Benzoquinoquinoxaline derivatives stabilize and cleave H-DNA and repress transcription downstream of a triplex-forming sequence.

Oligopyrimidine*oligopurine sequences with potential to form intramolecular triple helix structures (H-DNA) have been found mainly in high eukaryote genomes. However, the natural occurrence and function of H-DNA remains elusive largely because we lack appropriate reagents to demonstrate the formation of these structures in cells. We examined whether a triple-helix specific stabilizing compound, benzoquinoquinoxaline (BQQ), and its 1,10-phenanthroline derivative can be efficiently utilized to study the formation and stabilization of an intramolecular triple-helical DNA structure in growing Escherichia coli cells and in vitro. Cell uptake of BQQ was confirmed by fluorescence microscopy. A plasmid carrying an H-DNA forming sequence upstream of a reporter gene was used to assess the effects of H-DNA formation and stabilization in growing cells. The presence of the H-DNA forming sequence dramatically repressed beta-lactamase expression, and sub-growth-inhibitory doses of BQQ caused a further 40% reduction. Most importantly, repression was dependent on the triple-helix forming sequence and correlated with the addition of BQQ. As the abundance of the H-DNA forming plasmid was not affected by the addition of BQQ, the dose-dependent reduction at the protein level observed here is likely caused by repression of transcription. Finally, the triple-helix specific interaction of BQQ with the target DNA sequence was demonstrated using a triple-helix directed cleavage assay by BQQ-1,10-phenanthroline conjugate in vitro.

Assessing sources of nitrate contamination in the Shiraz urban aquifer (Iran) using the δ(15)N and δ(18)O dual-isotope approach.

Nitrate ([Formula: see text]) is one of the major threats to the quality of the drinking water taken from the Shiraz aquifer. This aquifer undergoes high anthropogenic pressures from multiple local urban (including uncontrolled sewage systems), agricultural and industrial activities, resulting in [Formula: see text] concentrations as high as 149 mg L(-1), well above the 50 mg L(-1) guideline defined by the World Health Organisation. We coupled here classical chemical and dual isotope (δ(15)N and δ(18)O of [Formula: see text]) approaches trying to characterize sources and potential processes controlling the budget of this pollutant. Chemical data indicate that nitrate in this aquifer is explained by distinct end-members: while mineral fertilizers isotopically show to have no impact, our isotope approach identifies natural soil nitrification and organic [Formula: see text] (manure and/or septic waste) as the two main contributors. Isotope data suggest that natural denitrification may occur within the aquifer, but this conclusion is not supported by the study of other chemical parameters.

Birth and death of orphan genes in Rickettsia.

The origin and evolution of the thousands of species-specific genes with unknown functions, the so-called orphan genes, has been a mystery. Here, we have studied the rates and patterns of orphan sequence evolution, using the Rickettsia as our reference system. Of the Rickettsia conorii orphans examined in this study, 80% were found to be short gene fragments or fusions of short segments from neighboring genes. We reconstructed the putative sequences of the full-length genes from which the short orphan fragments are thought to have originated. One of the genes thus reconstructed displays weak similarity to the ankyrin-repeat protein family, an identification that is strongly supported by comparative molecular modeling. Studies of the patterns of gene fragmentation underscore the importance of short repeated sequences as targets for recombination events that result in sequence loss and the formation of short, transient open reading frames. Our analysis demonstrates that gene sequences present in the common ancestor can be inferred even in cases when no full-length open reading frame is present in any of the contemporary species. Such reconstructions support the identification of lost protein functions and hint at important lifestyle changes.

Proliferation and deterioration of Rickettsia palindromic elements.

It has been suggested that Rickettsia Palindromic Elements (RPEs) have evolved as selfish DNA that mediate protein sequence evolution by being targeted to genes that code for RNA and proteins. Here, we have examined the phylogenetic depth of two RPEs that are located close to the genes encoding elongation factors Tu (tuf) and G (fus) in Rickettsia. An exceptional organization of the elongation factor genes was found in all 11 species examined, with complete or partial RPEs identified downstream of the tuf gene (RPE-tuf) in six species and of the fus gene (RPE-fus) in 10 species. A phylogenetic reconstruction shows that both RPE-tuf and RPE-fus have evolved in a manner that is consistent with the expected species divergence. The analysis provides evidence for independent loss of RPE-tuf in several species, possibly mediated by short repetitive sequences flanking the site of excision. The remaining RPE-tuf sequences evolve as neutral sequences in different stages of deterioration. Likewise, highly fragmented remnants of the RPE-fus sequence were identified in two species. This suggests that genome-specific differences in the content of RPEs are the result of recent loss rather than recent proliferation.

Deep origin of plastid/parasite ATP/ADP translocases.

Membrane proteins that transport ATP and ADP have been identified in mitochondria, plastids, and obligate intracellular parasites. The mitochondrial ATP/ADP transporters are derived from a broad-specificity transport family of eukaryotic origin, whereas the origin of the plastid/parasite ATP/ADP translocase is more elusive. Here we present the sequences of five genes coding for ATP/ADP translocases from four species of Rickettsia. The results are consistent with an early duplication and divergence of the five ATP/ADP translocases within the rickettsial lineage. A comparison of the phylogenetic depths of the mitochondrial and the plastid/parasite ATP/ADP translocases indicates a deep origin for both transporters. The results provide no evidence for a recent acquisition of the ATP/ADP transporters in Rickettsia via horizontal gene transfer, as previously suggested. A possible function of the two types of ATP/ADP translocases was to allow switches between glycolysis and aerobic respiration in the early eukaryotic cell and its endosymbiont.

Genome deterioration: loss of repeated sequences and accumulation of junk DNA.

A global survey of microbial genomes reveals a correlation between genome size, repeat content and lifestyle. Free-living bacteria have large genomes with a high content of repeated sequences and self-propagating DNA, such as transposons and bacteriophages. In contrast, obligate intracellular bacteria have small genomes with a low content of repeated sequences and no or few genetic parasites. In extreme cases, such as in the 650 kb-genomes of aphid endosymbionts of the genus Buchnera all repeated sequences above 200bp have been eliminated. We speculate that the initial downsizing of the genomes of obligate symbionts and parasites occurred by homologous recombination at repeated genes, leading to the loss of large blocks of DNA as well as to the consumption of repeated sequences. Further sequence elimination in these small genomes seems primarily to result from the accumulation of short deletions within genic sequences. This process may lead to temporary increases in the genomic content of pseudogenes and 'junk' DNA. We discuss causes and long-term consequences of extreme genome size reductions in obligate intracellular bacteria.