Synthesis of histones during sea urchin oogenesis (original) (raw)
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Developmental Biology, 1981
The rates of synthesis of the mRNA molecules coding for histones of sea urchin embryos were determined. At different stages during the early development of the sea urchin, Stmn&ocmtrotus purpuratus, embryos were labeled for brief periods with a radioactive nucleoside. The specific activity of the precursor nucleoside triphosphate pool was measured, and the accumulation of radioactivity into histone mRNA molecules was monitored by separation of the RNA on polyacrylamide gels. The rate of histone mRNA synthesis was found to increase dramatically after the 16cell stage, reaching a value of 80 X lo-i5 g/min/embryo at about the 128-cell stage and gradually declining to a value of 12 X lo-i5 g/min/embryo at the 300-cell stage. The maximal accumulation rates require that histone genes be transcribed at least once per minute.
A maternal store of histones in unfertilized sea urchin eggs is demonstrated by two independent criteria . Stored histones are identified by their ability to assemble into chromatin of male pronuclei of fertilized sea urchin eggs in the absence of protein synthesis, suggesting a minimum of at least 25 haploid equivalents for each histone present and functional in the unfertilized egg. In addition, electrophoretic analysis of proteins from acid extracts of unfertilized whole eggs and enucleated merogons reveals protein spots comigrating with cleavage stage histone standards, though not with other histone variants found in later sea urchin development or in sperm . Quantification of the amount of protein per histone spot yields an estimate of several hundred haploid DNA equivalents per egg of stored histone. The identity of some of the putative histones was verified by a highly sensitive immunological technique, involving electrophoretic transfer of proteins from the two-dimensional polyacrylamide gels to nitrocellulose filters. Proteins in amounts <2 x 10 -°p g can be detected by this method .
Microheterogeneity of late histones in larval stages of sea urchin development
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1990
The total histone complement of early plutei were compared with that of intermediate and late larvae of the sea urchin Tefrapygus niger. 2. Electrophoretic comparison indicates that there are quantitative and qualitative shifts of the five classes throughout late larval development. 3. The strong similarity in the amino acid composition of total histones isolated from early, intermediate and late plutei indicates that the observed electrophoretic heterogeneity is due to post-translational modifications.
Translational regulation of histone synthesis in the sea urchin strongylocentrotus purpuratus
Journal of Cell Biology, 1982
The pattern and schedule of histone synthesis in unfertilized eggs and early embryos of the sea urchin Strongylocentrotus purpuratus were studied using two-dimensional gel electrophoresis. After fertilization there is an abrupt change in the pattern of histone variant synthesis. Although both cleavage-stage-and c~-histone mRNA are stored in sea urchin eggs, unfertilized eggs synthesize only cleavage-stage (CS) variants. However, after fertilization, both CS and c~ messages are translated. Since histone mRNA isolated from unfertilized eggs can be translated in vitro, the synthesis of ~ histone subtypes appears to be under translational control. Although the synthesis of ~ subtypes is shown here to occur before the second S phase after fertilization, little or no c~ histone is incorporated into chromatin at this time. Thus, early chromatin is composed predominantly of CS variants probably recruited for the most part from the large pool of CS histones stored in the unfertilized egg.
Block of histone synthesis in isolated sea urchin cells actively synthesizing DNA
Developmental Biology, 1978
The synthesis of DNA and histones are normally coupled. Only a few exceptions have been found. Dissociated cells from sea urchin embryos stop DNA synthesis, but resume it after trypsin treatment. It was of interest, therefore, to examine whether the synthesis of histones is similarly affected. Sea urchin embryos were dissociated from swimming blastulae and the cells so obtained were divided into two batches, one of which was treated with trypsin. Normal embryos from the same culture were used as controls. It was found that, in the dissociated cells, histone synthesis ceases, but unlike DNA, its synthesis is not resumed after treatment with trypsin.
Nucleic Acids Research, 1979
Histone mRNAs at different stages of development were purified by hybridization with the cloned homologous histone genes. The electrophoretic patterns of oocytes, 2-4 blastomeres, 64 cells and morula histone mRNAs was found to be identical, whereas the electrophoretic pattern of mesenchyme blastula histone mRNA was aseddebr different. The cloned histone DNA of P.lividus was hybridized with the RNA of each stage. The Tm was 74°C in all cases except for the mesenchyme histone mRNAs whose Tm was 59°C, thus suggesting that at least two different clusters of histone genes are active in the course of the sea urchin development.
Developmental Biology, 1980
We have analyzed the transcriptional and translational products of recombinant plasmids containing the sea urchin histone genes following their injection into the germinal vesicle (GV or oocyte nucleus) of Xenopus laeuis oocytes. Plasmids pSp2 and pSplO2 contain the identical sea urchin DNA fragment with the Hl, H2B, and H4 histone protein coding regions and adjacent spacer sequences, but are inserted in different vectors. The plasmid, pRC 39, contains only that segment of the pSp2 eucaryotic fragment corresponding to the sea urchin H2B gene plus 125 base pairs of the "upstream" spacer sequences. The translational products produced under the direction of pSp2 and pSplO2 comigrate with authentic sea urchin Hl and H2B histones in a two-dimensional gel system. Since sea urchin H4 histone protein comigrates with endogenous Xenopus H4 histone. the synthesis of the sea urchin protein could not be confirmed. The transcriptional products observed in oocytes injected in the GV with plasmids pSp2 and pSplO2 hybridize to a radioactively labeled DNA probe produced from the Hl, H2B, and H4 sea urchin histone genes, and comigrate with several of the authentic sea urchin histone mRNAs on polyacrylamide gels under denaturing conditions. These results suggest that the injected sea urchin histone genes are being transcribed properly and are producing functional histone mRNAs in Xenopus oocytes. We calculate that the injected genes are being transcribed at approximately 0.1 transcript per gene per hour. This represents IO7 to 10" transcripts or approximately 0.1% of the total transcriptional activity of stage 6 oocytes. We do not detect proper transcriptional or translational products when plasmid pRC 39 is injected into the GVs of Xenopus oocytes. This result suggests that the eucaryotic fragment present in this plasmid may not contain the information for proper expression of the sea urchin H2B gene.