Detachment of the flagella of Chlamydomonas induces a rapid accumulation of mRNAs for tubulin and other flagellar proteins. Measurement of the rate of alpha and beta tubulin RNA synthesis during flagellar regeneration shows that deflagellation elicits a rapid, 4-7-fold burst in tubulin RNA synthesis. The synthesis rate peaks within 10-15 rain, then declines back to the predeflagellation rate. Redeflagellation of cells at times before the first flagellar regeneration is completed (and when cells have already accumulated elevated levels of tubulin RNA) induces another burst in tubulin RNA synthesis which is identical to the first in magnitude and duration. This finding indicates that the induction signal may act to simply reprogram the tubulin genes for a transient burst of maximal synthesis. Evidence is presented that the stability of the tubulin RNAs changes during regeneration. Stability changes include both an apparent stabilization during regeneration and accelerated decay following regeneration.The eucaryotic biflagellate, Chlamydomonas reinhardtii, undergoes flagellar regeneration after mechanical detachment of its flagella. The response to deflagellation involves a large increase in the synthesis of flagellar proteins (14, 33) effected by an increased accumulation (6-40-fold) of their messenger RNAs (15,20,29,31). Flagella are rapidly assembled, within 60-90 min, and subsequently flagellar-specific protein synthesis and mRNA abundance return to basal levels. Thus the response to deflagellation involves both an induction and a deinduction, the entire process spanning <3 h.Recent evidence from this laboratory indicates that the increase in alpha and beta tubulin mRNA levels is due, at least in part, to a transcriptional activation of the tubulin genes. Nuclei isolated from deflagellated cells support an average fivefold more transcription oftubulin RNA sequences than do nuclei from nondeflagellated cells (12). We were interested in obtaining more detailed information about the extent and duration of the transcriptional induction and its termination. In particular, we wanted to know the maximum level of induction and when it occurs, the timing of the deinduction, and whether it can be correlated with completion of flagellar assembly, and whether the activation of tubulin RNA synthesis is sufficient to account for the large increase in abundance observed.In this report, we describe the results of in vivo labeling experiments designed to elucidate the tubulin RNA synthetic and decay changes occurring throughout the regeneration process. MATERIALS AND METHODS Cell Growth and Preparation: Chlamydomonas reinhardtii cells,strain 21gr, were grown in a low phosphate variant of medium I (27) in which potassium phosphate salts have been reduced 20-fold (0.065 raM) and the media buffered with 10 mM HEPES. Cells were grown to ~1 x 106 celis/ml except where noted. For deflagellation and labeling, cells were concentrated to 1-2 x l07 cells/ml in spent media or in fresh media containing no phosphate. Cells were def...

After flagellar detachment in Chlamydomonas reinhardi, there is a rapid synthesis and accumulation of mRNAs for tubulin and other flagellar proteins. Maximum levels of these mRNAs (flagellar RNAs) are reached within 1 h after deflagellation, after which they are rapidly degraded to their predeflagellation levels. The degradation of alpha-and beta-tubulin RNAs was shown to be due to the shortening of their half-lives after accumulation (Baker et al., J. Cell Biol. 99:2074-2081. Deflagellation in the presence of protein synthesis inhibitors results in the accumulation of tubulin and other flagellar mRNAs by kinetics similar to those of controls. However, unlike controls, in which the accumulated mRNAs are rapidly degraded, these mRNAs are stabilized in cycloheximide. The stabilization by cycloheximide is specific for the flagellar mRNAs accumulated after deflagellation, since there is no change in the levels of flagellar mRNAs in nondeflagellated (uninduced) cells in the presence of cycloheximide. The kinetics of flagellar mRNA synthesis after deflagellation are shown to be the same in cycloheximide-treated and control cells by in vivo labeling and in vitro nuclear runoff experiments. These results show that protein synthesis is not required for the induced synthesis of flagellar mRNAs, and that all necessary transcriptional control factors are present in the cell before deflagellation, but that protein synthesis is required for the accelerated degradation of the accumulated flagellar mRNAs. Since cycloheximide prevents the induced synthesis and accumulation of flagellar proteins, it is possible that the product(s) of protein synthesis required for the accelerated decay of these mRNAs is a flagellar protein(s). The possibility that one or more flagellar proteins autoregulate the stability of the flagellar mRNAs is discussed.The cellular response to flagellar detachment in Chlamydomonas reinhardi includes a rapid and coordinate induction of the mRNAs for tubulin and other flagellar proteins (25,27,37,38). The accumulation of alpha-and beta-tubulin RNAs is accomplished by both a transient increase in transcription rate and an apparent stabilization of the tubulin transcripts during flagellar regeneration (3, 21). Maximum levels of mRNAs for tubulin and other flagellar proteins accumulate within 1 h, after which these RNAs are rapidly degraded (25,27,37,38). The half-life of tubulin mRNA during this deinduction is only about 20 min, or 2 to 3 times shorter than in nonregenerating, vegetative cells (3). These data suggest that a specific decay mechanism operates during deinduction which accelerates the return of the induced mRNAs to basal levels.In this report, we show that protein synthesis is not required for the activation of transcription and accumulation of tubulin and other induced mRNAs, but is necessary for the accelerated degradation of these RNAs after induction. MATERIALS AND METHODSCell growth and preparation. Chlamydomonas reinhardi vegetative cells, strain 21gr, were grown in medium I of Sager and Granic...

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In Chlamydomonas, the usual rapid degradation of tubulin mRNAs induced by flagellar amputation is prevented by inhibition of protein synthesis with cycloheximide. Evidence is presented that the ability of cycloheximide to stabilize alpha-tubulin mRNA depends on the time of addition. Addition of cycloheximide to cells before induction strongly stabilizes the induced mRNAs, while addition after their synthesis stabilizes them only transiently. Moreover, cycloheximide inhibition does not stabilize the same alpha-tubulin mRNA species in uninduced cells. These results suggest that cycloheximide is not acting to stabilize the induced alpha-tubulin mRNAs simply by preventing ribosome translocation. The stabilized state of tubulin mRNA was found to correlate with its occurrence on smaller polysomes but larger EDTA-released mRNP particles than the unstable state. A second effect of cycloheximide on the metabolism of induced tubulin mRNAs is to accelerate complete poly(A) removal. This effect of cycloheximide inhibition, unlike stabilization, occurs whenever cycloheximide is added to cells, and appears unrelated to stabilization. The effect is shown to be mRNA-specific; poly(A)-shortening on the rbcS2 mRNA is not altered in the presence of cycloheximide, nor do completely deadenylated molecules accumulate. Experiments in which cells were released from cycloheximide inhibition suggest that deadenylated alpha-tubulin mRNAs may be less stable than their polyadenylated counterparts during active translation.