Weak preservation of local neutral substitution rates across mammalian genomes

<p>Abstract</p> <p>Background</p> <p>The rate at which neutral (non-functional) bases undergo substitution is highly dependent on their location within a genome. However, it is not clear how fast these location-dependent rates change, or to what extent the substitution rate <it>patterns </it>are conserved between lineages. To address this question, which is critical not only for understanding the substitution process but also for evaluating phylogenetic footprinting algorithms, we examine ancestral repeats: a predominantly neutral dataset with a significantly higher genomic density than other datasets commonly used to study substitution rate variation. Using this repeat data, we measure the extent to which orthologous ancestral repeat sequences exhibit similar substitution patterns in separate mammalian lineages, allowing us to ascertain how well local substitution rates have been preserved across species.</p> <p>Results</p> <p>We calculated substitution rates for each ancestral repeat in each of three independent mammalian lineages (primate – from human/macaque alignments, rodent – from mouse/rat alignments, and laurasiatheria – from dog/cow alignments). We then measured the correlation of local substitution rates among these lineages. Overall we found the correlations between lineages to be statistically significant, but too weak to have much predictive power (<it>r</it>²<<it>5%</it>). These correlations were found to be primarily driven by regional effects at the scale of several hundred kb or larger. A few repeat classes (e.g. 7SK, Charlie8, and MER121) also exhibited stronger conservation of rate patterns, likely due to the effect of repeat-specific purifying selection. These classes should be excluded when estimating local neutral substitution rates.</p> <p>Conclusion</p> <p>Although local neutral substitution rates have some correlations among mammalian species, these correlations have little predictive power on the scale of individual repeats. This indicates that local substitution rates have changed significantly among the lineages we have studied, and are likely to have changed even more for more diverged lineages. The correlations that do persist are too weak to be responsible for many of the highly conserved elements found by phylogenetic footprinting algorithms, leading us to conclude that such elements must be conserved due to selective forces.</p

Substitution Patterns Are GC-Biased in Divergent Sequences across the Metazoans

The fastest-evolving regions in the human and chimpanzee genomes show a remarkable excess of weak (A,T) to strong (G,C) nucleotide substitutions since divergence from their common ancestor. We investigated the phylogenetic extent and possible causes of this weak to strong (W→S) bias in divergent sequences (BDS) using recently sequenced genomes and recombination maps from eight trios of eukaryotic species. To quantify evidence for BDS, we inferred substitution histories using an efficient maximum likelihood approach with a context-dependent evolutionary model. We then annotated all lineage-specific substitutions in terms of W→S bias and density on the chromosomes. Finally, we used the inferred substitutions to calculate a BDS score—a log odds ratio between substitution type and density—and assessed its statistical significance with Fisher's exact test. Applying this approach, we found significant BDS in the coding and noncoding sequence of human, mouse, dog, stickleback, fruit fly, and worm. We also observed a significant lack of W→S BDS in chicken and yeast. The BDS score varies between species and across the chromosomes within each species. It is most strongly correlated with different genomic features in different species, but a strong correlation with recombination rates is found in several species. Our results demonstrate that a W→S substitution bias in fast-evolving sequences is a widespread phenomenon. The patterns of BDS observed suggest that a recombination-associated process, such as GC-biased gene conversion, is involved in the production of the bias in many species, but the strength of the BDS likely depends on many factors, including genome stability, variability in recombination rate over time and across the genome, the frequency of meiosis, and the amount of outcrossing in each species

JCoDA: a tool for detecting evolutionary selection

<p>Abstract</p> <p>Background</p> <p>The incorporation of annotated sequence information from multiple related species in commonly used databases (Ensembl, Flybase, Saccharomyces Genome Database, Wormbase, etc.) has increased dramatically over the last few years. This influx of information has provided a considerable amount of raw material for evaluation of evolutionary relationships. To aid in the process, we have developed JCoDA (Java Codon Delimited Alignment) as a simple-to-use visualization tool for the detection of site specific and regional positive/negative evolutionary selection amongst homologous coding sequences.</p> <p>Results</p> <p>JCoDA accepts user-inputted unaligned or pre-aligned coding sequences, performs a codon-delimited alignment using ClustalW, and determines the dN/dS calculations using PAML (Phylogenetic Analysis Using Maximum Likelihood, yn00 and codeml) in order to identify regions and sites under evolutionary selection. The JCoDA package includes a graphical interface for Phylip (Phylogeny Inference Package) to generate phylogenetic trees, manages formatting of all required file types, and streamlines passage of information between underlying programs. The raw data are output to user configurable graphs with sliding window options for straightforward visualization of pairwise or gene family comparisons. Additionally, codon-delimited alignments are output in a variety of common formats and all dN/dS calculations can be output in comma-separated value (CSV) format for downstream analysis. To illustrate the types of analyses that are facilitated by JCoDA, we have taken advantage of the well studied sex determination pathway in nematodes as well as the extensive sequence information available to identify genes under positive selection, examples of regional positive selection, and differences in selection based on the role of genes in the sex determination pathway.</p> <p>Conclusions</p> <p>JCoDA is a configurable, open source, user-friendly visualization tool for performing evolutionary analysis on homologous coding sequences. JCoDA can be used to rapidly screen for genes and regions of genes under selection using PAML. It can be freely downloaded at <url>http://www.tcnj.edu/~nayaklab/jcoda</url>.</p

Distribution and phylogeny of the bacterial translational GTPases and the Mqsr/YgiT regulatory system

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Valgud on raku ehituskivideks ja eluks vajalike reaktsioonide katalüüsijateks. Bioinformaatika on meid varustanud võimsate järjestuste analüüsi vahenditega. Järjestuse sarnasuse alusel grupeeruvad valgud perekondadeks. Valguperekonna moodustavad homoloogsed järjestused ehk siis järjestused, mis pärinevad samast eellasjärjestusest. Tihti omavad samasse perekonda kuuluvad valgud ka sama või üksteisele lähedast funktsiooni. Meie teadmised valkude funktsioonidest pärinevad üksikutelt mudelorganismidelt. Tihti huvitab teadlasi kui universaalne või spetsiifiline on üks või teine kirjeldatud funktsioon. Kuidas ja millal evolutsiooni käigus tekib olemasolevast materjalist uute omadustega (uue funktsiooniga) valk läbi geeniduplikatsiooni? Kui tihti on sellised sündmused evolutsioonilises ajaskaalas aset leidud? Oma töös olen ma analüüsinud bakterite translatsioonilisi GTPaase (trGTPaas) ja mqsR/ygiT toksiin-antitoksiin (TA) süsteemi valke. Ühiseks nime¬¬tajaks mõlemale on valgusünteesi aparaat – mõlemad on seotud ribosoomiga ja sealtkaudu raku võimega sõltuvalt vajadusele toota valke. Küsimused, mida selles kontekstis on küsitud, saab laias laastus jagada kaheks: a) valguperekonna esindatusega seotud ja b) valguperekonna evolutsiooni ja funktsionaalse innovatsiooniga seotud. Translatsiooniliste GTPaaside puhul bakterites saame rääkida üheksast erinevast perekonnast – üheksast erinevast funktsioonide komplektist. Täisgenoomidele põhinev analüüs näitas, et üheksast trGTPaaside perekonnast on bakterites konserveerunud neli: IF2, EF-Tu, EFG ja LepA(EF4). Vaatamata sellele, et RF3’e on omistatud klassikalise valgusünteesi mudeli valguses kanooniline roll translatsiooni lõpetamisel, puudus RF3 geen ligikaudu 40% analüüsitud bakteri genoomides. Samas aga ebaselge funktsiooniga LepA osutus bakterite spetsiifiliseks trGTPaasiks. Eelnev analüüs tõi ka välja EFG paraloogide laia esinemise – paljud bakteri¬genoomid sisaldasid 2–3 üksteisest küllaltki erinevat (divergeerunud) EFG geeni. Lähem analüüs tõi välja, et kogu varieeruvuse EFG perekonnas võib jagada neljaks alamperekonnaks: EFG I, spdEFG1, spdEFG2 ja EFG II. Eksperimentaalselt on hästi iseloomustatud EFG I. Uuritud on ka spdEFG’sid ja leitud, et esimene neist omab translokaasi aktiivsust translatsioonil ja teine osaleb ribosoomide retsükleerimisel. Laialt levinud EFG II alamperekond on aga halvasti uuritud. Fülogeneetiline analüüs võimaldab püstitada hüpoteesi nelja EFG alamperekonna iidsest päritolust, st. nad on tekkinud ajalises skaalas enne (või samaaegselt) eukarüootse rakuvormi lahknemist arhedest ja bakteritest. Funktsionaalse innovatsiooni kandjaks EFG II valgus võib pidada eelkõige 12 positsiooni, mis on spetsiifiliselt konserveerunud just EFG II alamperekonnal. EFG II’e iseloomulikus kõrge divergentsuse taustal tõusevad need positsioonid esile GTPaasi domäänis, domäänis II ja neljandas domäänis. Konserveerunud muutused GTPaasi domäänis, millest osad on GTP’d siduvas G1 motiivis, võimaldavad teha järeldusi muutunud GTP sidumise ja hüdrolüüsi tingimuste kohta. Suurenenud laeng neljanda domääni lingu otsas, mis E. coli EFG’l siseneb A-saiti, võimaldab spekuleerida muutuse üle translokatsiooni keskkonnas. Konserveerunud muutused domään II piirkonnas viitavad muutunud interaktsioonile ribosoomi, domään I ja domään III vahel. EFG II alamperekonna fülogeneetiline ja järjestuste analüüs näitab selgelt hõimkonna/klassi spetsiifiliste alam-alamgruppide olemasolu. Need alam-alamgrupid erinevad teineteisest G2 motiivi konserveeruvuse ja insertsioonide/deletsioonide mustri alusel. See teine tase kirjeldab EFG II kui hõimkonna/klassi spetsiifilist faktorit. Mis on EFG II roll tegelikult ja kuidas ning millistes tingimustes ta komplementeerib EFG I, ootab alles vastuseid. Antud töö on loonud raamistiku tulevaste eksperimentide tarvis.Proteins are vital for the cell – they serve as building blocks and catalysts for many different reactions. Bioinformatics has equipped us with powerful analysis tools. According to sequence similarity, proteins can be grouped into families. Protein family is composed of homologous sequences, i. e. from sequences, which share a common ancestor. Proteins, which belong to the same family, perform their function in a similar way. Our knowledge about functional properties of proteins originates from experimental works performed with a limited number of model organisms. Scientists are often interested in the universality or specificity of one or another described protein and function. How often is gene duplication and following innovation the source for genes/proteins with a new function? How often such events take place in the evolutionary timescale? In my dissertation I have analyzed gene and protein sequences of translational GTPases (trGTPases) and mqsR/ygiT toxin-antitoxin of bacteria. Common denominator for both protein families is their connection to cells protein synthesis machinery. Two types of questions can be asked in this context: those that are related to a) the representation of specific proteins/function, and b) the evolution and functional innovation. In the case of trGTPases nine different protein families, i. e. presence or absence of nine different functional complexes in the cell were described. Analyzes carried on completed genome sequences of bacteria revealed four conserved families: IF2, EF-Tu, EFG, and LepA(EF4). Despite the fact that in the classical model of protein synthesis RF3 carries canonic role at the final step of translation, RF3 coding gene was found missing approximately in 40% of analyzed bacteria. Surprisingly, LepA, whose function is still not well understood, appears to be specific trGTPase for bacteria. The analysis also revealed a wide distribution of EFG paralogs – many bacteria contained two to three relatively diverged gene copies for EFG. The phylogenetic tree of EFG revealed four subfamilies: EFG I, spdEFG1, spdEFG2, and EFG II. The EFG I subfamily is experimentally well characterized. Also, spdEFG1 was found to act as translocase and spdEFG2 helps recycle ribosome, indicating functional split between co-occurring paralogs. However, little research has been done on widely distributed EFG II subfamily. Phylogenetic analyses, performed by us, enable to propose hypothesis about ancient origin of EFG subfamilies - they have appeared at the same timescale with (or even before) arousing eukaryotic life-forms. Functional innovation, common for the whole subfamily, is carried by 12 EFG II specific positions. In contrast to overall high divergeny, these conserved positions have spotlighted in the GTPase domain, and in the domain II and IV. Conserved changes in the GTPase domain, some of which are located in the G1 motif, indicate changed conditions in GTP binding and hydrolysis. Increased charge in protruding loop of the fourth domain, which inserts into A-site, enables us to speculate about changes in the local conditions of the A-site during translocation. Conserved changes in the domain II indicate changed interaction between EFG domains I, II, and III and the ribosome. Phylogenetic analysis of the EFG II subfamily reveals phyla/class specific sub-subgroups. These sub-subgroups differ from each other by conserved amino acids pattern of the G2 motif and insertion/deletion pattern detected from multiple sequence alignment. This another level characterizes EFG II as phyla/class specific factor. Further research should be conducted on what role EFG II actually performs and how it complements EFG I. Current study can serve as framework for future experiments

Twinscan: A Software Package for Homology-Based Gene Prediction

A complete mapping from genome to proteome would constitute a foundation for genome-based biology and provide targets for pharmaceutical and therapeutic intervention. This is one reason gene structure prediction has been a major subfield of computational biology for over 20 years. Many of the widely used gene prediction systems were developed in the 1990s and are unable to take advantage of the revolution in comparative genomics brought on by the sequencing of the entire genomes of an increasing numbers of vertebrates. Twinscan is a new system for high-throughput gene-structure prediction that exploits the patterns of conservation observed in alignments between a target genomic sequence and its homologous sequence in other organisms. The approach employs a symbolic conservation sequence that effectively combines many local alignments into a single global alignment. This has several important properties that make Twinscan particularly useful for high-throughput gene prediction. For mammals, Twinscan has been shown to be significantly more accurate and reliable by all measures than any non-comparative genomic method. Twinscan is based on, and includes as a component, the same hidden Markov model topology as Genscan, a popular non-homology based gene prediction program. Twinscan has an object-oriented design and is implemented in the C++ programming language. Twinscan’s three major components consist of probabilistic models of both the DNA sequence and the conservation sequence as well as a dynamic programming framework. Both the models and the computational structure are complicated aggregate classes. In this report, the design and implementation of Twinscan is described at the source-code level for the first time

Bridging In Vi Tro & In S Il Ico Techniques: Uncovering the Structural Characteristics and Light-Dependent Modifications of the Light-Response Btb Proteins in Arabidopsis Thaliana

The conjugation of the NEDD8 protein has been found to cause a wide range of changes to the overall function of a protein. The addition of NEDD8 to a conserved lysine on Cul proteins has been found to provide Cul with increased flexibility, while the transcription factor protein p53 was prevented from binding to DNA after NEDD8 conjugation. Using Arabidopsis thaliana, we investigated the potential conjugation of NEDD8 to the AtLRB1 and AtLRB2 proteins, which are both members of Cul3-RING E3 Ubiquitin Ligase complexes, using both in vitro and in silico techniques. The AtLRBs are negative regulators of the red light response pathway, interacting preferentially with Cul3 in red light conditions by an unknown mechanism. Our investigation into the amino-terminal portion of AtLRB2 (residues 1-144) found two conserved regions which contained high sequence identity with Cul1 and Rbx1, two members of Cullin-RING ligase complexes. These two conserved regions were also found to share a similar distribution and placement on both the native crystal structure of Cullin proteins in complex with Rbx1 and on the predicted AtLRB2 structural protein model. Furthermore, the region of Rbx1 sharing sequence similarity to the LRBs directly interacts with NEDD8. Preliminary in vitro results also suggest that purified AtLRB2 is modified by NEDD8, but the significance of this modification on the function of the LRB proteins is not yet known

Identification of Deleterious and Disease Alleles in a General Population and Preterm Labor Patients

With the recent advance in sequencing technology, there have been growing interests in developing new methods to predict disease-causing alleles in a personal genome by integrating functional evidences from sequence conservation, genome-wide association studies and the transcriptional regulatory network. However, even in protein-coding regions, it is not well understood how often and by what mechanism deleterious alleles disrupting strong sequence conservation can become common in population frequency and affect complex traits in humans. Moreover, in non-coding regions, even for known disease-causing genes, it is not clear how sequence conservation can be combined with functional genomic data to predict underlying disease-causing variants. To address the first question, I developed a new likelihood ratio test for sequence conservation to predict deleterious missense alleles in the human genome. By applying the new test to three personal genomes, I find that the presence of only 10% of common deleterious SNPs can be explained by false positives due to multiple hypothesis testing, violation of evolutionary model assumptions, recent gene duplication and relaxation of selective constraints on biological processes. Next, by applying the likelihood ratio test to a general human population, I find that both computationally predicted deleterious SNPs and known disease-associated alleles are enriched within genomic regions that have been influenced by positive selection in the recent past. The observed pattern agrees with the prediction that deleterious alleles can dragged along to higher-than-expected allele frequencies due to the genetic linkage with beneficial alleles by the hitchhiking effect. Second, I developed an integrative strategy to predict disease-causing non-coding variants in FSH receptor, a gene known to be associated with preterm birth, as a proof of principle. I sequenced protein-coding and conserved non-coding regions in preterm and term mothers, and conducted fine-mapping and transcription factor binding site analysis to narrow down the causal non-coding variants. Here, I find that in non-coding regions the causal variants can be resolved better by accounting for the expected effects of binding site mutations on the transcription regulatory network in addition to sequence conservation. These results indicate that the comparative genomics will provide the new opportunity to explore deleterious and disease-causing genetic variation at an unprecedentedly high resolution across the genome and in a population especially if functional genomics can be integrated with comparative genomics