The surface complex of Euglena gracilis. Involvement in cellular movements and biochemical and molecular analyses of the components

Detta är en avhandling från Biology building

Sammanfattning: The flagellate Euglena gracilis shows rhythms in phototaxis and cell shape changes between oblong and round. Artificial UV-B radiation of 0.5 W m-2 caused a loss in phototaxis, but the peak of activity occurred at the same time as for control cells. The transition from round to oblong cell shape was slower after UV-B radiation, but the amplitude between the roundest and the most oblong cell shape was unchanged. Nitrogen deficiency caused a total loss of phototaxis and the cells remained round all the time. The cells lost all their chlorophyll and were, therefore, photosynthetically inactive. Isolated plasma membranes were used to characterize the rhythms of some plasma membrane-bound enzymes. We wanted to see whether stress-induced changes of rhythms could be detected also at the molecular level. The plasma membranes were isolated using aqueous polymer two-phase partitioning. Three plasma membrane-located enzyme activities were analysed: Mg2+-dependent ATPase, 5´-nucleotidase and adenylyl cyclase. The specific activities of all three enzymes were decreased by UV-B radiation. Nitrogen deficiency reduced the activity of the Mg2+-dependent ATPase but increased the activities of the 5’-nucleotidase and adenylyl cyclase. All three enzymes showed diurnal rhythms that were affected by UV-B radiation. The rhythm of ATPase activity could be correlated with that of photosynthesis in both control and UV-B radiated cultures. Also, the rhythms of adenylyl cyclase activity and cell shape changes showed some similarities. An anti–amoeba actin antibody recognized a 47 kDa protein in isolated plasma membranes. Washing with 150 mM NaOH released both the 47 kDa polypeptide and b-tubulin from the membrane. Thus, both the 47 kDa polypeptide and b-tubulin have a peripheral association with the membrane, perhaps as part of a larger protein complex such as that of the cytoskeleton. DNase I activity was inhibited by the protein fraction that was released by NaOH, in which the 47 kDa polypeptide was enriched. The partial sequence of the actin gene was determined by reverse transcriptase-PCR of Euglena gracilis mRNA. A single PCR product was obtained and we were able to obtain 963 bases of unique sequence. Sequence comparison showed a homology with other actins of 69% to 80%. The construction of a phylogenetic tree based on actin sequences from different organisms placed Euglena gracilis in a cluster with Trypanosoma brucei and Leishmania major.

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