scholarly journals Isolation of Recombinant Adeno-Associated Virus Vector-Cellular DNA Junctions from Mouse Liver

1999 ◽  
Vol 73 (7) ◽  
pp. 5438-5447 ◽  
Author(s):  
Hiroyuki Nakai ◽  
Yuichi Iwaki ◽  
Mark A. Kay ◽  
Linda B. Couto
Keyword(s):  
Portal Vein ◽  
Dna Sequences ◽  
Mouse Liver ◽  
Host Genome ◽  
Vector System ◽  
Rrna Gene ◽  
Raav Vector ◽  
Adeno Associated Virus ◽  
Dna Junctions ◽  
Cellular Dna

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors allow for sustained expression of transgene products from mouse liver following a single portal vein administration. Here a rAAV vector expressing human coagulation factor F.IX (hF.IX), AAV-EF1α-F.IX (hF.IX expression was controlled by the human elongation factor 1α [EF1α] enhancer-promoter) was injected into mice via the portal vein or tail vein, or directly into the liver parenchyma, and the forms of rAAV vector DNA extracted from the liver were analyzed. Southern blot analyses suggested that rAAV vector integrated into the host genome, forming mainly head-to-tail concatemers with occasional deletions of the inverted terminal repeats (ITRs) and their flanking sequences. To further confirm vector integration, we developed a shuttle vector system and isolated and sequenced rAAV vector-cellular DNA junctions from transduced mouse livers. Analysis of 18 junctions revealed various rearrangements, including ITR deletions and amplifications of the vector and cellular DNA sequences. The breakpoints of the vector were mostly located within the ITRs, and cellular DNA sequences were recombined with the vector genome in a nonhomologous manner. Two rAAV-targeted DNA sequences were identified as the mouse rRNA gene and the α1 collagen gene. These observations serve as direct evidence of rAAV integration into the host genome of mouse liver and allow us to begin to elucidate the mechanisms involved in rAAV integration into tissues in vivo.

scholarly journals What Can Electrochemical Methods Offer in Determining DNA–Drug Interactions?

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3478
Author(s):  
Sandra Ramotowska ◽  
Aleksandra Ciesielska ◽  
Mariusz Makowski
Keyword(s):  
Dna Sequences ◽  
Electrode Materials ◽  
High Reliability ◽  
Electrochemical Methods ◽  
Biologically Active ◽  
New Drugs ◽  
Binding Modes ◽  
Voltammetric Techniques ◽  
Experimental Approaches ◽  
Cellular Dna

The interactions of compounds with DNA have been studied since the recognition of the role of nucleic acid in organisms. The design of molecules which specifically interact with DNA sequences allows for the control of the gene expression. Determining the type and strength of such interaction is an indispensable element of pharmaceutical studies. Cognition of the therapeutic action mechanisms is particularly important for designing new drugs. Owing to their sensitivity, simplicity, and low costs, electrochemical methods are increasingly used for this type of research. Compared to other techniques, they require a small number of samples and are characterized by a high reliability. These methods can provide information about the type of interaction and the binding strength, as well as the damage caused by biologically active molecules targeting the cellular DNA. This review paper summarizes the various electrochemical approaches used for the study of the interactions between pharmaceuticals and DNA. The main focus is on the papers from the last decade, with particular attention on the voltammetric techniques. The most preferred experimental approaches, the electrode materials and the new methods of modification are presented. The data on the detection ranges, the binding modes and the binding constant values of pharmaceuticals are summarized. Both the importance of the presented research and the importance of future prospects are discussed.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

Integration of viral DNA sequences in cells transformed by adenovirus 2 or SV40

1980 ◽  
Vol 210 (1180) ◽  
pp. 423-435 ◽  
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Viral Genome ◽  
Viral Dna ◽  
Viral Genomes ◽  
Viral Dnas ◽  
Cellular Dna ◽  
Viral Insertion ◽  
Viral Sequences ◽  
Heteroduplex Formation

We have cloned and propagated in prokaryotic vectors the viral DNA sequences that are integrated in a variety of cells transformed by adenovirus 2 or SV40. Analysis of the clones reveals that the viral DNA sequences sometimes are arranged in a simple fashion, collinear with the viral genome; in other cell lines there are complex arrangements of viral sequences in which tracts of the viral genome are inverted with respect to each other. In several cases the nucleotide sequences at the joints between cell and viral sequences have been determined: usually there is a sharp transition between cellular and viral DNAs. The viral sequences are integrated at different locations within the genomes of different cell lines; likewise there is no specific site on the viral genomes at which integration occurs. Sometimes the viral sequences are integrated within repetitive cellular DNA, and sometimes within unique sequences. In some cases there is evidence that the viral sequences along with the flanking cell DNA have been amplified after integration. The sequences that flank the viral insertion in the line of SV40-transformed rat cells known as 14B have been used as probes to isolate, from untransformed rat cells, clones that carry the region of the chromosome in which integration occurred. Analysis of the structure of these clones by restriction endonuclease digestion and heteroduplex formation shows that a rearrangement of cellular sequences has occurred, presumably as a consequence of integration.

Oncogenes in Laryngeal Cancer: Serial Passage of Transformed Cellular DNA

1985 ◽  
Vol 93 (3) ◽  
pp. 346-350 ◽  
Author(s):  
William H. Friedman ◽  
Barry N. Rosenblum ◽  
Paul Loewenstein ◽  
Helen Thornton ◽  
George Katsantonis ◽  
...  
Keyword(s):  
Laryngeal Cancer ◽  
Dna Sequences ◽  
Squamous Cell Cancer ◽  
Cell Cancer ◽  
Serial Passage ◽  
3T3 Cells ◽  
Human Dna ◽  
Cellular Dna ◽  
Nih 3T3 ◽  
Narrow Bands

DNA originally extracted from squamous cell cancer of the larynx has been serially passaged through transformed populations of NIH/3T3 mouse fibroblasts. The transformed foci were then harvested, cloned to volume, and incubated with a fresh population of NIH/3T3 cells in a second passage. Transforming efficiencies were enhanced by serial passage. In addition, Southern Blot analysis of the transformed foci revealed hybridization between transformant DNA and human probe DNA from the Alu family of conserved human DNA sequences. In the first passage this hybridization took the form of diffuse homology throughout the entire molecular weight distribution. The second-passage DNA showed “narrow bands” indicating the possibility that an oncogene has been identified in laryngeal cancer and that serial passage has eliminated contaminating human sequences. Repetitive transfection in third- and fourth-passage studies is now being completed.

scholarly journals Host Cell DNA Repair Pathways in Adeno-Associated Viral Genome Processing

2006 ◽  
Vol 80 (21) ◽  
pp. 10346-10356 ◽  
Author(s):  
Vivian W. Choi ◽  
Douglas M. McCarty ◽  
R. Jude Samulski
Keyword(s):  
Dna Repair ◽  
Dna Recombination ◽  
Recq Helicase ◽  
Cellular Mechanisms ◽  
Adeno Associated Virus ◽  
Mrn Complex ◽  
Dependent Vector ◽  
Cellular Dna ◽  
Dna Maintenance

ABSTRACT Recentstudies have shown that wild-type and recombinant adeno-associated virus (AAV and rAAV) genomes persist in human tissue predominantly as double-stranded (ds) circular episomes derived from input linear single-stranded virion DNA. Using self-complementary recombinant AAV (scAAV) vectors, we generated intermediates that directly transition to ds circular episomes. The scAAV genome ends are palindromic hairpin-structured terminal repeats, resembling a double-stranded break repair intermediate. Utilizing this substrate, we found cellular DNA recombination and repair factors to be essential for generating circular episomal products. To identify the specific cellular proteins involved, the scAAV circularization-dependent vector was used as a reporter in 19 mammalian DNA repair-deficient cell lines. The results show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient scAAV genome circularization. We further demonstrated that the scAAV genome requires ATM and DNA-PKCS, but not NBS1, to efficiently convert to a circular form in nondividing cells in vivo using transgenic mice. These studies identify specific pathways involved for further elucidating viral and cellular mechanisms of DNA maintenance important to the viral life cycle and vector utilizations.

scholarly journals Adeno-Associated Virus (AAV) Type 5 Rep Protein Cleaves a Unique Terminal Resolution Site Compared with Other AAV Serotypes

1999 ◽  
Vol 73 (5) ◽  
pp. 4293-4298 ◽  
Author(s):  
John A. Chiorini ◽  
Sandra Afione ◽  
Robert M. Kotin
Keyword(s):  
Dna Sequences ◽  
Cleavage Site ◽  
Inverted Terminal Repeat ◽  
Nonstructural Proteins ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Rep Proteins ◽  
A Genome ◽  
Viral Components

ABSTRACT Adeno-associated virus (AAV) replication depends on two viral components for replication: the AAV nonstructural proteins (Rep) intrans, and inverted terminal repeat (ITR) sequences incis. AAV type 5 (AAV5) is a distinct virus compared to the other cloned AAV serotypes. Whereas the Rep proteins and ITRs of other serotypes are interchangeable and can be used to produce recombinant viral particles of a different serotype, AAV5 Rep proteins cannot cross-complement in the packaging of a genome with an AAV2 ITR. In vitro replication assays indicated that the block occurs at the level of replication instead of at viral assembly. AAV2 and AAV5 Rep binding activities demonstrate similar affinities for either an AAV2 or AAV5 ITR; however, comparison of terminal resolution site (TRS) endonuclease activities showed a difference in specificity for the two DNA sequences. AAV2 Rep78 cleaved only a type 2 ITR DNA sequence, and AAV5 Rep78 cleaved only a type 5 probe efficiently. Mapping of the AAV5 ITR TRS identified a distinct cleavage site (AGTG TGGC) which is absent from the ITRs of other AAV serotypes. Comparison of the TRSs in the AAV2 ITR, the AAV5 ITR, and the AAV chromosome 19 integration locus identified some conserved nucleotides downstream of the cleavage site but little homology upstream.

scholarly journals How, when, and where relic DNA biases estimates of microbial diversity

2017 ◽  
Author(s):  
JT Lennon ◽  
ME Muscarella ◽  
SA Muscarella ◽  
BK Lehmkuhl
Keyword(s):  
Microbial Diversity ◽  
Dna Sequences ◽  
Species Abundance ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Soil Sediment ◽  
Species Abundance Distributions ◽  
Abundance And Diversity ◽  
Dna Pool ◽  
Biased Estimates

Extracellular or “relic” DNA is one of the largest pools of nucleic acids in the mbiosphere1,2. Relic DNA can influence a number of important ecological and evolutionary processes, but it may also bias estimates of microbial abundance and diversity, which has implications for understanding environmental, engineered, and host-associated ecosystems. We developed models capturing the fundamental processes that regulate the size and composition of the relic DNA pools to identify scenarios leading to biased estimates of biodiversity. Our models predict that bias increases with relic DNA pool size, but only when the species abundance distributions (SAD) of relic and intact DNA are distinct from one another. We evaluated our model predictions by quantifying relic DNA and assessing its contribution to bacterial diversity using 16S rRNA gene sequences collected from different ecosystem types, including soil, sediment, water, and the mammalian gut. On average, relic DNA made up 33 % of the total bacterial DNA pool, but exceeded 80 % in some samples. Despite its abundance, relic DNA had no effect on estimates of taxonomic and phylogenetic diversity, even in ecosystems where processes such as the physical protection of relic DNA are common and predicted by our models to generate bias. Rather, our findings are consistent with the expectation that relic DNA sequences degrade in proportion to their abundance and therefore may contribute minimally to estimates of microbial diversity.

scholarly journals DNA Palindromes with a Modest Arm Length of ≳20 Base Pairs Are a Significant Target for Recombinant Adeno-Associated Virus Vector Integration in the Liver, Muscles, and Heart in Mice

2007 ◽  
Vol 81 (20) ◽  
pp. 11290-11303 ◽  
Author(s):  
Katsuya Inagaki ◽  
Susanna M. Lewis ◽  
Xiaolin Wu ◽  
Congrong Ma ◽  
David J. Munroe ◽  
...  
Keyword(s):  
Mouse Liver ◽  
Integration Site ◽  
Marker Gene ◽  
Cpg Islands ◽  
Previous Method ◽  
Adeno Associated Virus ◽  
Transcription Start Sites ◽  
Vector Integration ◽  
Integration Sites

ABSTRACT Our previous study has shown that recombinant adeno-associated virus (rAAV) vector integrates preferentially in genes, near transcription start sites and CpG islands in mouse liver (H. Nakai, X. Wu, S. Fuess, T. A. Storm, D. Munroe, E. Montini, S. M. Burgess, M. Grompe, and M. A. Kay, J. Virol. 79:3606-3614, 2005). However, the previous method relied on in vivo selection of rAAV integrants and could be employed for the liver but not for other tissues. Here, we describe a novel method for high-throughput rAAV integration site analysis that does not rely on marker gene expression, selection, or cell division, and therefore it can identify rAAV integration sites in nondividing cells without cell manipulations. Using this new method, we identified and characterized a total of 997 rAAV integration sites in mouse liver, skeletal muscle, and heart, transduced with rAAV2 or rAAV8 vector. The results support our previous observations, but notably they have revealed that DNA palindromes with an arm length of ≳20 bp (total length, ≳40 bp) are a significant target for rAAV integration. Up to ∼30% of total integration events occurred in the vicinity of DNA palindromes with an arm length of ≳20 bp. Considering that DNA palindromes may constitute fragile genomic sites, our results support the notion that rAAV integrates at chromosomal sites susceptible to breakage or preexisting breakage sites. The use of rAAV to label fragile genomic sites may provide an important new tool for probing the intrinsic source of ongoing genomic instability in various tissues in animals, studying DNA palindrome metabolism in vivo, and understanding their possible contributions to carcinogenesis and aging.

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