GFP-linked cDNA sequences encoding peptides with putative cortical localizations
E. Marlo Nelsen and Joseph Frankel
Department of Biological Sciences
University of Iowa
Background Information
cDNA from Tetrahymena thermophila was inserted into a unique XhoI site located immediately 3' to the GFP coding region of a vegetative expression cassette situated in the 3' UTR of the rDNA in the processing vector pVGF1, a derivative of pD5H8, constructed by M.C. Yao and C.H. Yao. Aliquots of this GFP-linked cDNA library were transformed by standard electroporation (BTX) into conjugating cells of B2192 IV x CU428 VII. Paromomycin-resistant survivors were distributed into wells in microtiter plates and screened at 200x magnification of an inverted fluorescent microscope. Of an estimated 120,000 transformed clones screened in 67,000 microtiter wells, about 25% expressed GFP during vegetative growth. Approximately 75 clones showed localization of GFP in macronuclei or macronuclei plus micronuclei, 40 showed localization in intracytoplasmic organelles or aggregates, 2 lit up what appeared to be internal membranes and vesicles, and 11 showed cortical localizations. Cells showing cortical localizations were recloned in fresh microtiter plates and the site(s) of GFP localization were verified. Localized GFP expression was often transient, probably due to co-transformation with a second plasmid from the library. Dual transformation was proven in some cases.
Aliquots of cells with cortical localizations were frozen at -70o C. DNA was isolated from cells expressing cortical localizations and PCR-amplified using primers, designed in the Yao lab, derived from the regions flanking the cDNA insert. PCR products were cleaned using the Qiagen PCR-purification kit, and cloned using the Invitrogen A/T cloning kit. DNA from several white colonies was run on gels to check for heterogeneity of inserts, and was sequenced.
Of the 11 transformed cell clones with cortical localizations, one set of four clones and another set of two clones turned out to be identical in both sequence and localization, indicating recovery of identical inserts from the library in two or more independently-screened clones. Of the remaining five clones, four had little or no background glow in microscopy and unequivocal inserts; one (25-11H4) gave uniform inserts in AT cloning but had a strong background glow, raising the possibility of co-transformation by a second undetected insert. For another clone, the insert responsible for the cytological localization was almost certainly not recovered, so it is not included.
&nsbp;Scanned images of projection slides showing localization of GFP resulting from any of these inserts are available upon request to joseph-frankel@uiowa.edu.
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