This research was an attempt to characterize previously constructed mutants with a specifically altered psbO gene which encodes a 33 kDa protein active in photosynthesis. This polypeptide was believed to function in stabilization of manganese ions during photolysis of water at the photosystem II. The initial phase of this work was concerned with determining the manganese content of the genetically manipulated PS II particles of the photosynthetically active cyanobacteria.We found however, that the results of the isolation procedure for PS II particles of photosynthetically active cyanobacteria as described by Burnap et al. was not reproducible in our research organism. This prevented the chemical characterization of function of these particles as had been planned.In the second phase of the research sequencing of the mutated gene was to be performed for several clones in order to determine the kinds of specific alterations that had been made. The mutated genes had been cloned into both pUC1 20 and pPGV5 vectors which were transformed into Escherichia OR (EQQJi) and the cyanobacterium Synechococcus PCC 7942, respectively.Several attempts were mad o isolate plasmid DNA from both the transformed E QQJI and cyanobacterium. Isolation of pUC120 DNA was not achieved due to the toxicity of the 33 kDa protein product of the psbO gene in sgJj. The pPGV5 plasmid isolation was successful and PCR-sequencing was performed. However, the sequencing did not result in a readable sequence. Instead, banding patterns showed more than one nucleotide per lane. Since pPGV5 contains a strong constitutive promoter, a large amount of mutant protein was being produced. Our findings suggested that transformed cyanobacteria may have been under pressure to revert the altered gene to wild-type. Thus, upon growth of a single colony to a larger volume, a heterogeneous population of cells with different sizes of plasmids may have resulted. Restriction analysis of isolated plasmid DNA confirmed the presence of multiple-sized plasmid molecules. Therefore, this research has shown that the previously constructed mutants are not stable enough to characterize for alterations in manganese binding. / Department of Biology
| Identifer | oai:union.ndltd.org:BSU/oai:cardinalscholar.bsu.edu:handle/184550 |
| Date | January 1992 |
| Creators | Tzalis, Dimitrios |
| Contributors | Ball State University. Dept. of Biology., Vann, Carolyn N |
| Source Sets | Ball State University |
| Detected Language | English |
| Format | vii, 86 leaves : ill. ; 28 cm. |
| Source | Virtual Press |
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