UBC Theses and Dissertations
Histone synthesis during early development of Xenopus laevis (the South African clawed toad) Byrd, Earl William
Histones are the basic proteins complexed with DNA in the chromosomes of eucaryotic organisms. The synthesis of histones and their nuclear accumulation was followed during early embryogenesis in Xenopus laevis, the South African clawed toad. Histone synthesis was first examined in total cell homogenates of Xenopus laevis embryos. Electrophoretic and chromatographic experiments indicate that histone synthesis takes place during the cleavage and swimming embryo stages of development in Xenopus laevis. Furthermore, the major classes of histones (I, II and III and IV) made during these different stages appear to be qualitatively the same, as shown by electrophoresis on polyacrylamide gels and by Amberlite chromatography, and include histones similar to those found in adult somatic tissues of this amphibian. Experiments utilizing polyacrylamide gels show that these proteins coelectrophorese with known histone markers I, Ilb1, and III from trout testes and histone IV from peas. Individual basic proteins were purified from 8M urea-2M LiCl extracts of total embryo protein by carboxymethyl cellulose chromatography and separated by Amberlite chromatography. The amino acid composition of the Amberlite peaks from swimming embryos indicates that these peaks contain histones comparable to those of other vertebrate species. Rechromatography of the first lysine-rich peak on Amberlite with a very shallow guanidine hydrochloride gradient resolved this peak into a ribosomal contaminant and at least three lysine-rich fractions in swimming embryos, suggesting that there is microheterogeneity in Xenopus laevis histone I. The synthesis and nuclear accumulation of early embryonic histones was examined next. Histones were isolated from nuclear chromatin preparations of pre-gastrula embryos and swimming tadpoles and subjected to acrylamide gel electrophoresis and amino acid analysis. Histone synthesis was demonstrated by the incorporation of ¹⁴C02 into the amino acid residues of acid hydrolyzed proteins. Gel electrophoresis indicated that each of the major groups of histones was synthesized in pre-gastrula embryos, as well as in later stages, and incorporated into nuclear chromatin. These newly synthesized embryo histones were similar to those in adult Xenopus laevis, when compared by gel electrophoresis and amino acid analysis. ftctinomycin D pulse-labeling experiments utilizing the incorporation of ³H-lysine into partially dissociated blastulae showed a 20 fold decrease of histone synthesis in Actinomycin D treated embryos from that of sibling controls when these basic proteins were separated by Amberlite chromatography. These data suggest that histones are synthesized almost entirely from newly made mRNA after stages 5 to 6 in the embryo. On the whole, these observations on histone synthesis and the role of maternal template in pre- and post-gastrula Xenopus development are in general agreement with most of the findings in sea urchin embryos.
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