Electron microscopic mapping of deletions on a streptococcal plasmid carrying extraordinarily long inverted repeats

Deletions Δ101, Δ102, and Δ103 which occurred within the extraordinarily long inverted repeats of the self-ligated large EcoRI fragment of the Streptococcal MLS (macrolides, lincosamides, streptogramin B)-resistance plasmid pSM19035 led to the formation of plasmids pDB101, pDB102, and pDB103. Their molecular lengths were determined by contour length measurements to be 17.8, 17.4, and 13.9 kb, respectively. Electron microscopic examination of self-annealed molecules revealed stem-loop structures with inverted repeats comprising 41 to 91% of the mass of plasmids. Two unique sequences (US₁ and US₂) separated the inverted repeats in the case of pDB101 and pDB103, while in pDB102 the repeats were joined at one end and separated at the other by a unique sequence (US₂). The size of the unique sequence US₂ was identical for all three plasmids, and the location of the resistance determinant was determined by electron microscopic examination of self-annealed molecules of the recombinant plasmid pDB201. Mapping of the deletion termini, accomplished by combining electron microscopic and HindIII restriction data, suggested that deletions may occur at preferential sites.     

Simultaneous fine mapping of closely linked epistatic quantitative trait loci using combined linkage disequilibrium and linkage with a general pedigree

Causal mutations and their intra- and inter-locus interactions play a critical role in complex trait variation. It is often not easy to detect epistatic quantitative trait loci (QTL) due to complicated population structure requirements for detecting epistatic effects in linkage analysis studies and due to main effects often being hidden by interaction effects. Mapping their positions is even harder when they are closely linked. The data structure requirement may be overcome when information on linkage disequilibrium is used. We present an approach using a mixed linear model nested in an empirical Bayesian approach, which simultaneously takes into account additive, dominance and epistatic effects due to multiple QTL. The covariance structure used in the mixed linear model is based on combined linkage disequilibrium and linkage information. In a simulation study where there are complex epistatic interactions between QTL, it is possible to simultaneously map interacting QTL into a small region using the proposed approach. The estimated variance components are accurate and less biased with the proposed approach compared with traditional models.     

Identification of a gene, rgg, which regulates expression of glucosyltransferase and influences the Spp phenotype of Streptococcus gordonii Challis.

Streptococcus gordonii Challis was previously shown to give rise to phase variants expressing high (Spp+) or low (Spp-) levels of extracellular glucosyltransferase (GTF) activity. Here, shotgun cloning of an S. gordonii Spp+ chromosomal digest resulted in a chimeric plasmid (pAM5010) able to complement the Spp- phenotype. In addition, introduction of pAM5010 into an Spp+ strain resulted in a 10-fold increase in GTF expression. Deletion analysis of pAM5010 identified a 1.2-kb DNA segment which exhibited the same functional properties as pAM5010. Nucleotide sequence analysis of this region revealed a gene approximately 1 kb in size. The gene was designated rgg. Disruption of the chromosomal rgg gene open reading frame in an Spp+ strain resulted in strain DS512, which displayed an Spp(-)-like phenotype and had 3% of wild-type GTF activity. A plasmid containing the rgg gene was able to complement the DS512 phenotype and significantly increase GTF expression above wild-type levels. Sequence analysis and other data showed that the S. gordonii GTF determinant, designated gtfG, is located 66 bp downstream of the rgg gene. The sequence also revealed interesting inverted repeats which may play a role in the regulation of gtfG. We conclude that rgg positively regulates the expression of GTF and influences expression of the Spp phenotype.     

Sex pheromone cAD1 in Streptococcus faecalis: induction of a function related to plasmid transfer

In short matings between two donor strains with distinguishable isogenic conjugative plasmids (derivatives of pAD1), only the strain preexposed to the sex pheromone cAD1 behaved as a donor.     

Uncertainty/Safety Factors in Health Risk Assessment: Opportunities for Improvement

This paper presents the results of deliberations from participants who met on the second day of the Fourth Annual Workshop on the Evaluation of Uncertainty/Safety Factors in Health Risk Assessment. The group reviewed the previous day's presentations and implications for improvement in risk assessment. After much discussion, the group concluded that, in the short term, significant improvements could be made in the pharmacokinetic component of the inter-species uncertainty factor and developed a series of default options for this factor. These defaults consider route of exposure (oral or inhalation), and the form of the active compound (parent, metabolite, or very reactive metabolite). Several assumptions are key to this approach, such as a similar oral or inhalation bioavailability across species. We believe this method represents a useful default approach until more compound-specific information is available.