Project description:The genus Flaveria has been extensively used as a model to study the evolution of C4 photosynthesis as it contains both C3 and C4 species as well as a number of species that exhibit intermediate types of photosynthesis. The current phylogenetic tree of the Flaveria genus contains 21 of the 23 known Flaveria species and has been constructed using a combination of morphologicial data and three non-coding DNA sequences (nuclear encoded ETS, ITS and chloroplast encoded trnl-F). However, recent studies have suggested that phylogenetic trees inferred using a small number of molecular sequences may often be incorrect. Moreover, studies in other genera have often shown substantial differences between trees inferred using morphological data and those using molecular sequence. To provide new insight into the phylogeny of the genus Flaveria we utilize RNA-Seq data to construct a multi-gene concatenated phylogenetic tree of 17 Flaveria species. Furthermore, we use this new data to identify 14 C4 specific non-synonymous mutation sites, 12 of which (86%) can be independently verified by public sequence data. We propose that the data collection method provided in this study can be used as a generic method for facilitating phylogenetic tree reconstruction in the absence of reference genomes for the target species. 18 Flaveria sample including 11 species are sequenced, other three samples were also sequenced as out-group. In all, 21 samples.
Project description:The genus Flaveria has been extensively used as a model to study the evolution of C4 photosynthesis as it contains both C3 and C4 species as well as a number of species that exhibit intermediate types of photosynthesis. The current phylogenetic tree of the Flaveria genus contains 21 of the 23 known Flaveria species and has been constructed using a combination of morphologicial data and three non-coding DNA sequences (nuclear encoded ETS, ITS and chloroplast encoded trnl-F). However, recent studies have suggested that phylogenetic trees inferred using a small number of molecular sequences may often be incorrect. Moreover, studies in other genera have often shown substantial differences between trees inferred using morphological data and those using molecular sequence. To provide new insight into the phylogeny of the genus Flaveria we utilize RNA-Seq data to construct a multi-gene concatenated phylogenetic tree of 17 Flaveria species. Furthermore, we use this new data to identify 14 C4 specific non-synonymous mutation sites, 12 of which (86%) can be independently verified by public sequence data. We propose that the data collection method provided in this study can be used as a generic method for facilitating phylogenetic tree reconstruction in the absence of reference genomes for the target species.
Project description:A phylogenetic analysis of seven different species (human, mouse, rat, worm, fly, yeast, and plant) utilizing all (541) basic helix-loop-helix (bHLH) genes identified, including expressed sequence tags (EST), was performed. A super-tree involving six clades and a structural categorization involving the entire coding sequence was established. A nomenclature was developed based on clade distribution to discuss the functional and ancestral relationships of all the genes. The position/location of specific genes on the phylogenetic tree in relation to known bHLH factors allows for predictions of the potential functions of uncharacterized bHLH factors, including EST's. A genomic analysis using microarrays for four different mouse cell types (i.e. Sertoli, Schwann, thymic, and muscle) was performed and considered all known bHLH family members on the microarray for comparison. Cell-specific groups of bHLH genes helped clarify those bHLH genes potentially involved in cell specific differentiation. This phylogenetic and genomic analysis of the bHLH gene family has revealed unique aspects of the evolution and functional relationships of the different genes in the bHLH gene family. PMID: 18557763 We used microarrays to determine bHLH expression in 20d rat Sertoli cells. RNA samples from two control groups (Sertoli cells cultured for 72 h) are compared to two treated groups (Sertoli cells cultured for 72 h with cAMP).
Project description:A phylogenetic analysis of seven different species (human, mouse, rat, worm, fly, yeast, and plant) utilizing all (541) basic helix-loop-helix (bHLH) genes identified, including expressed sequence tags (EST), was performed. A super-tree involving six clades and a structural categorization involving the entire coding sequence was established. A nomenclature was developed based on clade distribution to discuss the functional and ancestral relationships of all the genes. The position/location of specific genes on the phylogenetic tree in relation to known bHLH factors allows for predictions of the potential functions of uncharacterized bHLH factors, including EST's. A genomic analysis using microarrays for four different mouse cell types (i.e. Sertoli, Schwann, thymic, and muscle) was performed and considered all known bHLH family members on the microarray for comparison. Cell-specific groups of bHLH genes helped clarify those bHLH genes potentially involved in cell specific differentiation. This phylogenetic and genomic analysis of the bHLH gene family has revealed unique aspects of the evolution and functional relationships of the different genes in the bHLH gene family. PMID: 18557763 We used microarrays to determine bHLH expression in 20d rat Sertoli cells.
Project description:This application is the second part of a BBSRC-funded grant to compare and contrast the plastid-signalling pathways disrupted by Norflurazon and far-red light treatment of Arabidopsis seedlings. The first application of this laboratory to GARNet's Affymetrix service (2002-08-25-17.41.49_McCormac) addressed the Norflurazon pathway; this application addresses the far-red pathway. The assembly of photosynthetic complexes in developing chloroplasts is critical to the establishment of the autotrophic plant. This requires light-mediated upregulation of both nuclear- and chloroplast-encoded genes. The expression of such photosynthetically-associated nuclear genes is also often dependant on a retrograde plastid signal which emanates from chloroplasts to modulate nuclear transcription. Extensive studies using the herbicide Norflurazon to knock-out the plastid signal (including this lab's previous Affymetrix application to GARNet) are identifying the affected gene sets. However, genetic studies have indicated the existence of more than one plastid signalling pathway. We have recently investigated a phytochrome A-mediated, far-red (FR) input pathway which blocks subsequent chloroplast development under white light (WL). This has also been found to inhibit the transcription of a small group of known nuclear-encoded plastidic proteins. Here we wish to establish how wide-reaching this FR-effect is on nuclear transcription, and will directly compare the affected gene groups with those identified from our earlier (and other's) studies with a Norflurazon treatment. In this array experiment we will compare RNA from seedlings grown in complete darkness (D) before transfer to WL, with that of seedlings preconditioned under a restricted wavelength FR source before exposure to WL. This comparison of FR- and Norflurazon-affected gene groupings will indicate whether the plastid signalling pathways are likely to be the same, overlapping or highly divergent. As well as wild-type seedlings, the gun1,gun5 mutant line is to be used as both these alleles are well established as alleviating the Norflurazon-inhibited pathway, but their affect on the FR-pathway is less clear. A single replicate of a phyA-null mutant line will also be included in order to differentiate between specific phytochromeA-mediated responses and other FR effects. It is envisaged that, in general, D- and FR-treated samples of the phyA mutant line will respond in the same way as each other and as the wild-type D-treated samples and, thus, the lack of a biological repeat in this case is not a major short-fall. The proposed experiment will consist of: wild-type: D-pretreated (x2 biological replicates) wild-type: FR-preconditioned (x2) gun1,gun5: D-pretreated (x2) gun1,gun5: FR-preconditioned (x2) phyA: D-pretreated (x1) phyA: FR-preconditioned (x1)
Project description:This application is the second part of a BBSRC-funded grant to compare and contrast the plastid-signalling pathways disrupted by Norflurazon and far-red light treatment of Arabidopsis seedlings. The first application of this laboratory to GARNet's Affymetrix service (2002-08-25-17.41.49_McCormac) addressed the Norflurazon pathway; this application addresses the far-red pathway. The assembly of photosynthetic complexes in developing chloroplasts is critical to the establishment of the autotrophic plant. This requires light-mediated upregulation of both nuclear- and chloroplast-encoded genes. The expression of such photosynthetically-associated nuclear genes is also often dependant on a retrograde plastid signal which emanates from chloroplasts to modulate nuclear transcription. Extensive studies using the herbicide Norflurazon to knock-out the plastid signal (including this lab's previous Affymetrix application to GARNet) are identifying the affected gene sets. However, genetic studies have indicated the existence of more than one plastid signalling pathway. We have recently investigated a phytochrome A-mediated, far-red (FR) input pathway which blocks subsequent chloroplast development under white light (WL). This has also been found to inhibit the transcription of a small group of known nuclear-encoded plastidic proteins. Here we wish to establish how wide-reaching this FR-effect is on nuclear transcription, and will directly compare the affected gene groups with those identified from our earlier (and other's) studies with a Norflurazon treatment. In this array experiment we will compare RNA from seedlings grown in complete darkness (D) before transfer to WL, with that of seedlings preconditioned under a restricted wavelength FR source before exposure to WL. This comparison of FR- and Norflurazon-affected gene groupings will indicate whether the plastid signalling pathways are likely to be the same, overlapping or highly divergent. As well as wild-type seedlings, the gun1,gun5 mutant line is to be used as both these alleles are well established as alleviating the Norflurazon-inhibited pathway, but their affect on the FR-pathway is less clear. A single replicate of a phyA-null mutant line will also be included in order to differentiate between specific phytochromeA-mediated responses and other FR effects. It is envisaged that, in general, D- and FR-treated samples of the phyA mutant line will respond in the same way as each other and as the wild-type D-treated samples and, thus, the lack of a biological repeat in this case is not a major short-fall. The proposed experiment will consist of: wild-type: D-pretreated (x2 biological replicates) wild-type: FR-preconditioned (x2) gun1,gun5: D-pretreated (x2) gun1,gun5: FR-preconditioned (x2) phyA: D-pretreated (x1) phyA: FR-preconditioned (x1) Experiment Overall Design: Number of plants pooled:300 seedlings