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Targeted Mutation of Genes Implicated in DNA Replication and Repair in Sulfolobus acidocaldarius
| Title: | Targeted Mutation of Genes Implicated in DNA Replication and Repair in Sulfolobus acidocaldarius |
| Author: | Runck, Laura Ann |
| Description: | The main goals of this research were to evaluate different experimental methods for gene cloning, disruption, and transfer to the Sulfolobus acidocaldarius genome, and to determine function of the genes in vivo. S. acidocaldarius and other hyperthermophilic archaea thrive at temperatures 80C and above which would typically render the DNA unstable. It is not well understood how the hyperthermophilic archaea such as Sulfolobus acidocaldarius maintain their DNA, but genomic analyses have found some homologs to known DNA repair and replication genes. These include genes implicated in nucleotide excision repair, photoreactivational repair, alternative excision repair, homologous recombinational repair, and DNA replication. Topoisomerase-mediated cloning of PCR products provided the most reliable cloning method, whereas disruption of cloned genes was more difficult. One cloned disruption (trpC::pyrE) made using restriction enzymes and ligase, and two linear PCR disruptions (phr::pyrE; uvde::pyrE) made by a direct PCR method, were tested for integration into the S. acidocaldarius genome. Analysis of successful transformants by PCR and sequencing indicate that all three disruptions have been integrated into the S. acidocaldarius via homologous recombination. In two cases the phenotypes have been confirmed. The trpC disruptant (DG251) is a tryptophan auxotroph and the phr disruptant (LR10) lacks photoreactivational repair. This work is significant because targeted gene disruption has not been reported for S. acidocaldarius and it provides more genetic tools for use in Sulfolobus spp. It also gives evolutionary insights into the diversity and similarities among the three domains of life, and even more specifically evolutionary insight into the molecular processes of the hyperthermophilic archaea. |
| Permanent Link: |
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1227205786
http://hdl.handle.net/2374.OX/105466 |
| Date: | 2008 |
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