and further in the F1 progeny. β-gal protein expression was detected in several tissues from both F0 and F1 animals. These results indicate that spermatozoa can reverse-transcribe exogenous RNA so as to generate transcriptionally competent sequences that are transmitted to offspring upon fertilization.

Electroporated sperm mediation of a gene transfer system for finfish and shellfish.

We investigated gene transfer in finfish and shellfish via electroporated sperm. The mobility of sperm, the fertilization rate, the hatching rate, gene transfer rate, and abnormality rate of derived embryos were primarily dependent on the voltage level and concentration of DNA during electroporation. Optimal conditions for sperm of each species of aquatic animals can be reached. Genome of the electroporated sperm was analyzed by PCR, and it was shown that an expected-sized product was amplified, corresponding to that of the transgene's amplification. Southern blotting also showed that a positive band located at the same position as the DNA fragment used for the transfer was found in the electroporated sperm after DNase treatment. When the genome isolated from embryos, larvae, juvenile, and adult individuals, all derived from sperm electroporated with foreign DNA molecules, was analyzed by PCR, the existence of foreign DNA was detected in some samples. The integration of the transferred DNA into the genome of transgenic samples was also shown by Southern blot analysis. There was a mosaic distribution of exogenous DNA in a wide variety of tissues analyzed. In addition to CAT activity being positive for the experimental larvae, the transferred GH gene was functional in transgenic finfish and shellfish and resulted in fast-growing transgenic varieties. The overall evidence strongly suggests that the use of electroporated sperm is the simplest yet most efficient approach to perform mass gene transfer in aquacultural animals, including marine mollusks.

Transformation of maize embryogenic calli mediated by ultrasonication and regeneration of fertile transgenic plants

An insecticidal protein gene isolated fromBacillus thuringiensis was transferred into maize by using ultrasonication. The fertile transgenic plants and their progeny were obtained. The Southern hybridization results indicated that the foreign gene had integrated into the maize genome. It has been found that the acoustic intensity and the duration of treatment are the important parameters influencing transformation efficiency by ultrasonication. The maximum relative transformation frequency of 34.1 % was achieved after 30 min of sonication at 0. 5 W/cm(2) acoustic intensity. With appropriate parameters the ultrasonication can make a number of micropores formed on the cell surface and minimize the treatment damage to the foreign DNA molecules, thus facilitating the DNA molecules to enter the cells.