No description available.

Les objectifs de la thèse étaient (i) de mettre en évidence les pressions de sélection qui s’exercent sur les gènes orthologues chez les végétaux et (ii) de cerner les caractéristiques structurales et fonctionnelles qu’ils partagent, notamment au niveau de leurs promoteurs, pour (iii) définir une nouvelle démarche d’empreinte phylogénétique. Pour s’appuyer sur des relations d’orthologie les moins ambiguës possibles, l’étude a été centrée sur les gènes ‘uniques’ définis par comparaison de séquences. Les gènes uniques forment 3 groupes structurellement, fonctionnellement et évolutivement distincts. Les gènes uniques spécifiques soit d’Arabidopsis thaliana soit d’Oryza sativa présentent des caractéristiques différentes des gènes uniques conservés, y compris au niveau de leur promoteur. D’une part, les gènes uniques spécifiques à une espèce sont préférentiellement des gènes codant pour des peptides excrétés et impliqués dans des fonctions de régulation. D’autre part, les gènes uniques conservés ont des caractéristiques qui les associent aux gènes impliqués dans les fonctions du métabolisme de base des cellules et qui évoluent lentement. Certains motifs potentiellement régulateurs ont été trouvés spécifiquement sur-representés dans leur promoteur. Par ailleurs, ces gènes, quand ils sont dupliqués, retournent rapidement à l’état unique, suggérant un fort désavantage sélectif à l’existence simultanée de deux copies peu divergentes de ces gènes. / The main objectives of this thesis were (i) to search for the selection pressure exerted on plant orthologous genes and (ii) to describe the structural and functional features they share in particular in their promoters, in order to (iii) define a novel phylogenetic footprinting approach. Pairs of unique genes, defined by sequence comparisons, have been used because they were considered as having the greater chance to be true orthologues. Plant unique genes form three groups of genes with different structural, functional and evolutionary features. The unique genes that are specific either to Arabidopsis thaliana or Oryza sativa have features that are different than those of conserved unique genes. On one hand, the species-specific unique genes code preferentially for excreted peptides implied in regulatory functions. On the other hand, the conserved unique genes have characteristics described in genes implied in basal metabolism of the cells and which evolve slowly. Some potential regulatory motifs have been found over-represented only in the promoter of these genes. Lastly, after duplication, these genes generally lose one duplicate, which suggest a strong negative selection against the co-existence of two not diverged copies of the same gene.

Triacylglycerol (TAG) is a class of lipid molecules composed of three fatty acyl chains esterified to a glycerol backbone. In plants, TAG is synthesized in various tissues and serves as a carbon and energy source. Oil biosynthesis is well understood in oilseeds however how plants store oil in non-seed tissue is yet to be determined. In Avocado (Persea americana), a basal angiosperm, TAG is exclusively accumulated in mesocarp tissue and therefore is emerging as a model system to uncover underlying mechanisms of TAG biosynthesis in tissues other than seed. The mesocarp of Avocado fruit contains ~60-70% of oil by dry weight. Recent transcriptome studies revealed that the TAG biosynthesis is transcriptionally regulated in non-seed tissues. In seed tissues, TAG biosynthesis is regulated by many seed maturation factors directly or indirectly through downstream transcription factor WRINKLED1 (WRI1). Transcriptome studies revealed that in addition to ortholog of WRI1, orthologs for WRI2 and WRI3 were also highly expressed in avocado mesocarp during the period of oil accumulation. Based on the transcriptome data, I hypothesize that putative WRI genes (WRI1, 2, 3) of avocado enhance oil content in nonseed tissues. Currently, cloning of Putative PaWRI 1, 2 and 3 genes into a binary vector, followed by agrobacterium-mediated transformation to generate transient and stable transient lines, is underway. Full-length cDNA for PaWRI genes (1 & 2) were amplified and cloned into pK34 entry vector followed by sequence confirmation. PaWRI genes (1 & 2) were subcloned into pB110 destination vector and will be transformed into agrobacterium for their integration into the plants. Cloning of WRI3 is still ongoing. Transient expression of putative PaWRI 1, 2 and 3 genes, will be validated using tobacco leaf assay, are expected to enhance oil accumulation in leaf tissues. Agrobacterium bearing PaWRI genes and a viral silencing protein (p19) will be co-infiltrated on to the underside of Nicotiana benthamiana leaves. Infiltrated plants will be placed in growth room with 16:8 light/dark cycle. Four days post infiltration, infected leaf areas will be harvested and TAG content and composition will be determined by gas chromatography coupled with flame ionization detector. Functional validation of these orthologs is expected to reveal the preferred WRI isoform that likely participates in regulation of oil biosynthesis in avocado mesocarp. Additionally, this work may also elucidate the differences between regulation of TAG accumulation in seed and non-seed tissues and identify new targets to enhance TAG biosynthesis in plants.

Triacylglycerol (TAG) is a class of lipid molecules composed of three fatty acyl chains esterified to a glycerol backbone. In plants, TAG is synthesized in various tissues and serves as a carbon and energy source. Oil biosynthesis is well understood in oilseeds however how plants store oil in non-seed tissue is yet to be determined. In Avocado (Persea americana), a basal angiosperm, TAG is exclusively accumulated in mesocarp tissue and therefore is emerging as a model system to uncover underlying mechanisms of TAG biosynthesis in tissues other than seed. The mesocarp of Avocado fruit contains ~60-70% of oil by dry weight. Recent transcriptome studies revealed that the TAG biosynthesis is transcriptionally regulated in non-seed tissues. In seed tissues, TAG biosynthesis is regulated by many seed maturation factors directly or indirectly through downstream transcription factor WRINKLED1 (WRI1). Transcriptome studies revealed that in addition to ortholog of WRI1, orthologs for WRI2 and WRI3 were also highly expressed in avocado mesocarp during the period of oil accumulation. Currently, cloning of Putative PaWRI 1, 2 and 3 genes into a binary vector, followed by agrobacterium-mediated transformation to generate transient and stable transient lines, is underway. Transient expression of putative PaWRI 1, 2 and 3 genes, using tobacco leaf assay, are expected to enhance oil accumulation in leaf tissues. Stable expression of PaWRI 1, 2, and 3 in Atwri-/- is expected to restore oil accumulation in seeds. TAG content and composition will be determined by gas chromatography coupled with flame ionization detector. Functional validation of these orthologs is expected to reveal the preferred WRI isoform that likely participates in regulation of oil biosynthesis in avocado mesocarp. Additionally, this work may also elucidate the differences between regulation of TAG accumulation in seed and non-seed tissues and identify new targets to enhance TAG biosynthesis in plants.

No description available.

Occidiofungin is an antifungal compound that targets the conserved cytoskeletal protein, actin. Despite >90% amino acid conservation between fungal actin proteins, sensitivity to occidiofungin has been shown to vary with C. albicans, F. oxysporum, and P. digitatum exhibiting a resistant profile relative to S. cerevisiae. To determine whether differences in the amino acid sequences of actin contribute to differences in occidiofungin susceptibility, we expressed the actin gene from these fungal organisms in the ACT1 S. cerevisiae shuffle strain. Functionality of actin gene products was determined by measuring growth kinetics, actin protein levels, nuclear position, and actin cable formation. Results demonstrated functional complementation for all actin orthologs. Analysis of occidiofungin susceptibility found that fungal actin ortholog expression resulted in a similar sensitivity profile as the wildtype S. cerevisiae. These findings suggest that amino acid differences in actin are not directly responsible for the resistance to occidiofungin identified for these fungal organisms.

Saccharomyces cerevisiae is an excellent experimental model organism to study various biological processes owing to its versatile genetics, biochemistry, and standard laboratory conditions. S. cerevisiae shows distinct biological responses under nutritional starvation conditions. S. cerevisiae undergoes dimorphic transition from a unicellular yeast form to a multicellular pseudohyphae (Gimeno et al., 1992) under nitrogen starvation, but in the complete absence of a fermentable carbon source, it undergoes gametogenesis called sporulation (Mitchell, 1994). While the signal transduction cascades and regulatory controls under nutritional starvation conditions are studied to great extent, the role of S. cerevisiae core RNA polymerase II (pol II) is not much understood. S. cerevisiae core RNA pol II consists of 12 subunits (Woychik and Hampsey, 2002), which is organized into a ten-subunit core and the Rpb4/7 subcomplex (Edwards et al., 1991). Rpb4/7 subcomplex is known to play important roles in stress survival (Choder 2004; Sampath and Sadhale, 2005.). S. cerevisiae rpb4 null diploid strains show reduced sporulation levels but exhibits a predisposition to pseudohyphal morphology (Pillai et al., 2003). Overexpression of Rpb7 partially rescues some of these defects (Sharma et al., 1999; Sheffer et al., 2001). Rpb7 is a highly conserved protein but Rpb4 is the least conserved amongst all RNA pol II subunits at the sequence level. Rpb4 and Rpb7 also affect different cellular functions, which are not directly dependent on each other. (a) Relative levels of RNA pol II subunits Rpb4 and Rpb7 differentially affect starvation response in Saccharomyces cerevisiae S. cerevisiae rpb4 null diploid strains show reduced sporulation levels as compared to wild type but exhibits pseudohyphal predisposition. Overexpression of RPB7 partially rescues the sporulation defect but results in an exaggeration of the pseudohyphae phenotype. We generated S. cerevisiae strains expressing different levels of Rpb4 and Rpb7 proteins in the same strains and analyzed their effect on sporulation and pseudohyphal morphology. We observed that sporulation is dependent on Rpb4 because sporulation level gradually increases with an increase in the Rpb4 protein level in the strain. Rpb7 reduces sporulation level but enhances pseudohyphal exaggeration in a dose-dependent manner. Rpb4 is dominant over Rpb7 in both the starvation responses because strain expressing an equimolar ratio of Rpb4 and Rpb7 protein exhibits RPB4+ phenotypes. (b) Domainal organization of Saccharomyces cerevisiae Rpb7 orthologs reflects functional conservation Rpb7 orthologs are known in eukaryotes and archaebacteria. The primary structure of Rpb7 is conserved. We chose Rpb7 orthologs from Candida albicans, Schizosaccharomyces pombe and Homo sapiens sapiens to investigate whether Rpb7 orthologs are also functionally conserved. We observed that all the orthologs tested are functionally conserved because they can complement the absence of RPB7 in S. cerevisiae. However, we uncovered functional differences amongst Rpb7 orthologs with respect to its function in rpb4 null strain and ess1 ts strain. Furthermore, we made N and C-terminal chimeric RPB7 constructs from these orthologs with S. cerevisiae Rpb7. These chimeras also can replace ScRpb7 in S. cerevisiae. However, functional differences were observed with each chimera pair in rpb4 null strain and ess1 ts strain, showing that the N and C-terminal domains of Rpb7 protein can be genetically dissected. The genetic observation on the domainal organization of Rpb7 orthologs is strengthened by the crystal structure of Rpb7 (Armache et al., 2005), which shows that Rpb7 is structurally organized into an N terminal RNP domain and a C terminal OB fold domain. (c) The Rpb7 subunit of Candida albicans RNA polymerase II induces lectin-mediated flocculation in Saccharomyces cerevisiae The Rpb7 ortholog of C. albicans is a conserved functional ortholog of ScRpb7. We observed that CaRpb7 induces Ca2+-dependent flocculation and agar-invasive growth in S. cerevisiae. CaRpb7 overexpression induces very high transcript levels of FLO1 and FLO11. We believe that the observed flocculation and agar-invasive phenotypes are due to Flo1 and Flo11 respectively, because Flo1 and Flo11 contribute mainly to cell-cell adhesion while Flo11 contributes mainly to cell-substrate adhesion (Verstrepen and Klis, 2006; Lo et al., 1998; Guo et al., 2000). Pathway analysis revealed that CaRpb7-induced flocculation is dependent on Mss11 transcriptional activator. Two-hybrid analysis revealed that CaRpb7 does not physically interact with transcriptional repressors known to repress FLO gene transcription, however genetic analysis revealed that CaRpb7 is epistatic to the repressor Sfl1. Rpb7 orthologs possess conserved domains with potential RNA binding ability (Orlicky et al., 1999) and ScRpb7 is known to play in mRNA stability (Lotan et al., 2007). The possibility of CaRpb7 specifically affecting the stability of FLO gene transcripts is being pursued.

Proteins are the functional units of cells. However, a major portion of the proteome does not have a known functional annotation. This dissertation explores protein -protein interactions, involving these uncharacterized or unknown function proteins. Initially, protein – protein interactions were tested and analyzed for paralogous proteins in Escherichia coli. To expand this concept further and to get an overview, protein – protein interactions were analyzed using ‘comparative interactomics’ for four pathogenic bacterial species including Escherichia coli, Yersinia pestis, Vibrio cholerae and Staphylococcus aureus. This approach was used to study unknown function protein pairs as well as to focus on uncharacterized hub proteins. The dissertation aims at using protein – protein interactions along with other research data about proteins as a possible approach to narrow down on functions of proteins.

In order to identify expressologs (orthologs exhibiting the highest expression profile ranking) among a variety of plant species, bioinformatic methods were used in order to first identify sequence orthologs and subsequently to rank these orthologs based on expression profile similarity. Analyses conducted on these data suggested that expressologs exhibited greater functional equivalency. A comparison of drought response in A. thaliana and Populus showed that expressologs exhibited a higher correlation when computed using stress data as opposed to developmental data. This suggested that the use of condition-specific data sets is more appropriate when examining specific conditions. Analysis was conducted in order to investigate the hypothesis that neutral evolution was a predominant factor in gene expression divergence. Some evidence was found for selection acting on expression pattern maintenance. Further analysis will be required in order to confirm the type of selection acting to maintain expression patterns across species.

In order to identify expressologs (orthologs exhibiting the highest expression profile ranking) among a variety of plant species, bioinformatic methods were used in order to first identify sequence orthologs and subsequently to rank these orthologs based on expression profile similarity. Analyses conducted on these data suggested that expressologs exhibited greater functional equivalency. A comparison of drought response in A. thaliana and Populus showed that expressologs exhibited a higher correlation when computed using stress data as opposed to developmental data. This suggested that the use of condition-specific data sets is more appropriate when examining specific conditions. Analysis was conducted in order to investigate the hypothesis that neutral evolution was a predominant factor in gene expression divergence. Some evidence was found for selection acting on expression pattern maintenance. Further analysis will be required in order to confirm the type of selection acting to maintain expression patterns across species.