For investigating the behaviour of minichromosomes in metaphase, cells were inoculated in minimal doLEU medium + 2% raff/gal and grown O/N at 25°C. Depending on the density of the culture 500µl to 1ml were harvested and washed twice with water. They were resuspended in 5ml of minimal doLEU medium + 2% glc and incubated for 2hours at 25°C, gently rotating. Due to the glucose containing medium expression of Cdc20p was repressed and cells were arrested in metaphase (due to the relative short time of medium switch not all of the cells were completely arrested). The minichromosomes were visualized by bound LacI-GFP. Its expression was induced by adding CuSO4 to a final concentration of 250µM for 15min to the cells. In the meantime coverslips were prepared by placing a 50µl drop of ConA solution (1mg/ml stock diluted 1:10) in the center and incubating it for 10min. After washing the coverslip with water or medium, 50µl of cell suspension was placed on the center and allowed to adhere for 10 to 15 min. The coverslip was washed carefully with medium (w/o CuSO4) to remove unattached cells. In case of
recovery experiments, cells were washed with medium containing 2% raff/gal. With vacuum grease a square in the size of the coverslip was marked on a glass slide. With foreceps the coverslip was carefully placed upside down on the glass slide, so that the vacuum grease was sealing the edges. The coverslip was then carefully pressed on the slide and extra liquid was removed with a tissue. After this cells were ready for imaging.
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Acknowledgements
I would like to thank all those people who accompanied me with their constant support during my studies and the work on this project.
First of all I am really grateful to Evi for supervising my work in the lab and being there with words and deeds whenever I needed help. With endless patience for all my problems she was guiding me through this year. Many thanks to Stefan for giving me the opportunity to work in his lab on this interesting project, for scientific advisory and the hard work of correcting my thesis. Due to a great working atmosphere I really enjoyed this year in the lab. Therefor many thanks to Fabi, Chrissi, Kathi, Tom and Peter. All of them where supporting me and sharing their experience and knowledge with me. Besides from technical assistance and scientific discussions I would also like to thank for the “off-topic” discussions. My colleagues became wonderful friends to me.
Many thanks to the girls Doris, Sandra, Johanna, Ivi, Janja, Barbara and Eva for hours of chatting, laughing, eating, drinking coffee, partying, having fun, discussing the past and looking ahead…
I am so grateful for all the help I got from my family. The constant support and love from my parents, my sister and my grandparents, is the basement for everything I have achieved so far. They are there for me, whenever I need them.
I want to thank Thomas for his endless patience, especially during the last weeks, for sharing my sorrows and happiness, for being there for me and for his unremitting love. Thank you for being my best friend.
Curriculum Vitae
Family name Wöhrer
First name Sophie
Date and place of birth 19.06.1986, Mödling
Nationality Austria
Academic career:
10/2004 – 10/2009 Studies of Molecular Biology University of Vienna, Austria
07-08/2008 International Biology Undergraduate Summer School BUSS2008
University of Zurich, ETH Zurich, Switzerland 07/2006 CEEPUS Biomedicine Student’s Council, one week
Summer University Zadar, Croatia 09/1996 – 06/2004 Real- und Aufbaugymnasium
Don Bosco Gymnasium, Unterwaltersdorf, Austria
Publications:
Eva Kiermaier, Sophie Woehrer, Yutian Peng, Karl Mechtler and Stefan Westermann. A Dam1-based artificial kinetochore is sufficient to promote chromosome segregation in budding yeast. Nature Cell Biology, 11(9), 1109-1115 (2009).