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• 1. [Article] Degradation by Cullin 3 and Effect on WNK Kinases Suggest a Role of KLHL2 in the Pathogenesis of Familial Hyperkalemic Hypertension

  Mutations in WNK1 and WNK4, and in components of the Cullin-Ring Ligase system, kelch-like 3 (KLHL3) and Cullin 3 (CUL3), can cause the rare hereditary disease, Familial Hyperkalemic Hypertension (FHHt). ...

  Citation × Citation Citation Abstract Copy Title: Degradation by Cullin 3 and Effect on WNK Kinases Suggest a Role of KLHL2 in the Pathogenesis of Familial Hyperkalemic Hypertension Author: Zhang, Chong, Meermeier, Nicholas P., Terker, Andrew, Blankenstein, Katharina I., Singer, Jeffrey D., Hadchouel, Juliette, Ellison, David, Yang, Chao-Ling Year: 2015 Mutations in WNK1 and WNK4, and in components of the Cullin-Ring Ligase system, kelch-like 3 (KLHL3) and Cullin 3 (CUL3), can cause the rare hereditary disease, Familial Hyperkalemic Hypertension (FHHt). The disease is characterized by overactivity of the renal sodium chloride cotransporter (NCC), which is phosphorylated and activated by the WNK-stimulated Ste20-type kinases, SPAK and OSR1. WNK kinases themselves can be targeted for ubiquitination and degradataion by the CUL3-KLHL3 E3 ubiquitin ligase complex. It is unclear, however, why there are significant differences in phenotypic severity among FHHt patients with mutations in different genes. It was reported that kelch-like 2 (KLHL2), a homolog of KLHL3, can also target WNK kinases for ubiquitation and degradation, and may play a special role in the systemic vasculature. Our recent study revealed the disease mutant CUL3 exhibits enhanced degradation of its adaptor protein KLHL3, potentially resulting in accumulation of WNK kinases secondarily. To investigate if KLHL2 plays a role in FHHt, we studied the effect of wild type and FHHt mutant CUL3 on degradation of KLHL2 and WNK kinase proteins in HEK293 cells. Although CUL3 facilitates KLHL2 degradation, the disease mutant CUL3 is more active in this regard. KLHL2 facilitated the degradation of wild type but not disease mutant WNK4 protein. These results suggest that KLHL2 likely plays a role in the pathogenesis of FHHt, and aggravates the phenotype caused by mutations in CUL3 and WNK4. Title: Degradation by Cullin 3 and Effect on WNK Kinases Suggest a Role of KLHL2 in the Pathogenesis of Familial Hyperkalemic Hypertension Author: Zhang, Chong, Meermeier, Nicholas P., Terker, Andrew, Blankenstein, Katharina I., Singer, Jeffrey D., Hadchouel, Juliette, Ellison, David, Yang, Chao-Ling Year: 2015 Mutations in WNK1 and WNK4, and in components of the Cullin-Ring Ligase system, kelch-like 3 (KLHL3) and Cullin 3 (CUL3), can cause the rare hereditary disease, Familial Hyperkalemic Hypertension (FHHt). The disease is characterized by overactivity of the renal sodium chloride cotransporter (NCC), which is phosphorylated and activated by the WNK-stimulated Ste20-type kinases, SPAK and OSR1. WNK kinases themselves can be targeted for ubiquitination and degradataion by the CUL3-KLHL3 E3 ubiquitin ligase complex. It is unclear, however, why there are significant differences in phenotypic severity among FHHt patients with mutations in different genes. It was reported that kelch-like 2 (KLHL2), a homolog of KLHL3, can also target WNK kinases for ubiquitation and degradation, and may play a special role in the systemic vasculature. Our recent study revealed the disease mutant CUL3 exhibits enhanced degradation of its adaptor protein KLHL3, potentially resulting in accumulation of WNK kinases secondarily. To investigate if KLHL2 plays a role in FHHt, we studied the effect of wild type and FHHt mutant CUL3 on degradation of KLHL2 and WNK kinase proteins in HEK293 cells. Although CUL3 facilitates KLHL2 degradation, the disease mutant CUL3 is more active in this regard. KLHL2 facilitated the degradation of wild type but not disease mutant WNK4 protein. These results suggest that KLHL2 likely plays a role in the pathogenesis of FHHt, and aggravates the phenotype caused by mutations in CUL3 and WNK4. Close

• 2. [Article] Antibacterial Gene Transfer Across the Tree of Life

  Though horizontal gene transfer (HGT) is widespread, genes and taxa experience biased rates of transferability. Curiously, independent transmission of homologous DNA to archaea, bacteria, eukaryotes, and ...

  Citation × Citation Citation Abstract Copy Title: Antibacterial Gene Transfer Across the Tree of Life Author: Metcalf, Jason A., Funkhouser-Jones, Lisa J., Brileya, Kristen A., Reysenbach, Anna-Louise, Bordenstein, Seth R. Year: 2014 Though horizontal gene transfer (HGT) is widespread, genes and taxa experience biased rates of transferability. Curiously, independent transmission of homologous DNA to archaea, bacteria, eukaryotes, and viruses is extremely rare and often defies ecological and functional explanations. Here, we demonstrate that a bacterial lysozyme family integrated independently in all domains of life across diverse environments, generating the only glycosyl hydrolase 25 muramidases in plants and archaea. During coculture of a hydrothermal vent archaeon with a bacterial competitor, muramidase transcription is upregulated. Moreover, recombinant lysozyme exhibits broad-spectrum antibacterial action in a dose-dependent manner. Similar to bacterial transfer of antibiotic resistance genes, transfer of a potent antibacterial gene across the universal tree seemingly bestows a niche-transcending adaptation that trumps the barriers against parallel HGT to all domains. The discoveries also comprise the first characterization of an antibacterial gene in archaea and support the pursuit of antibiotics in this underexplored group. Title: Antibacterial Gene Transfer Across the Tree of Life Author: Metcalf, Jason A., Funkhouser-Jones, Lisa J., Brileya, Kristen A., Reysenbach, Anna-Louise, Bordenstein, Seth R. Year: 2014 Though horizontal gene transfer (HGT) is widespread, genes and taxa experience biased rates of transferability. Curiously, independent transmission of homologous DNA to archaea, bacteria, eukaryotes, and viruses is extremely rare and often defies ecological and functional explanations. Here, we demonstrate that a bacterial lysozyme family integrated independently in all domains of life across diverse environments, generating the only glycosyl hydrolase 25 muramidases in plants and archaea. During coculture of a hydrothermal vent archaeon with a bacterial competitor, muramidase transcription is upregulated. Moreover, recombinant lysozyme exhibits broad-spectrum antibacterial action in a dose-dependent manner. Similar to bacterial transfer of antibiotic resistance genes, transfer of a potent antibacterial gene across the universal tree seemingly bestows a niche-transcending adaptation that trumps the barriers against parallel HGT to all domains. The discoveries also comprise the first characterization of an antibacterial gene in archaea and support the pursuit of antibiotics in this underexplored group. Close

• 3. [Article] Phenotypic Variation in the Model Organism, Danio Rerio

  Model organisms are used to study evolutionary conserved traits. Zebrafish (Danio rerio) are used a model organism because of their highly fecundity, external fertilization, and robust nature, making them ...

  Citation × Citation Citation Abstract Copy Title: Phenotypic Variation in the Model Organism, Danio Rerio Author: Champaigne, Rachel D, Brown, Kim H. Year: 2014 Model organisms are used to study evolutionary conserved traits. Zebrafish (Danio rerio) are used a model organism because of their highly fecundity, external fertilization, and robust nature, making them highly adaptable to environmental and genetic variation. In an effort to limit data variation that lies outside of topic interest, phenotypic measures of variation must be performed, understood, and taken into consideration for future studies. A common measurement of phenotypic variation in fish is in the maximum (Ucrit) swimming speeds. Inter and intra-strain variation in zebrafish Ucrit swimming speeds will be observed in a swim tunnel. Baseline values will be recorded from ten fish from each of two families in each of three strains: WIK, Tubigan and AB. Environmental variation in zebrafish raised (beginning at one month of age) in tanks with various volumes and densities of fish will also be recorded from each strain. Mitochondrial density in white muscle will be compared in each strain in each condition. Diet, take salinity, pH, temperature, and light exposure will all remain constant for all fish. Title: Phenotypic Variation in the Model Organism, Danio Rerio Author: Champaigne, Rachel D, Brown, Kim H. Year: 2014 Model organisms are used to study evolutionary conserved traits. Zebrafish (Danio rerio) are used a model organism because of their highly fecundity, external fertilization, and robust nature, making them highly adaptable to environmental and genetic variation. In an effort to limit data variation that lies outside of topic interest, phenotypic measures of variation must be performed, understood, and taken into consideration for future studies. A common measurement of phenotypic variation in fish is in the maximum (Ucrit) swimming speeds. Inter and intra-strain variation in zebrafish Ucrit swimming speeds will be observed in a swim tunnel. Baseline values will be recorded from ten fish from each of two families in each of three strains: WIK, Tubigan and AB. Environmental variation in zebrafish raised (beginning at one month of age) in tanks with various volumes and densities of fish will also be recorded from each strain. Mitochondrial density in white muscle will be compared in each strain in each condition. Diet, take salinity, pH, temperature, and light exposure will all remain constant for all fish. Close

• 4. [Article] Modeling Microvirus Capsid Protein Evolution Utilizing Metagenomic Sequence Data

  The Microviridae are increasingly becoming recognized as one of the most globally ubiquitous and highly diverse virus families, and as such, provide an advantageous model for studying virus evolution and ...

  Citation × Citation Citation Abstract Copy Title: Modeling Microvirus Capsid Protein Evolution Utilizing Metagenomic Sequence Data Author: Diemer, Geoffrey S., Stedman, Kenneth M. Year: 2016 The Microviridae are increasingly becoming recognized as one of the most globally ubiquitous and highly diverse virus families, and as such, provide an advantageous model for studying virus evolution and adaptation. Here, we utilize microvirus sequences from diverse physiochemical environments, including novel sequences from a high-temperature acidic lake, to chart the outcome of natural selection in the main structural protein of the virus. Each icosahedral microvirus virion is composed of sixty identical capsid proteins that interact along twofold, threefold and fivefold symmetry axis interfaces to encapsidate a small, circular, single-stranded DNA genome. Viable assembly of the virus is guided by scaffolding proteins, which coordinate inter-subunit contacts between the capsid proteins. Structure-based analysis indicates that amino acid sequence conservation is predominantly localized to the twofold axis interface. While preservation of this quaternary interface appears to be essential, tertiary and secondary structural features of the capsid protein are permissive to considerable sequence variation. Title: Modeling Microvirus Capsid Protein Evolution Utilizing Metagenomic Sequence Data Author: Diemer, Geoffrey S., Stedman, Kenneth M. Year: 2016 The Microviridae are increasingly becoming recognized as one of the most globally ubiquitous and highly diverse virus families, and as such, provide an advantageous model for studying virus evolution and adaptation. Here, we utilize microvirus sequences from diverse physiochemical environments, including novel sequences from a high-temperature acidic lake, to chart the outcome of natural selection in the main structural protein of the virus. Each icosahedral microvirus virion is composed of sixty identical capsid proteins that interact along twofold, threefold and fivefold symmetry axis interfaces to encapsidate a small, circular, single-stranded DNA genome. Viable assembly of the virus is guided by scaffolding proteins, which coordinate inter-subunit contacts between the capsid proteins. Structure-based analysis indicates that amino acid sequence conservation is predominantly localized to the twofold axis interface. While preservation of this quaternary interface appears to be essential, tertiary and secondary structural features of the capsid protein are permissive to considerable sequence variation. Close

• 5. [Article] The orientation of the Pyrococcus furiosus transcription factor TFB2 in the transcription initiation complex

  The hyperthermophile archaeon, Pyrococcus furiosus encodes two eukaryotic TFIIB family proteins, TFB1 and TFB2. TFB1 is very similar to TFIIB in terms of sequence homology and function, whereas TFB2 is ...

  Citation × Citation Citation Abstract Copy Title: The orientation of the Pyrococcus furiosus transcription factor TFB2 in the transcription initiation complex Author: Bhattarai, Arati Year: 2014 The hyperthermophile archaeon, Pyrococcus furiosus encodes two eukaryotic TFIIB family proteins, TFB1 and TFB2. TFB1 is very similar to TFIIB in terms of sequence homology and function, whereas TFB2 is unusual as it is missing highly conserved sequences in its N-terminal domain that are present in TFIIB and TFB1. Despite this, TFB2 is effective in transcription process, albeit with lower efficiency compared to TFB1. Other archaea also contain multiple TFBs, but unlike Pyrococcus furiosus TFB2, these multiple TFBs have higher sequence homology to each other and have similar transcription efficiencies. Photochemical cross-linking experiments have shown that the B-reader of TFB in archaea and TFIIB in eukaryotes is close to transcription start site and is very important in RNAP recruitment to promoter DNA and transcription start site selection. Thus the lack of the highly conserved B reader region in P. furiosus TFB2 presents the opportunity to further study the functional importance of this region. In this study several amino acids in N-terminal domain of TFB2 were mutated with photoactivable unnatural amino acid p-benzoyl L- phenylalanine (pBpa) and the proximity of TFB2 relative to DNA was determined by photochemical cross-linking experiments. The results showed that TFB2 interacts with DNA near the TATA box via its C-terminal domain, and interacts with both strands of DNA near the transcription start site via its divergent B-reader and the B-linker sequences. The B-reader loop region is close to transcription start site and interacts with the transcribed strand of promoter DNA while the B-linker strand cross-links with the non-transcribed strand. Some of the amino acids in between the B-reader loop and the B-linker strand region in TFB2 are seen to cross-link both the transcribed and the non-transcribed strand. Thus, despite the absence of strong homology to conserved B-reader and B-linker sequences, TFB2 is likely to interact with DNA in the transcription bubble and facilitate in transcription initiation. Title: The orientation of the Pyrococcus furiosus transcription factor TFB2 in the transcription initiation complex Author: Bhattarai, Arati Year: 2014 The hyperthermophile archaeon, Pyrococcus furiosus encodes two eukaryotic TFIIB family proteins, TFB1 and TFB2. TFB1 is very similar to TFIIB in terms of sequence homology and function, whereas TFB2 is unusual as it is missing highly conserved sequences in its N-terminal domain that are present in TFIIB and TFB1. Despite this, TFB2 is effective in transcription process, albeit with lower efficiency compared to TFB1. Other archaea also contain multiple TFBs, but unlike Pyrococcus furiosus TFB2, these multiple TFBs have higher sequence homology to each other and have similar transcription efficiencies. Photochemical cross-linking experiments have shown that the B-reader of TFB in archaea and TFIIB in eukaryotes is close to transcription start site and is very important in RNAP recruitment to promoter DNA and transcription start site selection. Thus the lack of the highly conserved B reader region in P. furiosus TFB2 presents the opportunity to further study the functional importance of this region. In this study several amino acids in N-terminal domain of TFB2 were mutated with photoactivable unnatural amino acid p-benzoyl L- phenylalanine (pBpa) and the proximity of TFB2 relative to DNA was determined by photochemical cross-linking experiments. The results showed that TFB2 interacts with DNA near the TATA box via its C-terminal domain, and interacts with both strands of DNA near the transcription start site via its divergent B-reader and the B-linker sequences. The B-reader loop region is close to transcription start site and interacts with the transcribed strand of promoter DNA while the B-linker strand cross-links with the non-transcribed strand. Some of the amino acids in between the B-reader loop and the B-linker strand region in TFB2 are seen to cross-link both the transcribed and the non-transcribed strand. Thus, despite the absence of strong homology to conserved B-reader and B-linker sequences, TFB2 is likely to interact with DNA in the transcription bubble and facilitate in transcription initiation. Close

• 6. [Article] Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene <i>vp3</i>

  Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae, where >90% of the viral genes do not ...

  Citation × Citation Citation Abstract Copy Title: Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene <i>vp3</i> Author: Iverson, Eric, Goodman, David A., Gorchels, Madeline E., Stedman, Kenneth M. Year: 2017 Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae, where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1) which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all ORFs. We demonstrate that almost half of the SSV1 genes are not essential for infectivity and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae. The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. Importance: Most of the putative genes in the spindle-shaped archaeal hyperthermophile Fuselloviruses have no clearly similar sequences to characterized genes. In order to determine which of these SSV genes are important for function we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known Fuselloviruses, vp3, allow the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of these unique, ubiquitous and extremely stable archaeal viruses. Title: Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene <i>vp3</i> Author: Iverson, Eric, Goodman, David A., Gorchels, Madeline E., Stedman, Kenneth M. Year: 2017 Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae, where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1) which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all ORFs. We demonstrate that almost half of the SSV1 genes are not essential for infectivity and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae. The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. Importance: Most of the putative genes in the spindle-shaped archaeal hyperthermophile Fuselloviruses have no clearly similar sequences to characterized genes. In order to determine which of these SSV genes are important for function we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known Fuselloviruses, vp3, allow the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of these unique, ubiquitous and extremely stable archaeal viruses. Close

• 7. [Article] Novel Cul3 Binding Proteins Function to Remodel E3 Ligase Complexes

  Background: Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, ...

  Citation × Citation Citation Abstract Copy Title: Novel Cul3 Binding Proteins Function to Remodel E3 Ligase Complexes Author: Wimuttisuk, Wananit, West, Mark, Davidge, Brittney, Yu, Kebing, Salomon, Arthur, Singer, Jeffrey D. Year: 2014 Background: Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as leucine-rich repeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein. Results: Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of Cul3-bound proteins that contain either the LRR or WD40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor. To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex. Conclusion: Our study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC/MS-MS analysis. Subsequently, our biochemical analysis revealed that some CLWs modify binding of BTB domain-containing proteins to the complex, causing degradation of the BTB domain-containing protein. As these CLWs were excluded from our list of substrates, we propose that CLWs serve as unique Cul3 binding proteins that provide an alternative regulatory mechanism for the complex. Title: Novel Cul3 Binding Proteins Function to Remodel E3 Ligase Complexes Author: Wimuttisuk, Wananit, West, Mark, Davidge, Brittney, Yu, Kebing, Salomon, Arthur, Singer, Jeffrey D. Year: 2014 Background: Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as leucine-rich repeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein. Results: Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of Cul3-bound proteins that contain either the LRR or WD40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor. To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex. Conclusion: Our study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC/MS-MS analysis. Subsequently, our biochemical analysis revealed that some CLWs modify binding of BTB domain-containing proteins to the complex, causing degradation of the BTB domain-containing protein. As these CLWs were excluded from our list of substrates, we propose that CLWs serve as unique Cul3 binding proteins that provide an alternative regulatory mechanism for the complex. Close

• 8. [Article] Using Phylogenetically-Informed Annotation (PIA) to search for light-interacting Genes in Transcriptomes from Non-Model Organisms

  Background: Tools for high throughput sequencing and de novo assembly make the analysis of transcriptomes (i.e. the suite of genes expressed in a tissue) feasible for almost any organism. Yet a challenge ...

  Citation × Citation Citation Abstract Copy Title: Using Phylogenetically-Informed Annotation (PIA) to search for light-interacting Genes in Transcriptomes from Non-Model Organisms Author: Speiser, Daniel L., Pankey, Molly S., Zaharoff, Alexander K., Battelle, Barbara A., Bracken-Grissom, Heather D., Breinholt, Jesse W., Bybee, Seth M., Cronin, Thomas W., Garm, Anders, Lindgren, Annie R., Patel, Nipam H., Porter, Megan L., Protas, Meredith E., Rivera, Ajna S., Serb, Jeanne M., Zigler, Kirk S., Crandall, Keith A., Oakley, Todd H. Year: 2014 Background: Tools for high throughput sequencing and de novo assembly make the analysis of transcriptomes (i.e. the suite of genes expressed in a tissue) feasible for almost any organism. Yet a challenge for biologists is that it can be difficult to assign identities to gene sequences, especially from non-model organisms. Phylogenetic analyses are one useful method for assigning identities to these sequences, but such methods tend to be time-consuming because of the need to re-calculate trees for every gene of interest and each time a new data set is analyzed. In response, we employed existing tools for phylogenetic analysis to produce a computationally efficient, tree-based approach for annotating transcriptomes or new genomes that we term Phylogenetically-Informed Annotation (PIA), which places uncharacterized genes into pre-calculated phylogenies of gene families. Results: We generated maximum likelihood trees for 109 genes from a Light Interaction Toolkit (LIT), a collection of genes that underlie the function or development of light-interacting structures in metazoans. To do so, we searched protein sequences predicted from 29 fully-sequenced genomes and built trees using tools for phylogenetic analysis in the Osiris package of Galaxy (an open-source workflow management system). Next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based Evolutionary Placement Algorithm (implemented in RAxML) to place sequences of potential LIT genes on to our pre-calculated gene trees. Finally, we implemented PIA in Galaxy and used it to search for LIT genes in 28 newly-sequenced transcriptomes from the light-interacting tissues of a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. Our new trees for LIT genes are available on the Bitbucket public repository (http://bitbucket.org/osiris_phylogenetics/pia/) and we demonstrate PIA on a publicly-accessible web server (http://galaxy-dev.cnsi.ucsb.edu/pia/). Conclusions: Our new trees for LIT genes will be a valuable resource for researchers studying the evolution of eyes or other light-interacting structures. We also introduce PIA, a high throughput method for using phylogenetic relationships to identify LIT genes in transcriptomes from non-model organisms. With simple modifications, our methods may be used to search for different sets of genes or to annotate data sets from taxa outside of Metazoa. Title: Using Phylogenetically-Informed Annotation (PIA) to search for light-interacting Genes in Transcriptomes from Non-Model Organisms Author: Speiser, Daniel L., Pankey, Molly S., Zaharoff, Alexander K., Battelle, Barbara A., Bracken-Grissom, Heather D., Breinholt, Jesse W., Bybee, Seth M., Cronin, Thomas W., Garm, Anders, Lindgren, Annie R., Patel, Nipam H., Porter, Megan L., Protas, Meredith E., Rivera, Ajna S., Serb, Jeanne M., Zigler, Kirk S., Crandall, Keith A., Oakley, Todd H. Year: 2014 Background: Tools for high throughput sequencing and de novo assembly make the analysis of transcriptomes (i.e. the suite of genes expressed in a tissue) feasible for almost any organism. Yet a challenge for biologists is that it can be difficult to assign identities to gene sequences, especially from non-model organisms. Phylogenetic analyses are one useful method for assigning identities to these sequences, but such methods tend to be time-consuming because of the need to re-calculate trees for every gene of interest and each time a new data set is analyzed. In response, we employed existing tools for phylogenetic analysis to produce a computationally efficient, tree-based approach for annotating transcriptomes or new genomes that we term Phylogenetically-Informed Annotation (PIA), which places uncharacterized genes into pre-calculated phylogenies of gene families. Results: We generated maximum likelihood trees for 109 genes from a Light Interaction Toolkit (LIT), a collection of genes that underlie the function or development of light-interacting structures in metazoans. To do so, we searched protein sequences predicted from 29 fully-sequenced genomes and built trees using tools for phylogenetic analysis in the Osiris package of Galaxy (an open-source workflow management system). Next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based Evolutionary Placement Algorithm (implemented in RAxML) to place sequences of potential LIT genes on to our pre-calculated gene trees. Finally, we implemented PIA in Galaxy and used it to search for LIT genes in 28 newly-sequenced transcriptomes from the light-interacting tissues of a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. Our new trees for LIT genes are available on the Bitbucket public repository (http://bitbucket.org/osiris_phylogenetics/pia/) and we demonstrate PIA on a publicly-accessible web server (http://galaxy-dev.cnsi.ucsb.edu/pia/). Conclusions: Our new trees for LIT genes will be a valuable resource for researchers studying the evolution of eyes or other light-interacting structures. We also introduce PIA, a high throughput method for using phylogenetic relationships to identify LIT genes in transcriptomes from non-model organisms. With simple modifications, our methods may be used to search for different sets of genes or to annotate data sets from taxa outside of Metazoa. Close