Host Genomic DNA Research Articles

Foamy virus biology and its application for vector development.

Spuma- or foamy viruses (FV), endemic in most non-human primates, cats, cattle and horses, comprise a special type of retrovirus that has developed a replication strategy combining features of both retroviruses and hepadnaviruses. Unique features of FVs include an apparent apathogenicity in natural hosts as well as zoonotically infected humans, a reverse transcription of the packaged viral RNA genome late during viral replication resulting in an infectious DNA genome in released FV particles and a special particle release strategy depending capsid and glycoprotein coexpression and specific interaction between both components. In addition, particular features with respect to the integration profile into the host genomic DNA discriminate FV from orthoretroviruses. It appears that some inherent properties of FV vectors set them favorably apart from orthoretroviral vectors and ask for additional basic research on the viruses as well as on the application in Gene Therapy. This review will summarize the current knowledge of FV biology and the development as a gene transfer system.

In Vitro HIV-1 Selective Integration into the Target Sequence and Decoy-Effect of the Modified Sequence

Although there have been a few reports that the HIV-1 genome can be selectively integrated into the genomic DNA of cultured host cell, the biochemistry of integration selectivity has not been fully understood. We modified the in vitro integration reaction protocol and developed a reaction system with higher efficiency. We used a substrate repeat, 5′-(GTCCCTTCCCAGT )n(ACTG GGAAGGGAC)n-3′, and a modified sequence DNA ligated into a circular plasmid. CAGT and ACTG (shown in italics in the above sequence) in the repeat units originated from the HIV-1 proviral genome ends. Following the incubation of the HIV-1 genome end cDNA and recombinant integrase for the formation of the pre-integration (PI) complex, substrate DNA was reacted with this complex. It was confirmed that the integration selectively occurred in the middle segment of the repeat sequence. In addition, integration frequency and selectivity were positively correlated with repeat number n. On the other hand, both frequency and selectivity decreased markedly when using sequences with deletion of CAGT in the middle position of the original target sequence. Moreover, on incubation with the deleted DNAs and original sequence, the integration efficiency and selectivity for the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary discontinuously with changes in manganese dichloride concentration in the reaction buffer, probably due to its influence on the secondary structure of substrate DNA. Finally, integrase was found to form oligomers on the binding site and substrate DNA formed a loop-like structure. In conclusion, there is a considerable selectivity in HIV-integration into the specified sequence; however, similar DNA sequences can interfere with the integration process, and it is therefore difficult for in vivo integration to occur selectively in the actual host genome DNA.

Identification of HIV integration sites in infected host genomic DNA

The integration of the Human Immunodeficiency Virus (HIV) genetic information into the host genome is fundamental for its replication and long-term persistence in the host. Isolating and characterizing the integration sites can be useful for obtaining data such as identifying the specific genomic location of integration or understanding the forces dictating HIV integration site selection. The methods outlined in this article describe a highly efficient and precise technique for identifying HIV integration sites in the host genome on a small scale using molecular cloning techniques and standard sequencing or on a massive scale using 454 pyrosequencing.

The technology of large-scale pharmaceutical plasmid purification by cetyltrimethylammonium bromide and Tritonx-114

Many methods for plasmid purification have been developed, and the whole process must be designed to remove the host RNA, protein, genomic DNA and endotoxin. Currently, plasmid is mostly purified by time-consuming chromatographies. As an alternative, a new plasmid purification technology with cetyltrimethylammonium bromide (CTAB) is described. After lysis with alkali, the CTAB was directly titrated into the supernatant for plasmid precipitation, then the coprecipitated pellets were dealt with 3 M KAc and TritonX-114. Quality detection showed that the purified plasmids were free from the contamination of host RNA. In 1 mg purified plasmid, the bacterial genomic DNA, host endotoxin and protein were less than 10 μg/ mg, 50 EU/ mg and 10 μg/mg, respectively. The ratio of OD260/OD280 was between 1.75 - 1.85, more than 90% of the prepared plasmid presented in the supercoiled form. Further test demonstrated that the pcDNAlacZ purified with CTAB and authoritative endotoxin-free plasmid Kit had the similar transfection efficiency in vivoand in vitro. CTAB can be used for plasmid purification; the main advantages of the DNAs purified with CTAB include the avoidance of animal-derived enzymes, toxic substance like chloroform and phenol. More attractive is that the whole process has the predominance of low cost. Key words: Plasmid, CTAB, TritonX-114, genomic DNA, RNA, endotoxin, protein, transfection.

Endogenous bornavirus elements in mammalian genome

Approximately 8% of our genome is made up of endogenous retroviral elements. Endogenous retrovirus is a fossil record of ancient retrovirus infection and, therefore, gives important insights into the evolutional relationship between retroviruses and their hosts. On the other hand, until recently, it has been believed that no endogenous non-retroviral viruses exist in animal genomes. We lately discovered endogenous elements homologous to the nucleoprotein of bornaviruses, a negative-strand RNA virus, in the genomes of many mammalian species, including humans. We also demonstrated that mRNA of extant mammalian bornavirus, Borna disease virus, is reverse-transcribed and integrated into the host genome DNA. These findings provided novel insights not only into the interaction between RNA viruses and their hosts, but also into the mechanism underlying the gain of novelty in mammalian genomes. In this review, we will briefly summarize our recent knowledge about endogenous bornavirus elements and also introduce some recent discoveries regarding endogenous elements of non-retroviral viruses in vertebrate genomes.

Development and in‐house validation of the event‐specific qualitative and quantitative PCR detection methods for genetically modified cotton MON15985

To implement genetically modified organism (GMO) labeling regulations, an event-specific analysis method based on the junction sequence between exogenous integration and host genomic DNA has become the preferential approach for GMO identification and quantification. In this study, specific primers and TaqMan probes based on the revealed 5'-end junction sequence of GM cotton MON15985 were designed, and qualitative and quantitative polymerase chain reaction (PCR) assays were established employing the designed primers and probes. In the qualitative PCR assay, the limit of detection (LOD) was 0.5 g kg(-1) in 100 ng total cotton genomic DNA, corresponding to about 17 copies of haploid cotton genomic DNA, and the LOD and limit of quantification (LOQ) for quantitative PCR assay were 10 and 17 copies of haploid cotton genomic DNA, respectively. Furthermore, the developed quantitative PCR assays were validated in-house by five different researchers. Also, five practical samples with known GM contents were quantified using the developed PCR assay in in-house validation, and the bias between the true and quantification values ranged from 2.06% to 12.59%. This study shows that the developed qualitative and quantitative PCR methods are applicable for the identification and quantification of GM cotton MON15985 and its derivates.

Resistance to novel drug classes

Understanding the mechanisms that underlie resistance development to novel drugs is essential to a better clinical management of resistant viruses and to prevent further resistance development and spread. Integrase inhibitors and CCR5 antagonists are the more recent antiretroviral classes developed. The HIV-1 integrase, responsible for the chromosomal integration of the newly synthesized double-stranded viral DNA into the host genomic DNA, represents a new and important target; and two integrase inhibitors (INIs), raltegravir and elvitegravir, have been shown promising results in clinical trials. Viral entry is also an attractive step for the development of new drugs against HIV variants resistant to current antiretroviral drugs, and two CCR5 antagonists have been designed to inhibit HIV-1 binding to R5 co-receptor and are under clinical investigation. Drug resistance to INIs occurs through the selection of mutations within HIV integrase. The kinetic of selection seems rapid and one mutation alone is able to confer resistance to integrase inhibitor, suggesting that this class of drug has a low genetic barrier. Two ways could explain the failure of the CCR5 antagonist class: a rapid outgrowth of pre-existing archived X4 virus or the selection of a resistance to CCR5 antagonists through amino acid changes in V3 loop.

Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries

To prepare a clinical-grade anti-caries DNA vaccine pGJA-P/VAX and explore its immune effect and protective efficacy against a cariogenic bacterial challenge. A large-scale industrial production process was developed under Good Manufacturing Practices (GMP) by combining and optimizing common unit operations such as alkaline lysis, precipitation, endotoxin removal and column chromatography. Quality controls of the purified bulk and final lyophilized vaccine were conducted according to authoritative guidelines. Mice and gnotobiotic rats were intranasally immunized with clinical-grade pGJA-P/VAX with chitosan. Antibody levels of serum IgG and salivary SIgA were assessed by an enzyme-linked immunosorbent assay (ELISA), and caries activity was evaluated by the Keyes method. pGJA-P/VAX and pVAX1 prepared by a laboratory-scale commercial kit were used as controls. The production process proved to be scalable and reproducible. Impurities including host protein, residual RNA, genomic DNA and endotoxin in the purified plasmid were all under the limits of set specifications. Intranasal vaccination with clinical-grade pGJA-P/VAX induced higher serum IgG and salivary SIgA in both mice and gnotobiotic rats. While in the experimental caries model, the enamel (E), dentinal slight (Ds), and dentinal moderate (Dm) caries lesions were reduced by 21.1%, 33.0%, and 40.9%, respectively. The production process under GMP was efficient in preparing clinical-grade pGJA-P/VAX with high purity and intended effectiveness, thus facilitating future clinical trials for the anti-caries DNA vaccine.

Characterization of the Exogenous Insert and Development of Event-specific PCR Detection Methods for Genetically Modified Huanong No. 1 Papaya

Genetically modified (GM) papaya (Carica papaya L.), Huanong No. 1, was approved for commercialization in Guangdong province, China in 2006, and the development of the Huanong No. 1 papaya detection method is necessary for implementing genetically modified organism (GMO) labeling regulations. In this study, we reported the characterization of the exogenous integration of GM Huanong No. 1 papaya by means of conventional polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR strategies. The results suggested that one intact copy of the initial construction was integrated in the papaya genome and which probably resulted in one deletion (38 bp in size) of the host genomic DNA. Also, one unintended insertion of a 92 bp truncated NptII fragment was observed at the 5' end of the exogenous insert. Furthermore, we revealed its 5' and 3' flanking sequences between the insert DNA and the papaya genomic DNA, and developed the event-specific qualitative and quantitative PCR assays for GM Huanong No. 1 papaya based on the 5' integration flanking sequence. The relative limit of detection (LOD) of the qualitative PCR assay was about 0.01% in 100 ng of total papaya genomic DNA, corresponding to about 25 copies of papaya haploid genome. In the quantitative PCR, the limits of detection and quantification (LOD and LOQ) were as low as 12.5 and 25 copies of papaya haploid genome, respectively. In practical sample quantification, the quantified biases between the test and true values of three samples ranged from 0.44% to 4.41%. Collectively, we proposed that all of these results are useful for the identification and quantification of Huanong No. 1 papaya and its derivates.

Inhibition of porcine endogenous retrovirus (PERV) replication by HIV-1 gene expression inhibitors

Porcine endogenous retrovirus (PERV) is persistently integrated into the host genomic DNA as a provirus and released from a variety of porcine cells. PERV infects a certain range of human cells, which is a major concern in xenotransplantation. Therefore, the use of viral gene expression inhibitors could be envisaged, if they reduce PERV production from porcine organs and minimize viral transmission to human recipients. In the present study, four HIV-1 gene expression inhibitors were examined for their inhibitory effect on PERV replication in porcine cells constitutively producing the virus. Among the compounds, the fluoroquinolone derivative K-37 and the bacterial product EM2487 displayed potent and selective inhibition of PERV replication in the cells mediated by the suppression of viral mRNA synthesis. Thus, retroviral gene expression inhibitors may be able to reduce the risk of PERV transmission.

The adsorption ofPseudomonas aeruginosabacteriophage φKMV is dependent on expression regulation of type IV pili genes

Pseudomonas aeruginosa bacteriophage phiKMV requires type IV pili for infection, as observed from the phenotypic characterization and phage adsorption assays on a phage infection-resistant host strain mutant. A cosmid clone library of the host (P. aeruginosa PAO1) genomic DNA was generated and used to select for a clone that was able to restore phiKMV infection in the resistant mutant. This complementing cosmid also re-established type IV pili-dependent twitching motility. The correlation between bacteriophage phiKMV infectivity and type IV pili, along with its associated twitching motility, was confirmed by the resistance of a P. aeruginosa PAO1DeltapilA mutant to the phage. Subcloning of the complementing cosmid and further phage infection analysis and motility assays suggests that a common regulatory mechanism and/or interaction between the ponA and pilMNOPQ gene products are essential for bacteriophage phiKMV infectivity.

Dicer Cuts the Kidney

We should occasionally stop, take a breath, and reflect about what is happening at the interface between medicine and science. Collectively, we are uncovering paradigms that are relevant to understanding disease at a frantic pace. One can only turn to wonder, in this moment of reflection, when

Development of a molecular marker for specific detection of Fusarium oxysporum f. sp. cubense race 4

Fusarium oxysporum f. sp. cubense is the causal agent of Panama disease of banana. A rapid and reliable diagnosis is the foundation of integrated disease management practices in commodity crops. For this diagnostic purpose, we have developed a reliable molecular method to detect Foc race 4 isolates in Taiwan. By PCR amplification, the primer set Foc-1/Foc-2 derived from the sequence of a random primer OP-A02 amplified fragment produced a 242 bp size DNA fragment which was specific to Foc race 4. With the optimized PCR parameters, the molecular method was sensitive and could detect small quantities of Foc DNA as low as 10 pg in 50 to 2,000 ng host genomic DNA with high efficiency. We also demonstrated that by using our PCR assay with Foc-1/Foc-2 primer set, Foc race 4 could be easily distinguished from other Foc races 1 and 2, and separated other formae speciales of F. oxysporum.

Characterization of temperate phage Che12 and construction of a new tool for diagnosis of tuberculosis

A temperate phage, Che12, able to infect Mycobacterium tuberculosis, was isolated from soil samples taken from tuberculosis sanatorium area in Chennai, India. The plaque morphology of this phage showed varying grades of turbidity on lawns of M. tuberculosis. The temperate nature of Che12 was established by super infection immunity. Phage integration into the host genomic DNA was confirmed by Southern hybridization using Che12 DNA as a probe. PCR amplification and sequencing of a part of the integrated phage genome in a M. tuberculosis lysogen also confirmed the temperate nature of Che12. The morphology of the phage particles was observed by electron microscopy, revealing similarities to other mycobacteriophages like L5, D29 and TM4. A luciferase reporter phage, phAETRC16, was constructed by cloning firefly luciferase gene into Che12. Infection of viable M. tuberculosis cells by phAETRC16 resulted in expression of luciferase leading to sustained light output. Che12, a true temperate phage infecting M. tuberculosis, is thus ideally suited for developing a diagnostic tool facilitating rapid diagnosis of M. tuberculosis.

The Temperate Marine Phage ΦHAP-1 of Halomonas aquamarina Possesses a Linear Plasmid-Like Prophage Genome

A myovirus-like temperate phage, PhiHAP-1, was induced with mitomycin C from a Halomonas aquamarina strain isolated from surface waters in the Gulf of Mexico. The induced cultures produced significantly more virus-like particles (VLPs) (3.73 x 10(10) VLP ml(-1)) than control cultures (3.83 x 10(7) VLP ml(-1)) when observed with epifluorescence microscopy. The induced phage was sequenced by using linker-amplified shotgun libraries and contained a genome 39,245 nucleotides in length with a G+C content of 59%. The PhiHAP-1 genome contained 46 putative open reading frames (ORFs), with 76% sharing significant similarity (E value of <10(-3)) at the protein level with other sequences in GenBank. Putative functional gene assignments included small and large terminase subunits, capsid and tail genes, an N6-DNA adenine methyltransferase, and lysogeny-related genes. Although no integrase was found, the PhiHAP-1 genome contained ORFs similar to protelomerase and parA genes found in linear plasmid-like phages with telomeric ends. Southern probing and PCR analysis of host genomic, plasmid, and PhiHAP-1 DNA indicated a lack of integration of the prophage with the host chromosome and a difference in genome arrangement between the prophage and virion forms. The linear plasmid prophage form of PhiHAP-1 begins with the protelomerase gene, presumably due to the activity of the protelomerase, while the induced phage particle has a circularly permuted genome that begins with the terminase genes. The PhiHAP-1 genome shares synteny and gene similarity with coliphage N15 and vibriophages VP882 and VHML, suggesting an evolutionary heritage from an N15-like linear plasmid prophage ancestor.

Plasmid purification by hydrophobic interaction chromatography using sodium citrate in the mobile phase

The most significant impurities in plasmid DNA (pDNA)-containing solutions after intermediate recovery are host RNA and genomic DNA. Hydrophobic interaction chromatography (HIC) with ammonium sulfate-based buffers has been used to explore the higher hydrophobicity of those impurities when compared with pDNA. Although successful at lab scale, the large amounts of ammonium sulfate involved constitute an important limitation in terms of waste treatment/disposal if HIC is to be used at process scales. In this work, different HIC ligands (phenyl, butyl, octyl), salts and salt concentrations were screened with the specific goal of replacing ammonium sulfate with an environmentally friendly salt. The combination of phenyl-Sepharose with sodium citrate at high concentrations (≥1.2 M) not only allowed the separation of pDNA from RNA, gDNA, endotoxins and protein, but also enabled the separation of supercoiled from open circle isoforms. Alternatively, by exploring negative HIC with the phenyl matrix and 1.0 M sodium citrate, it was possible to purify total pDNA from solutions obtained by tangential flow filtration and pre-conditioned with sodium citrate. At the highest loading tested (2 mL feed equivalent to ca. 1 mg pDNA per mL of bed) despite high-level removal of endotoxins (>98.5%) and proteins (>82%) the purity of recovered plasmid DNA determined by HIC-HPLC was just 73% (primarily due to traces of RNA and gDNA). Much higher purities (close to 100%) were achieved when lower loadings were employed. In summary, this study clearly shows that despite lower purification factors achieved with sodium citrate compared to ammonium sulfate-based HIC, replacing the latter salt with sodium citrate is indeed feasible.

Femtosecond laser: a new intradermal DNA delivery method for efficient, long‐term gene expression and genetic immunization

A femtosecond laser beam gene transduction (SG-LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 microg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg-specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNgamma, and IL-4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG-LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV-infected replicating cells. Tumor growth retardation was induced in vaccinated mice challenged with an HBsAg-expressing syngeneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.

Germline mutagenesis mediated by Sleeping Beauty transposon system in mice

Following the descovery of its transposition activity in mammalian culture systems, the Sleeping Beauty (SB) transposon has since been applied to achieve germline mutagenesis in mice. Initially, the transposition efficiency was found to be low in cultured systems, but its activity in germ cells was unexpectedly high. This difference in transposition efficiency was found to be largely dependent on chromosomal status of the host genomic DNA and transposon vector design. The SB transposon system has been found to be suitable for comprehensive mutagenesis in mice. Therefore, it is an effective tool as a forward genetics screen for tagged insertional mutagenesis in mice.

Effect of salt on purification of plasmid DNA using size-exclusion chromatography

In the present study, we compared the performances of size-exclusion chromatography for the purification of plasmid DNA when different concentrations (0.5 M, 1 M, 2 M, respectively) of two types of salt (NaCl and (NH 4) 2SO 4) are present in running buffers. Our experiment results displayed that it is not only the resolution of RNA but also those of supercoiled plasmid DNA and host's genomic DNA were increased greatly in the presence of high concentration of water-structure salt. We deduce that two separation modes may be involved in the process: The supercoiled plasmid DNA is influenced mainly by compaction effect and eluted in the size-exclusion mode; whereas, RNA and genomic DNA are influenced mainly by hydrophobic effect due to their stretched and loose structures and eluted in the interaction mode. This method led to an improved efficiency of size-exclusion chromatography.

Are phytoplankton population density maxima predictable through analysis of host and viral genomic DNA content?

Phytoplankton:virus interactions are important factors in aquatic nutrient cycling and community succession. The number of viral progeny resulting from an infection of a cell critically influences the propagation of infection and concomitantly the dynamics of phytoplankton populations. Host nucleotide content may be the resource limiting viral particle assembly. We present evidence for a strong linear correlation between measured viral burst sizes and viral burst sizes predicted from the host DNA content divided by the viral genome size, across a diversity of phytoplankton:viral pairs. An analysis of genome sizes therefore supports predictions of taxon-specific phytoplankton population density thresholds beyond which viral proliferation can trim populations or terminate phytoplankton blooms. We present corollaries showing that host:virus interactions may place evolutionary pressure towards genome reduction of both phytoplankton hosts and their viruses.