"Wu zhu yu", which is obtained from the dried unripe fruits of Tetradium ruticarpum (A. Jussieu) T. G. Hartley, has been used as a traditional Chinese medicine for treatment of headaches, abdominal colic, and hypertension for thousands of years. The present study was designed to assess the molecular genetic diversity among 25 collected accessions of T. ruticarpum (Wu zhu yu in Chinese) from different areas of China, based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers. Thirteen ISSR primers generated 151 amplification bands, of which 130 were polymorphic. Out of 165 bands that were amplified using 10 iPBS primers, 152 were polymorphic. The iPBS markers displayed a higher proportion of polymorphic loci (PPL = 92.5%) than the ISSR markers (PPL = 84.9%). The results showed that T. ruticarpum possessed high loci polymorphism and genetic differentiation occurred in this plant. The combined data of iPBS and ISSR markers scored on 25 accessions produced five clusters that approximately matched the geographic distribution of the species. The results indicated that both iPBS and ISSR markers were reliable and effective tools for analyzing the genetic diversity in T. ruticarpum.
Base Sequence ; Binding Sites ; DNA Fingerprinting ; DNA Primers ; metabolism ; DNA, Plant ; genetics ; isolation & purification ; Evodia ; classification ; genetics ; Genetic Markers ; genetics ; Genetic Variation ; Interspersed Repetitive Sequences ; genetics ; Phylogeny ; Polymorphism, Genetic ; Random Amplified Polymorphic DNA Technique ; Terminal Repeat Sequences ; genetics

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

To obtain the genome sequence of human bocavirus 2 (HBoV2), different regions of HBoV2 genome were amplified through PCR in fecal specimens which had been identified as single-positive for HBoV2 in 2010. A genome sequence of HBoV2 (HBoV2-NC, 5444 bp) was obtained after sequence assembly. The phylogenetic analysis showed that HBoV2-NC had the closest evolutionary relationship with HBoV2 Lanzhou strain. The predication of inverted terminal repeats of HBoV2-NC by DINAMelt showed that inverted terminal repeats were contained in HBoV2-NC 5' terminal, which had the typical stem-loop structure in other parvoviruses. Finally, some flanking sequences of HBoV2-NC were amplified by linker-PCR.
Base Sequence ; Gene Amplification ; Genome, Viral ; Human bocavirus ; chemistry ; classification ; genetics ; Humans ; Molecular Sequence Data ; Nucleic Acid Conformation ; Parvoviridae Infections ; virology ; Phylogeny ; RNA, Viral ; chemistry ; genetics ; 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

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

Cloning of flanking sequences of double-copy gene is a challenge in molecular biology. We developed a method to solve this problem by combining an optimized inverse PCR (iPCR) with TAIL-PCR. First, Southern blotting analysis was used to determine a proper restriction enzyme that could obtain proper-length restriction fragments that contained the target gene. Then optimized iPCR was performed to amplify the restriction fragments that contained the separated copies of the gene. Based on the obtained sequences, TAIL-PCR was performed to amplify further flanking regions of the gene. With this method, we obtained all of the EcoR I restriction fragments (2.2-5.1 kb) and Hind III restriction fragments (8.5-11.7 kb) of mitochondrial atpA gene in cytoplasmic male sterile (CMS) line and maintainer line of Upland cotton. The results showed that this method was an efficient approach to clone flanking sequences of double-copy gene.
Chromosome Walking ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Genes, Mitochondrial ; Genes, Plant ; genetics ; Gossypium ; genetics ; Plant Proteins ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Terminal Repeat Sequences

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

The genomic DNA extracted from chicken embryo fibroblasts (CEF) of SPF chickens from three chicken farms was used as template to amplify the ALV proviral DNA by PCR with four pairs of primers, high positive detection rates of gag - gene (29/46), pol - gene (27/46), env - gene (24/46) and LTR fragment (31/46) were achieved. Eight continuous and overlapping fragments were amplified from one DNA sample with 8 pairs of primers according to published sequences, then cloned into the TA vector and se quenced. The complete sequence of the whole genome of ALV strain SD0501 was established and analyzed with DNAstar software. Comparisons of SD0501 sequence with that of other representative endogenous avian virus strains demonstrated that the genomes of ALV were relatively conservative, the nucleotide identity of all the strains was over 99.1%, and env - gene was over 98.5%. However, a low identity was demonstrated among the representative strains of different subgroups, especially, the env - gene showed obvious difference, the corresponding identity was as low as 56.3% - 91.5%.
Animals ; Avian Leukosis Virus ; genetics ; Base Sequence ; Chick Embryo ; Genome, Viral ; Polymerase Chain Reaction ; Proviruses ; genetics ; Sequence Analysis, DNA ; Specific Pathogen-Free Organisms ; Terminal Repeat Sequences

OBJECTIVETo verify if mutated polyadenylation signal retroviruses can produce viral-host readthrough transcripts (Rth) and have the ability to transform human gastric epithelial GES-1 cells, and to discuss the new functions of retroviruses in gastric cancer related gene research.

METHODSThe polyadenylation signal-deficient retrovirus vector mutated by PCR site-directed mutagenesis was used to make polyadenylation signal-deficient retroviruses by PA317 packaging cells. The GES-1 cells were infected by the viruses and selected by G418. Viral-host readthrough RNAs were checked by Northern blot. The cell growth and soft agar assay were run to test the transformed cells.

RESULTSpolyadenylation signal-deficient retroviruses could be packaged by PA317 packaging cells. The viruses had the ability to infect GES-1 cells. Northern blot analysis of viral RNA from infected pools and individual G418-resistant clones demonstrated that mutation of consensus LTR polyadenylation signals generated Rth viral RNA in the infected GES-1 cells. Phenotypic analysis results showed that the GES-1 cells infected with plyadenylation signal mutant viruses tended to grow in a cluster manner. Pools of PA317 cells infected with mutant viruses were able to form colonies in soft agar with a higher efficiency than control or uninfected cells.

CONCLUSIONHost readthrough transcripts generated by polyadenylation signal mutant viruses may contribute to transformation GES-1 cell phenotypes. The mutant vectors and the method described in the present work may be useful as tools to trap and identify genes involved in retroviral insertion mediated cell transformation.

Animals ; Cell Line ; Cell Transformation, Neoplastic ; Epithelial Cells ; cytology ; metabolism ; virology ; Fibroblasts ; cytology ; virology ; Humans ; Mice ; Mutagenesis, Site-Directed ; RNA 3' Polyadenylation Signals ; genetics ; RNA, Viral ; metabolism ; Retroviridae ; genetics ; Stomach ; cytology ; Terminal Repeat Sequences