HiTRACE: High-throughput robust analysis for capillary electrophoresis

Motivation: Capillary electrophoresis (CE) of nucleic acids is a workhorse technology underlying high-throughput genome analysis and large-scale chemical mapping for nucleic acid structural inference. Despite the wide availability of CE-based instruments, there remain challenges in leveraging their full power for quantitative analysis of RNA and DNA structure, thermodynamics, and kinetics. In particular, the slow rate and poor automation of available analysis tools have bottlenecked a new generation of studies involving hundreds of CE profiles per experiment. Results: We propose a computational method called high-throughput robust analysis for capillary electrophoresis (HiTRACE) to automate the key tasks in large-scale nucleic acid CE analysis, including the profile alignment that has heretofore been a rate-limiting step in the highest throughput experiments. We illustrate the application of HiTRACE on thirteen data sets representing 4 different RNAs, three chemical modification strategies, and up to 480 single mutant variants; the largest data sets each include 87,360 bands. By applying a series of robust dynamic programming algorithms, HiTRACE outperforms prior tools in terms of alignment and fitting quality, as assessed by measures including the correlation between quantified band intensities between replicate data sets. Furthermore, while the smallest of these data sets required 7 to 10 hours of manual intervention using prior approaches, HiTRACE quantitation of even the largest data sets herein was achieved in 3 to 12 minutes. The HiTRACE method therefore resolves a critical barrier to the efficient and accurate analysis of nucleic acid structure in experiments involving tens of thousands of electrophoretic bands.Comment: Revised to include Supplement. Availability: HiTRACE is freely available for download at http://hitrace.stanford.ed

International Summer School, ‘ From Genome to Life’

This report from the International Summer School ‘From Genome to Life’, held at the Institute d'Etudes Scientifiques de Cargèse in Corsica in July 2002, covers the talks of the invited speakers. The topics of the talks can be broadly grouped into the areas of genome annotation, comparative and evolutionary genomics, functional genomics, proteomics, structural genomics, pharmacogenomics, and organelle genomes, epigenetics and RNA

The Structure and Evolution of Non-canonical Coiled coils

Coiled coils are ubiquitous protein structural elements which support a wide range of biological functions. They can serve as molecular spacers, oligomerization motifs, mechanical levers in membrane fusion, components of cytoskeleton as well as facilitate ion transport and signal transduction. Canonical coiled coils are regular, left-handed supercoiled bundles of two or more α-helices, with a characteristic heptad repeat pattern. However, other periodicities engendering different supercoils are possible. Insertion of two (nonads) or six (hexads) residues in a heptad repeat locally breaks the helices into short β-strands which assemble as a triangular structural element we call the β-layer. In the first project, we structurally characterized two hexad repeat families. Repetitive nonads and hexads yield a new structure, the α/β coiled coil, with regularly alternating α- and β-segments. Conversion of hexads to heptads by insertion of one residue per repeat gives a canonical coiled coil. Our results support previous data that novel backbone structures are possible within the allowed regions of Ramachandran space with minor mutations to a known fold. Secondly, we characterized the human paralogs MCUR1 and CCDC90B of a novel membrane protein family conserved in prokaryotes and mitochondria. The proteins were found to exhibit a conserved head-neck-stalk-anchor architecture, where a membrane-anchored trimeric coiled-coil stalk projects the N-terminal helical head domain via a β-layer neck. Cellular localization studies showed that prokaryotic and eukaryotic proteins localize to the cytoplasmic and inner mitochondrial membranes, respectively, with an N-in C-out topology. Using MCUR1, an essential regulator of Ca(2+) uptake through mitochondrial calcium uniporter (MCU), we studied the role of individual domains and found that the conserved head interacts directly with MCU. Ca(2+) binding destabilizes MCUR1 head domain, which then accelerates its conversion to β-amyloid fibrils. Finally, we studied the effect of frameshift resistant (FSR) repeat amplification on the structure and function of existing and novel proteins. This type of repetition comprising units of n∤3 base-pairs and lacking stop codons, encodes the same protein repeat of n residues in all three frames. We focused primarily on heptad FSR repeats which conform to coiled-coil periodicity and are significantly enriched in bacteria. Using cyanobacterium Microcystis aeruginosa, we investigated the in vivo expression of FSR repeat ORFs with proteome and transcriptome analysis and found that a number of them are highly transcribed, but undetectable at the protein level. Through biophysical and biochemical methods, we showed that FSR repeat insertion products are initially unstructured and mostly non-functional; however, they can obtain beneficial mutations over evolutionary time-scales to become more structured, giving rise to novel cellular functions

Ride the Tide: Observing CRISPR/Cas9 genome editing by the numbers

Targeted genome editing has become a powerful genetic tool for modification of DNA sequences in their natural chromosomal context. CRISPR RNA-guided nucleases have recently emerged as an efficient targeted editing tool for multiple organisms. Hereby a double strand break is introduced at a targeted DNA site. During DNA repair genomic alterations are introduced which can change the function of the DNA code. However, our understanding of how CRISPR works is incomplete and it is still hard to predict the CRISPR activity at the precise target sites. The highly ordered structure of the eukaryotic genome may play a role in this. The organization of the genome is controlled by dynamic changes of DNA methylation, histone modification, histone variant incorporation and nucleosome remodelling. The influence of nuclear organization and chromatin structure on transcription is reasonably well known, but we are just beginning to understand its effect on genome editing by CRISP

Iron uptake and homeostasis in the veterinary pathogen Rhodococcus equi: an integrated omics approach

Rhodococcus equi, a veterinary pathogen that causes pyogranulomatous pneumonia, can secrete low molecular weight chelators called siderophores to scavenge iron when its bioavailability is limited. When iron is plentiful, synthesis of siderophores and ferri–siderophore transport systems are repressed. Current literature on bacterial iron regulation and homeostasis indicates two distinct protein families of global iron-dependent transcriptional repressor: Fur and DtxR. Gram-negative bacteria produce Fur to regulate iron uptake genes and the biosynthesis of siderophores in response to the iron level in the cell. However, the Gram-positive Corynebacteriaceae produce DtxR-like proteins to regulate analogous genes. Much remains undefined with respect to rhodococcal siderophore biosynthesis and uptake. Detailed analysis of the R. equi 103S genome for genes related to iron homeostasis identified two potential metal regulatory genes each from the Fur and DtxR families: iron dependent regulatory protein (IdeR), Diphtheria toxin repressor (DtxR), Ferric uptake regulator A (FurA) and Ferric uptake regulator B (FurB). Bioinformatic analysis confirmed that this complement of genes was conserved throughout Rhodococcus and the Corynebacteriaceae in general. To investigate their individual roles in metal homeostasis, molecular cloning and gene expression was performed, to facilitate analysis of regulator-metal specificities. Each gene was cloned but over-expression for functional analysis could only be achieved for ideR; thus, a thorough systematic analysis could not be achieved. In order to address their individual roles, homology-based protein modelling was used, and comparisons made with characterised homologues from M. tuberculosis. The geometrical conservation of key ligand amino acid residues strongly suggests R. equi utilises ideR as an iron regulator; furB as a zinc regulator, dtxR as a manganese regulator and furA as an oxidative stress response protein. Most bacteria generate an exaggerated response to iron limitation in vitro, however R. equi produces very small siderophore yields s, which has complicated their characterisation. In-frame deletion of the putative metal regulator genes ideR, dtxR, furA and furB was attempted in order to address the hypothesis that de-repression might generate greater yields. All genes were deleted individually; a marked phenotypic difference was noted only for R. equi-ΔfurA, which significantly upregulated the catalase encoded by the neighbouring gene and was coincidentally hyper-resistant to hydrogen peroxide. Surprisingly, analysis of siderophore production in the mutants indicated no increase in yield. The thesis discusses the relevance of this observation to microbial ecology. The availability of these mutants in combination with their predicted metal specificities facilitates the design of experiments to define their individual roles in metal homeostasis beyond the scope of this thesis. The combination of ‘omic’ analyses was attempted here to initiate the ultimate definition of the complex molecular network associated with iron uptake. The genomic investigation informed hypothesis building for the other omic analyses. It suggested R. equi is capable of synthesising two siderophores, rhequibactin and rhequichelin; up to three had previously been postulated in the literature. Culture optimisation was required to deliver a robust experimental design to impose iron limitation in isolation from other stresses. Once medium composition and biomarker-indicated harvesting criteria were established, biomass and associated secretomes were produced en masse for integrated omics analysis. A comparative untargeted metabolomics study demonstrated an adapted iron-starved metabolome; strong siderophore candidates were then investigated using a targeted strategy. A strong candidate metabolite was identified by mass that appeared to be responsible for a heterobactin-like chromophore, however further biochemical characterisation has been elusive. Interestingly, the metabolite readily precipitates on complexation with iron, an observation also made for heterobactins. Secondly, a transcriptomic study was attempted to study the global gene expression under iron starvation, and the impact of the loss of the IdeR in the deletion mutant generated in this work. However, the RNA extraction proved particularly challenging likely due to difficulties arising from lysis of the mycolic acid-containing cell wall. In the absence of a high-quality transcriptome sample, the study did not advance further and other aspects of the study were prioritised. Finally, a comparative proteomic analysis into iron regulatory mechanisms associated with the rhodococcal cell wall was performed. Current literature deliberates how R. equi uses a range of strategies to overcome iron limitation through proposed uptake mechanisms associated with translocation across the cytoplasmic membrane via ABC transport systems, while no consideration has yet been made with regards to transport across the mycolic acid-containing cell wall structure. In this study no obvious candidate proteins for ferri-sideophore transport across the mycolate region were identified, therefore it is possible that R. equi utilises facilitated diffusion via a porin for entry of ferri-siderophore complexes into the pseudoperiplasm, where a substrate-binding lipoprotein may act as the primary receptor to facilitate cytosolic transfer through an ABC transport system

Cyanobacterial RNA polymerase: Structural features and acclimation to environmental change

Siirretty Doriast