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• 1. [Article] Growth Phase-Dependent Gene Regulation in Vivo in Sulfolobus Solfataricus

  Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene ...

  Citation × Citation Citation Abstract Copy Title: Growth Phase-Dependent Gene Regulation in Vivo in Sulfolobus Solfataricus Author: DeYoung, Melissa, Thayer, Michelle, Van der Oost, John, Stedman, Kenneth M. Year: 2011 Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene regulation in Archaea. SSV1- based lacS reporter gene constructs containing the Sulfolobus 16S/23S rRNA gene core promoter, the TF55a core promoter, or the native lacS promoter were tested in Sulfolobus solfataricus cells lacking the lacS gene. The 42-bp 16S/23S rRNA gene and 39-bp TF55a core promoters are sufficient for gene expression in S. solfataricus. However, only gene expression driven by the 16S/23S rRNA gene core promoter is dependent on the culture growth phase. This is the smallest known regulated promoter in Sulfolobus. To our knowledge, this is the first study to show growth phase-dependent rRNA gene regulation in Archaea. Title: Growth Phase-Dependent Gene Regulation in Vivo in Sulfolobus Solfataricus Author: DeYoung, Melissa, Thayer, Michelle, Van der Oost, John, Stedman, Kenneth M. Year: 2011 Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene regulation in Archaea. SSV1- based lacS reporter gene constructs containing the Sulfolobus 16S/23S rRNA gene core promoter, the TF55a core promoter, or the native lacS promoter were tested in Sulfolobus solfataricus cells lacking the lacS gene. The 42-bp 16S/23S rRNA gene and 39-bp TF55a core promoters are sufficient for gene expression in S. solfataricus. However, only gene expression driven by the 16S/23S rRNA gene core promoter is dependent on the culture growth phase. This is the smallest known regulated promoter in Sulfolobus. To our knowledge, this is the first study to show growth phase-dependent rRNA gene regulation in Archaea. Close

• 2. [Article] Consequences of miRNA misregulation on embryonic development and aging

  microRNAs (miRNAs), ~21-24 nucleotide-long RNAs that post-transcriptionally regulate gene expression, have rapidly become one of the most extensively studied mechanisms of the past decade. Since their ...

  Citation × Citation Citation Abstract Copy Title: Consequences of miRNA misregulation on embryonic development and aging Author: Franzosa, Jill A. microRNAs (miRNAs), ~21-24 nucleotide-long RNAs that post-transcriptionally regulate gene expression, have rapidly become one of the most extensively studied mechanisms of the past decade. Since their discovery as temporal regulators of post-embryonic development in C. elegans, miRNAs have been functionally implicated in almost every cellular process investigated to date. miRNAs are integral to the complex biological processes of embryonic development and aging. In this research, we sought to determine whether misregulation of miRNAs could be responsible for eliciting adverse effects during these two distinct developmental stages. First, to uncover the potential role of miRNAs in teratogenicity, we investigated whether miRNAs were involved in regulation of retinoic acid (RA) induced vertebrate axis defects. Global miRNA expression profiling revealed that RA exposure suppressed the expression of miR-19 family members during zebrafish somitogenesis. Bioinformatics analyses predict that miR-19 targets cyp26a1, a key RA detoxifying enzyme, and a physiological reporter assay confirmed that cyp26a1 is a bona fide target of miR-19. Transient knockdown of miR-19 phenocopied RA-induced body axis defects. In gain-of-function studies, exogenous miR-19 rescued the axis defects caused by RA exposure. Our findings indicate that the teratogenic effects of RA exposure result, in part, from repression of miR-19 and the subsequent misregulation of cyp26a1. This highlights a previously unidentified role of miR-19 in facilitating vertebrate axis development. Next, to explore whether age-related changes in miRNAs trigger deficits in regeneration capacity, we performed mRNA and small RNA sequencing on regenerating and non-regenerating caudal fin tissue from aged, adult and juvenile zebrafish. An unbiased approach identified cbx7 as the most abundant transcript with significantly increased expression in regenerative-competent adult and juvenile tissue and decreased expression in regenerative-compromised aged tissue. While cbx7 is a known regulator of aging, this is the first report of its role in tissue regeneration. A computational approach was used to discover mRNAs expressed during regeneration, which are potential targets of the significantly expressed miRNAs in regenerating tissue. miR-21 was one of the most abundant and significantly increased miRNAs in regenerating tissue and exhibited an aberrant age-dependent expression profile. Bioinformatics predicts miR-21 to target the 3' UTR of cbx7 and a reporter assay confirmed that miR-21 targets cbx7 in vivo. Transient knockdown of miR-21 inhibited tissue regeneration, suggesting a role for miRNA mediated regulation of cbx7 during regeneration. These findings reveal a novel, age-dependent regenerative function of cbx7 and emphasize the importance of miR-21 as a master regulator of vertebrate regenerative responses. This research, when combined, underscores the negative consequences misregulation of miRNAs has on embryonic development and aging. Title: Consequences of miRNA misregulation on embryonic development and aging Author: Franzosa, Jill A. microRNAs (miRNAs), ~21-24 nucleotide-long RNAs that post-transcriptionally regulate gene expression, have rapidly become one of the most extensively studied mechanisms of the past decade. Since their discovery as temporal regulators of post-embryonic development in C. elegans, miRNAs have been functionally implicated in almost every cellular process investigated to date. miRNAs are integral to the complex biological processes of embryonic development and aging. In this research, we sought to determine whether misregulation of miRNAs could be responsible for eliciting adverse effects during these two distinct developmental stages. First, to uncover the potential role of miRNAs in teratogenicity, we investigated whether miRNAs were involved in regulation of retinoic acid (RA) induced vertebrate axis defects. Global miRNA expression profiling revealed that RA exposure suppressed the expression of miR-19 family members during zebrafish somitogenesis. Bioinformatics analyses predict that miR-19 targets cyp26a1, a key RA detoxifying enzyme, and a physiological reporter assay confirmed that cyp26a1 is a bona fide target of miR-19. Transient knockdown of miR-19 phenocopied RA-induced body axis defects. In gain-of-function studies, exogenous miR-19 rescued the axis defects caused by RA exposure. Our findings indicate that the teratogenic effects of RA exposure result, in part, from repression of miR-19 and the subsequent misregulation of cyp26a1. This highlights a previously unidentified role of miR-19 in facilitating vertebrate axis development. Next, to explore whether age-related changes in miRNAs trigger deficits in regeneration capacity, we performed mRNA and small RNA sequencing on regenerating and non-regenerating caudal fin tissue from aged, adult and juvenile zebrafish. An unbiased approach identified cbx7 as the most abundant transcript with significantly increased expression in regenerative-competent adult and juvenile tissue and decreased expression in regenerative-compromised aged tissue. While cbx7 is a known regulator of aging, this is the first report of its role in tissue regeneration. A computational approach was used to discover mRNAs expressed during regeneration, which are potential targets of the significantly expressed miRNAs in regenerating tissue. miR-21 was one of the most abundant and significantly increased miRNAs in regenerating tissue and exhibited an aberrant age-dependent expression profile. Bioinformatics predicts miR-21 to target the 3' UTR of cbx7 and a reporter assay confirmed that miR-21 targets cbx7 in vivo. Transient knockdown of miR-21 inhibited tissue regeneration, suggesting a role for miRNA mediated regulation of cbx7 during regeneration. These findings reveal a novel, age-dependent regenerative function of cbx7 and emphasize the importance of miR-21 as a master regulator of vertebrate regenerative responses. This research, when combined, underscores the negative consequences misregulation of miRNAs has on embryonic development and aging. Close

• 3. [Article] Investigations of the isoprenoid nucleosides of pea root transfer ribonucleic acid

  Citation × Citation Citation Abstract Copy Title: Investigations of the isoprenoid nucleosides of pea root transfer ribonucleic acid Author: Babcock, Donner Franklin, 1942- Title: Investigations of the isoprenoid nucleosides of pea root transfer ribonucleic acid Author: Babcock, Donner Franklin, 1942- Close

• 4. [Article] 10 Biochemistry Lecture (Enzyme Regulation) from Kevin Ahern's BB 350.mp4

  Citation × Citation Citation Abstract Copy Title: 10 Biochemistry Lecture (Enzyme Regulation) from Kevin Ahern's BB 350.mp4 Author: Ahern, Kevin Title: 10 Biochemistry Lecture (Enzyme Regulation) from Kevin Ahern's BB 350.mp4 Author: Ahern, Kevin Close

• 5. [Article] 11 Biochemistry Lecture (Enzyme Regulation II) from Kevin Ahern's BB 350.mp4

  Citation × Citation Citation Abstract Copy Title: 11 Biochemistry Lecture (Enzyme Regulation II) from Kevin Ahern's BB 350.mp4 Author: Ahern, Kevin Title: 11 Biochemistry Lecture (Enzyme Regulation II) from Kevin Ahern's BB 350.mp4 Author: Ahern, Kevin Close

• 6. [Article] Genome Rearrangements Can Make and Break Small RNA Genes

  Small RNAs (sRNAs) are short, transcribed regulatory elements that are typically encoded in the intergenic regions (IGRs) of bacterial genomes. Several sRNAs, first recognized in Escherichia coli, are ...

  Citation × Citation Citation Abstract Copy Title: Genome Rearrangements Can Make and Break Small RNA Genes Author: Raghavan, Rahul, Kacharia, Fenil R., Millar, Jess A, Sislak, Christine Demko, Ochman, Howard Year: 2015 Small RNAs (sRNAs) are short, transcribed regulatory elements that are typically encoded in the intergenic regions (IGRs) of bacterial genomes. Several sRNAs, first recognized in Escherichia coli, are conserved among enteric bacteria, but because of the regulatory roles of sRNAs, differences in sRNA repertoires might be responsible for features that differentiate closely related species. We scanned the E. coli MG1655 and Salmonella enterica Typhimurium genomes for nonsyntenic IGRs as a potential source of uncharacterized, species-specific sRNAs and found that genome rearrangements have reconfigured several IGRs causing the disruption and formation of sRNAs. Within an IGR that is present in E. coli but was disrupted in Salmonella by a translocation event is an sRNA that is associated with the FNR/CRP global regulators and influences E. coli biofilm formation. A Salmonella-specific sRNA evolved de novo through point mutations that generated a σ70 promoter sequence in an IGR that arose through genome rearrangement events. The differences in the sRNA pools among bacterial species have previously been ascribed to duplication, deletion, or horizontal acquisition. Here, we show that genomic rearrangements also contribute to this process by either disrupting sRNA-containing IGRs or creating IGRs in which novel sRNAs may evolve. Title: Genome Rearrangements Can Make and Break Small RNA Genes Author: Raghavan, Rahul, Kacharia, Fenil R., Millar, Jess A, Sislak, Christine Demko, Ochman, Howard Year: 2015 Small RNAs (sRNAs) are short, transcribed regulatory elements that are typically encoded in the intergenic regions (IGRs) of bacterial genomes. Several sRNAs, first recognized in Escherichia coli, are conserved among enteric bacteria, but because of the regulatory roles of sRNAs, differences in sRNA repertoires might be responsible for features that differentiate closely related species. We scanned the E. coli MG1655 and Salmonella enterica Typhimurium genomes for nonsyntenic IGRs as a potential source of uncharacterized, species-specific sRNAs and found that genome rearrangements have reconfigured several IGRs causing the disruption and formation of sRNAs. Within an IGR that is present in E. coli but was disrupted in Salmonella by a translocation event is an sRNA that is associated with the FNR/CRP global regulators and influences E. coli biofilm formation. A Salmonella-specific sRNA evolved de novo through point mutations that generated a σ70 promoter sequence in an IGR that arose through genome rearrangement events. The differences in the sRNA pools among bacterial species have previously been ascribed to duplication, deletion, or horizontal acquisition. Here, we show that genomic rearrangements also contribute to this process by either disrupting sRNA-containing IGRs or creating IGRs in which novel sRNAs may evolve. Close

• 7. [Article] Antisense transcription is pervasive but rarely conserved in enteric bacteria

  Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent ...

  Citation × Citation Citation Abstract Copy Title: Antisense transcription is pervasive but rarely conserved in enteric bacteria Author: Raghavan, Rahul, Sloan, D. B., Ochman, H. Year: 2012 Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell's transcription machinery. IMPORTANCE Application of high-throughput methods has revealed the expression throughout bacterial genomes of transcripts encoded on the strand complementary to protein-coding genes. Because transcription is costly, it is usually assumed that these transcripts, termed antisense RNAs (asRNAs), serve some function; however, the role of most asRNAs is unclear, raising questions about their relevance in cellular processes. Because natural selection conserves functional elements, comparisons between related species provide a method for assessing functionality genome-wide. Applying such an approach, we assayed all transcripts in two closely related bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, and demonstrate that, although the levels of genome-wide antisense transcription are similarly high in both bacteria, only a small fraction of asRNAs are shared across species. Moreover, the promoters associated with asRNAs show no evidence of sequence conservation between, or even within, species. These findings indicate that despite the genome-wide transcription of asRNAs, many of these transcripts are likely nonfunctional. Title: Antisense transcription is pervasive but rarely conserved in enteric bacteria Author: Raghavan, Rahul, Sloan, D. B., Ochman, H. Year: 2012 Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell's transcription machinery. IMPORTANCE Application of high-throughput methods has revealed the expression throughout bacterial genomes of transcripts encoded on the strand complementary to protein-coding genes. Because transcription is costly, it is usually assumed that these transcripts, termed antisense RNAs (asRNAs), serve some function; however, the role of most asRNAs is unclear, raising questions about their relevance in cellular processes. Because natural selection conserves functional elements, comparisons between related species provide a method for assessing functionality genome-wide. Applying such an approach, we assayed all transcripts in two closely related bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, and demonstrate that, although the levels of genome-wide antisense transcription are similarly high in both bacteria, only a small fraction of asRNAs are shared across species. Moreover, the promoters associated with asRNAs show no evidence of sequence conservation between, or even within, species. These findings indicate that despite the genome-wide transcription of asRNAs, many of these transcripts are likely nonfunctional. Close

• 8. [Article] Platinum coordination to RNA

  xix, 111 p. : ill. (some col.)

  Citation × Citation Citation Abstract Copy Title: Platinum coordination to RNA Author: Chapman, Erich G., 1984- Year: 2011, 2010 xix, 111 p. : ill. (some col.) Title: Platinum coordination to RNA Author: Chapman, Erich G., 1984- Year: 2011, 2010 xix, 111 p. : ill. (some col.) Close

• 9. [Article] In vitro and in cellulo interactions of platinum and ruthenium anticancer metallodrugs with RNA

  xviii, 125 p. : ill. (some col.)

  Citation × Citation Citation Abstract Copy Title: In vitro and in cellulo interactions of platinum and ruthenium anticancer metallodrugs with RNA Author: Hostetter, Alethia A., 1981- Year: 2011, 2012 xviii, 125 p. : ill. (some col.) Title: In vitro and in cellulo interactions of platinum and ruthenium anticancer metallodrugs with RNA Author: Hostetter, Alethia A., 1981- Year: 2011, 2012 xviii, 125 p. : ill. (some col.) Close

• 10. [Article] Classifying Chemical Bioactivity by Coupling High-throughput Phenotypic Anchoring and Transcriptome Profiling in Zebrafish

  There are more than 87,000 chemicals in current use with little to no toxicity information available. Assessing such a large number of chemicals using traditional methods would take an unreasonable amount ...

  Citation × Citation Citation Abstract Copy Title: Classifying Chemical Bioactivity by Coupling High-throughput Phenotypic Anchoring and Transcriptome Profiling in Zebrafish Author: Haggard, Derik There are more than 87,000 chemicals in current use with little to no toxicity information available. Assessing such a large number of chemicals using traditional methods would take an unreasonable amount of time and money, and require the use a large number of animals. The incorporation of high-throughput in vivo model systems that can overcome limitations of in vitro screens while providing a rapid genome-wide view of chemical bioactivity are needed to help fully realize the vision of 21st century toxicity testing. In this dissertation, I employed transcriptomics using the high-throughput in vivo developmental zebrafish model to generate hypotheses regarding the mechanism of action of specific chemicals and to identify unique transcriptional signatures for a set of endocrine disrupting chemicals that can be used to classify the activity of known and unknown chemicals. We employed comparative transcriptomics in wild-type and ahr2-null zebrafish exposed to mono-substituted isopropylated triaryl phosphate (mITP), a component of the Firemaster 550 flame retardant mixture, and demonstrated that the cardiotoxic effects of mITP are likely mediated through inhibition of retinoic acid receptor signaling and not the aryl hydrocarbon receptor (AhR). Although mITP induced a transcriptional profile indicative of AhR activation in wild-type zebrafish, these signatures were not related to cardiotoxicity. I found that mITP exposure, independent of ahr2 status, decreased the expression of many Hox genes as well as enzymes responsible for retinoic acid metabolism, which are known to be regulated by retinoic acid receptors. Several of the dysregulated Hox genes are involved in cardiac cell lineage determination in early heart development, and in heart tube elongation and looping via cell recruitment in the second heart field. I also employed transcriptomics to investigate the mechanism of developmental toxicity of the antimicrobial agent, triclosan (TCS). Exposure to TCS resulted in robust transcriptome changes with a large number of transcripts being significantly decreased. Downstream functional analyses showed that many of the transcripts significantly affected by TCS are involved in liver functioning and development, suggesting that TCS is hepatotoxic in embryonic zebrafish. I also compared our transcriptomic analysis with the comprehensive in vitro bioactivity profile of TCS from the Environmental Protection Agency’s Toxicity Forecaster (ToxCast) program, and observed low concordance between these two screening strategies. Lastly, I conducted transcriptome profiling of 25 endocrine disrupting chemicals in order to identify discriminatory transcriptional signatures that would help classify chemicals with estrogen, androgen, or thyroid hormone activity. Clustered correlation analysis of the top 1000 significantly differentially expressed transcripts revealed four chemicals with highly similar and unique transcriptional profiles compared to the other 21 chemicals. These four chemicals included three known thyroid hormone receptor agonists, and one unknown chemical, which was later identified as a failed pharmaceutical with thyroid receptor agonist activity. I identified a panel of 27 transcripts as well as a unique pigmentation phenotype for these four chemicals which can be used in future chemical screens to detect other thyroid receptor agonist chemicals in zebrafish. Overall, the work presented here demonstrates the utility of phenotypically anchored whole genome transcriptomics in zebrafish to identify putative mechanisms of action for many chemicals. Furthermore, the information obtained from these types of analyses can be used to develop predictive models to classify the bioactivity of known and unknown chemicals, or can identify biomarkers that can be used in future high-throughput screens. Title: Classifying Chemical Bioactivity by Coupling High-throughput Phenotypic Anchoring and Transcriptome Profiling in Zebrafish Author: Haggard, Derik There are more than 87,000 chemicals in current use with little to no toxicity information available. Assessing such a large number of chemicals using traditional methods would take an unreasonable amount of time and money, and require the use a large number of animals. The incorporation of high-throughput in vivo model systems that can overcome limitations of in vitro screens while providing a rapid genome-wide view of chemical bioactivity are needed to help fully realize the vision of 21st century toxicity testing. In this dissertation, I employed transcriptomics using the high-throughput in vivo developmental zebrafish model to generate hypotheses regarding the mechanism of action of specific chemicals and to identify unique transcriptional signatures for a set of endocrine disrupting chemicals that can be used to classify the activity of known and unknown chemicals. We employed comparative transcriptomics in wild-type and ahr2-null zebrafish exposed to mono-substituted isopropylated triaryl phosphate (mITP), a component of the Firemaster 550 flame retardant mixture, and demonstrated that the cardiotoxic effects of mITP are likely mediated through inhibition of retinoic acid receptor signaling and not the aryl hydrocarbon receptor (AhR). Although mITP induced a transcriptional profile indicative of AhR activation in wild-type zebrafish, these signatures were not related to cardiotoxicity. I found that mITP exposure, independent of ahr2 status, decreased the expression of many Hox genes as well as enzymes responsible for retinoic acid metabolism, which are known to be regulated by retinoic acid receptors. Several of the dysregulated Hox genes are involved in cardiac cell lineage determination in early heart development, and in heart tube elongation and looping via cell recruitment in the second heart field. I also employed transcriptomics to investigate the mechanism of developmental toxicity of the antimicrobial agent, triclosan (TCS). Exposure to TCS resulted in robust transcriptome changes with a large number of transcripts being significantly decreased. Downstream functional analyses showed that many of the transcripts significantly affected by TCS are involved in liver functioning and development, suggesting that TCS is hepatotoxic in embryonic zebrafish. I also compared our transcriptomic analysis with the comprehensive in vitro bioactivity profile of TCS from the Environmental Protection Agency’s Toxicity Forecaster (ToxCast) program, and observed low concordance between these two screening strategies. Lastly, I conducted transcriptome profiling of 25 endocrine disrupting chemicals in order to identify discriminatory transcriptional signatures that would help classify chemicals with estrogen, androgen, or thyroid hormone activity. Clustered correlation analysis of the top 1000 significantly differentially expressed transcripts revealed four chemicals with highly similar and unique transcriptional profiles compared to the other 21 chemicals. These four chemicals included three known thyroid hormone receptor agonists, and one unknown chemical, which was later identified as a failed pharmaceutical with thyroid receptor agonist activity. I identified a panel of 27 transcripts as well as a unique pigmentation phenotype for these four chemicals which can be used in future chemical screens to detect other thyroid receptor agonist chemicals in zebrafish. Overall, the work presented here demonstrates the utility of phenotypically anchored whole genome transcriptomics in zebrafish to identify putative mechanisms of action for many chemicals. Furthermore, the information obtained from these types of analyses can be used to develop predictive models to classify the bioactivity of known and unknown chemicals, or can identify biomarkers that can be used in future high-throughput screens. Close