The synthesis of authentic sea urchin transcriptional and translational products by sea urchin histone genes injected into Xenopus laevis oocytes (original) (raw)
Related papers
Expression of sea urchin histone genes in the oocyte of Xenopus laevis
Journal of Molecular Biology, 1979
h22, the cloned repeat unit of Psammechinus miliaris histone genes, is transcribed and translated when inject.ed into Xenopus oocyte nuclei. Transcription is mediated by polymerase B. RNAs indist.inguishable from sea urchin histone mcsscngtr RNAs H2A and H2B are synthesized, but' a large portion of transcripts is polydisperse. From hybridization experiments it can be dcduccd that the H2A and H2B messenger RNAs hybridize rxclusively to the coding strand of the stfructural genes, whereas polydisperse RNA is homologous t,o both strands of the repeat unit. h22 transcripts are stable in t,he oocyt,e and direct the synthesis of sea urchin histone proteins H2A and H2B. h22 transcript,s were isolated separately from oocytr nuclei and cytoplasm. The cytoplasmic RNA contained virtually all of the sea urchin HZA and H2B ml%NAs, and some minor RNAs of discret,e size. On t.hf> other hand, polytlispcrse RNA and RNA complementary to the antisense strand of h22 DNA remained in the nucleus. Covalent circrdarit,y of the h22 DNA seemed to be an importa,nt, factor for the mode of transcriptSion, since only circular h22 and circular pCRI-h22, but not linearized h22, produced H2B mRKA. These findings camphasize special, new aspects for both DNA t,ranscription and RNA processing and transport wit,hin tho living cell.
Transcription of sea urchin histone genes in HeLa cells
Nucleic Acids Research, 1983
HeLa cells were transfected with recombinant DNAs containing the embryonic histone gene repeat of P.miliaris (h22) inserted in either orientation into a pBR-SV4O vector. After 2 to 3 days cytoplasmic RNA was analyzed for authentic sea urchin histone gene transcripts. The correct 5 1 termini of all five histone genes were detected, three (H2B, H2A and H3) at relatively high levels. In contrast, termination was largely aberrant with the correct 3' terminus of only the H2B mRNA found in significant amounts. The levels of histone gene transcription were dependent on the presence, but not the orientation, of SV4O DNA in the recombinant plasmid. The efficiency of initiation of transcription of the individual sea urchin histone genes in HeLa cells was very similar to that previously observed in Xenopus oocytes. The termination pattern, however, was quite different in that oocytes, all but the H3 gene terminate efficiently. The idiosyncrasies in termination efficiencies for these two heterologous transcription systems may reflect the presence of termination factors which are relatively species or even tissue specific and only some of which recognize the sea urchin "terminators" correctly.
Molecular and cellular biology, 1988
Sea urchin early histone genes are active in preblastula embryos; late histone genes are maximally expressed during subsequent stages of embryogenesis. We used the Xenopus laevis oocyte to assay for trans-acting factors involved in this differential regulation. Sea urchin nuclear proteins were prepared by extracting gastrula-stage chromatin successively with 0.45, 1, and 2 M NaCl. We injected three fractions into oocytes along with plasmids bearing sea urchin early and late H2b histone genes. While neither the 0 to 0.45 M nor the 1 to 2 M salt fraction affected H2b gene expression, the 0.45 to 1 M salt fraction stimulated early and late H2b mRNA levels significantly. Late H2b gene expression was stimulated preferentially when the early and late genes were coinjected into the same oocytes. This extract did not stimulate the accumulation of transcripts of injected herpesvirus thymidine kinase genes or of the sea urchin Spec 1 gene, suggesting that the stimulatory activity is not a gen...
Molecular and cellular biology, 1981
We have examined histone gene expression during the early stages of sea urchin embryogenesis. The five histone genes expressed at that time are contained in tandem repetitive segments. It has been suggested that adjacent coding regions and their intervening spacer sequences are transcribed into large polycistronic messenger ribonucleic acid (RNA) precursors. We have subcloned into pBR322 deoxyribonucleic acid (DNA) sequences mapping either in the coding region, the 5' spacer, or the 3' spacer of the H2B histone gene. These clones were used to produce radioiodinated hybridization probes. We measured the steady-state quantity of H2B messenger RNA as well as spacer-specific RNA in the total RNA from embryos taken at various stages of development from fertilization to hatching of blastulae (0 to 22 h post-fertilization). Small amounts of RNA hybridizing to both spacer probes could be found. However, we show that these RNAs form mismatched hybrids with the spacer DNA and therefor...
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.
Proceedings of the National Academy of Sciences, 1988
An early and a late histone H2B gene from the sea urchin Stronglyocentrotus purpuratus were linked in a single plasmid and injected into the eggs of the sea urchin Lytechinus pictus. The levels of transcripts of injected early and late genes and of endogenous early genes were monitored during development by a ribonuclease protection assay. Transcripts of both the injected and endogenous early genes peaked during the blastula stage and decreased severalfold by the mesenchyme blastula stage. Transcripts of the injected late gene became detectable at the blastula stage and increased in amount subsequently, until at least the early gastrula stage, 28 hr after fertilization. Thus, the pattern of expression of the injected early and late H2B genes is similar to that of their endogenous counterparts. These results show that DNA sequences regulating the temporal pattern of early and late H2B gene expression must lie within the cloned DNA segments; i.e., within 600 base pairs of the early H2...
Accumulation of histone repeat transcripts in the sea urchin egg pronucleus
Cell, 1981
RNA transcripts complementary to a genomic histone repeat are found in high concentration in sea urchin egg pronuclei. In situ hybridizations with the recombinant plasmid pCO2 indicate that the nuclear concentration is at least 25 to 50 fold higher than that in the cytoplasm. If nuclear transcripts are predominantly histone mRNAs, they comprise about 12% of the histone mRNA in eggs, or about 0.36 pg. After fertilization these molecules persist through pronuclear fusion but disappear from nuclei by mid 2-cell stage. A similar high nuclear concentration is not observed for polyadenylated mRNAs or for two individual abundant maternal mRNAs. The high steady-state concentration of nuclear histone repeat transcripts suggests that they have an unusually long lifetime in pronuclei of unfertilized sea urchin eggs.
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.
A histone H1 protein in sea urchins is encoded by a poly(A)+ mRNA
Proceedings of the National Academy of Sciences, 1988
Typical histone genes lack intervening sequences and encode small mRNAs (400-800 nucleotides) with short leader and trailer regions. Most histone mRNAs are not polyadenylylated but rather terminate in a highly conserved stem and loop structure. The early, late, and testis-specific histone genes of sea urchins, described to date, have this typical histone gene structure. We have identified an unusual Hi gene, H1-8, in sea urchins that encodes a poly(A)+ mRNA. This mRNA is one of a group of polyadenylylated transcripts homologous with Hi gene probes. The sequence of H1-b has been determined. H1-b encodes a different Hi protein. Although the temporal expression of H1-b mRNA is similar to that of other late Hi ((3 and y) mRNAs, its spatial distribution at the time of maximal accumulation is distinct and confirms that H1-b is regulated differently than other Hi genes.
High molecular weight RNA containing histone messenger in the sea urchin Paracentrotus lividus
Journal of Molecular Biology, 1980
Sea m-chin RNA extracted from early and mesenchyme blastula embryos and oocytes and fractionated on denaturing sucrose density gradients, was hybridized with histone DNA recombinants of Psammechi~us miliaris (clone Xh22) and of Paracentrotus lividus (clone pPH70). Histone sequences are found in the 9 S a.nd larger than 9 S regions of the formamide/sucrose density gradients. The melting of the RNA-DNA duplexes obtained by hybridization of polysomal and high molecular weight RNA of embryos of P. Zividu,s at the stage of early blastula, suggests a degree of heterogeneity in the high M, RNA. The high M, RNA contains at least four of the five histone gene sequences covalently linked.