The stability and translation of sea urchin histone messenger RNA molecules injected into Xenopus laevis eggs and developing embryos

Embryonic sea urchin histone mRNA was injected into eggs and developing zygotes of Xenopus. The functional stability of the mRNA was monitored by separating newly synthesized sea urchin histones from those of Xenopus. Just as when injected into Xenopus oocytes, sea urchin H1, H2A, and H2B mRNA molecules have a functional half-life of about 3 hr in the developing embryo. This suggests that the endogenous Xenopus histone mRNA is also unstable and has a number of implications for the amount of histone mRNA that is stored in the oocyte and the time at which histone genes should become active in development. The injected mRNA is translated with little, if any, greater efficiency in the egg than in the oocyte. However, Xenopus histone synthesis increases about 20- to 50-fold during the transition from oocyte to egg. The injection experiments therefore suggest that this increase is brought about primarily by the mobilization of stored mRNA, rather than an increase in the efficiency of histone synthesis.     

Positions of sea urchin (Strongylocentrotus purpuratus) histone genes relative to restriction endonuclease sites on the chimeric plasmids pSp2 and pSp17.

The positions of the several sea urchin histone genes on the eukaryotic fragments of the chimeric plasmids pSp2 and pSp17 have been mapped relative to the Eco RI and Hind III restriction endonuclease sites on the plasmids. Two principal mapping methods using the electron microscope have been used: (a) the R-loop procedure and a new modification thereof to map the genes on duplex DNA; (b) the gene 32-ethidium bromide technique to visualize RNA-DNA hybrids on single strands of DNA. It is known that there are two histone genes, H3 and H2A, on pSp17. There are two Eco RI sites at the two junctions of the procaryotic segment with the eucaryotic segment on the plasmid. We show, by an electron microscope method, that for H2A, with a length of 0.52 kilobases (kb), one end of the gene is situated 0.02 to 0.03 kb from one RI site, and that there is a Hind III site within this gene at about 0.13 kb from the end phe other RI site of this plasmid. The H4 gene lies between H2B and H1. The ms the incubation temperature is raised up to a temperature just below that at which strand dissociation of the duplex DNA occurs.     

The relative positions of sea urchin histone genes on the chimeric plasmids pSp2 and pSp17 as studied by electron microscopy

The relative positions of the sea urchin histone genes and the spacer regions on the chimeric plasmids pSp2 and pSp17 have been mapped by hybridizing total histone messenger RNA to single strands of the plasmid DNAs. The lengths and spacing between the several RNA:DNA duplex regions on the single strands of DNA were measured by the gene 32-ethidium bromide electron microscope mapping method. We find that the genes are interdigitated with spacer sequences of different lengths; that there are three coding sequences on pSp2, all on the same strand, with the relative order H1, H4, and B4; and that there are two coding sequences on pSp17, both on the same strand, corresponding to the messages denoted B1 and B2–B3, where B4, B1, and B2–3 are electrophoretically resolved components of histone mRNA, all of size intermediate between the larger H1 and the smaller H4 message.     

A regulatory sequence near the 3'' end of sea urchin histone genes.

The 3' flanking sequences of all five histone genes have been sequenced in the histone DNA clone h19 of the sea urchin Psammechinus miliaris. A large (23 bp) and a small (10 bp) conserved sequence was found by sequence comparison, some 29-40 bp downstream from the termination codon. 12 bases of the larger homology block show a dyad symmetry. The available sequences of clone h22 of the same species and those of the histone clones pSp2 and pSp17 of Strongylocentrotus purpuratus, another sea urchin species, fit well into this comparison. Two types of sequences are involved in the dyad symmetry; one is H1, H3 and H4 specific, the other is H2A and H2B specific. If these conserved sequences are transcribed, a hairpin loop could form in the RNA molecules. This secondary structure might serve as a recognition signal for a regulatory protein.     

The organization of sea urchin histone genes

Sucrose gradient analysis of total sea urchin DNA cleaved with theEcoRI andHind III restriction endonucleases and identification of histone coding gene sequences by hybridization with histone mRNA have elucidated the basic organization of the histone gene repeat unit. These data, plus results obtained by electrophoretic analysis of purified endonuclease-cleaved sea urchin histone DNA and hybridization with cRNA transcribed from the eucaryotic segment of constructed plasmid chimeras cloned in E. coli, show that the several DNA sequences coding for individual histone proteins are intermingled in a 7 kilobase (kb) repeat unit. Cleavage of total sea urchin DNA withEcoRI produces 2.2 and 4.8 kb fragments which are homologous with the two cloned fragments, and which are contained in a 7 kbHind III fragment. Cleavage with both enzymes reveals that the 2.2 kbEcoRI fragment contains aHind III site 0.15–0.2 kb from an end. RNA · DNA hybridization between chimeric plasmid DNA and purified individual mRNAs isolated from sea urchin embryo polyribosomes has been used to assign coding sequences to either the 2.2 or 4.8 kb region of the histone DNA repeat unit. A map of the histone genes is proposed.     

The sea urchin histone gene complement

The only eukaryotic mRNAs that are not polyadenylated are the replication-dependent histone mRNAs in metazoans. The sea urchin genome contains two sets of histone genes that encode non-polyadenylated mRNAs. One of these sets is a tandemly repeated gene cluster with a 5.6-kb repeat unit containing one copy of each of the five alpha-histone genes and is present as a single large cluster which spans over 1 Mb. There is a second set of genes, consisting of 39 genes, containing two histone H1 genes, 34 genes encoding core histone proteins (H2a, H2b, H3 and H4) and three genes expressed only in the testis. Unlike vertebrates where these genes are clustered, the sea urchin late histone genes, expressed in embryos, larvae and adults, are dispersed throughout the genome. There are also genes encoding polyadenylated histone mRNAs, which encode histone variants, including all variants found in other metazoans, as well as a unique set of five cleavage stage histone proteins expressed in oocytes. The cleavage stage histone H1 is the orthologue of an oocyte-specific histone H1 protein found in vertebrates.     

Histone variants during sea urchin development.

The sea urchin genome contains several histone gene families whose expression is regulated in a developmental and tissue-specific fashion. The Cleavage Stage (CS) histone subtype is synthesized in unfertilized eggs and in embryos until the third cell cycle. The Early (E) subtype is synthesized during embryogenesis from the 2-4 cell stage to blastula. The only variant produced from the mesenchyme blastula stage to adult is the Late (L) subtype. In addition, two "sperm-specific" histone genes (SpH1 and SpH2B) are expressed exclusively in testis and their corresponding products are incorporated in sperm chromatin. In this review I will describe in some detail what is known about the characteristics of the various histone subtypes, with special focus on the Sp variants, and discuss the possible meaning of the presence of these histone variants during sea urchin development.     

Separation and properties of an H2B histone variant from the sperm chromatin of the sea urchin Sphaerechinus granularis

The purification and the physico-chemical characterization of one of the two H2B histone variants from the sperm of the sea urchin Sphaerechinus granularis are reported. The molecule shows, in addition to a distinctive molecular weight value, an amino acid composition different both from that of calf thymus H2B histone and from those of H2B histones from chromatin of sperm and embryos of other sea urchins. Circular dichroism and fluorescence data are discussed in comparison to those of calf thymus H2B.     

Isolation and sequence analysis of sea urchin (Lytechinus pictus) histone H4 messenger RNA

Histone messenger RNAs isolated from early blastula stage Lytechinus pictus sea urchin embryos have been separated into discrete RNA bands on polyacrylamide gels. The most rapidly migrating of these molecules, the putative histone H4 mRNA, has been digested with T₁ ribonuclease to generate oligonucleotides for nucleotide sequence analysis. Many of these sequences are colinear with the highly conserved amino acid sequence of histone H4 protein as determined for both cows and peas.Histone H4 messenger RNA hybridizes in conditions of DNA excess to sea urchin DNA which is repeated approximately 470-fold. Despite this level of repetition the nucleotide sequence of the H4 messenger RNA reflects little evolutionary divergence within the H4 genes of L. pictus as judged by the stoichiometric yield of T₁ oligonucleotides and the hybridization and thermal stability of histone H4 mRNA-DNA hybrids.     

Characterization of histones from plutei larvae of the sea urchin Tetrapygus niger

• 1.1. Histones were isolated from plutei larvae of the sea urchin Tetrapygus niger and analysed electrophoretically. Individual histones were purified and their amino acid compositions were determined.
• 2.2. The electrophoretic analysis revealed that larval histones are microheterogeneous; H1 exhibits four subforms, the nucleosomal core histones H2A, H2B and H3 were resolved into three subforms each and H4 had two subforms.
• 3.3. The comparisons of the amino acid compositions of plutei larvae histones with data from the literature of homonimus late variants isolated from gastrulas of other sea urchin species, indicate that late histone variants are conserved proteins with a very slight degree of species specificity and with general features of classical histones.