Characterization of the guinea pig cytomegalovirus genome by molecular cloning and physical mapping.

Fragments of guinea pig cytomegalovirus (GPCMV) DNA produced by HindIII or EcoRI restriction endonuclease digestion were cloned into vectors pBR322 and pACYC184, and recombinant fragments representing ca. 97% of the genome were constructed. Hybridization of 32P-labeled cloned and gel-purified HindIII, EcoRI, and XbaI fragments to Southern blots of HindIII-, EcoRI-, and XbaI-cleaved GPCMV DNA verified the viral origin of cloned fragments and allowed construction of HindIII, EcoRI, and XbaI restriction maps. On the basis of the cloning and mapping experiments, the size of GPCMV DNA was calculated to include 239 kilobase pairs, corresponding to a molecular weight of 158 X 10(6). No cross-hybridization between any internal fragments was seen. We conclude that the GPCMV genome consists of a long unique sequence with terminal repeat sequences but without internal repeat regions. In addition, GPCMV DNA molecules exist in two forms. In the predominant form, the molecules demonstrate sequence homology between the terminal fragments; in the minor population, one terminal fragment is smaller by 0.7 X 10(6) daltons and is not homologous with the fragment at the other end of the physical map. The structural organization of GPCMV DNA is unique for a herpesvirus DNA, similar in its simplicity to the structure reported for murine cytomegalovirus DNA and quite dissimilar from that of human cytomegalovirus DNA.     

Conformational changes in IpaD from Shigella flexneri upon binding bile salts provide insight into the second step of type III secretion

Shigella flexneri uses its type III secretion apparatus (TTSA) to inject host-altering proteins into targeted eukaryotic cells. The TTSA is composed of a basal body and an exposed needle with invasion plasmid antigen D (IpaD) forming a tip complex that controls secretion. The bile salt deoxycholate (DOC) stimulates recruitment of the translocator protein IpaB into the maturing TTSA needle tip complex. This process appears to be triggered by a direct interaction between DOC and IpaD. Fluorescence spectroscopy and NMR spectroscopy are used here to confirm the DOC-IpaD interaction and to reveal that IpaD conformational changes upon DOC binding trigger the appearance of IpaB at the needle tip. Fo?rster resonance energy transfer between specific sites on IpaD was used here to identify changes in distances between IpaD domains as a result of DOC binding. To further explore the effects of DOC binding on IpaD structure, NMR chemical shift mapping was employed. The environments of residues within the proposed DOC binding site and additional residues within the "distal" globular domain were perturbed upon DOC binding, further indicating that conformational changes occur within IpaD upon DOC binding. These events are proposed to be responsible for the recruitment of IpaB at the TTSA needle tip. Mutation analyses combined with additional spectroscopic analyses confirm that conformational changes in IpaD induced by DOC binding contribute to the recruitment of IpaB to the S. flexneri TTSA needle tip. These findings lay the foundation for determining how environmental factors promote TTSA needle tip maturation prior to host cell contact.     

Resolving ancient radiations: can complete plastid gene sets elucidate deep relationships among the tropical gingers (Zingiberales)?

Background and Aims

Zingiberales comprise a clade of eight tropical monocot families including approx. 2500 species and are hypothesized to have undergone an ancient, rapid radiation during the Cretaceous. Zingiberales display substantial variation in floral morphology, and several members are ecologically and economically important. Deep phylogenetic relationships among primary lineages of Zingiberales have proved difficult to resolve in previous studies, representing a key region of uncertainty in the monocot tree of life.

Methods

Next-generation sequencing was used to construct complete plastid gene sets for nine taxa of Zingiberales, which were added to five previously sequenced sets in an attempt to resolve deep relationships among families in the order. Variation in taxon sampling, process partition inclusion and partition model parameters were examined to assess their effects on topology and support.

Key Results

Codon-based likelihood analysis identified a strongly supported clade of ((Cannaceae, Marantaceae), (Costaceae, Zingiberaceae)), sister to (Musaceae, (Lowiaceae, Strelitziaceae)), collectively sister to Heliconiaceae. However, the deepest divergences in this phylogenetic analysis comprised short branches with weak support. Additionally, manipulation of matrices resulted in differing deep topologies in an unpredictable fashion. Alternative topology testing allowed statistical rejection of some of the topologies. Saturation fails to explain observed topological uncertainty and low support at the base of Zingiberales. Evidence for conflict among the plastid data was based on a support metric that accounts for conflicting resampled topologies.

Conclusions

Many relationships were resolved with robust support, but the paucity of character information supporting the deepest nodes and the existence of conflict suggest that plastid coding regions are insufficient to resolve and support the earliest divergences among families of Zingiberales. Whole plastomes will continue to be highly useful in plant phylogenetics, but the current study adds to a growing body of literature suggesting that they may not provide enough character information for resolving ancient, rapid radiations.     

A Mixed Methods Approach to Exploring the Relationship between Norway Rat (Rattus norvegicus) Abundance and Features of the Urban Environment in an Inner-City Neighborhood of Vancouver,Canada

Urban rats (Rattus spp.) are among the most ubiquitous pest species in the world. Previous research has shown that rat abundance is largely determined by features of the environment; however, the specific urban environmental factors that influence rat population density within cities have yet to be clearly identified. Additionally, there are no well described tools or methodologies for conducting an in-depth evaluation of the relationship between urban rat abundance and the environment. In this study, we developed a systematic environmental observation tool using methods borrowed from the field of systematic social observation. This tool, which employed a combination of quantitative and qualitative methodologies, was then used to identify environmental factors associated with the relative abundance of Norway rats (Rattus norvegicus) in an inner-city neighborhood of Vancouver, Canada. Using a multivariate zero-inflated negative binomial model, we found that a variety of factors, including specific land use, building condition, and amount of refuse, were related to rat presence and abundance. Qualitative data largely supported and further clarified observed statistical relationships, but also identified conflicting and unique situations not easily captured through quantitative methods. Overall, the tool helped us to better understand the relationship between features of the urban environment and relative rat abundance within our study area and may useful for studying environmental determinants of zoonotic disease prevalence/distribution among urban rat populations in the future.     

The Mouse MC13 Mutant Is a Novel ENU Mutation in Collagen Type II,Alpha 1

Phenotype-driven mutagenesis experiments are a powerful approach to identifying novel alleles in a variety of contexts. The traditional disadvantage of this approach has been the subsequent task of identifying the affected locus in the mutants of interest. Recent advances in bioinformatics and sequencing have reduced the burden of cloning these ENU mutants. Here we report our experience with an ENU mutagenesis experiment and the rapid identification of a mutation in a previously known gene. A combination of mapping the mutation with a high-density SNP panel and a candidate gene approach has identified a mutation in collagen type II, alpha I (Col2a1). Col2a1 has previously been studied in the mouse and our mutant phenotype closely resembles mutations made in the Col2a1 locus.     

Carriage of Clostridium difficile by Wild Urban Norway Rats (Rattus norvegicus) and Black Rats (Rattus rattus)

Clostridium difficile is an important cause of enteric infections in humans. Recently, concerns have been raised regarding whether animals could be a source of C. difficile spores. Although colonization has been identified in a number of domestic species, the ability of commensal pests to serve as a reservoir for C. difficile has not been well investigated. The objective of this study was to determine whether urban rats (Rattus spp.) from Vancouver, Canada, carry C. difficile. Clostridium difficile was isolated from the colon contents of trapped rats and was characterized using ribotyping, toxinotyping, and toxin gene identification. Generalized linear mixed models and spatial analysis were used to characterize the ecology of C. difficile in rats. Clostridium difficile was isolated from 95 of 724 (13.1%) rats, although prevalence differed from 0% to 46.7% among city blocks. The odds of being C. difficile positive decreased with increasing weight (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.87), suggesting that carriage is more common in younger animals. The strains isolated included 9 ribotypes that matched recognized international designations, 5 identified by our laboratory in previous studies, and 21 “novel” ribotypes. Some strains were clustered geographically; however, the majority were dispersed throughout the study area, supporting environmental sources of exposure and widespread environmental contamination with a variety of C. difficile strains. Given that urban rats are the source of a number of other pathogens responsible for human morbidity and mortality, the potential for rats to be a source of C. difficile for humans deserves further consideration.