Dioon Lindl. (Zamiaceae): Perspectives from phylogeny and a population genetic study of Dioon edule

Dioon Lindl. (Zamiaceae) is a small genus restricted to Mexico (12 species) and Honduras (one species). Previous systematic studies have been unable to fully resolve species relationships within the genus. Phylogenetic analyses were conducted with data from several sources, including Restriction Fragment Length Polymorphisms from the chloroplast genome, morphology, two introns of the low copy nuclear gene S-adenosyl-L-homocysteine hydrolase (SAHH) and the 5.8S/ITS2 regions of the nuclear ribosomal DNA. The goals of the study were to construct a total evidence species level phylogeny and to explore current biogeographical hypotheses. None of the analyses performed produced a fully resolved topology. Dioon is comprised of two main lineages (the Edule and Spinulosum Clades), which represents an ancient divergence within the genus. The two introns of the nuclear gene SAHH offer additional evidence for the split into two lineages. Intron 2 contains a 18 bp deletion in the Spinulosum Clade, providing a synapomorphy for that group. The 5.8S/ITS2 regions were highly polymorphic and subsequently omitted from the combined analyses. In order to visualize congruence between morphology and molecular data, morphological characters were mapped onto the combined molecular tree. Current biogeographical hypotheses of a general northward pattern of migration and speciation are supported here. However, sister relationships within the Edule Clade are not fully resolved. Seven DNA microsatellite markers were developed to investigate patterns of genetic variation of seven populations of D. edule, a species restricted to Eastern Mexico. We found that most of the genetic variation lies within populations (Ho = 0.2166–0.3657) and that levels of population differentiation are low (Fst = 0.088); this finding is congruent with the breeding system of this species, dioicy. Four of the populations deviate from Hardy Weinberg Equilibrium and have a high number of identical genotypes, we suggest that this unexpected pattern is due to the life-history strategy of the species coupled with the few number of polymorphic loci detected in these populations. Our results are not congruent with earlier evidence from morphology and allozyme markers that suggest that the two northernmost populations represent a distinct entity that is recognized by some taxonomists as D. angustifolium.

Combination of 16S rRNA variable regions provides a detailed analysis of bacterial community dynamics in the lungs of cystic fibrosis patients

Chronic bronchopulmonary bacterial infections remain the most common cause of morbidity and mortality among patients with cystic fibrosis (CF). Recent community sequencing work has now shown that the bacterial community in the CF lung is polymicrobial. Identifying bacteria in the CF lung through sequencing can be costly and is not practical for many laboratories. Molecular techniques such as terminal restriction fragment length polymorphism or amplicon length heterogeneity-polymerase chain reaction (LH-PCR) can provide many laboratories with the ability to study CF bacterial communities without costly sequencing. The aim of this study was to determine if the use of LH-PCR with multiple hypervariable regions of the 16S rRNA gene could be used to identify organisms found in sputum DNA. This work also determined if LH-PCR could be used to observe the dynamics of lung infections over a period of time. Nineteen samples were analysed with the V1 and the V1_V2 region of the 16S rRNA gene. Based on the amplicon size present in the V1_V2 region, Pseudomonas aeruginosa was confirmed to be in all 19 samples obtained from the patients. The V1 region provided a higher power of discrimination between bacterial profiles of patients. Both regions were able to identify trends in the bacterial population over a period of time. LH profiles showed that the CF lung community is dynamic and that changes in the community may in part be driven by the patient's antibiotic treatment. LH-PCR is a tool that is well suited for studying bacterial communities and their dynamics.

Sequence-related structural DNA anomalies affect restriction enzyme activity and DNA polymerase I binding

DNA serves as a target molecule for several types of enzymes and may assume a wide variety of structural motifs depending upon the local sequence. The BssHII restriction site (GC)3 resides in a 9bp region of alternating pyrimidine and purine residues within the &phis;X174 genome. Such sequences are known to demonstrate non-canonical helical behavior under the appropriate conditions. The kinetics of BssHII cleavage was investigated in supercoiled and linear plasmid DNA, and in a 323bp DNA fragment obtained via amplification of &phis;X174. The rate of enzyme cleavage was enhanced in the supercoiled form and in the presence of 50μM cobalt hexamine. Similarly, cobalt hexamine was also found to enhance TaqI activity directly adjacent to the (GC)3 region. ^ Initial DNA polymerase I binding studies (including a gel mobility shift assay and a protection assay) indicated a notable interaction between DNA polymerase I and the BssHII site. An in-depth study revealed that equilibrium binding of DNA polymerase I to the T7 RNA polymerase promoter was comparable to that of the (GC)3 site, however the strongest interaction was observed with a cruciform containing region. Increasing the ionic strength of the solution environment, including the addition of DNA polymerase I reaction buffer significantly decreased the equilibrium dissociation constant values. ^ It is suggested that the region within or around the BssHII site experiences a conformational change generating a novel structure under the influence of supercoiled tension or 50μM cobalt hexamine. It is proposed that this transition may enhance enzyme activity and binding by providing an initial enzyme-docking site—the rate-limiting step in restriction enzyme kinetics. The high binding potential of DNA polymerase I for each of the motifs described, is hypothesized to be due to recognition of the structural DNA anomalies by the 3′–5′ exonuclease domain. ^