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The abundant retinal protein of the Chlamydomonas eye is not the photoreceptor for phototaxis and photophobic responses

Markus Fuhrmann, Alke Stahlberg, Elena Govorunova^*, Simone Rank and Peter Hegemann

Institut für Biochemie I, Universität Regensburg, 93040 Regensburg, Germany
^* Present address: Moscow state university, Biology Department, 119899 Moscow, Russia

View larger version (17K): [in a new window]   Fig. 1. Constructs for the potential triggering of cop-silencing in Chlamydomonas. The efficiency of different silencing constructs was determined by measuring the decreased opsin levels in transformants obtained with each plasmid. Construct pMF15 was designed to induce cosuppression by providing a cop gene containing a nonsense mutation early in the gene (*). The constructs pMF38-47 provided a single ‘antisense RNA’ to target cop-mRNA under the control of a strong promotor. Arrows show the antisense portions of the cop-gene. Numbers within black segments of the diagram correspond to the position of exons in the cop-gene.%PTGS is calculated as the number of clones showing a reduction in chlamyopsin of more than 20% divided by the number of tested clones (n). %r is the amount of residual Cop protein in the mutant with the lowest observed Cop-level in each experiment.

View larger version (38K): [in a new window]   Fig. 2. Efficient silencing of cop gene expression mediated by ‘RNAi’-like constructs. (a) DNA-constructs tested for their RNAi mediating activity. (b) Relative amounts of opsin in different pSR25 transformants compared to the wild-type as determined by densitometric measurements of the Cop-band intensity on western blots using immunodetection. (c) Comparison of purified eyespot membranes (S3-fraction) of recipient cw15 arg-A cells and the transformant copd12. (d,e) Comparison of cop-transcripts by RNA-blot analysis. (d) Equal amounts of mRNA from wt (CC2454) cells and pSR25 transformant 35 (copd) were fractionated on an agarose gel, blotted and probed with full length DIG-labeled cop-cDNA. (e) 5 µg wt mRNA and 11 µg of transformant mRNA were hybridized with DIG-labeled cop fragment comprising exon 1-3.

View larger version (18K): [in a new window]   Fig. 3. Characterization of flash-induced ion currents of individual copd mutant cells. Photocurrents of an untransformed cw15arg-A cell and a copd13 transformant. Photocurrents (IP1) were plotted versus the photon exposure of the flash (Q). No intensity dependent differences in the amplitude of the photoreceptor current IP1 were observed in three cell lines with different Cop content.

View larger version (21K): [in a new window]   Fig. 4. Characterization of flash-induced photophobic responses of individual copd mutant cells. Average actual speed changes of individual cells after application of light flashes with different intensities (100, 10, 1 and 0.1%). The observed maximal speed reduction (v) was plotted versus the photon exposure (Q). No intensity dependent differences in the stop response are observed between wt (CC2454) and copd35 (opsin content <5%).

View larger version (18K): [in a new window]   Fig. 5. Phototactic orientation measured with a light scattering assay on cell populations of opsin antisense transformants with normal and reduced levels of the expressed opsin. Light scattering changes induced in cell suspensions by a 20 second pulse of continuous 502 nm light (CC2454 copd35 with 5% opsin and copd14 with a wt-opsin level. Traces were recorded at fluence rates (from below): 261, 14.8, 7.4, 2.6 and 1.3x1016 photons m-2 s-1; each trace is a mean value of two measurements in separate cell samples. Photoorientation was calculated as the absolute value of the difference between the output voltage measured after 20 seconds of illumination and 20 seconds after the light was switched off. Data points are mean values±s.d. of two measurements in separate cell samples.