Differentiation of strains and isolates of chicken reovirus using PCR and heteroduplex mobility assay in amplified cDNA

The method of chicken reovirus strain differentiation was worked out on the basis of RT-PCR and heteroduplex mobility assay (HMA). The S3 gene cDNA (633-896 b.p.) of some Russian and Italian chicken reovirus isolates was amplified by RT-PCR. The analysis of these cDNA samples was carried out by HMA. The relation between nucleotide differences and relative mobility of compared cDNA heteroduplex was reflected by the regression curve. The equation of linear regression was derived (y = 91.726-0.89x; where y is the level of nucleotide difference of compared cDNA (%), x is the relative mobility of compared cDNA heteroduplex (%)). This method made it possible to take correct results within 5-35% of nucleotide difference in heteroduplex sequences.     

The Polymerase η Translesion Synthesis DNA Polymerase Acts Independently of the Mismatch Repair System To Limit Mutagenesis Caused by 7,8-Dihydro-8-Oxoguanine in Yeast

Reactive oxygen species are ubiquitous mutagens that have been linked to both disease and aging. The most studied oxidative lesion is 7,8-dihydro-8-oxoguanine (GO), which is often miscoded during DNA replication, resulting specifically in GC → TA transversions. In yeast, the mismatch repair (MMR) system repairs GO·A mismatches generated during DNA replication, and the polymerase η (Polη) translesion synthesis DNA polymerase additionally promotes error-free bypass of GO lesions. It has been suggested that Polη limits GO-associated mutagenesis exclusively through its participation in the filling of MMR-generated gaps that contain GO lesions. In the experiments reported here, the SUP4-o forward-mutation assay was used to monitor GC → TA mutation rates in strains defective in MMR (Msh2 or Msh6) and/or in Polη activity. The results clearly demonstrate that Polη can function independently of the MMR system to prevent GO-associated mutations, presumably through preferential insertion of cytosine opposite replication-blocking GO lesions. Furthermore, the Polη-dependent bypass of GO lesions is more efficient on the lagging strand of replication and requires an interaction with proliferating cell nuclear antigen. These studies establish a new paradigm for the prevention of GO-associated mutagenesis in eukaryotes.Eukaryotic genome stability can be compromised by changes at the nucleotide level, alterations in chromosome structure, or changes in chromosome number. Although such changes are responsible for many human diseases, including cancer, a low level of instability is necessary to provide the raw material for evolutionary processes. Changes at the nucleotide level generally occur during replication, either as errors made when copying an undamaged DNA template or during the bypass of DNA lesions. Many types of DNA lesions are due to reactive oxygen species (ROS), which are generated by exposure to physical and chemical mutagens, as well as by normal metabolic processes, such as aerobic respiration (12, 32). Although cells contain multiple antioxidants and other proteins that protect the genome from oxidative damage, ROS have been implicated as causal agents of many diseases and of aging (11, 50).The most common oxidized DNA lesion is 7,8-dihydro-8-oxoguanine, which is referred to here as a GO lesion. The mutagenic potential of this lesion is due to miscoding during DNA synthesis, with replicative DNA polymerases usually misinserting adenine across from the lesion to generate GO·A mispairs and ultimately GC → TA transversions (49). Studies examining the crystal structure of T7 DNA polymerase complexed with a GO·C base pair or a GO·A mispair indicate the basis of this mutagenic specificity. Whereas the GO·C structure physically resembles that of a mismatch, the GO·A mispair structurally resembles a normal Watson-Crick base pair and therefore is likely to escape polymerase-associated proofreading activity (6). A GO-containing nucleotide triphosphate (8-oxo-dGTP) can also be used by DNA polymerases during DNA synthesis, leading specifically to AT > CG transversion events (7).There are three major proteins in Escherichia coli that work independently to prevent GO-associated mutagenesis: MutM (Fpg), MutY, and MutT (36). MutM is a DNA glycosylase that removes GO lesions in the GO·C base pairs created by oxidation of guanine in normal G·C base pairs, while MutY is an adenine-DNA glycosylase that removes adenines from the GO·A mispairs created by incorporation of adenine opposite a GO lesion. If DNA replication occurs before MutM can remove the GO lesion from a GO·C base pair, the lesion will likely generate a GO·A mispair, which is then subjected to the A-specific activity of the MutY protein. Once MutY removes the adenine from the newly synthesized strand, a cytosine can be inserted opposite the lesion, giving MutM another opportunity to excise the GO base. MutT is an 8-oxo-dGTPase that degrades 8-oxo-dGTP, thereby greatly reducing its incorporation into DNA. The postreplicative mismatch repair (MMR) pathway has also been implicated in preventing GO-associated mutagenesis in E. coli by functioning as an alternative to MutY or by helping MutY identify and remove mismatched adenines from GO·A mispairs (3, 60).In the yeast Saccharomyces cerevisiae, the Ogg1 protein is the functional homolog of MutM (55) and thus removes GO lesions that are base paired with cytosine. The MMR machinery is functionally analogous to the MutY protein (37), excising adenines that are inserted opposite GO lesions during DNA replication. The mismatch recognition MutSα complex (a heterodimer of the Msh2 and Msh6 proteins) specifically recognizes GO·A mispairs and initiates removal of the portion of the newly synthesized strand containing the adenine (37). A homolog of MutT has yet to be identified in yeast, although one does exist in mammalian cells (23). It is possible that the MutT homolog has eluded discovery either because it is essential, because there is a redundant activity, or because 8-oxo-dGTP is not a significant mutagen in yeast.A third mechanism that limits GO-associated mutagenesis in yeast involves the translesion synthesis (TLS) polymerase, polymerase η (Polη), which is a member of the Y family of DNA polymerases and is encoded by the RAD30 gene (18, 61). Y family polymerases have a large active-site pocket that can accommodate structurally deformed bases, enabling them to insert a nucleotide opposite a lesion (29). Not only is such lesion bypass potentially error prone, the larger active-site pocket of TLS polymerases imparts very low fidelity when copying undamaged DNA. Polη, for example, is error prone when bypassing some lesions, such as abasic sites (17), but has relatively high fidelity when bypassing GO lesions, usually inserting a cytosine across from the lesion (18, 61). At GO lesions, Polη is 10-fold more accurate and efficient than Polδ (34). When given an undamaged DNA template, however, the base substitution error frequency of Polη in vitro is 3 orders of magnitude greater than that of a typical replicative polymerase (35). In addition to Polη, there are two other TLS polymerases in S. cerevisiae (Polζ and Rev1), but neither has been implicated in the bypass of GO lesions (10, 48).The most straightforward way for Polη to be involved in GO bypass would be for it to be recruited when a replicative polymerase stalls or leaves behind a gap. The replication-blocking potential of GO lesions, however, is unclear. Some in vitro studies have shown that replicative DNA polymerases stall or pause when encountering a template GO lesion (18, 47), but other studies have suggested that this is not the case (49). The currently accepted model is that Polη is specifically recruited to fill the gaps generated by MMR when it initiates correction of GO·A mispairs (18, 54). This model of MMR-Polη cooperation in preventing GO-associated mutagenesis is based on epistasis analysis performed using the CAN1 forward mutation assay (18). Although the relationship between msh2 and rad30 was concluded to be epistatic, the data are also consistent with an additive relationship and, hence, potentially independent roles of Msh2 and Polη in limiting GO-associated mutagenesis. How and why the MMR pathway might specifically recruit a generally error-prone polymerase to fill the gaps in what is normally an extremely accurate repair process is not obvious.In the present study, a SUP4-o forward-mutation system was used to reexamine the relationship between MMR and Polη in preventing GO-associated mutagenesis in yeast. To enhance the accumulation of GO lesions, all experiments were conducted in mutants defective in removing GO from GO·C base pairs (an ogg1 background). In addition, both msh2 and msh6 mutants were analyzed. In an msh6 background, loss of the MutSα heterodimer eliminates the correction of GO·A mispairs, while retention of MutSβ (a heterodimer of Msh2 and Msh3) allows continued correction of most insertion-deletion loops. Finally, mutation spectra, as well as mutation rates, were considered in order to focus specifically on GC → TA mutations. The results reported here demonstrate that Polη can function independently of MMR to prevent GO-associated mutagenesis, presumably through its ability to bypass these lesions in an error-free manner. The data further indicate that the Polη-dependent bypass of GO lesions is more efficient on the lagging strand of replication and that it requires interaction with proliferating cell nuclear antigen (PCNA).     

Disease development of Pythium root rot in bulbous Iris and Crocus

Iris bulbs and Crocus corms were planted at two planting dates in sandy soil infested with Pythium spp. At monthly intervals during the growing season root rot infection was assessed over 3 consecutive years and disease development curves were predicted for both crops. The disease development was remarkably different for Iris and Crocus and the curve shape was determined by the crop rather than by the Pythium species. Planting date had a significant effect on disease development in both crops. No correlation was found between disease development and soil temperature.     

Catalytically Inactive Protein Phosphatase 2A Can Bind to Polyomavirus Middle Tumor Antigen and Support Complex Formation with pp60c-src

Interaction between the heterodimeric form of protein phosphatase 2A (PP2A) and polyomavirus middle T antigen (MT) is required for the subsequent assembly of a transformation-competent MT complex. To investigate the role of PP2A catalytic activity in MT complex formation, we undertook a mutational analysis of the PP2A 36-kDa catalytic C subunit. Several residues likely to be involved in the dephosphorylation mechanism were identified and mutated. The resultant catalytically inactive C subunit mutants were then analyzed for their ability to associate with a cellular (B subunit) or a viral (MT) B-type subunit. Strikingly, while all of the inactive mutants were severely impaired in their interaction with B subunit, most of these mutants formed complexes with polyomavirus MT. These findings indicate a potential role for these catalytically important residues in complex formation with cellular B subunit, but not in complex formation with MT. Transformation-competent MT is known to associate with, and modulate the activity of, several cellular proteins, including pp60(c-src) family kinases. To determine whether association of MT with an active PP2A A-C heterodimer is necessary for subsequent association with pp60(c-src), catalytically inactive C subunits were examined for their ability to form complexes containing pp60(c-src) in MT-expressing cells. Two catalytically inactive C subunit mutants that efficiently formed complexes with MT also formed complexes that included an active pp60(c-src) kinase, demonstrating that PP2A activity is not essential in cis in MT complexes for subsequent pp60(c-src) association.     

Use of a crude extract or purified antigen from first‐instar cattle grubs,Hypoderma lineatum,for the detection of anti‐Hypoderma antibodies in free‐ranging cervids from southern Spain

During the 2003–2005 hunting seasons, a total of 120 Cervidae, including 39 red deer (Cervus elaphus hispanicus) and 81 fallow deer (Dama dama), were examined for subcutaneous myiasis. Animals were shot from January to June in southern Spain. Specific antibodies against Hypodermatinae (Diptera: Oestridae) were detected by indirect enzyme‐linked immunosorbent assay (iELISA) using a crude larval extract (CLE) and a purified antigen [hypodermin C (HC)] obtained from first instars of Hypoderma lineatum (De Villers) (Diptera: Oestridae). Hypoderma actaeon Brauer was the only species detected in this study, which represents the first confirmation of this species in fallow deer from Spain. The overall prevalence of animals presenting subcutaneous larvae (14.2%) was considerably lower than the prevalences determined by iELISA with CLE (43.3%) and HC (40.0%). Red deer showed a higher prevalence of Hypoderma than fallow deer. The concordance between larval examination during the hunting season and iELISA using both antigens was low, whereas the concordance between the CLE and HC ELISAs was good. Larval antigens obtained from H. lineatum constitute a good tool for the diagnosis of H. actaeon in Cervidae, especially when the hunting season does not coincide with the maximum presence of larvae on the back.     

What you should know about PR3-ANCA: Evidence for the role of T cells in the pathogenesis of systemic vasculitis

The pathogenesis of systemic vasculitis is complex and is likely to involve many mechanisms. There is a growing body of evidence that T cells may contribute to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Predominantly, T cells and monocytes are found in inflammatory infiltrates in patients with Wegener's granulomatosis (WG). The production of ANCA appears to be T-cell-dependent. T lymphocytes from the peripheral blood of patients with ANCA-associated vasculitis have been shown to proliferate in response to proteinase 3 (PR3). These and other findings outlined in this review indicate T-cell involvement, although further studies are still needed to elucidate the exact contribution of T cells to the pathogenesis of systemic vasculitis.     

Does allometry of a sexually selected ornamental trait vary with sexual selection intensity? A multi‐species test in damselflies

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