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PubMed2001

Specific nucleoprotein complexes within adenovirus capsids.

Adenoviral DNA was examined within capsids by dimethyl sulfate footprinting. Protein-DNA interactions were visualized through ligation-mediated PCR (LM-PCR). Signals for protein binding were found adjacent to both inverted terminal repeats (ITR). There were no indications of close protein binding at several other loci of the viral genome. Therefore, adenovirus type 5 seems to contain sequence- or locus-specific DNA binding proteins within the virion.

Adenoviridae↗
PubMed2005

Molecular characterization and chromosomal distribution of Galileo, Kepler and Newton, three foldback transposable elements of the Drosophila buzzatii species complex.

Galileo is a foldback transposable element that has been implicated in the generation of two polymorphic chromosomal inversions in Drosophila buzzatii. Analysis of the inversion breakpoints led to the discovery of two additional elements, called Kepler and Newton, sharing sequence and structural similarities with Galileo. Here, we describe in detail the molecular structure of these three elements, on the basis of the 13 copies found at the inversion breakpoints plus 10 additional copies isolated during this work. Similarly to the foldback elements described in other organisms, these elements have long inverted terminal repeats, which in the case of Galileo possess a complex structure and display a high degree of internal variability between copies. A phylogenetic tree built with their shared sequences shows that the three elements are closely related and diverged approximately 10 million years ago. We have also analyzed the abundance and chromosomal distribution of these elements in D. buzzatii and other species of the repleta group by Southern analysis and in situ hybridization. Overall, the results suggest that these foldback elements are present in all the buzzatti complex species and may have played an important role in shaping their genomes. In addition, we show that recombination rate is the main factor determining the chromosomal distribution of these elements.

Animals↗
PubMed2006

Natural history of transposition in the green alga Chlamydomonas reinhardtii: use of the AMT4 locus as an experimental system.

The AMT4 locus of the green alga Chlamydomonas reinhardtii, which we mapped to the long arm of chromosome 8, provides a good experimental system for the study of transposition. Most mutations that confer resistance to the toxic ammonium analog methylammonium are in AMT4 and a high proportion of spontaneous mutations are caused by transposon-related events. Among the 15 such events that we have characterized at the molecular level, 9 were associated with insertions of the retrotransposon TOC1, 2 with a small Gulliver-related transposon, and 1 with the Tcr1 transposon. We found that Tcr1 is apparently a foldback transposon with terminal inverted repeats that are much longer and more complex than previously realized. A duplication of Tcr1 yielded a configuration thought to be important for chromosomal evolution. Other mutations in AMT4 were caused by two mobile elements that have not been described before. The sequence of one, which we propose to call the Bill element, indicates that it probably transposes by way of a DNA intermediate and requires functions that it does not encode. The sequence of the other and bioinformatic analysis indicates that it derives from a miniature retrotransposon or TRIM, which we propose to call MRC1 (miniature retrotransposon of Chlamydomonas).

Animals↗
PubMed2006

Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition.

The Tol2 element is a naturally occurring active transposable element found in vertebrate genomes. The Tol2 transposon system has been shown to be active from fish to mammals and considered to be a useful gene transfer vector in vertebrates. However, cis-sequences essential for transposition have not been characterized. Here we report the characterization of the minimal cis-sequence of the Tol2 element. We constructed Tol2 vectors containing various lengths of DNA from both the left (5') and the right (3') ends and tested their transpositional activities both by the transient excision assay using zebrafish embryos and by analyzing chromosomal transposition in the zebrafish germ lineage. We demonstrated that Tol2 vectors with 200 bp from the left end and 150 bp from the right end were capable of transposition without reducing the transpositional efficiency and found that these sequences, including the terminal inverted repeats (TIRs) and the subterminal regions, are sufficient and required for transposition. The left and right ends were not interchangeable. The Tol2 vector carrying an insert of >11 kb could transpose, but a certain length of spacer, <276 but >18 bp, between the left and right ends was necessary for excision. Furthermore, we found that a 5-bp sequence, 5'-(A/G)AGTA-3', is repeated 33 times in the essential subterminal region. Mutations in the repeat sequence at 13 different sites in the subterminal region, as well as mutations in TIRs, severely reduced the excision activity, indicating that they play important roles in transposition. The identification of the minimal cis-sequence of the Tol2 element and the construction of mini-Tol2 vectors will facilitate development of useful transposon tools in vertebrates. Also, our study established a basis for further biochemical and molecular biological studies for understanding roles of the repetitive sequence in the subterminal region in transposition.

Animals↗
PubMed2006

Recombinant avian adeno-associated virus: transgene expression in vivo and enhancement of expression in vitro.

Recombinant avian adeno-associated viruses coding for the LacZ gene were used to inoculate embryonating chicken eggs, to assess the usefulness of the system for the expression of a transgene in vivo. The results obtained indicate significantly higher levels of expression of the reporter gene at various time intervals in the embryos inoculated with the recombinant virus in comparison with the mock-inoculated controls. At the embryo level, significant differences were evident at 120 hr postinoculation; hatched chicks showed transgene expression up to 14 days of age. In a second experiment, different cell-line cultures were transfected with plasmids encoding for a reporter gene flanked by the avian adeno-associated virus inverted terminal repeats (ITR), either alone or in the presence of the major nonstructural proteins of the virus (Rep 78/68) to assess the ability of these proteins and DNA elements to enhance gene expression. Results indicate that the inclusion of the viral ITR alone or during coexpression of the Rep proteins significantly enhances the expression of the transgene in all cell lines tested, as evidenced by the detection of the beta-galacrosidase protein through chemiluminescence reactions and staining of transfected monolayers.

Animals↗
PubMed2002

Transposase-mediated construction of an integrated adeno-associated virus type 5 helper plasmid.

Adeno-associated viruses (AAVs) are replication-defective parvoviruses that require helper virusfunctionsfor efficient productive replication. The AAVs are currently premier candidates as vectors for human gene therapy applications. In particular; much recent interest has been expressed concerning recombinant AAV serotype 5 (rAAV-5) vectors, as they appear to utilize cellular receptors distinctfrom those of the prototypical AAV serotype (AAV-2) and have been reported to have transduction properties in vivo that differ significantly from those of the prototype. One of the most popular current methodsfor the production of rAAVs involves co-transfection of human 293 cells with three plasmids: (i) an adenovirus (Ad)-derived helper plasmid containing Ad genes required for AAV replication, (ii) an AAV-derived plasmid encoding complementing AAV genes (ie., the viral rep and cap genes), and (iii) a target plasmid containing a transgene of interestflanked by AAV inverted terminal repeats (ITRs) that confer packaging and replication capabilities upon the ITR-flanked heterologous DNA. Here we describe novel plasmid reagents designed for convenient and efficient production of rAAV-S. An integrated helper plasmid containing all Ad genes requiredfor the efficient production of recombinant AAV as well as the complementing AAV genes on the same plasmid backbone, was constructed via transposase-mediated insertion into an Ad helper plasmid of a transposable element containing the AAV-5 rep and cap genes linked to a selectable marker This simple strategy can be used in the rapid and efficient construction of integrated helper plasmids derived from any reported AAV serotype for which a molecular clone exists.

Animals↗
PubMed2006

Chimeric herpes simplex virus/adeno-associated virus amplicon vectors.

Chimeric or hybrid herpes simplex virus type 1/adeno-associated virus amplicon vectors combine the large transgene capacity of HSV-1 with the potential for site-specific genomic integration and stable transgene expression of AAV. These chimeric vectors have been demonstrated to support transgene expression for significantly longer periods than standard HSV-1 amplicons. Moreover, HSV/AAV hybrid vectors can mediate integration at the AAVS1 pre-integration site on human chromosome 19 at a relatively high rate, although random integration has also been observed. One major remaining hurdle of HSV/AAV hybrid vectors is the low packaging efficiency and titers when AAV rep sequences are included in the amplicon vector. In the conditions prevalent during the replication/packaging of HSV/AAV hybrid amplicons into HSV-1 virions, in particular the presence of HSV-1 replication factors and AAV Rep protein, at least three different viral origins of DNA replication are active: the HSV-1 ori, the AAV inverted terminal repeats (ITRs), and the p5 promoter/ori driving expression of the AAV rep gene. A detailed understanding of the properties of these origins of DNA replication and the molecular mechanisms of interactions between them, may allow designing novel hybrid vectors that allow the efficient and precise integration of large transgenes in the human genome.

Dependovirus↗
PubMed1991

Nucleotide sequence of Rhizobium meliloti insertion sequence ISRm1: homology to IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens.

Nucleotide sequencing of Rhizobium meliloti insertion sequence ISRm1 showed that it is 1319 nucleotides long and includes 32/31 nucleotide terminal inverted repeats. Analysis of five different insertion sites using sequencing primers complementary to sequences within the left and right ends demonstrated that ISRm1 generates five bp direct repeats at the sites of insertion. Although ISRm1 has shown a target preference for certain short regions (hot spots), there was no apparent similarity in the DNA sequences near the insertion sites. On one strand ISRm1 contains two contiguous open reading frames (ORFs) spanning most of its length. ISRm1 was found to have over 50% sequence homology to insertion sequences IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens. Their sizes, the sequences of their inverted repeats, and the characteristics of their insertion sites are also comparable, indicating that ISRm1, IS2 and IS426 belong to a class of related insertion sequences. Comparison of the proteins potentially encoded by these insertion sequences showed that the two ORFs found in ISRm1 are also present in IS2 and IS426, suggesting that they may be functional genes.

Agrobacterium tumefaciens↗
PubMed1995

Sequence of a transposon identified as Tn1000 (gamma delta).

We report the complete sequence of a transposon found in a cosmid clone of a human DNA sequence. The transposon is identified as the Escherichia coli transposon Tn1000 (also known as gamma delta) on the basis of the identity of the restriction map of the new sequence with that previously recorded for Tn1000 and homology between parts of the new sequence and that of published fragments of Tn1000 sequence. The transposon, which comprises 5,981 nucleotides including two 35 bp inverted terminal repeat sequences (ITRs), contains three open reading frames. The sequence of the resolvase coding region (tnpR) is identical to that published by others. A second reading frame can be identified as the tnpA gene, coding for the transposase, on the grounds of its strong homology with the corresponding gene from transposon Tn3. The third reading frame has the potential to code for a protein of unknown function containing 698 amino acids.

Amino Acid Sequence↗
PubMed1998

Sites of recombinant adeno-associated virus integration.

Adeno-associated virus (AAV), a defective parvovirus, is considered a promising vector for the delivery of therapeutic genes to cells. Both wild-type and recombinant AAV display a wide tropism and integrate into the host genome, in the absence of helper virus, establishing a latent infection. A unique characteristic of wild-type AAV and a potential advantage for use as a delivery system for gene therapy is the site-specific integration of wild-type virus within a small region of chromosome 19, 19q13.3-qter (AAVS1), in up to 85% of cell lines infected with the virus. Although recombinant AAVs, containing only the inverted terminal repeats of wild-type virus, can integrate efficiently into the host genome, specificity for the AAVS1 site appears to be lost. To address this question, the integration characteristics of two recombinant AAVs lacking the rep and cap genes in HeLa cells were examined. Analysis of Southern blots indicated that none of twenty-six cell clones generated after infection with either one of the recombinant AAVs demonstrated integration within the AAVS1 locus on chromo-some 19. Analysis of five of the cell lines by fluorescent chromosome in situ hybridization confirmed the loss of chromosome 19 specificity. Each integration site mapped near a known fragile site and/or location of a proto-oncogene or growth regulatory gene. Retention of site-specific integration of wild-type AAV will require the inclusion of additional AAV-specific sequences within the recombinant vectors.

Dependovirus↗
PubMed1999

Molecular paleontology of transposable elements from Arabidopsis thaliana.

We report results of a comprehensive computer-assisted analysis of new transposable elements (TEs) from Arabidopsis thaliana. Our analysis revealed several previously unknown pogo- and En/Spm-like families and two novel superfamilies of DNA transposons, Arnold and Harbinger. One of the En/Spm-like families (Atenspm) was found to be involved in generating satellite arrays in paracentromeric regions. Of the two superfamilies reported, Harbinger is distantly related to bacterial IS5-like insertion elements, and Arnold contains DNA transposons without terminal inverted repeats (TIRs), which were never reported in eukaryotes before. Furthermore, we report a large number of young and diverse copia-like autonomous and nonautonomous retroelements and discuss their potential evolutionary relationship with mammalian retroviruses. The A. thaliana genome harbors copia-like retroelements which encode a putative env-like protein reported previously in the SIRE-1 retrotransposon from soybean. Finally, we demonstrate a nonrandom chromosomal distribution of the most abundant A. thaliana TEs clustered in the first half of chromosome II, which includes the centromeric region. The families of TEs from A. thaliana are relatively young, extremely diverse and much smaller than those from mammalian genomes. We discuss the potential factors determining similarities and differences in the evolution of TEs in mammals and A. thaliana.

Amino Acid Sequence↗
PubMed2000

Complete sequence and organization of Periplaneta fuliginosa densovirus genome.

The replicative form (RF) of the Periplaneta fuliginosa densovirus (PfDNV) genome was cloned and its complete nucleotide sequence was determined. The PfDNV genome is 5454 nucleotides (nt) in length with distal 201-nt long inverted terminal repeats (ITRs). The first 122 nt at the 5'-end and the terminal 122 nt at the 3'-end of both strands are palindromes with identical sequences, of which each can fold into a typical U-shaped hairpin structure. The coding regions of PfDNV genome are evenly distributed in the 5'-halves of both strands. PfDNV genome contains seven major open reading frames (ORFs), four of which on the plus DNA strand may encode non-structural (NS) proteins, while the others on the minus DNA strand may encode structural proteins. Two potential functional promoters (P3 and P97) were found within the ITRs on both strands. The ORF2 polypeptide contains a highly conserved NTP-binding domain of NS proteins of parvoviruses. The ORF5 polypeptide has significant homology to the conserved PGY region of coat proteins of parvoviruses. The ORF6 polypeptide has distinct homology to the structural polypeptides of insect parvoviruses. The organization of PfDNV genome was compared to those of other parvoviruses.

Amino Acid Sequence↗
PubMed1998

[Expression of secreted ligand domain of vaccinia growth factor encoded by vaccinia virus strain tian tan in E. coli and insect cells].

Vaccinia virus growth factor (VGF) is encoded by an early gene of vaccinia virus strain TianTan located near the border between the terminal inverted repeats and the internal region of the viral genome as one copy. Its relationship to members of epidermal growth factor superfamily was discovered when computer searches of protein sequences revealed that VGF has strong sequence homology with these growth factors. To further get insight into the biological activity of VGF, we cloned and expressed the secreted ligand domain of VGF (VGFsl) encoded by vaccinia virus strain TianTan in E. coli and insect cells. The results of biological activity test indicated that expressed products is capable of binding to the EGF receptor, and stimulating EGF receptor tyrosin autophosphorylation to a level greater than that of EGF.

Animals↗
PubMed1999

[Cloning of 0-17.5 mu and sequencing of 0-4.8 mu of the lefe and DNA of the Ad7 vaccine strain left end].

OBJECTIVE: Cloning of 0-17.5 mu DNA fragment of adenovirus 7 vaccine strain and sequencing of 0-4.8 mu fragment (1,737 bp). METHODS: Isolating and purifying Ad7 vaccine genome from the A549 cultured cells, putting 0.3-17.5 mu fragment into pAd7T plasmid, then sequencing the 0-4.8 mu fragment including inverted terminal repeats (ITR), packaging sequence and Ela region. RESULTS: We obtained 0-17.5 mu fragment of Ad7 vaccine strain genome and sequenced its left terminal 1,737 bp. Sequence analysis showed that the Ad7 vaccine strain Ela region encodes 6,300, 24,000 and 28,000 proteins. Compared with equivalent region of Ad7 Gomen strain, they share the homology of the nucleotide sequence 98.9%, 97.3%, 97.5% and the homology of the deduced amino acid sequence 96.6%, 96.5%, and 96.9% respectively. When compared with Ad7 Grider strain, they share the homology of the nucleotide sequence 100%, 99.7%, 99.7% and the homology of the deduced amino acid sequence 100%, 99.1%, and 99.2% respectively. CONCLUSION: The Ad7 vaccine strain left terminal 1,737 bp nucleotide sequence showed a high homology with corresponding region of Ad7 Gomen strain and Grider strain.

Adenoviridae↗
PubMed2003

Platelet-derived growth factor-producing cells immortalized from rat mesencephalon with SV40 large T antigen transduced by an AAV vector.

PURPOSE: Adeno-associated virus (AAV) can infect a wide variety of mammalian cell types and is capable of infecting both dividing and non-dividing cell populations. Here we report the construction of a recombinant AAV vector which expresses the SV40 large T protein (AAV-T) and the use of this vector to immortalize primary cells from embryonic rat mesencephalon. METHODS: The AAV-T vector was constructed by introducing the BamH1 fragment of the pCMV/SVE/Neo plasmid containing T antigen and SV40 regulatory elements into the JM48 plasmid containing the inverted terminal repeats of AAV. Neuronal cultures from E-12 rat mesencephalon were grown in defined media supplemented with basic fibroblast growth factor. These cells were infected with the AAV-T vector. RESULTS: A cell line (designated RMAT) and six subclones were established from these cultures through multiple passages. This cell line was immunoreactive for SV40 large T antigen and the cytoskeletal proteins nestin and vimentin. Morphological differentiation and expression of neurofilament 160 kDa were induced by exposure to dibutyrl cyclic AMP. Immunoassays performed to measure endogenous production of growth factors showed that RMAT cells produced high levels of platelet-derived growth factor (PDGF). CONCLUSIONS: AAV may be a useful vector for the transduction of oncogenes to produce cell lines.

1-Methyl-3-isobutylxanthine↗
PubMed2003

Construction of a recombinant vector based on AAV carrying human endothelial nitric-oxide synthase gene.

AIM: To construct an AAV based vector carrying human endothelial nitric-oxide synthase (eNOS) cDNA and study its expression in vitro for future gene therapy. METHODS: eNOS cDNA was inserted into the EcoR I site of pSNAV-1 containing the cytomegalovirus (CMV) promoter and inverted terminal repeat sequences of adeno-associated virus. The constructed vector was transfected into BHK and C2C12 cells. eNOS cDNA and mRNA were detected by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), respectively. RESULTS: By restriction enzyme digestion analysis, it was proved that eNOS cDNA was inserted into pSNAV-1 in a proper direction. PCR detection demonstrated that pSNAV-eNOS was transferred into both BHK and C2C12 cells. RT-PCR analysis showed that these pSNAV-eNOS transfected cells could express eNOS mRNA. CONCLUSION: pSNAV-eNOS was successfully constructed with the ability to express human eNOS mRNA in cultured mammalian cells.

Animals↗
PubMedDate not supplied

[A densovirus of German cockroach Blatella germanica: detection, nucleotide sequence and genome organization].

A new Blattella germanica densovirus (BgDNV, Parvoviridae: Densovirinae, Densovirus) was found. Virus DNA and cockroach tissues infected with BgDNV were examined by electron microscopy. Virus particles about 20 nm in diameter were observed both in the nucleus and in the cytoplasm of infected cells. Virus DNA proved to be a linear molecule sized about 1.2 microns. The complete BgDNV genome was sequenced and analyzed. Five ORF were detected: two coded for structural capsid proteins and were on one DNA strand, and three coded for regulatory proteins and were on the other strand. Potential promoters and polyadenylation signals were identified. Structural analysis was performed for terminal inverted repeats containing extended palindromes. The genome structure of BgDNV was compared with that of other Parvoviridae.

Amino Acid Sequence↗
PubMedDate not supplied

[Search for unique segments of the ectromelia virus genome by cross blot hybridization with DNA from other orthopoxviruses].

In order to identify ectromelia virus (EMV) genome regions which may contain genes responsible for the specific pathogenicity of this virus, blot cross-hybridization of EMV DNA with those of other orthopoxviruses was performed. Two hybridization schemes were employed: one of them included hybridization of labelled cloned fragments of EMV with digests of other viral DNAs, the other, reciprocal, consisted in hybridization of labelled total DNAs of various orthopoxviruses with digests of the region of EMV DNA adjacent to the right-terminal inverted repeat. It was demonstrated that the counterpart to an approximately 8-kilobase pair portion of EMV genome flanking the inverted repeat could be detected only in the cowpox virus genome but not in the genomes of vaccinia and rabbitpox viruses. XhoI-O and XhoI-K fragments of EMV DNA contained, along with genes found in other poxviruses, certain genes which appeared to be unique for EMV. It is postulated that some of these genes may determine the specific biological properties of EMV, including its pathogenicity for mice.

DNA, Viral↗