The relative contributions of newly synthesized and stored messages to Hl histone synthesis in interspecies hybrid echinoid embryos.

We have measured the ratio of incorporation of 3H-lysine into the maternal and paternal forms of Hl histones synthesized by the interordinal hybrid embryo which results from the fertilization of sand dollar eggs with sea urchin sperm. This ratio has been used to calculate the relative contributions of newly transcribed and stored Hl histones mRNA to the synthesis of Hl histone at five different stages of development. These calculations are based on the assumption that histone mRNA of both parental types is transcribed with equal efficiency from the genome and that these RNAs are translated with equal efficiency in the cytoplasm of the hybrid embryos. On this basis, we have estimated that the contribution of new mRNA represents 80% of total Hl histone synthesis at the 16--32 cell stage, 54% at the hatching blastula stage, 40% at the mesenchyme blastula stage, and 100% after gastrulation. These data are discussed in the light of presently known parameters of histone and histone mRNA synthesis in echinoderm embryos.     

Asynchronous appearance of newly synthesized histone H1 subfractions in HeLa chromatin

Incorporation of radioactive alanine into chromatin-bound subfractions of H1 histone was studied in HeLa cells synchronized by the double thymidine block technique. The subfractions were resolved into three chromatographic peaks by Biorex-70. In the period 5-7 h after release from the thymidine block, peaks I and III showed twice as much incorporation as they did in the period 1-3 h after release, whereas peak II showed three times the incorporation at 5-7 h that it did at 1- 3 h. Thus, the H1-histone subfraction in peak II appears in chromatin somewhat later in S phase than do the subfractions in Peaks I and III.     

Changing rates of histone mRNA synthesis and turnover in Drosophila embryos

The rates of synthesis and turnover of histone mRNA in Drosophila embryos were determined by hybridization of in vivo and in vitro labeled embryonic RNA to Drosophila histone DNA of the recombinant plasmid cDm500. There is a large store of maternal histone mRNA, equivalent to at least 7 X 10(7) copies of each of the five classes of histone mRNA per embryo. Embryonic synthesis of histone mRNA begins at 90 min after oviposition, making the histone genes among the first to be transcribed by embryonic nuclei. Embryonic histone mRNA accumulates rapidly during the blastoderm and gastrula stages. The peak in the rate of histone mRNA synthesis per embryo coincides with the peak in the rate of DNA synthesis per embryo, which occurs at 6 hr after oviposition. After 6 hr, as the rate of DNA synthesis per embryo decreases, the rate of histone mRNA synthesis and the total mass of histone mRNA per embryo both drop sharply. The rate of histone mRNA synthesis per gene falls more than 60 fold in the first 13 hr after oviposition, from 1.3 -2.5 copies per gene-min at 2 hr to 0.02-0.03 copies per gene-min at 13 hr. From measurements of the mass of histone mRNA per embryo and of the rate of accumulation of newly synthesized histone mRNA at a number of stages of early embryogenesis we determined that the cytoplasmic half-life of histone mRNA decreases approximately 7 fold during early Drosophila development, from 2.3 hr at blastoderm to 20 min by the end of gastrulation. Thus the level of expression of histone genes in Drosophila development is controlled not only by the size of the maternal mRNA pool and changes in the rate of histone mRNA synthesis, but also by changes in the rate of histone mRNA turnover.     

Organization and expression of H1 histone and H1 replacement histone genes

The H1 family is the most divergent subgroup of the highly conserved class of histone proteins [Cole: Int J Pept Protein Res 30:433–449, 1987]. In several vertebrate species, the H1 complement comprises five or more subtypes, and tissue specific patterns of H1 histones have been described. The diversity of the H1 histone family raises questions about the functions of different H1 subtypes and about the differential control of expression of their genes. The expression of main type H1 genes is coordinated with DNA replication, whereas the regulation of synthesis of replacement H1 subtypes, such as H1° and H5, and the testis specific H1t appears to be more complex. The differential control of H1 gene expression is reflected in the chromosomal organization of the genes and in different promoter structures. This review concentrates on a comparison of the chromosomal organization of main type and replacement H1 histone genes and on the differential regulation of their expression. General structural and functional data, which apply to both H1 and core histone genes and which are covered by recent reviews, will not be discussed in detail.     

Replacement of maternal 40S rRNA precursor by newly synthesized precursor in early embryos of Xenopus laevis

The amount of intact 40S rRNA precursor was followed by Northern hybridization in the course of the early embryogenesis of wild-type Xenopus laevis and its anucleolate mutant. The total amount of 40S rRNA precursor did not alter appreciably until the midblastula stage, decreased at the late blastula stage, and then increased. In the anucleolate mutant, in which no rRNA synthesis occurs, the 40S rRNA precursor decreased at the late blastula stage and disappeared after the gastrula stage. In the nuclear fraction of the wild type, the 40S rRNA precursor was detectable after the midblastula stage. Therefore, the 40S rRNA precursor in the pre-blastula embryos is maternal and decreases at the late blastula stage. New synthesis of 40S rRNA precursor apparently occurs after the midblastula stage.     

Developmental regulation of the synthesis of proteins encoded by stored mRNA in radish embryos

Major radish (Raphanus sativus L. cv National) proteins synthesized at the beginning of germination have been characterized by their migration in two-dimensional electrophoresis.     

Adaptive nature of interspecies variation of histone H1 in insects

Summary An electrophoretic analysis of histone H1 and its fragments was carried out for several orders of insects. A total of more than 500 histone H1 variants were examined. For some of them a study of general molecular structure was performed by the method of incomplete succinylation. The molecular length of the fragment containing the C-terminal domain presumably responsible for chromatin condensation was found to be highly variable. The variance of the logarithm of the electrophoretic mobility of H1, which reflects its molecular length, was estimated for seven insect orders. There was no relationship between this variance and the evolutionary age of an order. On the other hand, the variance turned out to correlate strongly with the recent species number in the order, indicating that the accumulation of variation in H1 molecular length was in line with the general intensity of adaptive processes in the orders. This result seems to provide evidence for an adaptive mode of the evolution of the molecular length of H1. The possible role of H1 variability in adaptive evolution is discussed.     

Kinetics of accumulation and depletion of soluble newly synthesized histone in the reciprocal regulation of histone and DNA synthesis

Procedures are presented which permit the identification and analysis of cellular histone that is not bound to chromatin. This histone, called soluble histone, could be distinguished from that bound to chromatin by the state of H4 modification and the lack of H2A ubiquitination. Changes in the levels of newly synthesized soluble histone were analyzed with respect to the balance between histone and DNA synthesis in hamster ovary cells. Pulse-chase protocols suggested that the chase of newly synthesized histone from the soluble fraction into chromatin may have two kinetic components with half-depletion times of about 1 and 40 min. When protein synthesis was inhibited, the pulse-chase kinetics of newly synthesized histone from the solubl fraction into chromatin were not significantly altered from those of the control. However, in contrast to the control, when protein synthesis was inhibited, DNA synthesis was also inhibited with kinetics similar to those of the chase of newly synthesized histone from the soluble fraction. There was a rapid decrease in the rate of DNA synthesis with a half-deceleration time of 1 min down to about 30% of the control rate, followed by a slower decrease with an approximate half-deceleration time of 40 min. When DNA synthesis was inhibited, newly synthesized histone accumulated in the soluble fraction, but H2A and H2B continued to complex with chromatin at a significant rate. Soluble histone in G1 cells showed the same differential partitioning of H4/H3 and H2A/H2B between the soluble and chromatin-bound fractions as was found in cycling cells with inhibited DNA synthesis. These results support a unified model of reciprocal regulatory mechanisms between histone and DNA synthesis in the assembly of chromatin.     

Individual regulation of the accumulation of H1 mRNA and core histone mRNAs in sea urchin embryos.

The relative cytoplasmic accumulation of the individual histone mRNAs in sea urchins was determined by gel analysis of 3H-labeled cytoplasmic RNA isolated from embryos of the early cleavage through the mesenchyme blastula stages. A number of separate determinations showed that H1 mRNA accumulates at a molar ratio of 0.5 or less compared with each of the H2 or H3 core histone mRNAs through approximately the first 12 h of embryonic development. After this time, the accumulation of H1 mRNA increases relative to the core histone mRNAs, and approximately equimolar amounts of the histone mRNAs are produced by about the 14-h stage. The equimolar synthesis of H1 mRNA appears to be transient, returning to 0.5-molar levels several hours later. The increase in H1 mRNA accumulation, relative to the core histone RNAs, is coincident with the transition from expression of the early (alpha) sea urchin histone gene set to the late histone genes. Since all five of the early histone genes occur in a 1:1 ratio within repeating units, the data suggest that the genes within a single repeat, or their immediate products, are individually regulated. Gel analysis of the proteins synthesized in vivo by embryos demonstrates that the pattern of synthesis of the histone proteins reflects the changing ratios of the histone mRNAs.     

H1 histone subtypes and subtype synthesis switches of normal and delobed embryos of Ilyanassa obsoleta

Histone H1 subtype complexity and H1 histone subtype synthesis switches were characterized during the development of normal embryos of the mud snail Ilyanassa obsoleta. The effect of the removal of the third polar lobe on the normal H1 pattern of synthesis was then investigated in the delobed embryo to determine if classical polar lobe effects are accompanied by a perturbation of these patterns. SDS-gel electrophoresis and fluorography of radiolabeled 5% perchloric acid-soluble nuclear extracts resolved six H1 proteins designated bands 1-6. Bands 1-5 migrate as a cluster of individual bands with similar mobilities. Band 6 has a substantially slower mobility. The synthesis of band 6 is predominant during the first 6 hr post-trefoil. During cleavage and gastrulation bands 1 and 2 are predominant while band 3, 4, and 5 become predominant during organogenesis. In addition, it has been found that removal of the polar lobe delays the off-switch of the early bands 6, 1, and 2 and the on-switch of the late bands 3, 4, and 5. This must result in a different H1 composition in the chromatin of the two embryo types. Cell number data of normal and delobed embryos reveal that the delay in subtype synthesis switching is not caused by an overall delay of cell division in the delobed embryo. However, the data indicate that a subpopulation of cells may not divide, or may divide late, in the delayed embryo. The data also suggest that the D cell lineage may be involved in the control of histone synthesis switching in the A, B, and C cell lineages.     

The synthesis and isolation of DNA complementary to histone mRNA from mouse embryos

A 9S poly(A)-RNA preparation isolated from mouse embryos was shown to stimulate the synthesis of histones in an ascites cell free extract. This RNA preparation was used for the synthesis of a highly labelled cDNA probe complementary to histone mRNA. Hybridisation of this cDNA probe to rRNA showed that 52% of the cDNA consisted of sequences complementary to rRNA. The histone mRNA specific cDNA was purified by hybridising the impure cDNA to rRNA followed by removal of the single-stranded histone cDNA by hydroxylapatite chromatography.     

Expression of mouse histone H1(0) promoter sequences following microinjection into Xenopus oocytes and developing embryos

Evidence from expression studies using transfected F9 teratocarcinoma stem cells indicates that the synthesis of the H1(0) histone is turned on very soon after the cells have been treated with retinoic acid, which causes them to differentiate into murine parietal endoderm. This increase in H1(0) at the time of commitment would allow a reorganization of the chromatin with a reprogramming of the gene activity of undifferentiated F9 cells to the differentiated state. The particular interest of the further development of this differentiation model is to answer the question whether this specific stimulation of expression can be induced only in homologous differentiation systems, or whether the identified H1(0) promoter sequences can also be specifically stimulated by heterologous factors. We therefore injected the mammalian H1(0) promoter sequences into Xenopus oocytes and fertilized eggs. The results of oocyte injection experiments indicate that H1(0) promoter sequence elements similar to those used in transfected F9 cells are specifically expressed in the oocyte. For analysis of H1(0) expression in Xenopus embryos we used promoter constructs ligated to beta-galactosidase sequences for microinjection. This procedure allows a particularly rapid and complete detection of expressed promoter clones within the differentiated tissues of the early Xenopus embryo.