Polyadenylation of histone mRNA in Xenopus oocytes and embryos.

Oogenesis of amphibians is an atypical situation in which histone mRNA is polyadenylated. The poly(A) tract on H4 mRNA has been examined by S1 nuclease analysis. Throughout oogenesis the poly(A) tract is very short, and nonexistent on some mRNA molecules. The poly(A) tract is completely removed during maturation of the oocyte, and is absent in embryos and cultured cells.

Are there major developmentally regulated H4 gene classes in Xenopus?

Primer extension analysis has been used to study the principal H4 mRNAs present at different developmental stages and in several adult tissues of Xenopus borealis and X. laevis. In X. borealis a single sequence class predominates in oocytes, tadpoles and cultured fibroblasts. There is also a polymorphic minor type which shows no developmental regulation. The primer extension bands obtained from adult liver and kidney RNA appear to be the same as ovary and therefore these tissues almost certainly contain the same major H4 mRNA species. This is confirmed by S1 mapping of the 3' end of the mRNA. Thus for H4 genes in X. borealis there is no evidence of the kind of switches in histone gene expression seen in sea urchins or certain protostomes. The situation in X. laevis is complicated by considerably higher gene variability both within and between individuals. Nevertheless, in this species, as in X. borealis, there seems to be no major developmental switch in the regulation of H4 gene expression, a conclusion that also holds for an H1C and an H3 gene.

Organization and expression of cloned histone gene clusters from Xenopus laevis and X. borealis.

We have isolated several clones containing Xenopus histone genes from genomic libraries of X. laevis and X. borealis DNA. Each genomic clone has been mapped and the positions of 26 histone genes in seven laevis clones and 5 histone genes in one borealis clone have been determined. In laevis, the histone gene clusters show considerable variation in gene order within a single individual. When the cloned DNAs were microinjected into the nucleus of Xenopus oocytes, expression of cloned genes at the transcriptional and translational level was readily detectable. Previously unknown histone variants were revealed by the microinjection experiments.

Paternal gene expression in developing hybrid embryos of Xenopus laevis and Xenopus borealis.

We have studied protein synthesis in the viable hybrid Xenopus laevis (female) X Xenopus borealis (male) using 2D gel electrophoresis. Fourteen borealis-specific proteins were studied. Two of these proteins appeared by the gastrula stage, five in the gastrula and the rest later. Where homologous laevis proteins were tentatively identified, androgenetic haploid hybrids were used to study whether the protein was encoded by stored maternal mRNA, and how long this mRNA persisted. The two proteins appearing in blastulae were probably initially coded by stored maternal mRNA. This was not detectable by the tailbud-tadpole stage, and presumably had been destroyed.

Actin synthesis during the early development of Xenopus laevis.

Cytoskeletal beta and gamma-actin are amongst the most rapidly made proteins of oocytes, blastulae and late embryonic stages of Xenopus laevis but, relative to other proteins, the rate of synthesis is low in the egg or cleaving embryo, although the messenger RNA is present in comparable amounts at the different stages. Actin synthesis therefore involves post-transcriptional regulation. alpha-Actin, the actin class characteristics of straited muscle cells, is first detectable in late gastrulae and it is an abundant newly synthetized protein from the neurula stage onwards. mRNA template activity for this protein is not detectable before the gastrula stage. Thus alpha-actin synthesis probably reflects new gene action, confined to part of the embryo, for alpha-actin only appears in the section which includes presumptive skeletal muscle cells. It therefore constitutes the earliest cyto-specific protein so far demonstrated in Amphibia. When tadpole tail poly(A)-containing mRNA is injected into oocytes and eggs alpha-actin synthesis is seen in both cases. Extensive evidence for the identification of the actins is presented. This is based on location of synthesis, DNase-I binding and partial peptide mapping.

Changes in protein synthesis during the development of Xenopus laevis.

Patterns of protein synthesis during the development of Xenopus were studied by two-dimensional gel electrophoresis. Up to the end of the blastula stage we find no newly synthesized proteins which are not already made in the oocyte. The first new proteins are seen during gastrulation, and they increase in number during neurulation. Some of these are restricted to the 'ectodermal' region, and some to the 'endodermal' region of embryos divided into two parts. These new, region-specific proteins include alpha-actin. When the oocyte matures the number of detectable newly synthesized proteins decreases, reaching a minimum in the unfertilized egg. Some, such as beta- and gamma-actin, re-appear at the end of cleavage. This could not be shown to be a recovery artifact. The relation of the total mRNA to these changes in protein synthesis was studied by translation in the lysed reticulocyte cell-free system. The mRNAs that code for oocyte proteins that cease synthesis in the unfertilized egg and re-appear in blastulae are nevertheless detectable in total RNA made from eggs. These proteins therefore seem to cease and resume synthesis through translational control. mRNAs for new proteins first appear after gastrulation, just when these proteins are first detected in vivo. This strongly suggests, though it does not prove, that new gene activity is involved. It is therefore likely that region-specific gene activity is already present by the gastrula stage of development, and has an impact on the most abundant kinds of proteins made in the embryo.