Homo-DNA templated chemistry and its application to nucleic acid sensing

We have investigated the homo-DNA templated Staudinger reduction of the profluorophore rhodamine azide and have applied this reaction to the detection of natural DNA with a hybrid homo-DNA/DNA molecular beacon. In this system the sensing and the reporting unit are bioorthogonal to each other which facilitates sequence design and increases fidelity.

Genetic relatedness within the genus Campylobacter inferred from rpoB sequences

The genus Campylobacter comprises 17 species, some of which are important animal and human pathogens. To gain more insight into the genetic relatedness of this genus and to improve the molecular tools available for diagnosis, a universal sequencing approach was established for the gene encoding the beta-subunit of RNA polymerase (rpoB) for the genus Campylobacter. A total of 59 strains, including the type strains of currently recognized species as well as field isolates, were investigated in the study. A primer set specific for Campylobacter species enabled straightforward amplification and sequencing of a 530 bp fragment of the rpoB gene. The 16S rRNA gene sequences of all of the strains were determined in parallel. A good congruence was obtained between 16S rRNA and rpoB gene sequence-based trees within the genus Campylobacter. The branching of the rpoB tree was similar to that of the 16S rRNA gene tree, even though a few discrepancies were observed for certain species. The resolution of the rpoB gene within the genus Campylobacter was generally much higher than that of the 16S rRNA gene sequence, resulting in a clear separation of most species and even some subspecies. The universally applicable amplification and sequencing approach for partial rpoB gene sequence determination provides a powerful tool for DNA sequence-based discrimination of Campylobacter species.

Analysis of gene order data supports vertical inheritance of the leukotoxin operon and genome rearrangements in the 5' flanking region in genus Mannheimia

BACKGROUND: The Mannheimia subclades belong to the same bacterial genus, but have taken divergent paths toward their distinct lifestyles. For example, M. haemolytica + M. glucosida are potential pathogens of the respiratory tract in the mammalian suborder Ruminantia, whereas M. ruminalis, the supposed sister group, lives as a commensal in the ovine rumen. We have tested the hypothesis that vertical inheritance of the leukotoxin (lktCABD) operon has occurred from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades by exploring gene order data. RESULTS: We examined the gene order in the 5' flanking region of the leukotoxin operon and found that the 5' flanking gene strings, hslVU-lapB-artJ-lktC and xylAB-lktC, are peculiar to M. haemolytica + M. glucosida and M. granulomatis, respectively, whereas the gene string hslVU-lapB-lktC is present in M. ruminalis, the supposed sister group of M. haemolytica + M. glucosida, and in the most ancient subclade M. varigena. In M. granulomatis, we found remnants of the gene string hslVU-lapB-lktC in the xylB-lktC intergenic region. CONCLUSION: These observations indicate that the gene string hslVU-lapB-lktC is more ancient than the hslVU-lapB-artJ-lktC and xylAB-lktC gene strings. The presence of (remnants of) the ancient gene string hslVU-lapB-lktC among any subclades within genus Mannheimia supports that it has been vertically inherited from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades, thus reaffirming the hypothesis of vertical inheritance of the leukotoxin operon. The presence of individual 5' flanking regions in M. haemolytica + M. glucosida and M. granulomatis reflects later genome rearrangements within each subclade. The evolution of the novel 5' flanking region in M. haemolytica + M. glucosida resulted in transcriptional coupling between the divergently arranged artJ and lkt promoters. We propose that the chimeric promoter have led to high level expression of the leukotoxin operon which could explain the increased potential of certain M. haemolytica + M. glucosida strains to cause a particular type of infection.

Statistical evaluation of alternative models of human evolution

An appropriate model of recent human evolution is not only important to understand our own history, but it is necessary to disentangle the effects of demography and selection on genome diversity. Although most genetic data support the view that our species originated recently in Africa, it is still unclear if it completely replaced former members of the Homo genus, or if some interbreeding occurred during its range expansion. Several scenarios of modern human evolution have been proposed on the basis of molecular and paleontological data, but their likelihood has never been statistically assessed. Using DNA data from 50 nuclear loci sequenced in African, Asian and Native American samples, we show here by extensive simulations that a simple African replacement model with exponential growth has a higher probability (78%) as compared with alternative multiregional evolution or assimilation scenarios. A Bayesian analysis of the data under this best supported model points to an origin of our species approximately 141 thousand years ago (Kya), an exit out-of-Africa approximately 51 Kya, and a recent colonization of the Americas approximately 10.5 Kya. We also find that the African replacement model explains not only the shallow ancestry of mtDNA or Y-chromosomes but also the occurrence of deep lineages at some autosomal loci, which has been formerly interpreted as a sign of interbreeding with Homo erectus.