Computer Science – Performance
Scientific paper
Jan 2002
adsabs.harvard.edu/cgi-bin/nph-data_query?bibcode=2002iaf..confe.869k&link_type=abstract
IAF abstracts, 34th COSPAR Scientific Assembly, The Second World Space Congress, held 10-19 October, 2002 in Houston, TX, USA.,
Computer Science
Performance
Scientific paper
gravity perception and transduction. Elucidation of cytoskeleton involvement in the above processes will be a great contribution into the fundamental biology, namely, in signaling mechanisms. One of the useful models for investigation of cell gravisensing are higher plants where the sites of gravity perception and reaction are spatially separated. From this point of view the localization of cytoskeletal elements at the different stages of plant development with the special emphasis on cytoskeleton dynamics during development of gravisensitive regions are of special interests. It should be noted that most of plant cytoskeletal researches are conducted by the means of immunohistochemical reactions with the application of monoclonal antibodies specific to specific cytoskeletal proteins. Different modifications of these methods are beneficial for the ground-based experiments, but for the performance onboard the latter represents quite routine and complicated technique, which requires time and special skills for astronauts. Besides, immunohistochemistry provides only static images of the cytoskeleton arrangement in fixed cells while its localization in living cells is needed for better understanding of cytoskeleton function. In this connection we propose new approach for cytoskeleton visualization onboard in particular, use of green fluorescent protein (GFP) from Aequorea victoria, which has the unique properties as a marker for protein localization in vivo. Recently appeared this method helped to obtain significant data on the cytoskeleton dynamics in living cells, including plant cells. The creation of chimaeric protein-GFP gene constructs, obtaining the transformed plant cells possessed protein-GFP in their cytoskeletal composition will allow receiving a simple and efficient model for screening of cytoskeleton functional status in microgravity. To realize this idea at present state of art it would be possible to produce a respective chimaeric plant tubulin gene constructs based on its own or pBIN35S promoters and mGFP4 (kindly provided by J. Haseloff). To search and obtain the most efficient constructions preliminary 3D-structural computer modeling of chaemeric genes will be done. It's planned to ligate the GFP modified gene with C-end sequence of TUA1 or TUB1 gene which is variable (approximately 45- 90 bp) in all - or -tubulin genes. After selection we have to examine the efficiency of function of chimaeric tubulin. It can be achieved by means of studying in vitro the inclusion of chimaeric protein in assembly of microtubules. For observation of cytoskeleton dynamics in transformed living cells it is planned to use a standard fluorescent microscope with fluorescein isothiocyanate filter set (HQ480/40 excitation filter, HQ535/50 emission filter, Q505LP dichroic mirror; Chroma Technology, Brattleboro, VT). No significant autofluorescence will be seen using this filter combination. Confocal images of GFP expression could be taken also on a Leica DM IBR inverted laser confocal microscope using a standard FITC filter providing excitation at 480-490 nm and emission at 530-540 nm. For the efficient application of the above technique onboard the special protocol has been working out with the inclusion of basic training course for crew members.
Blume Ya. B.
Kordyum Elizabeth L.
Nyporko A. I.
Shevchenko Vasilij G.
Yemets A. I.
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