Fig. 1. Analysis of Chlamydomonas cells expressing GFP-tagged SFA. (A) Gene structure of sfa (accession number AJ344366). The size and positions of the introns are indicated. The positions of the codons of the skip residues are marked by dots. Note the regular spacing of the introns. (B) Gene construct used for the expression of SFA-GFP. The arrow indicates the position of the start of translation. (C) Comparison of the SMAFs in control cells (control; anti-SFA staining) with the GFP signals observed in GFP3 and GFP8. Cells were fixed with methanol (-20°C) for 20 seconds. GFP3 expressed moderated amounts of SFA-GFP, forming cross-like structures similar to that observed in control cells by indirect immunofluorescence, whereas GFP8 cells overexpressed SFA-GFP, causing oversized fibers, when cultivated with a light/dark-cycle of 14/10 hours (GFP3-L and GFP8-L). Small fibers were present in GFP8 when cultivated in the dark for 2 days (GFP8-D). Bar, 10 µm. (D) Western blot of control (C) and GFP8 cells. The membrane strip on the left was stained with amidoblack and the positions of the standard proteins (Mr) are indicated on the left. Anti-SFA reacted with a band of 34 kDa in control and GFP8 cells, and a band of approximately 60 kDa in GFP8. (E) Details of strain GFP8. (a) Frequently one (indicated by arrowheads) of the four SFA-GFP fibers was much longer than the other three. (b,c) GFP signal (b) or indirect immunofluorescence with anti-SFA (c) of a GFP8 cell with a gap in one of the SFA-GFP fibers. Bars, 5 µm.

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