The long range goal of this project is to analyse the mechanism of storage of mRNAs in mouse oocytes, and their activation during early embryonic development. Using growing oocytes cultured and labeled in vitro, the flow of newly synthesized stable messages into the stored and translated compartments will be described. Fractionation procedures include low speed centrifugation (apparently stored ribosomes and mRNA are complexed in large superstructures, the lattices seen in the EM), and chromatography on oligo (dT) cellulose. It is proposed that stored messages carry extra sequences not present in translated messages, a difference arising by incomplete processing of hnRNA resulting in retention of some repetitive sequence transcripts. This hypothesis will be explored in several ways. The properties of stored and translated mRNAs will be compared with respect to their molecular weight distribution. The representation of specific mRNAs in the stored and translated compartments will be analysed using cDNA probes, and the molecular weight of the mRNA for one protein in the two compartments compared. The repetitive sequence (""""""""Alu"""""""" sequence) transcript content of mRNA of eggs, oocytes, and somatic cells will be analysed using labeled cloned """"""""Alu"""""""" probe on Northern blots or excess cloned """"""""Alu"""""""" sequence with labeled cellular RNAs. The stored and translated mRNAs will also be compared with respect to this parameter. The synthesis of U1 RNA (postulated to play a role in slicing) in growing oocytes will be measured and its relative amount of oocytes and eggs quantitated on Northern blots using cloned labeled probe. The distribution of U1 RNA in oocytes, eggs, and early embroys will be analysed using specific antibodies to U1-RNP particles.
