BACKGROUND: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. METHODS: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. RESULTS: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. CONCLUSION: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors.
Compensation and Redress ; DNA ; Genes, Viral ; HIV Long Terminal Repeat ; HIV* ; HIV-1* ; Humans* ; Ribonucleoproteins ; RNA ; Rodentia* ; TATA Box ; TATA-Box Binding Protein ; Terminal Repeat Sequences ; Trans-Activators ; Transcriptional Activation

BACKGROUND: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. METHODS: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. RESULTS: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. CONCLUSION: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors.
Compensation and Redress ; DNA ; Genes, Viral ; HIV Long Terminal Repeat ; HIV* ; HIV-1* ; Humans* ; Ribonucleoproteins ; RNA ; Rodentia* ; TATA Box ; TATA-Box Binding Protein ; Terminal Repeat Sequences ; Trans-Activators ; Transcriptional Activation

Long terminal repeat (LTR) retrotransposons are mobile DNA sequences that ubiquitously exist in eukaryotic genomes. They replicate themselves in the genome by copy-paste mechanism with RNA as medium. In higher plants, many active LTR retrotransposons have been applied to analyze molecular marker technology, genetic tagging, insertion mutation and gene function. Here, we systematically review the characteristics of plant active LTR retrotransposons, including their structures, copy numbers and distributions. We further analyzed the gag (group-specific antigen) and pol (polymerase) sequence features of different plants active LTR retrotransposons and the distribution patterns of the cis-acting elements in LTR regions. The results show that autonomous active LTR retrotransposons must contain LTR regions and code Gag, Pr, Int, Rt, Rh proteins. Both LTR regions are highly homologous with each other and contain many cis-regulatory elements; RVT and RNase_H1_RT domain are essential for Rt and Rh protein respectively. These results provide the basis for subsequent identification of plant active LTR retrotransposons and their functional analysis.
Genome, Plant ; Mutagenesis, Insertional ; Plants ; genetics ; Retroelements ; Terminal Repeat Sequences

BACKGROUND: Replication of defective adenoviral vectors can be used for gene transfer into a wide spectrum of replicating and nonreplicating cells. Accordingly, selective introduction of genes encoding for susceptibility to nontoxic drugs into proliferating tumor may be used to treat cancer. We investigated the efficacy of in vitro transduction of neuroblastoma cell with the herpes simplex virus thymidine kinase gene followed by administration of the antiviral drug ganciclovir. METHODS: The recombinant adenoviral vector ADV/TK carrying the HSV-TK gene, under the control of the promoter from Rous sarcoma virus long term terminal repeat was constructed. And 1 x 10(4) Neuro 2a cells were plated in 96 well cultured plates and infected with ADV/TK at multiplicity of infection of 0, 1, 10, and 100. Twenty-four hours later, the infected cells were treated with PBS or ganciclovir at a concentration of 10 g/ml. After 48hr, the surviving cells in 96 well plates were determined by MTT assay. RESULTS: After infection in vitro with ADV/TK at moi of 0, 1, 10, 100 and subsequent ganciclovir treatment, the percent survival rate of 1 x 10(4) Neuro 2a cells were 105%, 32%, 25%, and 15%. But the survival rate of 1 x 10(4) Neuro 2a cells with PBS treatment were 100%, 92%, 105%, 103%. CONCLUSION: We concluded that in vitro transduction of neuroblastoma cell with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir is very effective.
Cell Line* ; Ganciclovir* ; Neuroblastoma* ; Phosphotransferases ; Rous sarcoma virus ; Simplexvirus ; Survival Rate ; Terminal Repeat Sequences ; Thymidine Kinase

Transposable elements (TEs), particularly, long terminal repeat retrotransposons (LTR-RTs), are the most abundant DNA components in all plant species that have been investigated, and are largely responsible for plant genome size variation. Although plant genomes have experienced periodic proliferation and/or recent burst of LTR-retrotransposons, the majority of LTR-RTs are inactivated by DNA methylation and small RNA-mediated silencing mechanisms, and/or were deleted/truncated by unequal homologous recombination and illegitimate recombination, as suppression mechanisms that counteract genome expansion caused by LTR-RT amplification. LTR-RT DNA is generally enriched in pericentromeric regions of the host genomes, which appears to be the outcomes of preferential insertions of LTR-RTs in these regions and low effectiveness of selection that purges LTR-RT DNA from these regions relative to chromosomal arms. Potential functions of various TEs in their host genomes remain blurry; nevertheless, LTR-RTs have been recognized to play important roles in maintaining chromatin structures and centromere functions and regulation of gene expressions in their host genomes.
Evolution, Molecular ; Gene Silencing ; Genome, Plant ; genetics ; Plants ; genetics ; Retroelements ; genetics ; Terminal Repeat Sequences ; genetics

AAV-ITR gene expression mini vector is a double-strand or single-strand DNA that only contains inverted terminal repeats of adeno-associated virus, cis-elements and gene of interest and does not contain any other foreign DNA sequences. We prepared Bac-ITR-EGFP and Bac-inrep. Spodoptera frugiperda cells were infected with Bac-ITR-EGFP (P3) and Bac-inrep (P3). Up to 100 μg of AAV-ITR-EGFP gene expression mini vectors were extracted from 2 x 10(7) cells of Sf9 72 h after infection. The gel electrophoresis analysis shows that most forms of AAV-ITR-EGFP gene expression mini vector were monomer and dimer. The mini vector expression efficacy was examined in vitro with HEK 293T cells. The EGFP expression was observed at 24 h after transfection, and the positive ratio reached 65% at 48 h after transfection.
Animals ; Baculoviridae ; DNA, Single-Stranded ; Dependovirus ; Gene Expression ; Genetic Vectors ; HEK293 Cells ; Humans ; Sf9 Cells ; Terminal Repeat Sequences ; Transfection

The pathogenicity of a field isolate of Marek's disease virus (MDV) named GXY2 integrated with retroviral long terminal repeat (LTR) sequence from a chicken with MD tumors was evaluated. Experimental chickens were divided into group A, B, C, D and E. The later four groups were vaccinated on one-day-old with CVI988/Rispens for group B and D, with HVT for group C and E, while group A was taken as no-vaccinated control. On 8-day-old, group A, B and C were challenged with GXY2 by intra-abdominal injection, group D and E were kept as un-challenged control. All the birds were raised routinely until 82 days post-challenge (PC), died birds during the experiment and the slaughtered birds at the end of the experiment were necropsied and examined for gross lesions of MD and further confirmed by a developed polymerase chain reaction (PCR) based differential diagnosis technique for avian neoplastic diseases. The results showed that time of onset of MD death of group A, B and C were PC 25, 77 and 29 days with the incidences of visible MD visceral tumors. On PC 82 days, tumor incidences and mortalities of group A, B and C were 72%, 34.8% and 50%, 84%, 21.7% and 20%, respectively. The vaccination protection of CVI988/Rispense and HVT were 51.67% and 30.56% respectively. Among all the visceral organs, heart had the highest tumor incidences (23.5%), and then followed by liver (14.7%) and gizzard (10.3%). The weight-gain of unvaccinated birds was significantly depressed and severe dystrophy of thymus and bursa of Fabricius were also found. The results of the study demonstrated that isolate GXY2 possessed the ability of causing acute tumors and overcoming the protection of the vaccinations of either CVI988/Rispense or HVT.
Animals ; Chickens ; Mardivirus ; genetics ; pathogenicity ; Marek Disease ; pathology ; virology ; Polymerase Chain Reaction ; Retroviridae ; genetics ; Terminal Repeat Sequences ; genetics

Approximately 45% of the human genome is comprised of transposable elements (TEs). Results from the Human Genome Project have emphasized the biological importance of TEs. Many studies have revealed that TEs are not simply "junk" DNA, but rather, they play various roles in processes, including genome evolution, gene expression regulation, genetic instability, and cancer disposition. The effects of TE insertion in the genome varies from negligible to disease conditions. For the past two decades, many studies have shown that TEs are the causative factors of various genetic disorders and cancer. TEs are a subject of interest worldwide, not only in terms of their clinical aspects but also in basic research, such as evolutionary tracking. Although active TEs contribute to genetic instability and disease states, non-long terminal repeat transposons are well studied, and their roles in these processes have been confirmed. In this review, we will give an overview of the importance of TEs in studying genome evolution and genetic instability, and we suggest that further in-depth studies on the mechanisms related to these phenomena will be useful for both evolutionary tracking and clinical diagnostics.
DNA ; DNA Transposable Elements* ; Gene Expression ; Gene Expression Regulation ; Genome* ; Genome, Human ; Human Genome Project ; Humans ; Terminal Repeat Sequences

Adeno-associated virus (AAV) has many advantages for gene therapy over other vector systems. However, after the production of recombinant AAV (Raav) vectors, the biological titration of rAAV stocks is still cumbersome. Different investigators used laboratory-specific methods or internal reference standards that may limit preclinical and clinical applications. The inverted terminal repeats (ITR) sequences are the only cis-regulated viral elements required for rAAV packaging and remain within viral vector genomes. ITR is the excellent target sequences for qPCR quantification of rAAV titer. In this study, we developed a novel qPCR strategy to quantify rAAVs' vector genome titer via targeting the ITR2 or ITR2-CMV element. In conclusion, the method is fast and accurate for the titration of rAAV2-derived vector genomes. It will promote the standardization of rAAV titration in the future.
Dependovirus ; classification ; genetics ; Genetic Vectors ; genetics ; Genome, Viral ; genetics ; Polymerase Chain Reaction ; methods ; Recombination, Genetic ; Serotyping ; Terminal Repeat Sequences ; genetics ; Transduction, Genetic

OBJECTIVETo evaluate the anti-viral effects of a plasmid expressing an inverted-repeat RNA targeting dengue virus type-2 (DENV-2) pre-membrane (prM) gene.

METHODSuckling mice were inoculated with live DENV-2 in the brain. The total RNA was extracted from the brain tissue of the infected mice, and the prM gene fragments were amplified by RT-PCR and then subcloned into XhoI/EcoR I of the pcDNA3.1(+) plasmid in antisense orientation to construct the plasmid pcDNA-asprM. DENV-2 prM sequences were also subcloned into pMD18-T-vector in sense orientation to construct the plasmid pMD18-T- prM. pcDNA-irRNA was constructed by inserting in sense orientation the prM fragment isolated from pMD18-T-prM into the NheI/Kpn I of pcDNA-asprM. The plasmid pcDNA-irRNA was transfected into BHK-21 cells and the anti-viral effects were analyzed by semi-quantitative PCR and real-time PCR.

RESULTSTransfection with the plasmid pcDNA-irRNA caused a reduction of NS3 mRNA expression level by 28% in BHK-21 cells following a 96-h challenge with DENV-2 as compared to the cells without plasmid transfection (positive control). The viral copies in pcDNA-irRNA-transfected cells was 1.44-fold lower than those in the positive control cells following a 72-h virus challenge, and the mRNA expression levels of NS1 were also significantly lower in the transfected cells at 96 h after viral challenge (P<0.05) as shown by real-time quantitative PCR.

CONCLUSIONThe inverted-repeat RNA for DENV-2 prM gene silencing can suppress DENV-2 replication in BHK-21 cells, which provides a basis for developing dengue virus gene vaccine.

Animals ; Base Sequence ; Cells, Cultured ; Cricetinae ; Dengue Virus ; physiology ; Gene Silencing ; Mice ; Mice, Inbred Strains ; RNA, Viral ; genetics ; Terminal Repeat Sequences ; Viral Envelope Proteins ; genetics ; Virus Replication ; genetics