Bochum, Universiẗat, Diss., 2000.

Bochum, Universiẗat, Diss., 2000.

Tübingen, Universiẗat, Diss., 2002. / Dateiformat: tgz, Dateien im PDF-Format.

Stuttgart, Universiẗat, Diss., 2002.

Konstanz, University, Diss., 2002.

Kaiserslautern, Techn. Universiẗat, Diss., 2003.

Four temperature-sensitive DNA deficient mutants of Paramecium aurelia have been isolated through a new application of UV light photolysis of 5-bromouracil substituted DNA, following nitrosoguanidine mutagenesis! Bromouracil was introduced by bacterial vector to achieve specific incorporation into DNA. Non mutant cells presumably incorporated 5-bromouracil at the restrictive temperature, mutants did not. The selection procedure took a month to complete, requiring two successive sexual generations. There were two lethal periods for wild type cells in the screening system; the first occurs during the irradiation of BU substituted macronuclei by UV light, the second, following autogamy (a result of homozygosis of UV light damage to the bromouracil labelled micronuclei). From 1300 isolated survivors from the screening procedure, 29 lines were classed as temperature-sensitive. Approximately 14 percent of these were ts DNA deficient (the ratio of restrictive to permissive temperature incorporation was 10 percent or less). Protein synthesis, and food-vacuole formation at the restrictive temperature deviated no more than 50 percent from the normal level. . The selection procedure was shown to be efficient in retrieving ts DNA deficient mutants. A genetically marked ts DNA mutant (isolated through the selection system earlier) was recovered at an enrichment of 7500 fold over the wild type (mean seven runs). The methods of production of bromouracil substituted bacteria have been standardized, and the durations of various parameters of the selection system have been set. For example, the length of UV light exposure following bromouracil substitution, and the length of restrictive temperature pretreatment (Equilibration time before bromouracil treatment) have been optimized. The relative effects of parts of the screening procedure are presented along with discussion of the results, and suggestions for future applications of the isolated Paramecium lines. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

published_or_final_version / Zoology / Master / Master of Science

Paramecium, a ciliate, is an important model for studying Ca2+ signaling and understanding chemoreception and signal transduction. There are several proteins, such as plasma membrane calcium ATPases (PMCAs)/ calcium pumps, SERCA pumps, calmodulin and Ca2+ channels that play an important role in maintaining intracellular Ca2+ level and signaling in Paramecium. Isoform 2 of PMCA has been identified in both the cilia and pellicle membranes of Paramecium, the activity of which leads to hyperpolarization. Plasma and ciliary membrane of Paramecium is made up of a variety of sterols and sphingolipids which constitute lipid rafts, demonstrated by the presence of detergent resistant membranes and their distribution in sucrose and Optiprep density gradients. PMCAs are important markers of lipid rafts and PMCA 2 is found to be localized in lipid rafts of both the cilia and somatic membrane of Paramecium. Methyl-β-cyclodextrin treatment can remove up to 42% of sterols from pellicle membranes but only about 12% from cilia. Sterol depletion of pellicle perturbs the distribution of PMCA 2 and other raft proteins in pellicle which is not observed in cilia as evident from western blot analysis and immunomicroscopic studies. There is evidence that selection of gradient medium for study of lipid rafts and its associated proteins is very important in Paramecium. Glutamate receptors and adenylyl cyclase, the upstream molecules of the signal transduction pathway through PMCA have also been identified in cellular cilia, indicating that these raft molecules forms a platform for signaling in Paramecium cilia.

Thesis (M. Phil.)--University of Hong Kong, 1988.