Population structure of the Japanese eel, Anguilla japonica

Mitochondrial DNA (mtDNA) sequences that include (a) a part of the cytochrome b gene, (b) two tRNA genes, and (c) a part of the noncoding D-loop region of 31 Anguilla japonica (Japanese eel) and 1 A. marmorata collected from Taiwan, Japan, and mainland China were determined to evaluate the population structure of Japanese eel. Among 30 genotypes identified from the 31 Japanese eel mtDNAs sequenced, there are 58 variable sites, predominantly clustered at the D-loop region. The phylogenetic tree constructed by the unweighted pair-group method with arithmetic mean shows neither significant genealogical branches nor geographic clusters. Furthermore, the sequence-statistics test reveals little, if any, significant genetic differentiation. These results indicate that the 31 Japanese eels might come from a single population. Analysis of sequence variation in mtDNA by using the relationship between the number of segregating sites and the average number of nucleotide differences under the neutral mutation hypothesis reveals that neutral mutation acts as a major factor influencing the evolutionary divergence of the Japanese eel mitochondrial genome sequenced, especially in the noncoding region.      

Molecular Analysis of Geographic Patterns of Eukaryotic Diversity in Antarctic Soils

We describe the application of molecular biological techniques to estimate eukaryotic diversity (primarily fungi, algae, and protists) in Antarctic soils across a latitudinal and environmental gradient between approximately 60 and 87°S. The data were used to (i) test the hypothesis that diversity would decrease with increasing southerly latitude and environmental severity, as is generally claimed for “higher” faunal and plant groups, and (ii) investigate the level of endemicity displayed in different taxonomic groups. Only limited support was obtained for a systematic decrease in diversity with latitude, and then only at the level of a gross comparison between maritime (Antarctic Peninsula/Scotia Arc) and continental Antarctic sites. While the most southerly continental Antarctic site was three to four times less diverse than all maritime sites, there was no evidence for a trend of decreasing diversity across the entire range of the maritime Antarctic (60 to 72°S). Rather, we found the reverse pattern, with highest diversity at sites on Alexander Island (ca. 72°S), at the southern limit of the maritime Antarctic. The very limited overlap found between the eukaryotic biota of the different study sites, combined with their generally low relatedness to existing sequence databases, indicates a high level of Antarctic site isolation and possibly endemicity, a pattern not consistent with similar studies on other continents.     

SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries

High-density single-nucleotide polymorphism (SNP) arrays have revolutionized the ability of genome-wide association studies to detect genomic regions harboring sequence variants that affect complex traits. Extensive numbers of validated SNPs with known allele frequencies are essential to construct genotyping assays with broad utility. We describe an economical, efficient, single-step method for SNP discovery, validation and characterization that uses deep sequencing of reduced representation libraries (RRLs) from specified target populations. Using nearly 50 million sequences generated on an Illumina Genome Analyzer from DNA of 66 cattle representing three populations, we identified 62,042 putative SNPs and predicted their allele frequencies. Genotype data for these 66 individuals validated 92% of 23,357 selected genome-wide SNPs, with a genotypic and sequence allele frequency correlation of r = 0.67. This approach for simultaneous de novo discovery of high-quality SNPs and population characterization of allele frequencies may be applied to any species with at least a partially sequenced genome.     

膝关节挫伤的磁共振影像表现

目的 :探讨磁共振短时的反转恢复序列 (STIR)在膝关节骨挫伤中的临床应用。方法 :通过 32例膝关节外伤病例在常规SE序列、FSE序列和STIR序列中的影像表现 ,分析STIR序列的优越性。结果 :32例共 45个骨挫伤病灶 ,T1W发现 38个 (占 84% ) ,T2W发现 37个 (占 82 % ) ,STIR序列病灶全部显示( 1 0 0 % )。结论 :STIR序列对骨挫伤的敏感性较高 ,能显示微小的骨髓水肿 ,充血及骨小梁的微骨折及其周围的骨软骨、关节囊的细微变化 ,对膝关节外伤具有较高价值。     

Detection of an activating c-kit mutation by real-time PCR in patients with anaphylaxis

A well-characterised gain-of-function point mutation within exon 17 of the c-kit proto-oncogene known as Asp816Val is present in patients with mastocytosis. Activation of mast cells through this receptor primes them for IgE-dependent activation, and patients with mastocytosis are at increased risk of anaphylaxis. We hypothesised that the Asp816Val mutation is associated with a history of anaphylaxis in the general population. A mismatch amplification real-time PCR assay was developed and validated to test for the Asp816Val mutation. Subjects were recruited to four subject groups: normal non-atopics, atopics without anaphylaxis, food-induced anaphylactics and non-food anaphylactics. Blood samples collected from forty subjects were tested for the presence of Asp816Val. Thirteen subjects were found to carry the mutation; normals (2/9), atopics (2/10), food anaphylactics (5/11) and non-food anaphylactics (4/10). Statistical analysis of the data determined that there was no significant difference between the numbers of subjects found to carry the Asp816Val mutation in each of the groups although a trend towards an increased occurrence in anaphylactics was observed. In summary, the hypothesis that the presence of the Asp816Val mutation is linked to the occurrence of anaphylaxis was not supported, but interestingly, we have shown for the first time Asp816Val may occur more frequently than previously reported within the general population.     

Solar UV-B radiation inhibits the growth of Antarctic terrestrial fungi

We tested the effects of solar radiation, and UV-B in particular, on the growth of Antarctic terrestrial fungi. The growth responses to solar radiation of five fungi, Geomyces pannorum, Phoma herbarum, Pythium sp., Verticillium sp., and Mortierella parvispora, each isolated from Antarctic terrestrial habitats, were examined on an agar medium in the natural Antarctic environment. A 3-h exposure to solar radiation of >287 nm reduced the hyphal extension rates of all species relative to controls kept in the dark. Pythium sp. cultures exposed to solar radiation for 1.5 h on five consecutive days were most sensitive to radiation of >287 nm, but radiation of >313 nm also inhibited growth to a lesser extent. Radiation of >400 nm had no effect on hyphal growth relative to controls kept in the dark. Short-wave solar UV-B radiation of between 287 and 305 nm inhibited the growth of Pythium sp. hyphae on and below the surface of the agar medium after 24 h, but radiation of > or =345 nm only reduced the growth of surface hyphae. Similar detrimental effects of UV-B on surface and, to a lesser extent, submerged hyphae of all five fungi were shown in the laboratory by using artificial UV-B from fluorescent lamps. A comparison of growth responses to solar radiation and temperature showed that the species that were most resistant to UV radiation grew fastest at higher temperatures. These data suggest that solar UV-B reduces the growth of fungi on the soil surface in the Antarctic terrestrial environment.     

A novel Antarctic microbial endolithic community within gypsum crusts

A novel endolithic microbial habitat is described from a climatically extreme site at Two Step Cliffs, Alexander Island, Antarctic Peninsula (71 degrees 54'S, 68 degrees 13'W). Small endolithic colonies (<3 mm in diameter) are found within the translucent gypsum crust that forms on the surface of sandstone boulders. Gypsum crusts are found on ice-free rocks throughout the Antarctic and therefore offer potential colonization sites at more inhospitable locations, including sites at higher latitudes. Cyanobacterial, bacterial and fungal components were cultured from the crust material and have been identified as Chloroglea sp., Sphingomonas sp. and Verticillium sp. respectively. A non-cultured, black-pigmented fungus was also found. Cyanobacterial primary productivity is low: at depths of 1.2 and 2.5 mm within the crust, estimates of possible cell divisions per year were < 38 and four respectively. This microniche is proposed to provide protection from desiccation, rapid temperature variation and UV radiation flux while allowing penetration of photosynthetically active radiation (PAR) for utilization by phototrophs. The endolithic communities are less extensive than those of the Dry Valleys, continental Antarctica, probably owing to only recent deglaciation (<7000 year ago).     

Interaction between the IncHI1 plasmid R27 coupling protein and type IV secretion system: TraG associates with the coiled-coil mating pair formation protein TrhB

Assemblies of plasmid-encoded proteins direct the conjugative transfer of plasmid DNA molecules between bacteria. These include the membrane-associated mating pair formation (Mpf) complex necessary for pilus production and the cytoplasmic relaxosome required for DNA processing. The proposed link between these distinct protein complexes is the coupling protein (the TraG family of proteins). Interactions between the coupling protein and relaxosome components have been previously characterized and we document here, for the first time, a direct interaction between the coupling protein and an Mpf protein. Using the adenylate cyclase bacterial two-hybrid (BTH) system, we present in vivo evidence that the IncHI1 plasmid R27-encoded proteins TraG and TrhB interact. This interaction was verified through a co-immunoprecipitation reaction. We have also been able to delineate the interaction domain of TrhB to TraG by showing a positive interaction using the first 220 amino acids of TrhB (452 aa). TrhB has a proline-rich domain from amino acids 135-173 which may serve to facilitate protein interactions and/or periplasmic extension. TrhB self association was detected using far-Western, co-immunoprecipitation, and also BTH analysis, which was used to define the homotypic interaction domain, comprising a predicted coiled-coil region at residues 77-124 of TrhB. These data support a model in which the coupling protein interacts with an Mpf component to target the transferring DNA strand held by the relaxosome to the transmembrane Mpf complex.     

Characterization of the double-partitioning modules of R27: correlating plasmid stability with plasmid localization

Plasmid R27 contains two independent partitioning modules, designated Par1 and Par2, within transfer region 2. Par1 is member of the type I partitioning family (Walker-type ATPase), and Par2 is a member of the type II partitioning family (actin-type ATPase). Stability tests of cloned Par1 and Par2 and insertional disruptions of Par1 and Par2 within R27 demonstrated that Par1 is the major stability determinant whereas Par2 is the minor stability determinant. Creation of double-partitioning mutants resulted in R27 integrating into the chromosome, suggesting that at least one partitioning module is required for R27 to exist in the extrachromosomal form. Using the lacO/LacI-green fluorescent protein (GFP) system, we labeled and visualized R27 and R27 partitioning mutants (Par1(-) and Par2(-)) under different growth conditions in live Escherichia coli cells. Plasmid R27 was visualized as the discrete GFP foci present at the mid- and quarter-cell regions in >99% of the cells. Time lapse experiments demonstrated that an increase in R27 plasmid foci resulted from focus duplication in either the mid- or quarter-cell regions of E. coli. Both R27 Par(-) variants gave a high percentage of plasmidless cells, as suggested by a uniform GFP signal, and cells with GFP patterns scattered throughout the entire cell, suggesting that plasmid molecules are randomly distributed throughout the cytoplasm. Those cells that did contain R27 Par(-) with one or two discrete foci had localization patterns that were statistically different from those formed with wild-type R27. Therefore, these results suggest that partitioning-impaired plasmids are characterized by individual and clustered plasmids that are randomly located within the host cytoplasm.