Sequence Organization of the Poly(A) RNA Synthesized and Accumulated in Lampbrush Chromosome Stage Xenopus laevis Oocytes

The poly(A) RNA of Xenopus laevis oocytes was extracted, renatured and spread for electron microscopy. As reported for the poly(A) RNA of sea urchin eggs (Costantini et al., 1980), the Xenopus oocyte RNA renatures extensively, revealing the presence of complementary, interspersed repetitive sequences on different RNA molecules. About 68% of the mass of the poly(A) RNA in late ovarian oocytes is included in multimolecular structures after renaturation, and a similar fraction of the poly(A) RNA of mid-lampbrush stage oocytes also contains interspersed repeat sequences. In contrast, less than 15% of the poly(A) RNA molecules of stage 41 tadpoles retains the capacity for RNA duplex formation under the same conditions. Poly(A) RNAs displaying interspersed sequence organization are located in the oocyte cytoplasm. This was shown using manually enucleated oocytes. Lampbrush stage oocytes were injected with [3H]GTP, allowed to incorporate for 48 hours, enucleated, and the poly(A) [3H]RNA extracted. The fraction of the labelled poly(A) RNA that could be attributed to mitochondrial transcription was ≤ 16%, according to measurements made with cloned mitochondrial DNA. By use of a cellulose column that separates RNA-containing duplex regions, the labelled RNA was shown to anneal almost completely with the poly(A) RNA already accumulated in the cytoplasm of the oocytes. The newly synthesized poly(A) RNA exported from lampbrush chromosome stage oocyte nuclei also displays an interspersed sequence organization. RNA synthesized in vitro in isolated germinal vesicles was reacted with six cloned complementary DNAs that contain maternal poly(A) RNA sequences, and all of these probes were represented in the transcription products. Thus the amphibian lampbrush chromosome stage oocyte nucleus synthesizes and transports to the cytoplasm poly(A) RNAs that, are similar in structure and sequence content to the maternal poly(A) RNA stored in the oocyte at the termination of oogenesis. We conclude that this poly(A) RXA turns over slowly, thereby maintaining an approximately constant steady state throughout oogenesis. However, the maternal poly(A) RNA pool is so large that the rate of synthesis required for this process demands the intense and widespread level of transcription observed in lampbrush chromosomes