AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region human genome. Understanding integration may aid developing gene therapy systems with predictable sites. Using gel mobility-shift assay, we have identified sequence within locus on chromosome 19 which specifically bound by Rep78 and Rep68 proteins. This Rep recognition GCTC repeating motif very similar sequences inverted terminal repeats genome are also Rep68. Cloned...

The effects of several forms agricultural use native fescue (Festuca scabrella) grasslands upon populations passerine birds were studied in the Hand Hills southern Alberta 1970.Censuses avian carried out on twelve 40-acre (16.2 ha) study plots as well two 10-mi (16.1 km) long roadside routes. vegetation and along routes was described.Native grasslands, undisturbed for 3 years, supported a community consisting Baird's sparrow (Ammodramus bairdii), Sprague's pipit (Anthus spragueii), savannah...

Lung diseases such as cystic fibrosis (CF) might be treated by gene therapy using viral vectors delivered to the airway. One potential vector is defective human parvovirus, adeno-associated virus (AAV). We examined AAV p5 transcription promoter for expression in immortalized cell lines derived from airway (IB3-1) or pancreas (CFPAC-1) of CF patients. expressing prokaryotic genes cat (pAAVp5cat) neo (pAAVp5neo) were evaluated after introduction into IB3-1 CFPAC-1 cells lipofection. In...

Transcriptional activation of the murine Cyp1a-1 (cytochrome P(1)450) gene by inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (dioxin) requires aromatic hydrocarbon (Ah) receptor and interaction an inducer-receptor complex with one or more Ah-responsive elements (AhREs) located about 1 kb upstream from transcriptional initiation site. We find that treatment mouse hepatoma Hepa-1 cells 2-aminopurine, inhibitor protein kinase activity, inhibits CYP1A1 mRNA induction TCDD well...

We have sequenced the human CYP1A2 (cytochrome P3450) gene, 1,906 basepairs (bp) of 5′ flanking region, and 113 bp 3′ region. The gene spans almost 7.8 kilobases, comprising seven exons six introns. transcriptional start site was determined by both primer extension S1 mapping. Including first noncoding exon 55 bp, entire mRNA is 3,121 in length, open reading frame, starting with nucleotide 10 2, encodes 515 amino acids (mol wt = 58,294). Between CYP1A1 P1450) genes, 4, 6, especially 5 are...

Incubation with cucumber phloem exudate in vitro results a dramatic decrease the electrophoretic mobility of Hop stunt viroid. UV cross-linking and combination size exclusion ion exchange chromatography indicate that this phenomenon reflects previously unsuspected ability protein 2, dimeric lectin most abundant component exudate, to interact RNA. In light its demonstrated move from cell via plasmodesmata as well long distances phloem, our suggest 2 may facilitate systemic movement viroids...

To investigate the role of individual structural domains in viroid pathogenicity and replication, a series interspecific chimeras was constructed by exchanging terminal left (TL) and/or (P) between tomato apical stunt (TASVd) citrus exocortis (CEVd) viroids. All six tested were replicated stably tomato, symptoms exhibited infected plants intermediate those induced parental Quantitative comparisons symptom development progeny accumulation revealed that: (i) TL domain TASVd contains...

The primary structures of the tomato planta macho and apical stunt viroids have been determined, probable secondary are proposed. Both can assume rodlike conformation with extensive base-pairing characteristic all known viroids. Sequence homologies between two (75%) members potato spindle tuber viroid group (73-83%) indicate that they both belong to this group. Comparative sequence analysis reveals striking similarities ends transposable genetic elements. These similarities, presence...

The Rep78 and Rep68 proteins of adeno-associated virus (AAV) bind to the AAV terminal repeat hairpin DNA are required for viral replication. We have expressed a series mutant rep genes from human immunodeficiency type 1 long promoter in 293 cells an vitro transcription-translation system. Mutant were analyzed binding resolution functions. Deletion amino acid residues 523 through 621 Rep 78 had no effect on these Amber truncated at either 237 or 243 showed detectable activity. A frameshift...

Replication of the palindromic inverted terminal repeats (ITRs) adeno-associated virus type 2 requires several functions viral nonstructural Rep proteins. These include binding to ITR, nicking double-stranded replication intermediate at resolution site (trs), and then strand displacement synthesis from nick. This report demonstrates ability both recombinant fusion maltose-binding protein (MBP)-Rep68 delta produced in Escherichia coli wild-type (wt) Rep68 bind a linear truncated form 57 with...

Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related AAV DNA replication. Rep78 likely be involved targeted integration of into chromosome 19, full characterization these is important for exploiting this phenomenon use as a vector gene therapy. To obtain sufficient quantities facilitating biochemical properties rep open reading frame was cloned...

The rep gene of adeno-associated virus type 2 (AAV) encodes four overlapping Rep proteins that are involved in regulation and replication the virus. We studied here mRNA transcribed from AAV p5 p19 promoters, using transient expression human 293 cells followed by Northern (RNA) blot analysis mRNA. transcript larger proteins, Rep78 Rep68, while smaller Rep52 Rep40. A plasmid (pNTC3) containing entire genome with an amber mutation accumulated higher levels than a wild-type genome. Addition...

Transcript levels from the P5 promoter of adeno-associated virus type 2 (AAV) are negatively regulated by AAV Rep78 and Rep68 proteins in absence helper virus. We have identified a Rep-responsive negative cis element between TATA box transcription start site using 5' 3' deletions fused to chloramphenicol acetyltransferase gene. This contains four imperfect GAGC repeats similar Rep recognition sequences (RRSs) inverted terminal preferred integration locus chromosome 19. Band shift analyses...

Summary Sporadic outbreaks of potato yellow vein disease (PYVD) were first observed in the early 1940's by growers Antioquia, Colombia. Long known to be transmitted greenhouse whitefly (Trialeurodes vaporariorum) , precise identity its causal agent (presumably viral nature) has remained obscure. Here, we present evidence that a closterovirus with bipartite genome, virus (PYVV), is associated PYVD. Electrophoretic analysis revealed diseased tissue contains 4–5 disease‐specific dsRNAs ranging...

Significant levels of extracellular glutathione (GSH) were detected in aerobically grown cultures some strains Salmonella typhimurium LT-2 and Escherichia coli K-12, B, B/r but not nine freshly isolated clinical isolates E. coli. Cultures S. generally contained less total GSH (intracellular plus external) than did cultures. TA1534 about 2 mM intracellular exported 30% its GSH. The external concentration increased logarithmically during exponential growth peaked at 24 microM...

The aphid Myzus persicae (Sulz.) was shown to transmit potato spindle tuber viroid (PSTVd) clone DTO-33 from source plants doubly infected with leafroll virus (PLRV) and PSTVd. Transmission of the persistent type did not occur when insects were allowed feed on singly plants. Only low levels PSTVd associated purified PLRV virions, but its resistance digestion micrococcal nuclease indicates that RNA is encapsidated within particles. Epidemiological surveys carried out at three locations in...

The adeno-associated virus type 2 (AAV) Rep68 protein produced in Escherichia coli as a fusion with maltose-binding (MBP-Rep68 delta) has previously been shown to possess DNA-DNA helicase activity, does the purified wild-type Rep68. In present study, we demonstrate that MBP-Rep68 delta also catalyzes unwinding of DNA-RNA hybrid. delta-mediated activity required ATP hydrolysis and presence Mg2+ ions was inhibited by high ionic strength. efficiency comparable its activity. However, lacked...

The adeno-associated virus type 2 (AAV) Rep78 and Rep68 proteins are required for viral replication. These encoded by unspliced spliced transcripts, respectively, from the p5 promoter of AAV therefore have overlapping amino acid sequences. share a variety activities including endonuclease, helicase, ATPase ability to bind hairpin DNA. part sequence which is identical in contains consensus helicase motifs that conserved among parvovirus replication proteins. In present study, we mutated...

Potato spindle tuber viroid can be separated into two fractions by polyacrylamide gelelectrophoresis in the presence of formamide and urea. One fraction contains predominantly circular molecules; second almost exclusively linear molecules. The purity was estimated electron microscopy formaldehyde-denatured length distributions denatured molecules were determined. Both found to infectious.

The replication of many viral and subviral pathogens as well the amplification certain cellular genes proceeds via a rolling circle mechanism. For potato spindle tuber (PSTVd) related viroids, possible role circular (-)strand RNA template for synthesis (+)strand progeny is unclear. Infected plants appear to contain only multimeric linear RNAs, attempts initiate infection with (-)PSTVd RNAs generally have failed. To examine critically infectivity monomeric viroid we developed ribozyme-based...

A viroid has been isolated from tomato plants affected by Indian bunchy top disease of (Lycopersicon esculentum Mill.). In dot blot hybridization assays with 32P-labelled cRNA probes specific for the detection various viroids, viriod was shown to be most closely related citrus exocortis (CEVd). Sequence determination showed that consists 372 nucleotides and confirmed its close relationship CEVd. The viroid, which we propose acronym CEVd-t, differs Australian CEVd strains B 36 47 nucleotides,...