Xenopus oocytes contain several mRNAs that are mobilized into polysomes only at the completion of meiosis (maturation) or at specific times following fertilization. To investigate the mechanisms that control translation during early development, we have focused on an mRNA, termed G10, that is recruited for translation during oocyte maturation. Coincident with its translation, the poly(A) tail of this message is elongated from -90 to 200 adenylate residues. To identify the cis sequence that is required for this cytoplasmic adenylation and recruitment, we have synthesized wild-type and deletion mutant G10 mRNAs with SP6 polymerase. When injected into oocytes that subsequently were induced to mature with progesterone, wild-type G10 mRNA, but not mutant transcripts lacking a 50-base sequence in the 3'-untranslated region, was polyadenylated and recruited for translation. The 50-base sequence was sufficient to confer polyadenylation and translation when fused to globin mRNA, which does not normally undergo these processes during oocyte maturation. Further mutational analysis of this region revealed that a U-rich sequence 5' to the AAUAAA hexanucleotide nuclear polyadenylation signal, as well as the hexanucleotide itself, were both required for polyadenylation and translation. The 50-base cis element directs polyadenylation, but not translation per se, as a transcript that terminates with 3'-deoxyadenosine (cordycepin) is not recruited for translation. The available data suggest that the dynamic process of polyadenylation, and not the length of the poly(A) tail, is required for translational recruitment during oocyte maturation.

Genes whose expression is restricted to oogenesis and early development may have important functions in these processes. Northern analysis showed that Xenopus B4 mRNA is expressed in oogenesis and embryogenesis through to the neurula stage. Immunocytochemistry with anti-B4 antibodies showed that B4 protein is only detectable in preneurula stages; it is localized to nuclei and is associated with metaphase chromosomes. Immunoblotting revealed approximately constant levels of B4 protein per embryo for the first 2 days of development. Thus, as the number of nuclei increases during early development, the amount of B4 protein per nucleus is diluted out. Sequencing of two B4 cDNA clones revealed that the predicted B4 translation product is a 29-kD protein with 29% identity with histone HI, distributed over the entire length of its sequence. The B4 protein also has certain other H1 protein characteristics--a tripartite structure consisting of a mainly hydrophobic central domain flanked by an amino-terminal segment and a long hydrophilic carboxyterminal tail containing a tandemly repeated amino acid motif. However, in contrast to histone HI mRNA, B4 mRNA has a classic polyadenylation signal, is polyadenylated, and lacks the histone HI 3' noncoding consensus sequence involved in RNA processing.

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A cDNA clone, pXlgC20, was isolated from a library constructed from poly(A)+ RNA from stage 10 X. laevis gastrulae. This sequence hybridizes with up to nine different RNA species ranging in size from 1600 to 3500 nucleotides, regularly spaced at intervals of about 230 nucleotides. Clone pXlgC20 contains two complete repeats of a 228 bp sequence as well as part of a third repeat, all adjacent and in the same orientation. One possible translational reading frame in pXlgC20 completely spans the repeat sequences, coding for a protein composed of tandem 76 amino acid units. The amino acid sequence of each unit completely matches that of human ubiquitin. Ubiquitin is translated in the form of a multimeric precursor molecule containing several units. We show that genomic DNA fragments exist that contain at least 12 of these units in tandem and propose that the different mRNA size classes vary in their number of ubiquitin coding sequences.

Unfertilized eggs of many species contain large amounts of maternal mRNA that are used to support protein synthesis during the first few hours of development, before the onset ofembryonic transcription. We have examined the accumulation of nonpolysomal maternal RNAs -in polysomes after fertilization in Xenopus laevis by measuring the distributions of specific sequences in nonpolysomal and polysomal fractions. In an arbitrary selection of 18 maternal sequences that are largely nonpolysomal in the full-grown oocyte, 13 became enriched in polysomes by the 16-cell cleavage stage. One sequence accumulated only 50% in polysomes at this time, while four sequences became polysomal later than the 16-cell stage. Several RNA sequences decreased in titer during early embryogenesis and were rare during organogenesis. Sequences that are mobilized rapidly and efficiently into polysomes shortly after fertilization and whose cellular concentrations are highest in embryos before organogenesis may provide genetic information for developmental functions restricted to very early embryogenesis. These experiments serve to identify such sequences in Xenopus.Our own approach toward identifying developmentally important genes in Xenopus is to isolate genes (or gene probes) in the absence of any information concerning their functions by constructing synthetic cDNA libraries from poly(A)+ RNA obtained from various stages of development. Subsequently, genes with potential relevance for early development are selected for further study on the basis of their developmental expression pattern. We specifically looked for genes whose expression (at the level of RNA) is restricted to oogenesis and very early embryogenesis-i.e., before organogenesis-and whose transcripts accumulate in polysomes immediately after fertilization. Previous work has established titers, polysomal distributions, and tissue localizations of both maternal and embryo-restricted sequences during embryogenesis as well as titers of maternal RNAs during oogenesis (4-6, 18, 19). In this communication we describe (i) the mobilization of many maternal RNA species into polysomes after fertilization and (ii) maternal RNA sequences that decrease in titer during the first half day of development, before the beginning of organogehesis.