使用林鹳微卫星引物对东方白鹳基因组DNA进行交叉扩增

利用林鹳11个微卫星位点的引物对东方白鹳进行交叉扩增。经过PCR体系的优化,在11个位点中有6个得到清晰的扩增条带,其余5位点得不到确切的扩增产物。对上述6个位点的扩增产物进行克隆测序分析,发现其中4个位点上的扩增产物含有微卫星重复序列,而另外两个位点中无重复单元。通过基因分型对上述4个微卫星位点进行多态性分析后发现其中的WSμ13,WSμ17位点分别为高度多态和中度多态位点,而另外两个位点则无多态性。同时还对影响交叉扩增结果成功率及微卫星位点多态性的因素进行了分析和总结。     

Characterization of 35 novel microsatellite DNA markers from the duck (Anas platyrhynchos) genome and cross-amplification in other birds

In order to study duck microsatellites, we constructed a library enriched for (CA)n, (CAG)n, (GCC)n and (TTTC)n. A total of 35 pairs of primers from these microsatellites were developed and used to detect polymorphisms in 31 unrelated Peking ducks. Twenty-eight loci were polymorphic and seven loci were monomorphic. A total of 117 alleles were observed from these polymorphic microsatellite markers, which ranged from 2 to 14 with an average of 4.18 per locus. The frequencies of the 117 alleles ranged from 0.02 to 0.98. The highest heterozygosity (0.97) was observed at the CAUD019 microsatellite locus and the lowest heterozygosity (0.04) at the CAUD008 locus, and 11 loci had heterozygosities greater than 0.50 (46.43%). The polymorphism information content (PIC) of 28 loci ranged from 0.04 to 0.88 with an average of 0.42. All the above markers were used to screen the polymorphism in other bird species. Two markers produced specific monomorphic products with the chicken DNA. Fourteen markers generated specific fragments with the goose DNA: 5 were polymorphic and 9 were monomorphic. But no specific product was detected with the peacock DNA. Based on sequence comparisons of the flanking sequence and repeat, we conclude that 2 chicken loci and 14 goose loci were true homologous loci of the duck loci. The microsatellite markers identified and characterized in the present study will contribute to the genetic map, quantitative traits mapping, and phylogenetic analysis in the duck and goose.     

Simultaneously reconstructing viral cross-species transmission history and identifying the underlying constraints

The factors that determine the origin and fate of cross-species transmission events remain unclear for the majority of human pathogens, despite being central for the development of predictive models and assessing the efficacy of prevention strategies. Here, we describe a flexible Bayesian statistical framework to reconstruct virus transmission between different host species based on viral gene sequences, while simultaneously testing and estimating the contribution of several potential predictors of cross-species transmission. Specifically, we use a generalized linear model extension of phylogenetic diffusion to perform Bayesian model averaging over candidate predictors. By further extending this model with branch partitioning, we allow for distinct host transition processes on external and internal branches, thus discriminating between recent cross-species transmissions, many of which are likely to result in dead-end infections, and host shifts that reflect successful onwards transmission in the new host species. Our approach corroborates genetic distance between hosts as a key determinant of both host shifts and cross-species transmissions of rabies virus in North American bats. Furthermore, our results indicate that geographical range overlap is a modest predictor for cross-species transmission, but not for host shifts. Although our evolutionary framework focused on the multi-host reservoir dynamics of bat rabies virus, it is applicable to other pathogens and to other discrete state transition processes.     

Microsatellite size homoplasies and null alleles do not affect species diagnosis and population genetic analysis in a fungal species complex

The suitability of 13 microsatellite loci for species diagnosis and population genetics in 11 species of the Phialocephala fortinii s.l.-Acephala applanata species complex (PAC) was assessed. Two data sets were compared to test possible biases in species typing and clone detection resulting from null alleles and size homoplasies. The first data set was based on fragment lengths derived from a multiplex polymerase chain reaction (PCR) assay and the second data set was received from singleplex PCR at lower stringency and sequencing. Most null alleles observed in the multiplex PCR assay could be amplified during singleplex PCR under less stringent conditions. Size homoplasies resulting from mutations in flanking regions and differences in microsatellite structures were observed. For example, Phialocephala uotolensis possessed a (CT)(13) in addition to the (GT)(x) motif at locus mPF_0644. Despite the occurrence of null alleles and size homoplasies, species diagnosis and population genetic analysis studies were not affected. These markers will facilitate studies on population biology, ecology and biogeography of PAC species.     

Genetic relationships in the peregrine falcon (Falco peregrinus) analysed by microsatellite DNA markers

Microsatellite DNA markers were developed from a peregrine falcon (Falco peregrinus) and genetic relationships among peregrine falcons in southern Norway were analysed using the markers. The genomic DNA library was screened for the presence of dinucleotide microsatellite repeats. Twelve loci revealed polymorphism through the initial analysis of 24 unrelated peregrine falcons, and Mendelian inheritance was confirmed in two peregrine falcon families bred in captivity. The estimated mean probability of identical genotypes in two unrelated individuals was 3 x 10-8, and the combined exclusion probability for parentage testing was 0.99 and 0.94 for one or both parents unknown, respectively. The markers were used to investigate the parentage of peregrine broods from the same nest site from different breeding seasons, and subsequently the nest-site fidelity of the breeding peregrines. High nest-site fidelity was found by studying pairwise comparisons of relatedness (rxy) estimates among chicks at six nest sites from three different breeding seasons. Cross-species amplifications showed that most loci also appeared to amplify polymorphic products in the gyrfalcon (F. rusticolus), merlin (F. columbarius), hobby (F. subbuteo) and kestrel (F. tinnunculus), demonstrating that the loci will provide powerful genetic markers in these falcons too.     

Adaptation of HIV-1 to its human host

Human immunodeficiency virus type 1 (HIV-1) originated from three independent cross-species transmissions of simian immunodeficiency virus (SIVcpzPtt) infecting chimpanzees (Pan troglodytes troglodytes) in west central Africa, giving rise to pandemic (group M) and non-pandemic (groups N and O) clades of HIV-1. To identify host-specific adaptations in HIV-1 we compared the inferred ancestral sequences of HIV-1 groups M, N and O to 12 full length genome sequences of SIVcpzPtt and four of the outlying but closely related SIVcpzPts (from P. t. schweinfurthii). This analysis revealed a single site that was completely conserved among SIVcpzPtt strains but different (due to the same change) in all three groups of HIV-1. This site, Gag-30, lies within p17, the gag-encoded matrix protein. It is Met in SIVcpzPtt, underwent a conservative replacement by Leu in one lineage of SIVcpzPts but changed radically to Arg on all three lineages leading to HIV-1. During subsequent diversification this site has been conserved as a basic residue (Arg or Lys) in most lineages of HIV-1. Retrospective analysis revealed that Gag-30 had reverted to Met in a previous experiment in which HIV-1 was passaged through chimpanzees. To examine whether this substitution conferred a species specific growth advantage, we used site-directed mutagenesis to generate variants of these chimpanzee-adapted HIV-1 strains with Lys at Gag-30, and tested their replication in both human and chimpanzee CD4+ T lymphocytes. Remarkably, viruses encoding Met replicated to higher titers than viruses encoding Lys in chimpanzee T cells, but the opposite was found in human T cells. Taken together, these observations provide compelling evidence for host-specific adaptation during the emergence of HIV-1 and identify the viral matrix protein as a modulator of viral fitness following transmission to the new human host.     

Similarities in Structure and Expression between Mouse P-Cadherin,Chicken B-Cadherin and Frog XB/U-Cadherin

By immunological methods, we show that the monoclonal antibody 6D5 which reacts specifically with Xenopus laevis XB/U-cadherin, also binds to mouse P-cadherin and to chicken B-cadherin but not to the respective E-cadherins (L-CAM) or other “classical” cadherins in these species. In the first extracellular domain, three amino acid residues are identified that are shared by frog XB/U-cadherin, chicken B-cadherin and mammalian P-cadherins but not by the other “classical” cadherins. With few exceptions, the other cadherins possess residues at these positions that are also characteristic of each type of cadherin. Moreover, the expression patterns of P-, B-, and XB/U-cadherin in mouse, chicken and frog are more similar to each other than they are to those of the E-cadherins, L-CAM or other classical cadherins. Taken together, our results suggest that mammalian P-cadherins, chicken B-cadherin and frog XB/U-cadherin are closely related, if not homologous, molecules. A number of differences in the expression patterns between P-, B-, and XB/U-cadherin indicate that these molecules assume differential morphogenetic roles in different species.     

Separating Pharmacokinetic and Pharmacodynamic Components in Empirical Adjustment Factor Distributions

For a particular chemical, one can treat the chemical-by-chemical variation in relative doses for equal toxicity in experimental animals and humans as a characterization of the likelihoods of extrapolation factors of different magnitudes. An emerging approach to noncancer risk assessment is to use such empirical distributions in place of fixed Uncertainty Factors. This paper discusses dividing the overall variation into two sub-distributions representing pharmacokinetic (PK) and pharmacodynamic (PD) contributions to the variation among chemicals in the animal-to-human toxicologically equivalent dose. If a physiologically based pharmacokinetic model (PBPK model) is used to derive a compound specific adjustment factor (CSAF) for the pharmacokinetic component, the deconvolution of the PK and PD components allows one to remove the PK component (to be replaced with the CSAF), while retaining the uncertainty in pharmacodynamics that PBPK models do not address. One must then add back the uncertainty in the PBPK determination of the CSAF (which may be considerable). A candidate criterion for whether one can use an uncertain PBPK model is whether the generic uncertainty about cross-species pharmacokinetics (reflected in the PK component of the overall empirical distribution) is larger than the chemical-specific uncertainty in the determination of kinetically equivalent doses in experimental animals and humans.     

Cross-amplification of microsatellites from the codling moth Cydia pomonella to three other species of the tribe Grapholitini (Lepidoptera: Tortricidae)

This study examined cross-species amplification of 33 microsatellite markers, previously developed for Cydia pomonella, in three related fruit moth species of the same tribe (Grapholitini), namely Grapholita molesta, Grapholita funebrana and Grapholita lobarzewskii. Eight microsatellite loci yielded polymorphic products for G. molesta, nine for G. funebrana and 11 for G. lobarzewskii. At all these loci, the number of alleles ranged between four and 11 in G. molesta, and between four and nine in G. funebrana and G. lobarzewskii each. The successful cross-amplified loci can be used for research on population genetics and gene flow of the three target species.