Further studies on phosphorylation and the thiol/disulphide ratio of histones in growth and development

1. The proportion of thiol groups in the total thiol+disulphide of histone extracts from fertilized eggs from Echinus and Psammechinus was increased during periods of structural alterations in the nucleus. 2. The probable start of DNA synthesis in the fertilized eggs coincided with periods of maximum thiol content. 3. Histone extracts from rat liver and regenerating liver were predominantly in the thiol form and no significant variations could be detected during the first 30hr. after partial hepatectomy. 4. An assay system was developed to follow the phosphorylation believed to be associated with the arginine-rich histone F3. Phosphorylation increased by 50% at 1 and 2hr. after partial hepatectomy. The phosphate content also increased during the period of DNA synthesis. 5. The increased phosphorylation found 1hr. after partial hepatectomy was not prevented by actinomycin or prior irradiation. 6. The phosphate content of histone F1 was very high in livers from foetal rats and declined in neonatal rats similarly to the decline in DNA synthesis.     

The pentose phosphate cycle is regulated by NADPH/NADP ratio in rat liver

The changes in the activity of the pentose phosphate cycle produced by the activation or inhibition of different NADPH-consuming pathways have been studied. The inhibition of fatty acid synthesis by kynurenate produced to the same extent, inhibition of the pentose phosphate cycle activity and an increase (about twofold) in the NADPH/NADP ratio. The addition of ter-butyl-hydroperoxide or paraquat, which is metabolized via NADPH-consuming pathways, produced the activation of the pentose phosphate cycle and a decrease in the NADPH/NADP ratio (about threefold). The plot of the NADPH/NADP ratio versus the pentose phosphate cycle activity gave a straight line with a regression index of 0.999. The regulation of the pentose phosphate cycle mainly by the intracellular NADPH/NADP ratio is discussed.     

Chromatin structure through the cell cycle. Studies with regeneration rat liver.

Liver nuclei were prepared through the first cell cycle in partially hepatectomized young rats showing 30% parenchymal cell synchrony. To determine if nucleosome structure altered during this period, liver nuclei from sham-operated rats were compared with nuclei isolated at various times after partial hepatectomy. These nuclei were exposed to deoxyribonuclease I (EC 3.1.4.5), deoxyribonuclease II (EC 3.1.4.6) or micrococcal nuclease (EC 3.1.4.7) and the nucleosome-associated DNA length was ascertained. In no case was a difference in the DNA lengths associated with nucleosome structure observed. Differences were observed with regard to the histones and their relative association with nuclear material. When nuclei from normal rat livers were incubated in hypo-osmolar medium 9% of histone 1 and 4% of the other histones were released. These released histones, unlike those remaining bound to the nuclei, showed high [3H]adenosine and [3H]acetate uptakes in vivo. [32P]P1 uptake was also much greater into released than bound histones 1 and 3, but was not different for histone2A. At 3.5-4.5 h after partial hepatectomy, the release of histone 1 was trebled and that of histone 4 doubled. By 13.5 h, when phosphorylation of the bound forms of histones 2A and especially 1 was increased, no further changes in histone release in hypo-osmolar medium were found. The released histones from partially hepatectomized livers had indistinguishable [3H]adenosine uptakes from controls. The roles are discussed of phosphorylation and ADP-ribosylation in labilizing histone binding.     

Changes in proportion of thiol to disulphide in acid-soluble nuclear proteins of Echinus during the first cell cycle

1. DNA synthesis in Echinus esculentus eggs kept at 10°C takes place just after fusion, 0.75–1.5h after fertilization, and at telophase at about 2.67–3.33h after fertilization. 2. An increase in the thiol/thiol+disulphide ratio in acid extracts from washed nuclear fractions of the eggs is found at fusion, at early stages of mitosis and at telophase. When DNA is being synthesized, the relative amount of thiol in the extracts increases. 3. There are at least five thiol-containing histones in the acid extract together with a diffusible thiol peptide containing methyl-lysine and 3-methylhistidine and a thiol-containing acidic protein.     

Stimulating effect of histones on DNA synthesis in the regenerating rat liver]

Effect of exogenous histones, nuclear globulins and acid proteins on DNA synthesis is studied in regenerating liver of rats in which the synthesis of their own proteins and thus DNA replication are inhibited by cycloheximide. In these conditions histones from regenerating rat liver are found to stimulate 3H-thymidine incorporation into DNA of hepatectomyzed rat liver. Nuclear globulins and acid proteins from regenerating liver, and histones from intact liver produced no stimulating effect on DNA sythesis.     

Variations in the carotenoid content of Chlamydomonas reinhardii throughout the cell cycle.

Synchronous cultures of Chlamydomonas reinhardii have been examined for the total amounts of carotenoid and chlorophyll present throughout a 12 hrs light -- 4 hrs dark life cycle. Variations in the carotenoid distribution at different points within the cell cycle have been found. During the greater part of the light period all major carotenoids increased at a proportionally similar rate. However, the increases in lutein and violaxanthin preceded those in beta-carotene and neoxanthin by some 2 hrs and that in loroxanthin, and algal xanthophyll, by abour 3 hrs. A marked drop in total carotenoid accumulation, corresponding to similar temporary falling away in the accumulation of beta-carotene, lutein and violaxanthin occurred at 9 hrs. The correspondence of this with the established drop in RNA accumulation and the break-up of the nucleolus was pointed out. Considerable redistribution among the carotenoids occurred during the dark period, notably the amount of beta-carotene increased relative to the total xanthophylls. The full significance of these results can not be estimated in the absence of comparative data on related organisms.     

Immunobiochemical evidence for the loss of sperm specific histones during male pronucleus formation in monospermic zygotes of sea urchins

To obtain information on the remodeling of sperm chromatin during male pronuclei formation, we have followed the sperm specific histones (SpH) that form the nucleosomal core by Western immunoblot analysis with polyclonal antibodies directed against the core SpH. The results obtained indicate that the complete set of SpH is absent from zygote chromatin at the beginning of the first S phase. The disappearance of SpH is not coincidental for the five histone classes: SpH4 and SpH3 are lost 5-15 min post insemination (p.i.), SpH2B and SpH2A disappear 20-40 min p.i., and SpH1 is progressively diminished up to 30 min p.i. This order of sperm chromatin remodeling is not affected by the inhibition of protein synthesis by emetine, indicating that the factor(s) responsible for SpH disappearance are present in unfertilized eggs. The lost SpH's are not replaced by newly synthesized CS variants, since the basic proteins synthesized de novo during male pronuclei formation are not incorporated into chromatin remaining in the cytoplasm. These newly synthesized proteins are different from the CS variants as judged by their electrophoretic migration.     

Studies on histones and nuclear phosphoproteins of rat liver and Morris hepatoma.

1. The chemical composition of chromatins obtained from Buffalo rat liver and Morris hepatoma strain 5123 was investigated. 2. The presence of an additional subfraction within the very lysine-rich histone (fl) was states in both kinds of tissues. It amounted to about 8% and 5% of fl in rat liver and Morris hepatoma, respectively. 3. Nuclear phosphoproteins (phenol-soluble) from normal and tumour tissues in polyacrylamide gel in SDS showed some qualitative differences in the range of molecular weights of about 40 000 - 70 000 and over 100 000 daltons.     

Identification of a cysteine protease responsible for degradation of sperm histones during male pronucleus remodeling in sea urchins

We have identified a 60-kDa cysteine protease that is associated with chromatin in sea urchin zygotes. This enzyme was found to be present as a proenzyme in unfertilized eggs and was activated shortly after fertilization. At a pH of 7.8–8.0, found after fertilization, the enzyme degraded the five sperm-specific histones (SpH), while the native cleavage-stage (CS) histone variants remained unaffected. Based on its requirements for reducing agents, its inhibition by sulfhydryl blocking compounds and its sensitivity to the cysteine-type protease inhibitors (2S,3S)-translator-epoxysuccinyl-L-leucyl-amido-3-methylbutane-ethyl-ester (E-64 d), cystatin and leupeptin, this protease can be defined as a cysteine protease. Consistently, this protease was not affected by the serine-type protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and pepstatin. The substrate selectivity and pH modulation of the protease activity strongly suggest its role in the removal of sperm-specific histones, which determines sperm chromatin remodeling after fertilization. This suggestion was further substantiated by the inhibition of sperm histones degradation in vivo by E-64 d. Based on these three lines of evidence, we postulate that this cysteine protease is responsible for the degradation of sperm-specific histones which occurs during male pronucleus formation. J. Cell. Biochem. 67:304–315, 1997. © 1997 Wiley-Liss, Inc.