Towards a comprehensive picture of alloacceptor tRNA remolding in metazoan mitochondrial genomes

Remolding of tRNAs is a well-documented process in mitochondrial genomes that changes the identity of a tRNA. It involves a duplication of a tRNA gene, a mutation that changes the anticodon and the loss of the ancestral tRNA gene. The net effect is a functional tRNA that is more closely related to tRNAs of a different alloacceptor family than to tRNAs with the same anticodon in related species. Beyond being of interest for understanding mitochondrial tRNA function and evolution, tRNA remolding events can lead to artifacts in the annotation of mitogenomes and thus in studies of mitogenomic evolution. Therefore, it is important to identify and catalog these events. Here we describe novel methods to detect tRNA remolding in large-scale data sets and apply them to survey tRNA remolding throughout animal evolution. We identify several novel remolding events in addition to the ones previously mentioned in the literature. A detailed analysis of these remoldings showed that many of them are derived from ancestral events.     

Towards fully automated structure-based function prediction in structural genomics: a case study

As the global Structural Genomics projects have picked up pace, the number of structures annotated in the Protein Data Bank as hypothetical protein or unknown function has grown significantly. A major challenge now involves the development of computational methods to assign functions to these proteins accurately and automatically. As part of the Midwest Center for Structural Genomics (MCSG) we have developed a fully automated functional analysis server, ProFunc, which performs a battery of analyses on a submitted structure. The analyses combine a number of sequence-based and structure-based methods to identify functional clues. After the first stage of the Protein Structure Initiative (PSI), we review the success of the pipeline and the importance of structure-based function prediction. As a dataset, we have chosen all structures solved by the MCSG during the 5 years of the first PSI. Our analysis suggests that two of the structure-based methods are particularly successful and provide examples of local similarity that is difficult to identify using current sequence-based methods. No one method is successful in all cases, so, through the use of a number of complementary sequence and structural approaches, the ProFunc server increases the chances that at least one method will find a significant hit that can help elucidate function. Manual assessment of the results is a time-consuming process and subject to individual interpretation and human error. We present a method based on the Gene Ontology (GO) schema using GO-slims that can allow the automated assessment of hits with a success rate approaching that of expert manual assessment.     

Towards miniaturization of a structural genomics pipeline using micro-expression and microcoil NMR

In structural genomics centers, nuclear magnetic resonance (NMR) screening is in increasing use as a tool to identify folded proteins that are promising targets for three-dimensional structure determination by X-ray crystallography or NMR spectroscopy. The use of 1D ¹H NMR spectra or 2D [¹H,¹⁵N]-correlation spectroscopy (COSY) typically requires milligram quantities of unlabeled or isotope-labeled protein, respectively. Here, we outline ways towards miniaturization of a structural genomics pipeline with NMR screening for folded globular proteins, using a high-density micro-fermentation device and a microcoil NMR probe. The proteins are micro-expressed in unlabeled or isotope-labeled media, purified, and then subjected to 1D ¹H NMR and/or 2D [¹H,¹⁵N]-COSY screening. To demonstrate that the miniaturization is functioning effectively, we processed nine mouse homologue protein targets and compared the results with those from the “macro-scale” Joint Center of Structural Genomics (JCSG) high-throughput pipeline. The results from the two pipelines were comparable, illustrating that the data were not compromised in the miniaturized approach.     

Towards higher-throughput membrane protein production for structural genomics initiatives

Integral membrane proteins present unparalleled challenges for structural genomics programs. Samples from this class of proteins are not only difficult to produce in quantities sufficient for analysis by X-ray diffraction or NMR, but their hydrophobic properties add extra dimension to their purification and subsequent crystallization. New systems that seek to tackle the production problems are in development. In our laboratory, one such strategy exploits the unique physiology of the Rhodobacter species of photosynthetic bacteria where we have designed an overexpression system that coordinates the heterologous production of targeted hydrophobic proteins with nascent, unfilled membranes that can be used to harbor them. In this study, we describe the means by which purification of recombinant membrane proteins produced in such a fashion can be purified efficiently from Rhodobacter membranes using relatively higher-throughput, semi-automated methods. These protocols utilize a state-of-the-art FPLC system for affinity chromatography, followed by gel filtration or ion exchange chromatography to enhance purity for crystallization attempts. The Rhodobacter expression system coupled with the semi-automation of purification steps represents an advance towards the development of a strategy for obtaining structures for membrane proteins at a more rapid pace.     

Towards a comprehensive structural variation map of an individual human genome

Background

Several genomes have now been sequenced, with millions of genetic variants annotated. While significant progress has been made in mapping single nucleotide polymorphisms (SNPs) and small (<10 bp) insertion/deletions (indels), the annotation of larger structural variants has been less comprehensive. It is still unclear to what extent a typical genome differs from the reference assembly, and the analysis of the genomes sequenced to date have shown varying results for copy number variation (CNV) and inversions.

Results

We have combined computational re-analysis of existing whole genome sequence data with novel microarray-based analysis, and detect 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing of the first published personal genome. We estimate a total non-SNP variation content of 48.8 Mb in a single genome. Our results indicate that this genome differs from the consensus reference sequence by approximately 1.2% when considering indels/CNVs, 0.1% by SNPs and approximately 0.3% by inversions. The structural variants impact 4,867 genes, and >24% of structural variants would not be imputed by SNP-association.

Conclusions

Our results indicate that a large number of structural variants have been unreported in the individual genomes published to date. This significant extent and complexity of structural variants, as well as the growing recognition of their medical relevance, necessitate they be actively studied in health-related analyses of personal genomes. The new catalogue of structural variants generated for this genome provides a crucial resource for future comparison studies.     

Patterns of protein-fold usage in eight microbial genomes: A comprehensive structural census

Eight microbial genomes are compared in terms of protein structure. Specifically, yeast, H. influenzae, M. genitalium, M. jannaschii, Synechocystis, M. pneumoniae, H. pylori, and E. coli are compared in terms of patterns of fold usage—whether a given fold occurs in a particular organism. Of the ∼340 soluble protein folds currently in the structure databank (PDB), 240 occur in at least one of the eight genomes, and 30 are shared amongst all eight. The shared folds are depleted in all-helical structure and enriched in mixed helix-sheet structure compared to the folds in the PDB. The top-10 most common of the shared 30 are enriched in superfolds, uniting many non-homologous sequence families, and are especially similar in overall architecture—eight having helices packed onto a central sheet. They are also very different from the common folds in the PBD, highlighting databank biases. Folds can be ranked in terms of expression as well as genome duplication. In yeast the top-10 most highly expressed folds are considerably different from the most highly duplicated folds. A tree can be constructed grouping genomes in terms of their shared folds. This has a remarkably similar topology to more conventional classifications, based on very different measures of relatedness. Finally, folds of membrane proteins can be analyzed through transmembrane-helix (TM) prediction. All the genomes appear to have similar usage patterns for these folds, with the occurrence of a particular fold falling off rapidly with increasing numbers of TM-elements, according to a “Zipf-like” law. This implies there are no marked preferences for proteins with particular numbers of TM-helices (e.g. 7-TM) in microbial genomes. Further information pertinent to this analysis is available at http://bioinfo.mbb.yale.edu/genome. Proteins 33:518–534, 1998. © 1998 Wiley-Liss, Inc.     

Structural characterization of genomes by large scale sequence-structure threading: application of reliability analysis in structural genomics

Background  

We establish that the occurrence of protein folds among genomes can be accurately described with a Weibull function. Systems which exhibit Weibull character can be interpreted with reliability theory commonly used in engineering analysis. For instance, Weibull distributions are widely used in reliability, maintainability and safety work to model time-to-failure of mechanical devices, mechanisms, building constructions and equipment.     

Survey of simple sequence repeats in completed fungal genomes

The use of simple sequence repeats or microsatellites as genetic markers has become very popular because of their abundance and length variation between different individuals. SSRs are tandem repeat units of 1 to 6 base pairs that are found abundantly in many prokaryotic and eukaryotic genomes. This is the first study examining and comparing SSRs in completely sequenced fungal genomes. We analyzed and compared the occurrences, relative abundance, relative density, most common, and longest SSRs in nine taxonomically different fungal species: Aspergillus nidulans, Cryptococcus neoformans, Encephalitozoon cuniculi, Fusarium graminearum, Magnaporthe grisea, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Ustilago maydis. Our analysis revealed that, in all of the genomes studied, the occurrence, abundance, and relative density of SSRs varied and was not influenced by the genome sizes. No correlation between relative abundance and the genome sizes was observed, but it was shown that N. crassa, the largest genome analyzed had the highest relative abundance of SSRs. In most genomes, mononucleotide, dinucleotide, and trinucleotide repeats were more abundant than the longer repeated SSRs. Generally, in each organism, the occurrence, relative abundance, and relative density of SSRs decreased as the repeat unit increased. Furthermore, each organism had its own common and longest SSRs. Our analysis showed that the relative abundance of SSRs in fungi is low compared with the human genome and that longer SSRs in fungi are rare. In addition to providing new information concerning the abundance of SSRs for each of these fungi, the results provide a general source of molecular markers that could be useful for a variety of applications such as population genetics and strain identification of fungal organisms.     

Challenges of structural genomics: bioinformatics

Structural genomics projects bring us many challenges, many of which were not anticipated when such initiatives were first planned and introduced. For instance, the huge amount of data generated within the project must be collected, displayed and analyzed to reap the benefits of this huge investment. Projects at the San Diego based Joint Center for Structural Genomics provide an example of how data can be managed within a structural genomics project, and how results can be presented on the web, as well as highlight some of the issues concerning data analysis.     

Towards a functional resource-based concept for habitat: a butterfly biology viewpoint

The habitat is the basic unit for developments in life history, population dynamics, landscape ecology and conservation of organisms. It is frequently treated as a particulate, invariant and homogeneous entity (a patch). Here we examine the implications of using this concept of habitat in butterfly biology. In doing so, we suggest the alternative approach of applying a functional resource-based concept of habitat. This recognises the fundamental requirements of organisms, consumables and utilities, the latter describing suitable environmental conditions as well as essential substrates. We argue that a resource-based concept is critical for butterfly conservation and call for the development of a resource database on butterfly biology .     

Invisible genomes: the genomics revolution and patenting practice

In the mid-1990s, the company Human Genome Sciences submitted three potentially revolutionary patent applications to the US Patent and Trademark Office, each of which claimed the entire genome sequence of a microorganism. The patent examiners, however, objected to these applications, and after negotiation they were eventually re-written to resemble more traditional gene patents. In this paper, which is based on a study of the patent examination files, we examine the reasons why these patent applications were unsuccessful in their original form. We show that with respect to utility and novelty, the patent attorney's case built on an understanding of the genome as a computer-related invention. The patent examiners did not object to the patenting of complete genome sequences as computer-related inventions on moral grounds or in terms of the distinction between a discovery and an invention. Instead, their objections were based on classification, rules and procedure. Rather than patent examiners having a notion of a genome that should not be patented, the notion of a 'genome', and the ways in which it may be different from a 'gene', played no role in these debates. We discuss the consequences of our findings for patenting in the biosciences.     

Mycobacterium tuberculosis: a model system for structural genomics

Over the past five years, genomics has had a major impact on Mycobacterium tuberculosis research. With the publication of the sequences of two virulent strains (H37Rv and CDC1551) and three closely related sequences, M. tuberculosis is becoming a model system for proteomics and structural genomics initiatives. Together with the promise of structures of proteins with novel folds, high-resolution structures of drug targets are providing the basis for rational inhibitor design, with the goal of the development of novel anti-tuberculars. In addition, this work is aiding scientists in the quest for an effective vaccine against this persistent pathogen.     

Activation of nuclear receptors: a perspective from structural genomics

Crystal structures of more than two dozen different nuclear receptor ligand binding domains have defined a simple paradigm of receptor activation, in which agonist binding induces the activation function-2 (AF-2) helix to form a charge clamp for coactivator recruitment. Recent structural studies present a surprising contrast. Activation of the mouse LRH-1 receptor is independent of a bound agonist despite its large ligand binding pocket, whereas the activation of the Drosophila DHR38 receptor is dependent on ecdysteroids even though the receptor lacks a ligand binding pocket. These new findings shed light on the diverse structural mechanisms that nuclear receptors have evolved for activation, and have important implications in their respective signaling pathways.     

Comprehensive genome analysis of 203 genomes provides structural genomics with new insights into protein family space

We present an analysis of 203 completed genomes in the Gene3D resource (including 17 eukaryotes), which demonstrates that the number of protein families is continually expanding over time and that singleton-sequences appear to be an intrinsic part of the genomes. A significant proportion of the proteomes can be assigned to fewer than 6000 well-characterized domain families with the remaining domain-like regions belonging to a much larger number of small uncharacterized families that are largely species specific. Our comprehensive domain annotation of 203 genomes enables us to provide more accurate estimates of the number of multi-domain proteins found in the three kingdoms of life than previous calculations. We find that 67% of eukaryotic sequences are multi-domain compared with 56% of sequences in prokaryotes. By measuring the domain coverage of genome sequences, we show that the structural genomics initiatives should aim to provide structures for less than a thousand structurally uncharacterized Pfam families to achieve reasonable structural annotation of the genomes. However, in large families, additional structures should be determined as these would reveal more about the evolution of the family and enable a greater understanding of how function evolves.     

TargetDB: a target registration database for structural genomics projects

TargetDB is a centralized target registration database that includes protein target data from the NIH structural genomics centers and a number of international sites. TargetDB, which is hosted by the Protein Data Bank (RCSB PDB), provides status information on target sequences and tracks their progress through the various stages of protein production and structure determination. A simple search form permits queries based on contributing site, target ID, protein name, sequence, status and other data. The progress of individual targets or entire structural genomics projects may be tracked over time, and target data from all contributing centers may also be downloaded in the XML format. AVAILABILITY: TargetDB is available at http://targetdb.pdb.org/     

Target selection for structural genomics: a single genome approach

We describe our strategy for selecting targets for protein structure determination in context of structural genomics of a single genome. In the course of target selection, we have studied two of the smallest microbial genomes, Mycoplasma genitalium and Mycoplasma pneumoniae. To our surprise, we found that only 71 Mycoplasma genes or their orthologues can be considered as easy targets for high-throughput structural studies--far fewer than expected. We discuss the methods and criteria used for target selection and the reasons explaining rarity of easy targets. First, despite the common opinion that protein folds can be predicted for only 30-50% of genes, the number of "truly unknown" structures is less than one-third. Second, due to the different codon usage, two thirds of Mycoplasma proteins cannot be directly expressed in E. coli in high-throughput manner and require substitution by their homologues from other organisms. Third, membrane or large multi-domain proteins are difficult targets because of solubility and size issues and often require identification and structure determination of protein domains. Finally, we propose different approaches to address the difficult targets.