Biology Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2749
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Item Archaeal Transcriptional Regulation of Catabolic Carbon Monoxide Dehydrogenase in Methanosarcina species(2009) Anderson, Kimberly Lynn; Sowers, Kevin R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In Archaea, the basal transcription machinery is eukaryotic-like, but some components, such as activator and repressor proteins, are bacteria-like. To further gain knowledge into cellular processes of Archaea, the genome of Methanosarcina thermophila was searched for helicase genes. A homolog of yeast RAD25, a gene with helicase and nucleotide excision repair (NER) abilities, was isolated. M. thermophila rad25 has the domains for helicase activity, but the C-terminal end is truncated, indicating that this protein mostly likely does not function in NER. After overexpression, helicase activity assays of Rad25 indicated that it might have helicase activity; however, there appeared to be contaminating proteins in the purification, so it was not possible to assign the activity only to Rad25. Additional work is necessary to characterize this protein. To investigate transcription, catabolic gene regulation was studied, specifically regulation of carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) from Methanosarcina species. The regions upstream of the transcriptional start site, as well as the 5' leader region of cdhA, were investigated for trans factors and cis elements that might be involved in regulation. Experiments revealed that regulation of cdhABCDE does not appear to involve trans factors upstream of the transcriptional start site. However, deletion analysis indicated that the 5' leader region does have a role in regulation. Comparing the protein levels to the mRNA levels revealed there was no significant difference between the two, indicating that translational regulation was not a factor. Other experiments ruled out differential mRNA stability as a factor in regulation. A region located between +358 and +405 was important in transcriptional regulation, indicating that regulation occurred at the level of transcription elongation. A model for regulation of catabolic CODH/ACS by differential elongation is proposed. Although 5' leader regions identified for other archaeal genes have been postulated to be involved in regulation, this was the first study to demonstrate a regulatory role by an archaeal leader sequence for differential elongation. Identifying regulatory mechanism(s) of catabolic genes such as CODH/ACS is critical for understanding the regulatory strategies employed by the methanoarchaea to efficiently direct carbon and electron flow during biomass conversion to methane.Item Expression of recombinant proteins in the methane-producing archaeon Methanosarcina acetivorans(2005-12-16) MacAuley, Sheridan Rose; Sowers, Kevin R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recombinant protein expression is a necessary tool for the investigation of proteins in the post-genomic era. While many systems exist for recombinant protein expression in organisms of the eukaryotic and eubacterial domains, few to none are available in the Archaea. A recombinant protein expression system using the methanogenic archaeon Methanosarcina acetivorans was developed which uses the highly regulated cdh promoter and allows expression of recombinant protein with optional 6xHis protein fusions to facilitate rapid purification. A protocol for high-density mass cultivation of M. acetivorans in a stainless steel bioreactor configured as a pH-auxostat was developed. The cdh promoter and alternate promoters were analyzed in attempt to enhance expression of recombinant proteins. The protein expression system was tested on several proteins: the Methanocaldococcus jannaschii prolyl tRNA synthetase, the M. acetivorans prolyl tRNA synthetase, the Methanosarcina thermophila carbonic anhydrase, and the Dehalococcoides ethenogenes tricholorethylene dehalogenase.Item REGULATION AND DISTRIBUTION OF TRANSPOSONS IN HYPERTHERMOPHILIC ARCHAEA(2004-04-30) Kanoksilapatham, Wirojne; Robb, Frank T; BiologyThe genus Pyrococcus consists of approximately 12 described species that are Archaea with optimal growth temperatures near to 100 oC. Pyrococcus furiosus and Pyrococcus woesei were both isolated from marine sediments in the same Italian Vulcano Island site. Insertion sequences (ISs) were identified in the genomes of P. furiosus and P. woesei. Specific primers to differentiate between the types of IS elements were developed. Nucleotide sequence of the rDNA operon (AY519654) from P. woesei was sequenced for the first time. Extensive co-linearity between the genomes of the P. woesei and P. furiosus was demonstrated using radio-labeled IS element sequence probes. A recent transposition event that disrupted the napA gene of P. woesei was revealed. The presence of a type I IS element was observed in the same context in the genomes of P. furiosus and P. woesei. The strains were proposed to be sub sp. of P. furiosus, based on their identical rDNA operon sequence and the presence of IS element markers. The ISs have putative archaeal promoters with a 5' TATA box "boxA". Full length IS mRNAs were detected. Evidence for programmed translational frameshift sites that might limit the translation of the full length transposase was found. Two functional programmed translational frameshift sites were identified in E. coli. The mechanism of frameshifting identified in context 79 in the tnp-I sequence appears to be a novel -2 mechanism, in that the shift site is part of a -1 frameshifting motif. The possible role of a putative ribosome binding site (rbs) observed in context 79, that correlates with high frameshift levels, is discussed.