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The relaxin-like peptide family consists of relaxin-1, 2 and 3, and the insulin-like peptides (INSL)-3, 4, 5 and 6. The evolution of this family has been controversial; points of contention include the existence of an invertebrate relaxin and the absence of a ruminant relaxin. Using the known members of the relaxin peptide family, all available vertebrate and invertebrate genomes were searched for relaxin peptide sequences. Contrary to previous reports an invertebrate relaxin was not found; sequence similarity searches indicate the family emerged during early vertebrate evolution. Phylogenetic analyses revealed the presence of potential relaxin-3, relaxin and INSL5 homologs in fish; dating their emergence far earlier than previously believed. Furthermore, estimates of mutation rates suggested that the expansion of the family (i.e. the emergence of INSL6, INSL4 and relaxin-1) during mammalia was driven by positive Darwinian selection. In contrast, relaxin-3 is constrained by strong purifying selection, implying a highly conserved function. (For complete abstract open document)

Tuberculosis is one of the leading causes of mortality globally. Although this disease has been around for many generations, treatment and management of the disease remains a daunting challenge. M. tb, is one of the most famous tuberculosis causing organisms, however there are many other mycobacterial strains and species that are also responsible for human mortality, globally. Not all mycobacterial species, however, are disease causing. It is only a few strains such as M. tb H37Rv, M. tb CDC1551, M. tb F11 and M. bovis which are responsible for causing disease. The rest are relatively harmless. What are the genetic differences between these virulent and avirulent strains that dictate a strain's behavior? The answers to these and many other questions lie hidden within the genomes of these organisms. Due to the great advances in DNA sequencing techniques, it is now now possible to more quickly and cheaply, sequence whole bacterial genomes in a single experimental run (High-throughput sequencing). Comparative genomics is therefore extremely relevant and important to be able to handle the dubious amounts of genomic data being poured into our public databases. Several comparative genomics environments already exist on the web today, however the goal of this project is to produce a web-based, comparative genomics environment which not only incorporates basic comparative genomics functions but also, novel tools such as the Seqword Genome Browser (SWGB) and the Mycobacterial Comparison Project (MCP). Using these tools, some interesting comparative genomics findings regarding certain strains of Mycobacteria are made. We reveal several genomic islands within M. avium and M. tb H37Rv. It is shown that certain genes which are usually found to be conserved among other bacteria, tend to be rather divergent among the mycobacteria. 'Mutational hotspots' containing many DNA replication genes are observed to have higher mutation rates relative to the rest of the genome which perhaps accounts for the slow-growth rate of these bacteria. By looking at the genetic profile of PE-PGRS genes in mycobacteria it was shown that M. tb H37Rv and M. tb F11 were actually closer for several genes than when compared to strain H37Ra. The finding was unexpected as H37Ra is known to be derived from H37Rv. These findings are extremely important in the area of TB research as it is of extreme importance to be able to trace areas of greater or lower selection within mycobacteria. Automated sequence comparison such as this is also important for tracking drug resistance markers and other features within mycobacteria so that more focused research can be carried out. The built system was tested and validated with mycobacteria, however, the system is flexible and designed with the intent of inclusion of any prokaryotic organism. It is hoped that systems such as these, and other advances in sequence comparison technology in the future, will provide the understanding needed to better control and cure diseases in the future. / Thesis (PhD)--University of Pretoria, 2010. / Biochemistry / unrestricted

Aquaculture has potential to support the food supply of increasing world population. Flavobacterial diseases pose a serious problem in wild and aquacultured fish stocks throughout the world. Flavobacterium columnare, F. branchiophilum, and F. psychrophilum are well-known Flavobacterium species that cause important fish losses. Recently, new Flavobacterium species, isolated from diseased fish, have been reported, but their virulence mechanisms are not clear. Thus, the goal of this study was to understand pathogenicity of Flavobacterium species. To this goal, 86 Flavobacterium genomes were analyzed by comparative genomics. Predicted virulence genes were identified for all genomes. For each species, unique and shared virulence genes were determined. For all genomes, unique and common predicted antibiotic resistance genes were identified as well. Secreted proteins are important virulence factors. Thus, all encoded secretion and related systems were determined. By using different genomics approaches, F. columnare genomovar I (highly virulent to cold-water fish species like trout) and genomovar II (extremely virulent to warm-water fish species such as catfish and tilapia) genomes were analyzed, and transposon mutants using Tn4351 in six F. columnare genomovar II strain 94-081 were generated. The hemolysin and glycine cleavage protein mutants had 15% and 10% mortalities, respectively while wild-type strain caused 100% mortality. Potential virulence genes, unique proteins, and other genomic features of F. columnare genomovars were determined. Mutants targeting unique genes in valine-leucine-isoleucine biosynthesis pathway were constructed. The virulence of Fcol(DELTA)leuD and Fcol(DELTA)ilvD mutants exhibited reduced virulence.

Transcriptomic analyses from across eukaryotes indicate that most of the genome is transcribed at some point in the developmental trajectory of an organism. One class of these transcripts is termed long intergenic noncoding RNAs (lincRNAs). Recently, attention has focused on understanding the evolutionary dynamics of lincRNAs, particularly their conservation within genomes. Here, we take a comparative genomic and phylogenetic approach to uncover factors influencing lincRNA emergence and persistence in the plant family Brassicaceae, to which Arabidopsis thaliana belongs. We searched 10 genomes across the family for evidence of >5000 lincRNA loci from A. thaliana. From loci conserved in the genomes of multiple species, we built alignments and inferred phylogeny. We then used gene tree/species tree reconciliation to examine the duplication history and timing of emergence of these loci. Emergence of lincRNA loci appears to be linked to local duplication events, but, surprisingly, not whole genome duplication events (WGD), or transposable elements. Interestingly, WGD events are associated with the loss of loci for species having undergone relatively recent polyploidy. Lastly, we identify 1180 loci of the 6480 previously annotated A. thaliana lincRNAs (18%) with elevated levels of conservation. These conserved lincRNAs show higher expression, and are enriched for stress-responsiveness and cis-regulatory motifs known as conserved noncoding sequences (CNSs). These data highlight potential functional pathways and suggest that CNSs may regulate neighboring genes at both the genomic and transcriptomic level. In sum, we provide insight into processes that may influence lincRNA diversification by providing an evolutionary context for previously annotated lincRNAs.

Alternative splicing (AS) is a common post-transcriptional process in eukaryotic organisms, by which multiple distinct functional transcripts are produced from a single gene. Because of its potential role in expanding transcript diversity, interest in alternative splicing has been increasing over the last decade, ever since the release of the human genome draft showed it contained little more than the number of genes of a worm. Although recent studies have shown that 94% human multi-exon genes undergo AS while aberrant AS may cause disease or cancer, evolution of AS in eukaryotic genomes remains largely unexplored mainly due to the lack of comparable AS estimates. In this thesis I built a Eukaryote Comprehensive & Comparable Alternative Splicing Events Database (ECCASED) based on the analyses of over 30 million Expressed Sequence Tag (ESTs) for 114 eukaryotic genomes, including protists (22), plants (20), fungi (23), metazoan (non-vertebrates, 29) and vertebrates (20). Using this database, I addressed two main questions: 1) How does alternative splicing relate to gene duplication (GD) as an alternative mechanism to increase transcript diversity? and 2) What is the contribution of alternative splicing to eukaryote transcript diversity? I found that the previous “interchangeable model” of AS and gene duplication is a by-product of an existing relation between gene expression breadth, AS and gene family size. I also show that alternative splicing has played a key role in the expansion of transcript diversity and that this expansion is the best predictor reported to date of organisms complexity assayed as number of cell types. In addition, by comparing alternative splicing patterns in cancer and normal transcript libraries I found that cancer derived transcript libraries have increased levels of “noisy splicing”.

Muscadinia rotundifolia est une espèce de la famille des Vitaceae. C’est un sous-genre du genre Vitis, le deuxième sous-genre étant celui des Euvitis qui comprend l’espèce cultivée Vitis vinifera (2n=38). M. rotundifolia (2n=40) est une source de résistance aux maladies très importante pour l’amélioration de la vigne. Son génome commence seulement à être décrit avec deux cartes génétiques récemment publiées. Ma thèse a consisté à utiliser des ressources génomiques chez M. rotundifolia cv Regale (banque BAC, collection de séquence d’extrémités de BAC ou BES et séquences de BACs) pour caractériser le génome de cette espèce en comparaison avec celui de V. vinifera. Les résultats obtenus ne montrent pas de différence importante entre les génomes des deux espèces en termes de composition du génome en bases (GC%), en séquences codantes ou en éléments répétés. De même, à une échelle globale, la famille de gènes NBS-LRR semble être similaire en termes de nombre et de balance entre les sous-familles. A une échelle plus fine cependant (carte physique et séquences de BAC), des remaniements relativement importants sont observés dans des régions portant cette famille de gènes, aboutissant parfois à des contenus différents en gènes, de région normalement homologues : duplication différentielles de gènes, présence/absence de gènes. / Muscadinia Rotundifolia is a species of the Vitaceae family. It is a sub-genus of the Vitis genus along with the Euvitis sub-genus, which the cultivated species Vitis vinifera belongs to. M. rotundifolia (2n=40) is a very important source of resistance to diseases in grapevine breeding programs. Its genome is only starting to be described with the recent publication of two genetic maps. The present study aimed at using M. rotundifolia cv Regale genomic resources (BAC library, BAC end sequences or BES, BAC sequences) in order to characterize the genome of this species in comparison with the genome of V. vinifera. The results showed that there is no striking difference between the two species in term of base composition (GC %), repeats frequency and gene space. The NBS LRR gene family also seems to be globally quite similar between the two species in terms of numbers and balance between subfamilies. At a finer scale (physical map and BAC sequence), frequent rearrangements are observed in genomic regions carrying the NBS-LRR gene family sometimes clearly associated with a different gene content between the two species in homologous regions: differential gene duplication, presence/absence of genes.

Using rice chromosome 1 pseudomolecule as a reference, a minimal tiling path for the euchromatic arms of sorghum chromosome 3 was constructed, in which 23 contigs contain an estimated 57.56 Mb of DNA. A total of 409 EST-STS markers and 255 genetic markers have been mapped onto the euchromatic arms providing excellent integration of the genetic and physical maps. A total of 21 contigs containing 9 ESTSTS and 35 genetic markers have been constructed across the heterochromatin block of sorghum chromosome 3 which comprise 16.57 Mb of DNA. Macrocolinearity between sorghum chromosome 3 and rice chromosome 1 was examined based on the mapped EST-STS markers. Approximately 85% of the EST-STS markers were colinear between these two homeologous chromosomes. Estimates of recombination were also determined, which indicates the existence of recombination cold and hot spots. Microcolinearity between sorghum chromosome 3 and rice chromosome 1 was examined at two different levels. In one case, overlapping sorghum BAC pools orthologous to a 5.1 Mb region of rice chromosome 1 were constructed and sequence skimmed. Alignment of the sorghum skim sequences to the TIGR rice gene models revealed ~62% colinearity between the two orthologous regions. In addition, colinearity between sorghum chromosome 3 and rice chromosome 5 was detected within this region which is likely due to the segmental homology between rice chromosome 1 and rice chromosome 5. Microcolinearity between sorghum and rice was also examined by comparing 2 fully sequenced sorghum chromosome 3 BAC clones to the orthologous region of rice chromosome 1. In this analysis, ~65% colinearity was detected for sorghum BAC 82G24 and ~59% colinearity was detected for sorghum BAC 181g10. Microcolinearity was largely confined to gene coding regions and sequences of exons displayed the highest percent identities. Small-scale gene rearrangements were also detected. Finally, RT-PCR analysis was carried out between a set of colinear and non-colinear genes from sorghum and rice to determine whether the loss of colinearity between orthologous genes resulted in a change in transcriptional regulation. No direct link between loss of colinearity and expression pattern was detected in these experiments.

Sequencing technologies have improved significantly in the past 10 years and the staggering number of genome sequences available has led to a migration from single-gene phylogenetics to multigene phylogenetics. A protocol was developed here to compare fungal genomes through BLAST to determine which BLAST statistics may best represent phylogenetic information. The results suggested that levels of sequence identity, relative to the query length, may be useful for predicting whether a gene will yield a well-resolved and consistent tree. Moreover, it was found that about 40% of the genes in a typical filamentous fungal genome may lead to a well-resolved and concordant tree topology that also matched an 18S rDNA derived topology; but for consistent results, multigene trees with a minimum of five genes should be used. An additional script to rapidly identify regions within genes that can be easily amplified was then developed and tested on eight genes. The genes were successfully amplified and several resultant amplicon trees matched the 18S rDNA topology. / NSERC

Alternative pre-messenger RNA splicing affects a majority of human genes and plays important roles in development and disease. Alternative splicing (AS) events conserved since the divergence of human and mouse are likely of primary biological importance, but relatively few such events are known. Here we describe sequence features that distinguish exons subject to evolutionarily conserved AS, which we call 'alternative-conserved exons' (ACEs) from other orthologous human/mouse exons, and integrate these features into an exon classification algorithm, ACEScan. Genome-wide analysis of annotated orthologous human-mouse exon pairs identified ~2,000 predicted ACEs. Alternative splicing was verified in both human and mouse tissues using an RT-PCR-sequencing protocol for 21 of 30 (70%) predicted ACEs tested, supporting the validity of a majority of ACEScan predictions. By contrast, AS was observed in mouse tissues for only 2 of 15 (13%) tested exons which had EST or cDNA evidence of AS in human but were not predicted ACEs, and was never observed for eleven negative control exons in human or mouse tissues. Predicted ACEs were much more likely to preserve reading frame, and less likely to disrupt protein domains than other AS events, and were enriched in genes expressed in the brain and in genes involved in transcriptional regulation, RNA processing and development. Our results also imply that the vast majority of AS events represented in the human EST databases are not conserved in mouse, and therefore may represent aberrant, disease- or allele-specific, or highly lineage-restricted splicing events.