In 1975, Elongation Factor P (EF-P) protein was first discovered in the bacterium Escherichia coli. EF-P is believed to facilitate the translation of proteins by stimulating peptide bond synthesis for a number of different aminoacyl-tRNA molecules in conjunction with the 70S ribosome peptidyl transferase. Known eukaryotic homologs, eukaryotic translation initiation factor 5A (eIF-5A) of EF-P exist but with very low sequence conservation. Nevertheless, because of the high sequence similarities seen between bacterial EF-Ps and its low sequence similarity with eIF-5A, there is interest in the pharmaceutical industry of developing a novel antibacterial drug that inhibits EF-P. Of 322 completely sequenced bacterial genomes stored in GenBank, only one organism lacked an EF-P protein. Interestingly, sixty-six genomes were discovered to carry a duplicate copy of efp. The EF-P sequences were then used to construct a protein phylogenetic tree, which provided evidence of horizontal and vertical gene transfer as well as gene duplication. To lend support to these findings, EF-P GC content, codon usage, and nucleotide and amino acid sequences were analyzed with positive and negative controls. The adjacent 10 kb upstream and downstream regions of efp were also retrieved to determine if gene order is conserved in distantly related species. While gene order was not preserved in all species, two interesting trends were seen in some of the distantly related species. The EF-P gene was conserved beside Acetyl-CoA carboxylase genes, accB and accC in certain organisms. In addition, some efp sequences were flanked by two insertion sequence elements. Evidence of gene duplication and horizontal transfers of regions were also observed in the upstream and downstream regions of efp. In combination, phylogenetic, sequence analyses, and gene order conservation confirmed evidence of the complex history of the efp genes, which showed incongruencies relative to the universal phylogenetic tree. To determine how efp is regulated, the upstream regions of efp were used to try to predict motifs in silico. While statistically significant motifs were discovered in the upstream regions of the orthologous efp genes, no conclusive similarities to known binding sites such as the sigma factor binding sites or regulatory protein binding sites were observed. This work may facilitate and enhance the understanding of the regulation, conservation, and role of EF-P in protein translation.

In 1975, Elongation Factor P (EF-P) protein was first discovered in the bacterium Escherichia coli. EF-P is believed to facilitate the translation of proteins by stimulating peptide bond synthesis for a number of different aminoacyl-tRNA molecules in conjunction with the 70S ribosome peptidyl transferase. Known eukaryotic homologs, eukaryotic translation initiation factor 5A (eIF-5A) of EF-P exist but with very low sequence conservation. Nevertheless, because of the high sequence similarities seen between bacterial EF-Ps and its low sequence similarity with eIF-5A, there is interest in the pharmaceutical industry of developing a novel antibacterial drug that inhibits EF-P. Of 322 completely sequenced bacterial genomes stored in GenBank, only one organism lacked an EF-P protein. Interestingly, sixty-six genomes were discovered to carry a duplicate copy of efp. The EF-P sequences were then used to construct a protein phylogenetic tree, which provided evidence of horizontal and vertical gene transfer as well as gene duplication. To lend support to these findings, EF-P GC content, codon usage, and nucleotide and amino acid sequences were analyzed with positive and negative controls. The adjacent 10 kb upstream and downstream regions of efp were also retrieved to determine if gene order is conserved in distantly related species. While gene order was not preserved in all species, two interesting trends were seen in some of the distantly related species. The EF-P gene was conserved beside Acetyl-CoA carboxylase genes, accB and accC in certain organisms. In addition, some efp sequences were flanked by two insertion sequence elements. Evidence of gene duplication and horizontal transfers of regions were also observed in the upstream and downstream regions of efp. In combination, phylogenetic, sequence analyses, and gene order conservation confirmed evidence of the complex history of the efp genes, which showed incongruencies relative to the universal phylogenetic tree. To determine how efp is regulated, the upstream regions of efp were used to try to predict motifs in silico. While statistically significant motifs were discovered in the upstream regions of the orthologous efp genes, no conclusive similarities to known binding sites such as the sigma factor binding sites or regulatory protein binding sites were observed. This work may facilitate and enhance the understanding of the regulation, conservation, and role of EF-P in protein translation.

Mitochondrial genomes provide a valuable dataset for phylogenetic studies, in particular of metazoan phylogeny because of the extensive taxon sample that is available. Beyond the traditional sequence-based analysis it is possible to extract phylogenetic information from the gene order. Here we present a novel approach utilizing these data based on cyclic list alignments of the gene orders. A progressive alignment approach is used to combine pairwise list alignments into a multiple alignment of gene orders. Parsimony methods are used to reconstruct phylogenetic trees, ancestral gene orders, and consensus patterns in a straightforward approach. We apply this method to study the phylogeny of protostomes based exclusively on mitochondrial genome arrangements. We, furthermore, demonstrate that our approach is also applicable to the much larger genomes of chloroplasts.

A seqüência completa do genoma mitocondrial de uma espécie pode ajudar no mapeamento de restrição e desenho de primers para PCR. Estes poderão servir para amplificação e posterior seqüenciamento de regiões específicas de outras espécies e populações relacionadas, para estudos filogenéticos e de dinâmica populacional. Até o momento, temos na literatura a seqüência completa do DNA mitocondrial (DNAmt) de um único himenóptero, Apis mellifera, espécie que é endêmica do Velho Mundo. Nenhum genoma mitocondrial de uma espécie de abelha nativa do Brasil foi até o momento descrito. Com a devastação crescente dos ecossistemas, há a perda de espécies de abelhas ainda pouco estudadas, e talvez até outras ainda não conhecidas. Entre os meliponíneos, há espécies-chave de diversos ecossistemas brasileiros, tendo portanto uma enorme importância ecológica. No decorrer deste projeto, foram amplificados via PCR e seqüenciados 77% do genoma mitocondrial da abelha sem ferrão Melipona bicolor (Apidae, Meliponini), contendo todos os 13 genes mitocondriais codificadores para proteínas, 18 dos 22 genes para RNAt e os dois genes para RNAr (sendo um integral e o outro parcialmente seqüenciado). Além do seqüenciamento, foram realizados neste trabalho: análise da organização do genoma (conteúdo e ordem gênica); análise da tradução dos genes para proteínas e código genético; análise de outras características moleculares (freqüência das bases, códons utilizados, iniciação e terminação de genes, freqüência de aminoácidos etc); e comparação das características acima mencionadas com o genoma mitocondrial de A. mellifera e também com outros insetos. O viés para o uso de bases A+T, bastante evidente em A. mellifera, mostrou-se ainda mais acentuado em M. bicolor. Foram encontradas diferenças no tamanho e composição dos genes. Pelo menos nove rearranjos na ordem gênica mitocondrial foram observados entre as duas espécies de abelhas, um fenômeno raro entre organismos tão próximos. Considerando que essas espécies compartilham um comportamento intrigante, a eussocialidade, esses rearranjos podem servir como um excelente marcador para estudar a origem e a evolução desse comportamento no grupo. / The complete sequence of the mitochondrial genome of a species may help on restriction mapping and to design PCR primers. These can be useful to amplify and sequence specific regions from other species and analyze populations, in phylogenetic and demographic studies. So far, there was reported on literature the mtDNA complete sequence for only one hymenopteran, Apis mellifera, endemic from the Old World. No mitocondrial genome of a Brazilian native bee was ever described. With the increasing devastation of natural environments, several bee species can be led to extinction, including those poorly studied and maybe some unknown species. The meliponines (stingless bees) include key species to several Brazilian ecosystems, so they play an important ecological role. In this project, we have PCR amplified and sequenced 77% of the mitochondrial genome of the stingless bee Melipona bicolor (Apidae, Meliponini). The sequenced region contains all of the 13 mitochondrial protein-coding genes, 18 of 22 tRNA genes, and both rRNA genes (one of them was only partially sequenced). Besides sequencing, this work consisted of: analysis of genome organization (gene content and order); analysis of gene translation and genetic code; analysis of other molecular features (base frequencies, codon usage, gene initiation and termination, amino acid frequencies etc.); and comparison of the characteristics mentioned above with A. mellifera mitocondrial genome and also other insects. The highly biased A+T content is a typical characteristic of A. mellifera mitochondrial genome, and it is even more extreme on M. bicolor mtDNA. There are length and compositional differences on genes between M. bicolor and A. mellifera. At least nine gene order rearrangements were observed by comparing the mtDNA of these species, what is a rare event on closely related organisms. Considering that both species share an intriguing behavior, eusociality, these gene rearrangements may be used as an excellent marker to study the origin and evolution of that behavior on bees.

A seqüência completa do genoma mitocondrial de uma espécie pode ajudar no mapeamento de restrição e desenho de primers para PCR. Estes poderão servir para amplificação e posterior seqüenciamento de regiões específicas de outras espécies e populações relacionadas, para estudos filogenéticos e de dinâmica populacional. Até o momento, temos na literatura a seqüência completa do DNA mitocondrial (DNAmt) de um único himenóptero, Apis mellifera, espécie que é endêmica do Velho Mundo. Nenhum genoma mitocondrial de uma espécie de abelha nativa do Brasil foi até o momento descrito. Com a devastação crescente dos ecossistemas, há a perda de espécies de abelhas ainda pouco estudadas, e talvez até outras ainda não conhecidas. Entre os meliponíneos, há espécies-chave de diversos ecossistemas brasileiros, tendo portanto uma enorme importância ecológica. No decorrer deste projeto, foram amplificados via PCR e seqüenciados 77% do genoma mitocondrial da abelha sem ferrão Melipona bicolor (Apidae, Meliponini), contendo todos os 13 genes mitocondriais codificadores para proteínas, 18 dos 22 genes para RNAt e os dois genes para RNAr (sendo um integral e o outro parcialmente seqüenciado). Além do seqüenciamento, foram realizados neste trabalho: análise da organização do genoma (conteúdo e ordem gênica); análise da tradução dos genes para proteínas e código genético; análise de outras características moleculares (freqüência das bases, códons utilizados, iniciação e terminação de genes, freqüência de aminoácidos etc); e comparação das características acima mencionadas com o genoma mitocondrial de A. mellifera e também com outros insetos. O viés para o uso de bases A+T, bastante evidente em A. mellifera, mostrou-se ainda mais acentuado em M. bicolor. Foram encontradas diferenças no tamanho e composição dos genes. Pelo menos nove rearranjos na ordem gênica mitocondrial foram observados entre as duas espécies de abelhas, um fenômeno raro entre organismos tão próximos. Considerando que essas espécies compartilham um comportamento intrigante, a eussocialidade, esses rearranjos podem servir como um excelente marcador para estudar a origem e a evolução desse comportamento no grupo. / The complete sequence of the mitochondrial genome of a species may help on restriction mapping and to design PCR primers. These can be useful to amplify and sequence specific regions from other species and analyze populations, in phylogenetic and demographic studies. So far, there was reported on literature the mtDNA complete sequence for only one hymenopteran, Apis mellifera, endemic from the Old World. No mitocondrial genome of a Brazilian native bee was ever described. With the increasing devastation of natural environments, several bee species can be led to extinction, including those poorly studied and maybe some unknown species. The meliponines (stingless bees) include key species to several Brazilian ecosystems, so they play an important ecological role. In this project, we have PCR amplified and sequenced 77% of the mitochondrial genome of the stingless bee Melipona bicolor (Apidae, Meliponini). The sequenced region contains all of the 13 mitochondrial protein-coding genes, 18 of 22 tRNA genes, and both rRNA genes (one of them was only partially sequenced). Besides sequencing, this work consisted of: analysis of genome organization (gene content and order); analysis of gene translation and genetic code; analysis of other molecular features (base frequencies, codon usage, gene initiation and termination, amino acid frequencies etc.); and comparison of the characteristics mentioned above with A. mellifera mitocondrial genome and also other insects. The highly biased A+T content is a typical characteristic of A. mellifera mitochondrial genome, and it is even more extreme on M. bicolor mtDNA. There are length and compositional differences on genes between M. bicolor and A. mellifera. At least nine gene order rearrangements were observed by comparing the mtDNA of these species, what is a rare event on closely related organisms. Considering that both species share an intriguing behavior, eusociality, these gene rearrangements may be used as an excellent marker to study the origin and evolution of that behavior on bees.

This thesis presents a novel approach to reconstructing ancestral genomes of a number of descendant species related by a phylogeny. Traditional methods face challenges due to cycles of whole genome doubling followed by fractionation in plant lineages. In response, the thesis proposes a new approach that first accumulates a large number of candidate gene adjacencies specific to each ancestor in a phylogeny. A subset of these which to produces long ancestral contigs are chosen through maximum weight matching. The strategy results in more complete reconstructions than existing methods, and a number of quality measures are deployed to assess the results. The thesis also presents a new computational technique for estimating the ancestral monoploid number of chromosomes, involving a "g-mer" analysis to resolve a bias due to long contigs and gap statistics to estimate the number. The method is applied to a set of phylogenetically related descendant species, and the monoploid number is found to be 9 for all rosid and asterid orders. Additionally, the thesis demonstrates that this result is not an artifact of the method, by deriving a monoploid number of approximately 20 for the metazoan ancestor. The reconstructed ancestral genomes are functionally annotated and visualized through painting ancestral projections on descendant genomes and highlighting syntenic ancestor-descendant relationships. The proposed method is applied to genomes drawn from a broad range of plant orders. The Raccroche pipeline reconstructs ancestral gene orders and chromosomal contents of the ancestral genomes at all internal vertices of a phylogenetic tree, and constructs chromosomes by counting the frequencies of ancestral contig co-occurrence on the extant genomes, clustering these for each ancestor, and ordering them. Overall, this thesis presents a significant contribution to the field of ancestral genome reconstruction, offering a new approach that produces more complete reconstructions and provides valuable insights into the evolutionary process giving rise to the gene content and order of extant genomes.

The evolutionary origin and the phylogeny of higher metazoan taxa is still under debate although considerable progress has been made in the past 20 years. Metazoa represents a monophyletic group of highly diverse animals including Bilateria, Cnidaria, Porifera, Ctenophores, and Placozoa. Bilateria comprises the majority of metazoans and consists of three major clades: Deuterostomia, Spiralia (= Lophotrochozoa sensu lato), and Ecdysozoa, whereas the sister group taxa Spiralia and Ecdyzozoa form the monophyletic clade Protostomia. Molecular data have profoundly changed the view of the bilaterian tree of life. One of the main questions concerning bilaterian phylogeny is the on-going debate about the evolution of complexity in Bilateria. It was assumed that the last common ancestor of Deuterostomia, Ecdysozoa and Spiralia had a segmented and coelomate body organization resembling that of an annelid. On the contrary, the traditional view is the evolution of Bilateria from a simple body organization towards more complex forms, assuming that the last common ancestor of Bilateria resembles a platyhelminth-like animal without coelomic cavities and segmentation. To resolve this question, it is necessary to unravel the phylogenetic relationships within Bilateria. By using mitogenomic and phylogenomic data, this thesis had a major contribution to clarify phylogenetic relationships within problematic metazoan taxa: (1) the phylogeny of Deuterostomia, (2) the questionable monophyly of Platyzoa, and first assumptions concerning the phylogeny of Gnathostomulida, Gastrotricha and Polycladida, (3) phylogenetic relationships within annelid taxa, especially Terebelliformia, Diurodrilidae, and Syllidae, with new insights into the evolution of mitochondrial gene order, and (4) new insights into the evolution of annelids, especially the interstitial ones, as well as the colonization of the interstitial realm.

Indiana University-Purdue University Indianapolis (IUPUI) / My thesis comprises of two individual projects: 1) we have developed a database for operon prediction using high-throughput sequencing datasets for bacterial genomes. 2) Genomics and mechanistic insights of convergent transcription in bacterial genomes. In the first project we developed a database for the prediction of operons for bacterial genomes using RNA-seq datasets, we predicted operons for bacterial genomes. RNA-seq datasets with different condition for each bacterial genome were taken into account and predicted operons using Rockhopper. We took RNA-seq datasets from NCBI with distinct experimental conditions for each bacterial genome into account and analyzed using tool for operon prediction. Currently our database contains 9 bacterial organisms for which we predicted operons. User interface is simple and easy to use, in terms of visualization, downloading and querying of data. In our database user can browse through reference genome, genes present in that genome and operons predicted from different RNA-seq datasets. Further in the second project, we studied the genomic and mechanistic insights of convergent transcription in bacterial genomes. We know that convergent gene pairs with overlapping head-to-head configuration are widely spread across both eukaryotic and prokaryotic genomes. They are believed to contribute to the regulation of genes at both transcriptional and post-transcriptional levels, although factors contributing to their abundance across genomes and mechanistic basis for their prevalence are poorly understood. In this study, we explore the role of various factors contributing to convergent overlapping transcription in bacterial genomes. Our analysis shows that the proportion of convergent overlapping gene pairs (COGPs) in a genome is affected due to endospore formation, bacterial habitat, oxygen requirement, GC content and the temperature range. In particular, we show that bacterial genomes thriving in specialized habitats, such as thermophiles, exhibit a high proportion of COGPs. Our results also conclude that the density distribution of COGPs across the genomes is high for shorter overlaps with increased conservation of distances for decreasing overlaps. Our study further reveals that COGPs frequently contain stop codon overlaps with the middle base position exhibiting mismatches between complementary strands. Further, for the functional analysis using cluster of orthologous groups (COGs) annotations suggested that cell motility, cell metabolism, storage and cell signaling are enriched among COGPs, suggesting their role in processes beyond regulation. Our analysis provides genomic insights into this unappreciated regulatory phenomenon, allowing a refined understanding of their contribution to bacterial phenotypes.