Regeneration of detached flagella by Chlamydomonas reinhardtii, requires the coordinate induction of synthesis of a large number of flagellar proteins. Following induction, flagellar protein mRNAs are specifically destroyed in a translation-dependent manner. This proposal addresses the mechanism and specificity of the regulated degradation. Chimeric mRNAs, composed of parts of the regulated alpha-tubulin mRNA, and an unregulated stable mRNA will be expressed from an alpha-tubulin promotor to determine the sequence specificity of degradation. An in vitro degradation extract, which reproduces degradation specificity observed in vivo, will be expoited to localize and characterize the trans-acting factors involved in degradation. The translation dependence of poly(A) metabolism and the effect of altered poly(A) metabolism on flagellar mRNA function and stability will be investigated by identifying the sequences which direct the rate of poly(A) loss, and altering those sequences. The finding that a probable poly(A)- binding protein (PABP) is coinduced by deflagellation suggests this protein may be involved in regulation of the coinduced mRNAs. The sequence for the Chlamydomonas PABP will be isolated from a lambda- library. Elimination of induction of this protein will be attempted by coinducing an antisense version, and the effect on coinduced mRNA stability and translation evaluated.
