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No description available.

The 80 S cytoplasmic ribosome is the largest of three populations of ribosomes responsible for protein synthesis in plants. It is comprised of two RNA/protein subunits of unequal size: the small (40 S) subunit selects messages to be translated and performs proofreading, while the large (60 S) subunit has peptidyl transferase acitivity, adding new amino acids to the growing polypeptide. In the model flowering plant <i>Arabidopsis thaliana</i> (hereafter <i>Arabidopsis</i>), four ribosomal RNAs and 81 ribosomal proteins (r-proteins) assemble to form the 80S ribosome. Although the <i>Arabidopsis</i> ribosome contains only a single copy of each of the 81 r-proteins (with the exception of small number of acidic phophoproteins), all r-proteins are encoded from multi-gene families containing two or more expressed members. Herein, I investigated r-protein paralogy in Arabidopsis via specific examination of a two member gene family, RPL23a. By analyzing patterns of reporter gene expression driven by full-length and truncated regulatory regions, I was able to identify a core promoter that is largely conserved between paralogs. Regulation was found to be complex, involving transcriptional, post-transcriptional and translational components. The effects of knocking-out a single RPL23a paralog (RPL23aB) were determined. Results indicated that this paralog is broadly dispensable, and Arabidopsis does not compensate for its loss at the transcriptional level. Subcellular localization was investigated by tagging RPL23aA/B with fluorescent proteins, demonstrating that RPL23aA is targeted to nucleolus more efficiently than RPL23aB, possibly due to a stronger nucleolar localization signal. RNA-interference was used to individually silence RPL23a paralogs to characterize functional overlap. Results showed that RPL23aA, and not RPL23aB, is required for normal development. Mutants with reduced levels of RPL23aA develop a pointed first leaf phenotype that I postulate may be due to disruption of miRNA-mediated degradation of specific auxin response genes. Lastly, the 26 S proteasome was inhibited to determine the importance of protein turnover in regulating RPL23a levels. Findings suggest that proteasome-mediated degradation of RPL23a is essential for preventing accumulation of unincorporated r-proteins. Overall, results indicate that the Arabidopsis RPL23a paralogs have diverged from each other: RPL23aA has become the predominant paralog, while RPL23aB functions in an anciliary capacity and/or is undergoing neofunctionalization.

The 80 S cytoplasmic ribosome is the largest of three populations of ribosomes responsible for protein synthesis in plants. It is comprised of two RNA/protein subunits of unequal size: the small (40 S) subunit selects messages to be translated and performs proofreading, while the large (60 S) subunit has peptidyl transferase acitivity, adding new amino acids to the growing polypeptide. In the model flowering plant <i>Arabidopsis thaliana</i> (hereafter <i>Arabidopsis</i>), four ribosomal RNAs and 81 ribosomal proteins (r-proteins) assemble to form the 80S ribosome. Although the <i>Arabidopsis</i> ribosome contains only a single copy of each of the 81 r-proteins (with the exception of small number of acidic phophoproteins), all r-proteins are encoded from multi-gene families containing two or more expressed members. Herein, I investigated r-protein paralogy in Arabidopsis via specific examination of a two member gene family, RPL23a. By analyzing patterns of reporter gene expression driven by full-length and truncated regulatory regions, I was able to identify a core promoter that is largely conserved between paralogs. Regulation was found to be complex, involving transcriptional, post-transcriptional and translational components. The effects of knocking-out a single RPL23a paralog (RPL23aB) were determined. Results indicated that this paralog is broadly dispensable, and Arabidopsis does not compensate for its loss at the transcriptional level. Subcellular localization was investigated by tagging RPL23aA/B with fluorescent proteins, demonstrating that RPL23aA is targeted to nucleolus more efficiently than RPL23aB, possibly due to a stronger nucleolar localization signal. RNA-interference was used to individually silence RPL23a paralogs to characterize functional overlap. Results showed that RPL23aA, and not RPL23aB, is required for normal development. Mutants with reduced levels of RPL23aA develop a pointed first leaf phenotype that I postulate may be due to disruption of miRNA-mediated degradation of specific auxin response genes. Lastly, the 26 S proteasome was inhibited to determine the importance of protein turnover in regulating RPL23a levels. Findings suggest that proteasome-mediated degradation of RPL23a is essential for preventing accumulation of unincorporated r-proteins. Overall, results indicate that the Arabidopsis RPL23a paralogs have diverged from each other: RPL23aA has become the predominant paralog, while RPL23aB functions in an anciliary capacity and/or is undergoing neofunctionalization.

Brucella melitensis is the etiological agent of brucellosis, a zoonotic disease characterized by abortions in ruminant animals and a chronic debilitating disease in humans. Despite genome sequencing, little is known about the genetic elements behind Brucella s ability to survive and cause disease. Regulatory networks provide the ability to adapt to changing environments by initiating expression from specific regulons to provide adjustments to metabolism and mechanisms that enhance survival. Little detail is known about transcriptional networks that exist in Brucella, but are of great interest because they could provide information about genetic loci that contribute to virulence and intracellular survival. Transposon mutagenesis identified gene loci that are indispensable for the intracellular replication of B. melitensis, including virulence genes, metabolic defects, and transcriptional regulators. Two transcriptional regulators of interest were identified, MucR and VjbR. VjbR is a LuxR homologue and is associated with the regulation of virulence genes in a density dependent manner in a number of bacterial pathogens, and is consistent with VjbR regulation of virulence genes in B. melitensis. Microarray analysis of vjbR and a potential activating signal C12-HSL revealed that both regulate numerous putative virulence genes, including adhesins, proteases, protein secretion/translocation components, potential effector proteins, lipoproteins, a hemolysin and stress survival aids. This analysis also revealed that C12-HSL is not an activating signal of VjbR, but instead acts to suppress VjbR activity. MucR is a transcriptional regulator shown to regulate exopolysaccharide synthesis in the closely related Rhizobiales. Microarray analysis of a mucR mutant in B. melitensis suggested that MucR contributes to the regulation of nitrogen metabolism and iron sequestering/storage. MucR was also found to regulate genes involved in stress response, regulating several proteases that may contribute to enhanced survival and virulence of the organism. This work identified approximately 1,000 genetic loci that may be important to the survival of B. melitensis, revealing potential virulence genes and metabolic defects. Interruption of the VjbR regulon could be a potential chemotherapeutic target for the treatment of brucellosis. Furthermore, this work describes the functions of two gene deletions that are being evaluated as novel attenuated vaccines.

Identification and characterization of regions influencing the precise spatial and temporal expression of genes is critical to our understanding of gene regulatory networks. Connecting transcription factors to the cis-regulatory elements that they bind and regulate remains a challenging problem in computational biology. The rapid accumulation of whole genome sequences and genome-wide expression data, and advances in alignment algorithms and motif-finding methods, provide opportunities to tackle the important task of dissecting how genes are regulated. Genes exhibiting similar expression profiles are often regulated by common transcription factors. We developed a method for identifying statistically over-represented regulatory motifs in the promoters of co-expressed genes using weight matrix models representing the specificity of known factors. Application of our methods to yeast fermenting in grape must revealed elements that play important roles in utilizing carbon sources. Extension of the method to metazoan genomes via incorporation of comparative sequence analysis facilitated identification of functionally relevant binding sites for sets of tissue-specific genes, and for genes showing similar expression in large-scale expression profiling studies. Further extensions address alternative promoters for human genes and coordinated binding of multiple transcription factors to cis-regulatory modules. Sequence conservation reveals segments of genes of potential interest, but the degree of sequence divergence among human genes and their orthologous sequences varies widely. Genes with a small number of well-distinguished, highly conserved non-coding elements proximal to the transcription start site may be well-suited for targeted laboratory promoter characterization studies. We developed a “regulatory resolution” score to prioritize lists of genes for laboratory gene regulation studies based on the conservation profile of their promoters. Additionally, genome-wide comparisons of vertebrate genomes have revealed surprisingly large numbers of highly conserved non-coding elements (HCNEs) that cluster nearby to genes associated with transcription and development. To further our understanding of the genomic organization of regulatory regions, we developed methods to identify HCNEs in insects. We find that HCNEs in insects have similar function and organization as their vertebrate counterparts. Our data suggests that microsynteny in insects has been retained to keep large arrays of HCNEs intact, forming genomic regulatory blocks that surround the key developmental genes they regulate.

Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins, which are proposed to have roles in essential trace metal homoeostasis and in the detoxification of metal ions. The genes encoding MTs have been isolated from a wide range of eukaryotes, although MT genes have not previously been isolated from prokaryotes. The polymerase chain reaction (PGR) was initially used to isolate a prokaryotic MT gene fragment from Synechococcus PCC 6301. PGR fragments were amplified using inosine-containing primers designed from the amino acid sequence of a prokaryotic MT. Subsequent cloning and nucleotide sequence analysis revealed that the deduced amino acid sequence of the PGR product corresponded to the amino acid sequence of the prokaryotic MT. The amplified product was thus part of the gene encoding the MT, and was designated smtA. The same primers used in the initial amplification were subsequently utilised for anchored PGR, to amplify the remainder of the coding region and the 3' and 5' flanking regions of the smtA gene. A genomic library was produced from Synechococcus PGG 7942 DNA and screened using the PGR products described above as probes. A genomic clone was isolated, nucleotide sequence analysis revealed the structure of the smt locus, two open reading frames, smtA and smtB, arranged in a divergent orientation about the smt operator/promoter region. The operator/promoter region contains the transcriptional and translational signals for the two genes and three regions that are candidate sites for interaction of regulatory proteins. The transcript start sites of the two genes were mapped within the operator/promoter region by primer extension analysis. An increase in the relative abundance of transcripts of both smt genes was studied in response to various metal ions in a series of northern blots. Inhibitor studies confirmed that the smtA gene is regulated at the transcriptional level. The 5' flanking region of the smtA gene conferred metal specific induction of the reporter gene lacZ. SmtB has sequence similarity to several prokaryotic regulatory proteins and contains a putative helix-turn-helix structural domain. Deletion analysis suggests that SmtB is a repressor of smtA expression. Subsequent work has confirmed that SmtB is a trans-acting repressor of expression from the smt operator/promoter.

Identification and characterization of regions influencing the precise spatial and temporal expression of genes is critical to our understanding of gene regulatory networks. Connecting transcription factors to the cis-regulatory elements that they bind and regulate remains a challenging problem in computational biology. The rapid accumulation of whole genome sequences and genome-wide expression data, and advances in alignment algorithms and motif-finding methods, provide opportunities to tackle the important task of dissecting how genes are regulated. Genes exhibiting similar expression profiles are often regulated by common transcription factors. We developed a method for identifying statistically over-represented regulatory motifs in the promoters of co-expressed genes using weight matrix models representing the specificity of known factors. Application of our methods to yeast fermenting in grape must revealed elements that play important roles in utilizing carbon sources. Extension of the method to metazoan genomes via incorporation of comparative sequence analysis facilitated identification of functionally relevant binding sites for sets of tissue-specific genes, and for genes showing similar expression in large-scale expression profiling studies. Further extensions address alternative promoters for human genes and coordinated binding of multiple transcription factors to cis-regulatory modules. Sequence conservation reveals segments of genes of potential interest, but the degree of sequence divergence among human genes and their orthologous sequences varies widely. Genes with a small number of well-distinguished, highly conserved non-coding elements proximal to the transcription start site may be well-suited for targeted laboratory promoter characterization studies. We developed a “regulatory resolution” score to prioritize lists of genes for laboratory gene regulation studies based on the conservation profile of their promoters. Additionally, genome-wide comparisons of vertebrate genomes have revealed surprisingly large numbers of highly conserved non-coding elements (HCNEs) that cluster nearby to genes associated with transcription and development. To further our understanding of the genomic organization of regulatory regions, we developed methods to identify HCNEs in insects. We find that HCNEs in insects have similar function and organization as their vertebrate counterparts. Our data suggests that microsynteny in insects has been retained to keep large arrays of HCNEs intact, forming genomic regulatory blocks that surround the key developmental genes they regulate.

Identification and characterization of regions influencing the precise spatial and temporal expression of genes is critical to our understanding of gene regulatory networks. Connecting transcription factors to the cis-regulatory elements that they bind and regulate remains a challenging problem in computational biology. The rapid accumulation of whole genome sequences and genome-wide expression data, and advances in alignment algorithms and motif-finding methods, provide opportunities to tackle the important task of dissecting how genes are regulated. Genes exhibiting similar expression profiles are often regulated by common transcription factors. We developed a method for identifying statistically over-represented regulatory motifs in the promoters of co-expressed genes using weight matrix models representing the specificity of known factors. Application of our methods to yeast fermenting in grape must revealed elements that play important roles in utilizing carbon sources. Extension of the method to metazoan genomes via incorporation of comparative sequence analysis facilitated identification of functionally relevant binding sites for sets of tissue-specific genes, and for genes showing similar expression in large-scale expression profiling studies. Further extensions address alternative promoters for human genes and coordinated binding of multiple transcription factors to cis-regulatory modules. Sequence conservation reveals segments of genes of potential interest, but the degree of sequence divergence among human genes and their orthologous sequences varies widely. Genes with a small number of well-distinguished, highly conserved non-coding elements proximal to the transcription start site may be well-suited for targeted laboratory promoter characterization studies. We developed a “regulatory resolution” score to prioritize lists of genes for laboratory gene regulation studies based on the conservation profile of their promoters. Additionally, genome-wide comparisons of vertebrate genomes have revealed surprisingly large numbers of highly conserved non-coding elements (HCNEs) that cluster nearby to genes associated with transcription and development. To further our understanding of the genomic organization of regulatory regions, we developed methods to identify HCNEs in insects. We find that HCNEs in insects have similar function and organization as their vertebrate counterparts. Our data suggests that microsynteny in insects has been retained to keep large arrays of HCNEs intact, forming genomic regulatory blocks that surround the key developmental genes they regulate. / Medicine, Faculty of / Medical Genetics, Department of / Graduate