Fig. 9. Characterization of Chlamydomonas cells transformed with pCB-AS1. (A) The construct used to reduce the expression of SFA. The solid bars represent introns of 71 bp at +4 and of 90 bp at +91; genomic and complementary DNA of SFA were joined 35 bp after the second intron. (B) Nothern blot documenting the expression of the antisense construct in AS5 and AS8. A transcript of 900 bp was detected in cells 45 minutes after a heat shock of 60 minutes (T105). The transcript was not detected before (T0) or 3 hours after heat shock (T240), or in a sample isolated from cells maintained in the dark for 24 hours (24hD). Equal amounts of total RNA (30 µg) were loaded as documented by the ethidium bromide-stained gel. (C) Western blots of control (C) cells and various AS-strains. Equal amounts of cytoskeletons isolated from AS8 and control cells were loaded and stained either with amidoblack (left membrane strip) or probed with anti-SFA (middle membrane strip). Right: membrane strip comparing SFA in insoluble fractions from AS3 and AS5 with that of control cells. Prolonged development of membrane strips visualized some residual SFA in the antisense strains (arrow). (D) AS8 cells permeabilized with -20°C methanol and stained with anti-tubulin (6-11B-1) and anti-SFA. Open arrowheads: uniflagellate cells; arrow: stronger SFA signal. Bar, 5 µm.

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