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Amber suppression in the archaebacterium Haloferax volcanii

University of British Columbia

The purpose of this project was to test whether amber suppression can occur in Haloferax volcanii or not, and if so, to construct a H. volcanii strain that can suppress amber mutations. To achieve this goal, a putative amber suppressor was constructed from the tyrosine transfer RNA of H. volcanii. Its ability to recognize an amber stop codon and to restore the wild type function of an amber mutated gene was tested. The gene coding for tyrosine tRNA of H. volcanii DS2 was cloned and sequenced. Site-directed mutagenesis was carried out to change the anticodon of the tRNA gene from GUA to CUA, which recognizes the tyrosine codon UAU and UAC, and the amber stop codon UAG respectively. The hisC gene of wild type H. volcanii was obtained and recloned into pGEM7(-). Site directed mutagenesis was carried out to change the DNA sequence of its first tyrosine codon (TAC) to an amber stop codon (TAG). I attempted to replace the wild type hisC gene in the H. volcanii WFD11 genome with the hisC gene carrying the amber mutation. However, although the construct carrying the hisC(Am) gene and an antibiotic resistance marker integrated into the genome at the correct place, displacement of the wild type gene through reverse recombination did not occur. An attempt to test amber suppression in H. volcanii was carried out. The hisC(Am) gene was introduced into the genome of a mutant strain of H. volcanii WR256 (his⁻, arg⁻) with the antibiotic selection marker mevinolin. The transformants were then transformed again with a plasmid that carries the putative amber suppressor (the tRNATY1 gene with the mutated amber anticodon) and the other antibiotic selection marker novobiocin. Transformants were then selected with both antibiotics and then tested for restoration of histidine auxotrophy. All transformants still required histidine for growth. Southern hybridization showed that the hisC(Am) gene was not integrated into the genome. Mevinolin resistance in the transformants was due to a double crossover recombination event of the antibiotic resistance gene into the genome to replace the wild type gene. Therefore I was not able to conclude whether amber suppression can occur in H.volcanii or not.

Thesis/Dissertation

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2009-02-24

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http://hdl.handle.net/2429/4972

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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