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UBC Theses and Dissertations
A puhA gene deletion and plasmid complementation system for facile site directed mutagenesis studies of the reaction center H protein of Rhodobacter Sphaeroides Chen, Xiaoyi
Abstract
The development of a Rhodobacter sphaeroides deletion/plasmid complementation system of the puhA gene (which encodes the reaction center [RC] heavy [H] subunit) for expression of site directed mutants of the RC H protein is described. The mutant strain ΔPUHA was constructed by introduction of a translationally in-frame deleted puhA allele at the chromosomal puhA gene site, and evaluated in plasmid complementation experiments. Strain ΔPUHA was unable to grow under photosynthetic conditions. Absorption spectroscopy showed this strain has a reduction in the amount of the light-harvesting I (LHI) complex. SDS-PAGE analysis of chromatophore proteins of strain ΔPUHA confirmed the absence of the RC H protein band. When ΔPUHA was complemented in trans with the wild type puhA gene in plasmids, photosynthetic growth and the RC H protein band in SDS-PAGE were restored. The results of comparisons of the properties of strains with different types of chromosomal puhA gene disruptions in complementation experiments are consistent with the idea that expression of one or more genes located 3' of puhA is required for optimal RC levels and photosynthetic growth. Since the ΔPUHA translationally in-frame deletion does not seem to interfere with transcription through and beyond the residual puhA sequences, this strain allows facile evaluation of the consequences of plasmid-borne RC H mutations in an otherwise wild type genetic background. The role of the RC H subunit in photosynthesis is discussed.
Item Metadata
Title |
A puhA gene deletion and plasmid complementation system for facile site directed mutagenesis studies of the reaction center H protein of Rhodobacter Sphaeroides
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1997
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Description |
The development of a Rhodobacter sphaeroides deletion/plasmid
complementation system of the puhA gene (which encodes the reaction center [RC]
heavy [H] subunit) for expression of site directed mutants of the RC H protein is
described. The mutant strain ΔPUHA was constructed by introduction of a
translationally in-frame deleted puhA allele at the chromosomal puhA gene site, and
evaluated in plasmid complementation experiments. Strain ΔPUHA was unable to
grow under photosynthetic conditions. Absorption spectroscopy showed this strain has a reduction in the amount of the light-harvesting I (LHI) complex. SDS-PAGE
analysis of chromatophore proteins of strain ΔPUHA confirmed the absence of the RC
H protein band. When ΔPUHA was complemented in trans with the wild type puhA gene in plasmids, photosynthetic growth and the RC H protein band in SDS-PAGE were restored. The results of comparisons of the properties of strains with different types of chromosomal puhA gene disruptions in complementation experiments are consistent with the idea that expression of one or more genes located 3' of puhA is required for optimal RC levels and photosynthetic growth. Since the ΔPUHA
translationally in-frame deletion does not seem to interfere with transcription through
and beyond the residual puhA sequences, this strain allows facile evaluation of the
consequences of plasmid-borne RC H mutations in an otherwise wild type genetic
background. The role of the RC H subunit in photosynthesis is discussed.
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Extent |
4064453 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-03-25
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0087974
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1997-11
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.