Sequence analysis and evolution of sea urchin (Lytechinus pictus and Strongylocentrotus purpuratus) histone H4 messenger RNAs

The putative histone H4 (F2a₁) mRNA has been isolated from early blastula Strongylocentrotus purpuratus sea urchin embryos. Nucleotide sequences of oligonucleotides obtained by digestion of this RNA with T₁ ribonuclease have been obtained and many are found to be colinear with the amino acid sequence of histone H4 protein. The sequences obtained from the H4 mRNAs of S. pnrpuratus have been compared with those obtained from Lytechinus pictus (Grunstein & Schedl, 1976). The two mRNAs for this highly conserved protein have undergone considerable divergence of the sort that would be predicted from the degeneracy of the genetic code. 11.5% of the bases have undergone substitution at a rate calculated to be 3 × 10^?9 base changes · codon^?1 · year^?1.     

Localization of sequences coding for histone messenger RNA in the chromosomes of Drosophila melanogaster

In situ hybridization of sea urchin (Psammechinus miliaris, Lytechinus pictus and Strongylocentrotus purpuratus) histone messenger RNA has been used to map complementary sequences on polytene chromosomes from Drosophila melanogaster. The sea urchin RNA hybridizes to the polytene regions from 39D3 through 39E1-2, including both of these bands (39D2 may also be included). This region is identical to the one which hybridizes most heavily with non-polyadenylated cytoplasmic RNA from D. melanogaster tissues. Sea urchin mRNAs coding for several individual histones each hybridize across the entire region from 39D3 (or D2) through 39E1-2, as would be expected if the individual mRNA sequences are interspersed. In view of the apparently even distribution of sequences complementary to histone mRNA within the 39D3-39E1-2 region, the significance of the several polytene bands in this region remains an open question. Biochemical characterization of the hybrids between sea urchin histone mRNA and D. melanogaster DNA suggests that sea urchin mRNAs for several of the histone classes have some portions which retain enough sequence homology with the D. melanogaster sequences to form hybrids, although the hybrids have base pair mismatches. In situ hybridization of chromosomes in which region 39D-E is ectopically paired show no evidence of sequence homology in the chromosome region with which 39D-E is associated.     

Size and sequence homology of masked maternal and embryonic histone messenger RNAs.

The messenger RNAs for five classes of histone proteins are shown by competitive RNA-DNA hybridization to be stored in the unfertilized egg of the sea urchin, Lytechinus pictus. The masked mRNAs for f2b, f2a2, f3 and f2al histones migrate in polyacrylamide slab gels with the same mobility as the histone mRNAs that are synthesized after fertilization and are found engaged in protein synthesis on polysomes. The masked maternal and embryonic mRNAs for histone f2a1 are identical in mobility when analyzed in a gel system capable of resolving differences estimated as small as 4–5 nucleotides in length. We conclude that these histone mRNAs synthesized during oogenesis and inactive prior to fertilization are not activated during embryogeny by alteration in their molecular size.     

Quantitation of the accumulation of histone messenger RNA during oogenesis in Xenopus leavis

The accumulation of messenger RNA coding for histone H3 in oogenesis of Xenopus laevis was studied by quantitative hybridization techniques, using a cloned genomic DNA fragment as a probe. This probe was isolated from cloned Xenopus histone DNA and contains most of the H3 coding sequences. Histone H3 mRNA accumulation was found to be completed before the maximum lampbrush stage. Hybridization of RNA blots with DNA probes containing genes for histones H2A, H2B, and H4 suggests the same accumulation pattern for the mRNAs coding for these histones as for histone H3 mRNA. The amount of H3 mRNA in the mature oocyte was established to be 130 ± 68 pg, i.e., about 5 × 10⁸ copies.     

Developmental studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus

Chromatin was isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus. The isolated chromatin shows less absorptivity ratio of 230 nm : 260 nm and possesses less protein than does embryonic chromatin. The ratio of histone : DNA is 1.02; nonhistone : DNA 0.13; RNA : DNA 0.04. Sperm chromatin melts in two steps with T_ms 70°C and 84°C in 2.5 × 10⁻⁴, M EDTA in contrast to embryonic chromatin with a single T_m = 72°C. Disc electrophoresis of basic proteins of sperm revealed one minor component with extremely fast mobility and three major components. The one with the slowest mobility is characteristic of sperm. The embryo has in turn its characteristic histone which also migrated slowly in disc electrophoresis. Both of these unique histone fractions are selectively extracted from chromatin by 5% perchloric acid. Amino acid analyses of these chromatographically purified unique fractions show that both contain a large amount of lysine, while that from sperm, in addition, contains also a large amount of arginine. Minimal molecular weights of 33,000 for sperm and 16,200 for embryo unique histone were estimated from these analyses. Sperm chromatin supports a level of RNA synthesis in vitro with exogeneously supplied RNA polymerase about 2% that of the corresponding free DNA.     

Developmental changes in chromatin proteins of the sea urchin from blastula to mature larva.

Chromatin proteins of the sea urchin Lytechinus pictus from blastula to fully grown feeding larva were analyzed by polyacrylamide-gel electrophoresis. Two subspecies of histone protein changed during development. Histone f1g increased progressively relative to f1m and within a few days after the larvae began to feed constituted >99% of the f1 fraction. Two slower-moving subfractions of histone f2b disappeared in the same interval, being replaced by a single fastermoving species. The electrophoretic profile of nonhistone protein changed quantitatively and qualitatively with the largest change, a relative decrease of high molecular weight bands and a new pattern of middle molecular weight bands, also occurring several days after feeding began. The profiles remained virtually the same during subsequent development though the cell number increased at least tenfold. The onset of feeding thus seems to be correlated with major changes in chromatin proteins.     

Expression of maternal and paternal histone genes during early cleavage stages of the echinoderm hybrid Strongylocentrotus purpuratus × Lytechinus pictus

Interspecific hybrids of the sea urchins Strongylocentrotus purpuratus (♀) and Lytechinus pictus (♂) were used to estimate the contributions of the maternal and paternal genomes to histone mRNA synthesis during early development. Radiolabeled histone mRNAs from the two sea urchin species were identified by hybridization to cloned histone genes from both S. purpuratus and L. pictus and shown to be electrophoretically distinguishable. The synthesis of maternal and paternal histone mRNA in these hybrid embryos is evident as early as the two-cell stage. By at least the 16-cell stage, both maternal and paternal histone mRNAs are associated with polysomes. The relative amounts of the maternal and paternal histone mRNAs synthesized by the zygote appear to be similar.     

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.     

Histone mRNA in eggs and embryos of Strongylocentrotus purpuratus

Histone messenger RNA is detectable in both the maternal RNA which is stored in the unfertilized sea urchin egg and in the RNA species which are synthesized $\text{de}$$\text{novo}$ after fertilization. Hybridization competition experiments show that sequences similar to pulse-labeled 912S RNA from morulae are present in total RNA from unfertilized eggs as well as that from later stages. The proportion of histone mRNA in cellular RNA increases after fertilization, reaching a maximum at the morula stage. Although these messengers are still present in hatched blastulae and gastrulae, they represent a smaller proportion of total RNA compared with earlier stages.     

Sequence complexity of heterogeneous nuclear RNA in sea urchin embryos.

The sequence complexity of heterogeneous nuclear RNA is sea urchin gastrulas was measured by RNA-driven hybridization reactions with nonrepetitive sea urchin DNA. 28.5% of the sequence complexity of the genome is represented in the nuclear RNA. This amounts to 1.74 X 10(8) nucleotides of diverse sequence, more than 10 times the nucleotide complexity of the polysomal messenger RNA extracted from sea urchin embryos at the same stage. The complex set of nuclear RNA sequences driving this hybridization reaction was shown to be the same as the rapidly labeled hnRNA, using pulse-labeled nuclear RNA as driver.     

High molecular weight RNA containing histone messenger in the sea urchin Paracentrotus lividus

Sea urchin RNA extracted from early and mesenchyme blastula embryos and oocytes and fractionated on denaturing sucrose density gradients, was hybridized with histone DNA recombinants of Psammechinus miliaris (clone λh22) and of Paracentrotus lividus (clone pPH70). Histone sequences are found in the 9 S and 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. lividus at the stage of early blastula, suggests a degree of heterogeneity in the high M_r RNA. The high M_r RNA contains at least four of the five histone gene sequences covalently linked.     

Reiteration frequency of histone coding sequences in man

A “10S”-RNA fraction with the characteristics of histone messenger was isolated from pulse labeled synchronized HeLa cells. Two properties indicate that this fraction consists of histone messenger sequences. First, its appearance in the cell cytoplasm is dependent upon continued DNA replication; and second, it is capable of cross hybridization with isolated histone DNA of sea urchin. The rate of hybridization of this RNA with an excess of human DNA suggests that there are 10–20 copies of each histone coding sequence in the human haploid genome.     

Adaptation of cultured human lymphoblasts to growth in citrulline

DNA synthesis is initiated in unfertilized sea urchin eggs (Strongylocentrotus purpuratus and Lytechinus pictus) by exposing them to NH₄OH-sea water (ordinary sea water titrated to pH 9–9.1 with NH₄OH). The eggs are considered to be unfertilized eggs by visual and electro-biological criteria and because they can later be fertilized and then do give visible and electrobiological fertilization reactions. The incorporation of ³H-thymidine proceeds in rounds, the magnitude increasing in successive rounds. It is also reported that the treatment with NH₄OH activates the uptake of thymidine by the eggs, although the internal thymidine builds up more slowly in unfertilized eggs treated with NH₄OH than it does in fertilized eggs. The magnitude of the incorporation of exogenously supplied labelled thymidine into DNA is lower in the NH₄OH-treated unfertilized eggs than in normal fertilized eggs. This difference is not attributed to differences in the amount of DNA synthesized and the explanation is sought in thymidine uptake and nucleotide pathways.     

The structural organization and DNA sequence of a wheat histone H4 gene.

Some wheat histone H4 genes have been cloned from a Charon 4 wheat genomic DNA library using sea urchin histone H4 DNA as a probe. DNA sequence analysis of a cloned gene showed that the deduced amino acid sequence of wheat histone H4 protein was identical to that of pea. The 5' end of wheat histone H4 mRNA was mapped on the cloned gene by the S1-procedure. Southern blotting analysis of the genomic DNA indicated that histone H4 genes were reiterated 100 to 125 times per hexaploid wheat genome.     

Electron spin resonance studies on the conformation and interactions of histories containing cysteine. Histones H3 from calf thymus and H4 from sea urchin sperm.

In this study the spin-label method has been used to obtain information about conformational properties of regions containing cysteine of histone H3 from calf thymus, histone H4 from sperm of the sea urchin Arbacia lixula, and the histone complex H3–H4. It has been found that the microenvironments of histone H3 causing immobilization of the spin labels are sensitive to variations in ionic strength of dilute solutions of phosphate buffer, are partially destroyed by urea, and fully destroyed by proteolytic enzymes. The interaction of spin-labeled histone H3 with histone H4 induces an increase of immobilization of the spin label, indicating an increase in rigidity at the cysteine region of histone H3. The use of a series of spin labels of variable length for histone H3 gives an estimate of 0.8–1.0 nm for the apparent depth of the spin label binding site, a value which does not change upon interaction of histone H3 with H4. Histone H4 from A. lixula sperm causes a similar immobilization of the spin label. As for histone H3, immobilization increases with the ionic strength, and the structures are destroyed by urea and proteolytic enzymes. Upon mixing with histone H3, however, the extent of immobilization appears only slightly changed, and together with sedimentation velocity results, these studies suggest that the spin label attached to histone H4 prevents the complex formation.     

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.