xPat1a is degraded whereas xPat1b is newly synthesized upon meiotic maturation. (A) Meiotic maturation time course scored as percentage of GVBD. Groups of stage VI oocytes treated with progesterone were sampled at different times, corresponding to maturation status scored in percentage of GVBD. The oocytes were analyzed by Western blot and probed with the indicated antibodies. (B) The N-terminal 1–331 amino acids mediate xPat1a degradation. mRNAs encoding for different MS2-tagged portions of the protein as indicated were injected in stage VI oocytes, subsequently matured into eggs by the addition of progesterone. Lysates were prepared from oocytes and eggs and analyzed by Western blotting with an MS2 antibody. Note that region 1–331 runs aberrantly in the SDS-PAGE gel. (*)Nonspecific band, serving as a loading control. (C) xPat1b mRNA is polyadenylated upon meiotic maturation. The 32[P]-labeled 3′ proximal 180 nt of xPat1b 3′ UTR and the 3′ proximal 65 nt of cyclin B1 3′ UTR were injected in stage VI oocytes (−) and subsequently matured into eggs (+) by the addition of progesterone. Input lanes contain uninjected RNA. 32[P]-labeled ØX174-HindIII fragments served as size markers.
