Estimation of the number of SNP genetic markers required for parentage verification

Formulae were developed to compute exclusion probabilities for parentage confirmation for any number of diallelic markers under the assumption that the minor allele frequency (MAF) varied among markers, but has a uniform distribution. Three scenarios were analysed: a progeny with (1) a single putative parent; (2) two putative parents; and (3) one actual parent and one putative parent. Exclusion probabilities were computed for minimum values for the MAFs of 0.1, 0.2 and 0.3, and required either one or at least two conflicts for exclusion. The numbers of markers required to obtain 99% exclusion probabilities based on a single conflict for the three minimum MAFs were 54, 45 and 39 for scenario 1; 17, 16 and 15 for scenario 2; and 28, 25 and 24 for scenario 3. The requirement of at least two conflicts for exclusion increased the number of markers required by approximately 45% for all three scenarios and all three minimum MAFs. The results obtained by the analytical formulae were very close to results obtained by simulation and to values in the literature for specific marker sets.     

Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli

Bacteria use type III secretion systems (TTSS) to translocate effector proteins into host cells. Better understanding of the TTSS and its effectors' functions will require assays to measure their activities in vivo and in real time. We designed a real-time, high-throughput translocation assay that utilizes fusions of effector genes to the beta-lactamase reporter gene, positioned under the effector's native promoter and chromosomal location. Using this assay, we simultaneously and quantitatively analyzed the translocation kinetics of six core enteropathogenic E. coli effectors, EspF, EspG, EspH, EspZ, Map, and Tir. A distinct order in the efficiencies of effector translocation was observed. Translocation efficiency was determined by multiple factors, including the intrabacterial effector concentration, effector-chaperone interactions, the efficiency of bacterial attachment to the host cells, and possibly also by a translocation autoinhibition mechanism. The described real-time translocation assay could be easily adapted for varied applications in the study of bacterial pathogenesis.     

Identification of the endostyle as a stem cell niche in a colonial chordate

Stem cell populations exist in "niches" that hold them and regulate their fate decisions. Identification and characterization of these niches is essential for understanding stem cell maintenance and tissue regeneration. Here we report on the identification of a novel stem cell niche in Botryllus schlosseri, a colonial urochordate with high stem cell-mediated developmental activities. Using in vivo cell labeling, engraftment, confocal microscopy, and time-lapse imaging, we have identified cells with stemness capabilities in the anterior ventral region of the Botryllus' endostyle. These cells proliferate and migrate to regenerating organs in developing buds and buds of chimeric partners but do not contribute to the germ line. When cells are transplanted from the endostyle region, they contribute to tissue development and induce long-term chimerism in allogeneic tissues. In contrast, cells from other Botryllus' regions do not show comparable stemness capabilities. Cumulatively, these results define the Botryllus' endostyle region as an adult somatic stem cell niche.     

Pattern of settlement and natural chimerism in the colonial urochordate Botryllus schlosseri

Colonies of the cosmopolitan urochordate Botryllus schlosseri that share one or both alleles at a single allorecognition locus (Fu/HC) and come into tissue contacts, may fuse and form a mixed entity, a chimera. Botryllus populations worldwide exhibit unprecedented extensive polymorphism at this locus, a result that restricts fusions to kin encounters. This study aims to compare spatiotemporal configurations in source and introduced B. schlosseri populations, residing on natural and man-made substrata, respectively. By using four microsatellite loci, we tested genetic consanguinity of colonies settled naturally along spatial vectors on both, natural (native populations) and man-made (introduced) substrates. Four populations were studied. Results revealed that B. schlosseri colonies, on both substrate types, assemble in groups of relatives that share similar microsatellite profiles. We suggest that this pattern of settlement promotes the formation of chimeras, which evoke conflicting interactions: cooperation between different somatic cell lines that constitute the colonial soma and competition between germ cells that inhabit the chimera gonads. Under natural conditions, the chimera may allow genetic flexibility that depends on joint genomic fitness of its partners. This is probably one of the life history characteristics that led to the worldwide distribution success of this species.     

Small RNAs encoded within genetic islands of Salmonella typhimurium show host-induced expression and role in virulence

The emergence of pathogenic strains of enteric bacteria and their adaptation to unique niches are associated with the acquisition of foreign DNA segments termed ‘genetic islands’. We explored these islands for the occurrence of small RNA (sRNA) encoding genes. Previous systematic screens for enteric bacteria sRNAs were mainly carried out using the laboratory strain Escherichia coli K12, leading to the discovery of ~80 new sRNA genes. These searches were based on conservation within closely related members of enteric bacteria and thus, sRNAs, unique to pathogenic strains were excluded. Here we describe the identification and characterization of 19 novel unique sRNA genes encoded within the ‘genetic islands’ of the virulent strain Salmonella typhimurium. We show that the expression of many of the island-encoded genes is associated with stress conditions and stationary phase. Several of these sRNA genes are induced when Salmonella resides within macrophages. One sRNA, IsrJ, was further examined and found to affect the translocation efficiency of virulence-associated effector proteins into nonphagocytic cells. In addition, we report that unlike the majority of the E. coli sRNAs that are trans regulators, many of the island-encoded sRNAs affect the expression of cis-encoded genes. Our study suggests that the island encoded sRNA genes play an important role within the network that regulates bacterial adaptation to environmental changes and stress conditions and thus controls virulence.     

Quantitative trait, genetic, environmental, and geographical distances among populations of the C4 grass Trachypogon plumosus in Neotropical savannas

Venezuelan savannas are exposed to land‐use changes and biological invasions which compromise their persistence and function. The native C₄ grass Trachypogon plumosus is the most important component of the savannas under diverse combinations of climate and soils, suggesting substantial interpopulation variation. We examined quantitative traits and isozyme variation of nine populations of this grass and related these estimates to geographical and environmental features of sampled locations. Isozyme diversity estimates were based on 10 polymorphic enzyme systems whereas 21 quantitative traits, from field and controlled growth conditions, were evaluated. Distance matrices for quantitative traits, isozyme, geographical and environmental data were subjected to clustering analysis. Correspondence between quantitative trait distance and genetic distance, and their association to geographical and environmental distances were analysed with Mantel tests. All quantitative traits differed significantly among populations. The average Q_ST calculated for eight quantitative traits measured in the greenhouse was 0.157. Isozyme diversity differed significantly among populations. About 28% of total isozyme variation occurred among populations. Significant positive associations were detected between environmental, quantitative field traits, and geographical distance as well as between the later and genetic distances. Genetic distances did not correspond significantly with quantitative traits nor did environmental distances. Ecologically meaningful associations were detected between field quantitative traits, environmental, and geographical data using cluster analysis. Our results support the hypothesis that processes of the neutral type are mainly responsible for the variation patterns observed in T. plumosus populations in Venezuelan savannas. Variation observed for quantitative traits among populations seems to be due to the effect of environmental conditions on phenotypically plastic traits, and not the result of directional selection favouring different phenotypes in different populations.     

Interspecific interactions among species of the coral genus Porites from Okinawa, Japan

Analysis of field established xenogeneic interactions among five Porites species from Sesoko Island, Okinawa, revealed a transitive type of hierarchy as: P. rus > P. cylindrica > P. lobata > P. australiensis> P. lutea. Out of the 111 interspecific encounters studied, in only 5.4% reciprocal interactions were recorded, and in a single case, the opposite directionality of hierarchy was documented. Allogeneic encounters were also observed. A single major effector mechanism, an overgrowth (together with secondary outcomes such as the formation of small points of rejection, bleaching and pink color formation along a narrow peripheral belt of contacting tissues), was the only response in all 10 xenogeneic and 5 allogeneic combinations. In some massive colonies, a long contacting line of up to 50 cm was established. No sign for allelopathy, stand-off or rejection from a distance (i.e., by sweeper tentacles, sweeper polyps) was observed. Results are discussed with the accumulated data on Porites species from different reefs, worldwide, confirming that this genus is commonly lower in the hierarchy of xenogeneic interactions.     

A cell line with decreased sensitivity to the methyl mercury-induced stimulation of alpha-amanitin sensitive RNA synthesis in isolated nuclei

1. In nuclei isolated from cells of the B50 rat neuroblastoma line the stimulatory effect of methyl mercury on alpha-amanitin-sensitive RNA synthesis is very much reduced compared to the stimulatory effect in HeLa nuclei (see: Frenkel G. D. and Randles K. (1982) Specific stimulation of alpha-amanitin-sensitive RNA synthesis in isolated HeLa nuclei by methyl mercury. J. biol. Chem. 257, 6275-6279). 2. The stimulatory effect of another mercury compound, p-hydroxymercuribenzoate, was also much less pronounced in the B50 nuclei. 3. Similar results were obtained with nuclei isolated from B50 cells which had been induced to differentiate by exposure to dibutaryl cyclic AMP. 4. Nuclei isolated from cells of another rat neuroblastoma line (B35), and nuclei from cells of a human neuroblastoma line both exhibited levels of stimulation similar to that of HeLa nuclei. 5. The B50 and HeLa cells were also compared as to their sensitivity to other effects of methyl mercury.