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.

As part of a study of glycolysis during early development we have examined the pattern of expression of enolase isoenzymes in Xenopus laevis. In addition, the nucleotide sequence of a cDNA clone coding for the complete amino acid sequence of one enolase gene (ENO1) in X. laevis was determined. X. laevis ENO1 shows highest homology to mammalian non-neuronal enolase. Analysis of enolase isoenzymes in X. laevis by non-denaturing electrophoresis on cellulose acetate strips revealed five isoenzymes. One form was present in all tissues tested, two additional forms were expressed in oocytes, embryos, adult liver and adult brain, and two further forms were restricted to larval and adult muscle. Since enolase is a dimer, three different monomers (gene products) could account for the observed number of isoenzymes. This pattern of enolase isoenzyme expression in X. laevis differs from that of birds and mammals. In birds and mammals the most acidic form is neuron-specific and there is only one major isoenzyme expressed in the liver. RNAase protection experiments showed the presence of ENO1 mRNA in oocytes, liver and muscle, suggesting that it codes for a non-tissue-restricted isoenzyme. ENO1 mRNA concentrations are high in early oocytes, decrease during oogenesis and decrease further after fertilization. Enolase protein, however, is maintained at high concentrations throughout this period.

Steady state kinetics and inhibition by a dipyridodiazepinone of the reverse transcriptase from human immunodeficiency virus type 1 (HIV) were studied using a heteropolymeric RNA template with a sequence from the authentic initiation site on the HIV genome. For addition of the first deoxynucleotide to primer, kcat/KM is 0.05 (nM-min)-1 and KM is 10 nM. When all 4 deoxynucleotide triphosphates are present and processive synthesis occurs, catalysis is less efficient; kcat/KM = .0077 (nM-min)-1 and KM = 100 nM for dATP. These results are consistent with a rate determining conformation change involved in translocation of the enzyme along the template. Inhibition by the dipyridodiazepinone BI-RG-587 is noncompetitive with respect to both nucleotide and template-primer; this compound decreases Vmax but does not affect KM. Thus, this inhibitor binds to a site distinct from the substrate binding sites with Ki of 220 nM. Inhibition by BI-RG-587 results in a uniform decrease in amount of products of all lengths rather than a shift from longer to shorter products, suggesting the inhibitor does not affect processivity of reverse transcriptase.

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