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Complementation of chimeric eEF1A in S. Cerevisiae.

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posted on 30.07.2012, 00:14 by Sandra Eltschinger, Eva Greganova, Manfred Heller, Peter Bütikofer, Michael Altmann

(A) Sequence alignment of conserved amino acid motifs separating domains I and II (upper panel) and II and III (lower panel) of eEF1A from different sources. To generate chimeric constructs, synthetic SpeI (triangle) or BamHI (arrow) cloning sites were introduced. (B) Schematic representation of chimeric constructs. Arrows and triangles indicate the positions of the cloning sites which were removed by site-directed mutagenesis to reconstruct the original eEF1A sequences. Numbers in brackets correspond to clones shown in Fig. 3C. (C) Complementation of S. cerevisiae strain TKY102 with chimeric eEF1A constructs. Upper panels: Yeast cells growing on plates after transformation with different chimeric constructs. Middle and bottom panels: Counterselection for the loss of endogenous eEF1A on plates containing 5-FOA at 25 or 30°C. Left panels: Complementation assays with chimeric constructs carrying cloning sites causing a N329K mutation in the case of the artificial BamHI-site (separating domains II and III) or I254T/G255S mutations in the case of the artificial SpeI-site (separating domains I and II). Right panels: complementation assays with chimeric constructs after reconstructing wild type eEF1A sequence motifs. (D) Growth properties of S. cerevisiae complemented with chimeric eEF1A constructs. (1) positive control with yeast eEF1A; (2) Chimeric yeast constructs carrying human domain III - without or with N329K mutation; (4) Chimeric yeast constructs carrying T. brucei domain III - without or with N329K mutation; (8) Chimeric yeast eEF1A construct carrying humain domain II.