Genome analysis of DNA repair genes in the alpha proteobacterium <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

Background  

The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria.     

Sixty years of genome biology

Sixty years after Watson and Crick published the double helix model of DNA''s structure, thirteen members of Genome Biology''s Editorial Board select key advances in the field of genome biology subsequent to that discovery.April 25th 2013 is the sixtieth anniversary of the infamous Watson and Crick Nature paper describing a model for the structure of DNA, published 25 April 1953: the now infamous ''double helix'' [1]. Two accompanying papers from Rosalind Franklin, Maurice Wilkins and colleagues leant experimental support to the proposed structure in the form of X-ray diffraction data [2,3], as described elsewhere in this issue of Genome Biology [4]. The model was a landmark discovery in the history of modern science, and was notable for its cross-disciplinary importance: the question addressed was of immense biological importance, but it was physicists and chemists whose expertise and techniques were needed in order to arrive at an answer. One of these physicists, Ray Gosling, describes the unveiling of Watson and Crick''s double helix structure as a ''eureka'' moment [4]: its simplicity and elegance were striking, and not only explained the X-ray diffraction data but also the mode of replication of life itself. It is rare for a scientific discovery to achieve such an iconic status, to pervade popular culture and the public consciousness, as well as to become an emblem of scientific inquiry - as exemplified by Genome Biology''s double helix-inspired logo. Although Avery had already shown DNA to be the genetic material [5], it took the convincing simplicity of Watson and Crick''s double helix for this notion to widely take hold, in place of theories favoring proteins. The discovery, therefore, had many important implications, and set the scene for future breakthroughs in the field of genome biology.To celebrate sixty years of such discoveries, we asked a jury composed of Genome Biology Editorial Board members to select key advances in the field since 25 April 1953. The brief was to choose a development that was either the most important or the most surprising, or that had the most personal impact, and to briefly summarize why. A number of selections focused on technological advances - from restriction mapping through microarrays and high-throughput sequencing. These technologies have clearly done much to inform our understanding of the biology of genomes. The most popular choice, however, was the discovery of introns. Much like the double helix, this discovery had something of the ''X factor'' to it: biologists trained in the post-intron era may take the concept of gene fragmentation for granted, but at the time it was a truly radical and paradigm-shifting idea. The sense of surprise made a strong impression on those old enough to remember the discovery, and one of the groups involved went so far as to describe it as ''amazing'' in the title of their paper [6].     

Mutations within lncRNAs are effectively selected against in fruitfly but not in human

Background

Previous studies in Drosophila and mammals have revealed levels of long non-coding RNAs (lncRNAs) sequence conservation that are intermediate between neutrally evolving and protein-coding sequence. These analyses compared conservation between species that diverged up to 75 million years ago. However, analysis of sequence polymorphisms within a species'' population can provide an understanding of essentially contemporaneous selective constraints that are acting on lncRNAs and can quantify the deleterious effect of mutations occurring within these loci.

Results

We took advantage of polymorphisms derived from the genome sequences of 163 Drosophila melanogaster strains and 174 human individuals to calculate the distribution of fitness effects of single nucleotide polymorphisms occurring within intergenic lncRNAs and compared this to distributions for SNPs present within putatively neutral or protein-coding sequences. Our observations show that in D.melanogaster there is a significant excess of rare frequency variants within intergenic lncRNAs relative to neutrally evolving sequences, whereas selection on human intergenic lncRNAs appears to be effectively neutral. Approximately 30% of mutations within these fruitfly lncRNAs are estimated as being weakly deleterious.

Conclusions

These contrasting results can be attributed to the large difference in effective population sizes between the two species. Our results suggest that while the sequences of lncRNAs will be well conserved across insect species, such loci in mammals will accumulate greater proportions of deleterious changes through genetic drift.     

PDZ Domains: Targeting signalling molecules to sub-membranous sites

PDZ (also called DHR or GLGF) domains are found in diverse membraneassociated proteins including members of the MAGUK family of guanylate kinase homologues, several protein phosphatases and kinases, neuronal nitric oxide synthase, and several dystrophin-associated proteins, collectively known as syntrophins. Many PDZ domain-containing proteins appear to be localised to highly specialised submembranous sites, suggesting their participation in cellular junction formation, receptor or channel clustering, and intracellular signalling events. PDZ domains of several MAGUKs interact with the C-terminal polypeptides of a subset of NMDA receptor subunits and/or with Shaker-type K⁺ channels. Other PDZ domains have been shown to bind similar ligands of other transmembrane receptors. Recently, the crystal structures of PDZ domains, with and without ligand, have been determined. These demonstrate the mode of ligand-binding and the structural bases for sequence conservation among diverse PDZ domains.     

The functional repertoires of metazoan genomes

Metazoan genomes are being sequenced at an increasingly rapid rate. For each new genome, the number of protein-coding genes it encodes and the amount of functional DNA it contains are known only inaccurately. Nevertheless, there have been considerable recent advances in identifying protein-coding and non-coding sequences that have remained constrained in diverse species. However, these approaches struggle to pinpoint genomic sequences that are functional in some species but that are absent or not functional in others. Yet it is here, encoded in lineage-specific and functional sequence, that we expect physiological differences between species to be most concentrated.     

Eukaryotic signalling domain homologues in archaea and bacteria. Ancient ancestry and horizontal gene transfer.

Phyletic distributions of eukaryotic signalling domains were studied using recently developed sensitive methods for protein sequence analysis, with an emphasis on the detection and accurate enumeration of homologues in bacteria and archaea. A major difference was found between the distributions of enzyme families that are typically found in all three divisions of cellular life and non-enzymatic domain families that are usually eukaryote-specific. Previously undetected bacterial homologues were identified for# plant pathogenesis-related proteins, Pad1, von Willebrand factor type A, src homology 3 and YWTD repeat-containing domains. Comparisons of the domain distributions in eukaryotes and prokaryotes enabled distinctions to be made between the domains originating prior to the last common ancestor of all known life forms and those apparently originating as consequences of horizontal gene transfer events. A number of transfers of signalling domains from eukaryotes to bacteria were confidently identified, in contrast to only a single case of apparent transfer from eukaryotes to archaea.     

A Nano-MgO and Ionic Liquid-Catalyzed ‘Green’ Synthesis Protocol for the Development of Adamantyl-Imidazolo-Thiadiazoles as Anti-Tuberculosis Agents Targeting Sterol 14α-Demethylase (CYP51)

In this work, we describe the ‘green’ synthesis of novel 6-(adamantan-1-yl)-2-substituted-imidazo[2,1-b][1,3,4]thiadiazoles (AITs) by ring formation reactions using 1-(adamantan-1-yl)-2-bromoethanone and 5-alkyl/aryl-2-amino1,3,4-thiadiazoles on a nano material base in ionic liquid media. Given the established activity of imidazothiadiazoles against M. tuberculosis, we next examined the anti-TB activity of AITs against the H₃₇Rv strain using Alamar blue assay. Among the tested compounds 6-(adamantan-1-yl)-2-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazole (3f) showed potent inhibitory activity towards M. tuberculosis with an MIC value of 8.5 μM. The inhibitory effect of this molecule against M. tuberculosis was comparable to the standard drugs such as Pyrazinamide, Streptomycin, and Ciprofloxacin drugs. Mechanistically, an in silico analysis predicted sterol 14α-demethylase (CYP51) as the likely target and experimental activity of 3f in this system corroborated the in silico target prediction. In summary, we herein report the synthesis and biological evaluation of novel AITs against M. tuberculosis that likely target CYP51 to induce their antimycobacterial activity.     

Issues in predicting protein function from sequence

Identifying homologues, defined as genes that arose from a common evolutionary ancestor, is often a relatively straightforward task, thanks to recent advances made in estimating the statistical significance of sequence similarities found from database searches. The extent by which homologues possess similarities in function, however, is less amenable to statistical analysis. Consequently, predicting function by homology is a qualitative, rather than quantitative, process and requires particular care to be taken. This review focuses on the various approaches that have been developed to predict function from the scale of the atom to that of the organism. Similarities in homologues' functions differ considerably at each of these different scales and also vary for different domain families. It is argued that due attention should be paid to all available clues to function, including orthologue identification, conservation of particular residue types, and the co-occurrence of domains in proteins. Pitfalls in database searching methods arising from amino acid compositional bias and database size effects are also discussed.