Conserved ortholog set (COS) markers are evolutionary conserved, single-copy genes, identified from large databases of express sequence tags (ESTs). They are of particular use for constructing syntenic genetic maps among species. In this study, we identified a set of 1,813 putative single-copy COS markers between spruce and loblolly pine, then designed primers for 931 of these markers and tested these primers with DNA from spruce, pine, and Douglas fir. Of these 931 primers, 56% (524) amplified a product in both spruce and pine, and 71% (373) of these were single-banded; 224 amplicons were singlebanded in all three species. Even though these COS markers were selected from large EST databases, a substantial proportion (20-30%) of amplicons displayed multiple bands or smears, suggesting significant paralogy. Sequencing of three single-banded amplicons showed high nucleotide similarities among 29 conifer species, suggesting orthology of single-banded amplicons. Screening for COS marker polymorphism in two pedigrees of white spruce and two pedigrees of loblolly pine revealed an average informativeness of 36% for spruce and 24% for pine (e.g., at least one parent was heterozygous for a single-nucleotide polymorphism within the entire amplified product). This corresponds to an average nucleotide heterozygosity of 0.05% and 0.03%, respectively, which is considerably lower than that found in other studies of spruce and pine. Thus, the advantages of COS markers for constructing syntenic maps are offset by their lower polymorphism.
Abstract:The immunogenicity of a peptide-protein conjugate developed by linking a peptide mimic DRPVPY of the Group A Streptococcus cell-wall polysaccharide (GAS-CWPS), to tetanus toxoid (TT) was examined. BALB/c mice were immunized three times subcutaneously following homologous or heterologous prime/boost strategies at 4-or 6-week intervals in two different experiments. DRPVPY-TT, CWPS-TT, heat-killed, pepsin-treated GAS bacteria (with exposed polysaccharide) and TT, were used as immunogens with alum as adjuvant. Antibody titers were determined by ELISA with GAS bacteria (with exposed polysaccharide) and DRPVPY-linked to bovine serum albumin (BSA, DRPVPY-BSA) as solid phase antigens. All mice primed with DRPVPY-TT developed high IgG antipeptide and anti-GAS titers. The binding of polyclonal anti-peptide antibodies to GAS could be inhibited by purified CWPS, synthetic oligosaccharides corresponding to CWPS, DRPVPY-BSA, DRPVPY and DRPVP, as assessed by competitive-inhibition ELISA. Anti-oligosaccharide titers were also obtained upon titration of anti-peptide sera with synthetic oligosaccharide-BSA conjugates. All mice primed with CWPS-TT and mice primed and boosted with GAS developed IgG anti-peptide titers. These data demonstrate conclusively the cross-reactivity of the immune responses and support the hypothesis of antigenic mimicry of the GAS-CWPS by the hexapeptide DRPVPY. However, mice boosted with DRPVPY-TT, after 6-8 weeks, showed a decrease in IgG anti-GAS titers, but an increase in IgG anti-peptide titers, suggesting carrier-induced suppression of the response to polysaccharide. Strategies are outlined for further refinement of a DRPVPY conjugate as a surrogate of the cell-wall polysaccharide for use in vaccines against Group A Streptococcus.
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