Decay-accelerating factor CD55 is identified as the receptor for echovirus 7 using CELICS, a rapid immuno-focal cloning method.

Using an anti-receptor mAb that blocks the attachment of echovirus 7 and related viruses (echoviruses 13, 21, 29 and 33), we have isolated a complementary DNA clone that encodes the human decay-accelerating factor (CD55). Mouse cells transfected with the CD55 clone bind echovirus 7, and this binding is blocked by the anti-receptor mAb. The method used (CELICS) allows rapid and direct cloning of genes encoding cell surface receptors. It is based on episomal replication and high efficiency expression of complementary DNA clones in the vector pCDM8 in COS or WOP cells, in conjunction with a sensitive immuno-focal screen that uses antibody probes linked to beta-galactosidase. Receptor positive cells were identified by a colour change and isolated individually using a micromanipulator. DNA extracted from a small number of cells was then cloned directly in Escherichia coli.     

Substitutions in the pheromone-responsive Gbeta protein of Saccharomyces cerevisiae confer a defect in recovery from pheromone treatment.

The pheromone-responsive Galpha protein of Saccharomyces cerevisiae, Gpa1p, stimulates an adaptive mechanism that downregulates the mating signal. In a genetic screen designed to identify signaling elements required for Gpa1p-mediated adaptation, a large collection of adaptive-defective (Adp-) mutants were recovered. Of the 49 mutants characterized thus far, approximately three-quarters exhibit a dominant defect in the negative regulation of the pheromone response. Eight of the dominant Adp- mutations showed tight linkage to the gene encoding the pheromone-responsive Gbeta, STE4. Sequence analysis of the STE4 locus in the relevant mutant strains revealed seven novel STE4 alleles, each of which was shown to disrupt proper regulation of the pheromone response. Although the STE4 mutations had only minor effects on basal mating pathway activity, the mutant forms of Gbeta dramatically affected the ability of the cell to turn off the mating response after exposure to pheromone. Moreover, the signaling activity of the aberrant Gbetagamma subunits was suppressed by G322E, a mutant form of Gpa1p that blocks the pheromone response by sequestering Gbetagamma, but not by E364K, a hyperadaptive form of Gpa1p. On the basis of these observations, we propose that Gpa1p-mediated adaptation involves the binding of an unknown negative regulator to Gbetagamma.     

Delayed prezygotic isolating mechanisms: evolution with a twist

Assortative mating characterizes the situation wherein reproducing individuals pair according to similarity. Usually, the impetus for this bias is attributed to some type of mate choice conferring benefits (e.g., increased fitness or genetic compatibility) and, thereby, promoting speciation and phenotypic evolution. We investigate, by computer simulation of an evolving deme-structured snail population, the ramifications ensuing from passive assortative mating wherein couples exhibiting opposite shell coil direction phenotypes experience a physical constraint on mating success: putative mating partners inhabiting stout dextral and sinistral shells are unable to exchange sperm. Because shell coil chirality genotype is encoded at a single locus by shell coil alleles that are inherited maternally, snails containing sinistral alleles can present the typical dextral phenotype. Consequently, the incidence of a sinistral allele in as few as one snail can be manifested as prezygotic reproductive isolation within a deme in a subsequent generation. However, because the efficacy of achieving this type of prezygotic reproductive isolation is affected by shell form, the likelihood and product of single-gene speciation should be determined by deme interaction (migration) and composition (morphological distribution). We test this hypothesis and show how stochastic migration interacts with passive assortative mating yielding morphologically induced prezygotic reproductive isolation to produce new species phenotypes. The results show that demes can achieve rapid macroscopic phenotypic transformation and indicate that sympatric speciation might be more plausible than naturalists recognize conventionally.     

Diverse lineages of pathogenic Leptospira species are widespread in the environment in Puerto Rico,USA

BackgroundLeptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide, especially in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources are rare. Furthermore, understanding of environmental Leptospira containing virulence associated genes and possibly capable of causing disease is incomplete, which may convolute leptospirosis diagnosis, prevention, and epidemiology.Methodology/Principal findingsWe collected environmental samples from 22 sites in Puerto Rico during three sampling periods over 14-months (Dec 2018-Feb 2020); 10 water and 10 soil samples were collected at each site. Samples were screened for DNA from potentially pathogenic Leptospira using the lipL32 PCR assay and positive samples were sequenced to assess genetic diversity. One urban site in San Juan was sampled three times over 14 months to assess persistence in soil; live leptospires were obtained during the last sampling period. Isolates were whole genome sequenced and LipL32 expression was assessed in vitro.We detected pathogenic Leptospira DNA at 15/22 sites; both soil and water were positive at 5/15 sites. We recovered lipL32 sequences from 83/86 positive samples (15/15 positive sites) and secY sequences from 32/86 (10/15 sites); multiple genotypes were identified at 12 sites. These sequences revealed significant diversity across samples, including four novel lipL32 phylogenetic clades within the pathogenic P1 group. Most samples from the serially sampled site were lipL32 positive at each time point. We sequenced the genomes of six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species likely belonging to a new serogroup.Conclusions/SignificanceDiverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of these lineages is unknown but several were consistently detected for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira diversity and should improve leptospirosis surveillance and diagnostics.     

The thrombin E192Q-BPTI complex reveals gross structural rearrangements: implications for the interaction with antithrombin and thrombomodulin.

Previous crystal structures of thrombin indicate that the 60-insertion loop is a rigid moiety that partially occludes the active site, suggesting that this structural feature plays a decisive role in restricting thrombin's specificity. This restricted specificity is typified by the experimental observation that thrombin is not inhibited by micromolar concentrations of basic pancreatic trypsin inhibitor (BPTI). Surprisingly, a single atom mutation in thrombin (E192Q) results in a 10(-8) M affinity for BPTI. The crystal structure of human thrombin mutant E192Q has been solved in complex with BPTI at 2.3 A resolution. Binding of the Kunitz inhibitor is accompanied by gross structural rearrangements in thrombin. In particular, thrombin's 60-loop is found in a significantly different conformation. Concomitant reorganization of other surface loops that surround the active site, i.e. the 37-loop, the 148-loop and the 99-loop, is observed. Thrombin can therefore undergo major structural reorganization upon strong ligand binding. Implications for the interaction of thrombin with antithrombin and thrombomodulin are discussed.     

Use of bovine EST data and human genomic sequences to map 100 gene-specific bovine markers

A system to use bovine EST data in conjunction with human genomic sequence to improve the bovine linkage map over the entire genome or on specific chromosomes was evaluated. Bovine EST sequence was used to provide primer sequences corresponding to bovine genes, while human genomic sequence directed primer design to flank introns and produce amplicons of appropriate size for efficient direct sequencing. The sequence tagged sites (STS) produced in this way from the four sires of the MARC reference families were examined for single nucleotide polymorphisms (SNPs) that could be used to map the corresponding genes. With this approach, along with a primer/extension mass spectrometry SNP genotyping assay, 100 ESTs were placed on the bovine genetic linkage map. The first 70 were chosen at random from bovine EST–human genomic comparisons. An additional 30 ESTs were successfully mapped to bovine Chromosome 19 (BTA19), and comparison of the resulting BTA19 map to the position of the corresponding human orthologs on the HSA17 draft sequences revealed differences in the spacing and order of genes. Over 80% of successful amplicons contained SNPs, indicating that this is an efficient approach to generating EST-associated genetic markers. We have demonstrated the feasibility of constructing a linkage map based on SNPs associated with ESTs and the plausibility of utilizing EST, comparative mapping information, and human sequence data to target regions of the bovine genome for SNP marker development.     

An alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi (Lates calcarifer)

Desaturase and elongase are two key enzyme categories in the long-chain polyunsaturated fatty acid (LCPUFA) pathway that convert dietary α-linolenic acid (18:3n-3) to docosahexaenoic acid (22:6n-3). The Δ6 desaturase is considered as rate limiting in the conversion. In a previous study in barramundi we demonstrated that the desaturase had a low Δ6 activity but noted that the enzyme also possessed Δ8 ability that utilised 20-carbon fatty acids. This observation suggests that an alternative pathway may exist in the barramundi via elongases to form 20-carbon metabolites from 18:3n-3 to 20:3n-3 and then Δ6/8 desaturase to 20:4n-3. Cloning of the barramundi elongation of very long-chain fatty acid gene (ELOVL) and heterologous expression of the corresponding elongase were performed to examine activity with regard to time course, substrate concentration and substrate preference. Results revealed that the barramundi elongase showed a broad range of substrate specificity including 18-carbon PUFA (including 18:3n-3 and 18:2n-6), 20- and 22-carbon LCPUFA, with greater activity towards omega-3 (n-3) than n-6 fatty acids. The findings from this study provide molecular evidence for an alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi.