Changes in chromatin of Daucus carota cells during embryogenesis

Cells of carrot (Daucus carota var. Rote Riesen) were cultured on media inductive and non-inductive for embryogenesis and analyzed for differences in their chromosomal proteins and chromatin template activity. Non-histone proteins were prepared from dehistonized chromatin and their properties were investigated. Non-histone proteins proved to be acidic and associated easily with calf thymus histone. Non-histone proteins were able to counteract the inhibitory effect of histone on DNA-directed RNA synthesis in vitro. Almost the same rate of restoration occurred regardless of the interaction between DNA and protein, when sufficient amounts of non-histone proteins were added. However, once the histone-DNA complex was established, the restoration by non-histone proteins at comparably lower concentration was poor. Another acidic protein, bovine serum albumin, had no effect on histone inhibited RNA synthesis. Also non-histone proteins enhanced the chromatin directed RNA synthesis more than 100%. The template activity of chromatin changed after the inductive treatment of embryo formation and induced cells showed higher template activity than non-indiiced controls after embryo cells were formed. Histone components were the same in inductive and non-inductive cells. On the other hand, there was a correlation between template activity and the stimulation by non-histone proteins of histone-inhibited RNA synthesis.     

Non-histone proteins of soluble nucleoproteins released from mouse myeloma nuclei by mild micrococcal nuclease digestion

Mono- and dinucleosomes preferentially cleaved from mouse myeloma chromatin by very mild micrococcal nuclease digestion at 0 degree C are soluble and are released from nuclei under near-physiological conditions in which normal nucleosomes containing Hl are insoluble. These nucleosomes are highly enriched in RNA, high-mobility-group proteins and a unique subset of other non-histone proteins. They are nearly devoid of histone Hl and contain DNA significantly less methylated than whole myeloma DNA, indicating that they comprise a subset of genomic sequences. Previously we have shown that this fraction is enriched in transcribed DNA sequences. Non-histone proteins that co-sedimented with readily solubilized nucleosomes included many of the most basic, low-to-moderate molecular weight chromosomal proteins. Many of these proteins were also preferentially acetylated in vivo. The residual, pelleted chromatin was highly enriched in high molecular weight proteins (greater than 60 000), and very depleted in medium molecular weight proteins. Readily solubilized nucleoproteins sedimenting like mononucleosomes were partly resolved by electrophoresis, under non-denaturing conditions, into several subfractions differing significantly in non-histone protein contents. Methods described here should be useful for identifying and isolating non-histone proteins bound to nucleosomes and other chromatin regions that are structurally and functionally unique.     

The characterization of the non-histone chromosomal proteins of the main classes of nuclei from rat brain fractionated by zonal centrifugation

1. Non-histone chromosomal proteins were isolated from the cell nuclei of whole rat brain and nuclei from different types of brain cells. 2. Brain nuclei were fractionated by zonal centrifugation into five zones deriving from five main categories of brain cells. These are the neuronals, astrocytes I, astrocytes II, oligodendrocytes I and oligodendrocytes II. 3. The non-histone chromosomal proteins were analysed by (a) sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, (b) electrofocusing electrophoresis and (c) two-dimensional electrophoresis. The results of this analysis showed a limited specific pattern of non-histone chromosomal proteins from the different classes of nuclei. Differences were found to exist between the proteins from neuronal and glial nuclei. In particular one polypeptide band with mol.wt. 10000 and pI8.5 was found to be present in the non-histone protein fractions of neuronal nuclei, and absent from the corresponding fractions of nearly all the other classes of nuclei. 4. Two other classes of nuclear proteins, buffered-saline-soluble and 0.35m-NaCl-soluble, were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis along with the non-histone chromosomal. The similarities and differences among these groups of proteins are discussed. 5. The patterns of non-histone chromosomal proteins during development were investigated by studying them in two age groups of animals: in infant rats (10 days old) and adult rats. The polypeptide that was found to be specific for the proteins of neuronal nuclei of adult rats is present in all the classes of nuclei of infant rats.     

The composition of liver chromatin proteins from embryos, chicks and adult chickens

The chemical composition of chromatin from the livers of 12-, 15- and 19-day-old embryos, of 1-day-old chicks and of adult chickens was analysed. The process of embryonic development is accompanied by an increase in non-histone chromatin proteins and chromatin RNA, as well as in the phosphorus content of chromatin phosphoproteins. The amount of these components decreases in the livers of 1-day-old chicks and adults. Two-dimensional polyacrylamide gel electrophoresis of acid-soluble chromatin proteins showed an increase in the amount of the H1 histone in 19-day-old embryos and adult chickens. Non-histone proteins of embryo liver chromatin showed a high content of the fraction of Mr of about 40 000; this was not the case for adult chickens. The non-histone protein fraction of Mr of about 120 000, characteristic of adult chicken liver proteins, was not found in the livers of 12- and 15-day-old embryos. Non-histone chromatin proteins isolated from the livers of animals of different age exhibited also quantitative differences.     

综述: 非组蛋白甲基化修饰的研究进展

非组蛋白的赖氨酸和精氨酸残基上的甲基化修饰已经被证明是一种普遍的蛋白质翻译后修饰方式,在生命活动中发挥重要作用．甲基化修饰方式的多样性以及它们与其他修饰之间的交互作用(crosstalk)复杂但精细地调控了基因表达、蛋白质活性及稳定性、DNA复制及基因组稳定性、RNA加工等多种功能．本文将对非组蛋白的甲基化修饰特征进行总结,归纳近些年来已报道的甲基化修饰酶、修饰位点及这些位点的生物学功能,并将特别阐述不同蛋白质修饰之间的交互作用,概述鉴定非组蛋白甲基化修饰的方法．     

Stimulation of non-histone chromosomal protein synthesis in simian virus 40-infected simian cells.

The pattern of synthesis of non-histone chromosomal proteins in simian virus (SV) 40-infected African green monkey kidney cells was analyzed by polyacryl-amide gel electrophoresis to see whether the changes in chromosomal protein metabolism are involved in the viral-induced synthesis of cellular DNA and mRNA. During the prereplicative phase of infection, the rate of histone synthesis was decreased until 15 h postinfection, whereas that of non-histone protein synthesis was increased after 5 h postinfection and reached a maximum at 10 to 15 h postinfection when viral-induced synthesis of cellular DNA and mRNA began to be observed. Stimulation of non-histone protein synthesis was also observed in the infected cells treated with cytosine arabinoside and was dependent on the multiplicity of infection. Stimulation occurred in almost all species of non-histone proteins. These results suggest that the stimulation of non-histone protein synthesis is caused by an early SV40 function and occurs prior to the viral-induced synthesis of cellular DNA and mRNA. During the replicative phase of infection, a marked increase in the rate of synthesis was observed in the non-histone proteins with molecular weights of about 48,000, 35,000, and 23,000, which were subsequently found to be SV40 capsid proteins.     

Chromosomal protein synthesis during erythropoiesis in the mouse spleen

Induced erythropoiesis in the mouse spleen was employed to study chromosomal protein synthesis during erythroid cell development. Splenic erythropoiesis occurring after phenylhydrazine induced hemolysis can be divided into an early phase during which nuclear RNA polymerase activity and RNA production are maximal and a late phase in which hemoglobin synthesis and DNA accumulation are maximal. Chromatin was isolated from splenic tissue during both the early and late phases of erythropoiesis as well as from non-anemic animals. The total protein content of chromatin from the early erythroid phase was greater than that of chromatin from the late erythroid phase or from non-anemic controls. The increase was due to a coordinate increase in the concentration of both histone and nonhistone proteins. During late erythropoiesis, the concentration of each returned to pre-anemic levels. Total histone synthesis increased 2.6-fold during early erythropoiesis as compared with the pre-anemic state and remained elevated in late erythropoiesis. The increase in histone synthesis was due to an increase in the synthesis of all five major histone proteins. Nonhistone protein synthesis was more active than that of histones in the pre-anemic spleen and rose only slightly during early erythropoiesis, returning to preanemic levels during late erythropoiesis. Fractionation of nonhistone proteins on SDS-urea polyacrylamide gels revealed complex patterns with significant differences between the pattern of erythroid spleen non-histone proteins and that of the pre-anemic spleen. Analysis of the incorporation of ³H-valine into the non-histone proteins indicated that during early erythropoiesis there was a generalized increase in nonhistone protein synthesis. During the late erythroid phase, the decline in non-histone protein synthesis was most marked for the higher molecular weight proteins.     

Non-histone chromatin protein fractions associated with ‘active’ chromatin in embryonic chicken liver

Non-histone chromatin proteins synthesized during chicken embryonic liver development were labeled with [3H]tryptophan and [3H]methionine and characterized by electrophoresis. During embryonic development protein/DNA ratio in chromatin was low (1.30-1.62) but synthesis of non-histone protein was high. Especially one characteristic fraction K (MW 18 000), tightly bound with DNA was preferentially associated with DNAase II sensitive, active transcribed sequences. In 7-day old and adult chicken synthesis of all non-histone proteins was low, fraction K was absent or synthesized only in small amounts in association with non-active sequences, however protein/DNA ratio in chromatin was high (2.30-2.33).     

Proteins from different classes of liver nuclei in normal and thioacetamide-treated rats

1. In normal rats the amounts of each of the main types of nuclear protein, i.e. soluble proteins, histones, non-histone chromosomal proteins and residual proteins, vary within the different classes of rat liver nuclei fractionated by zonal centrifugation. 2. Heterogeneity is observed in the non-histone chromosomal proteins prepared from different classes of liver nuclei. These differences were observed by analysis of the proteins both by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and electrofocusing electrophoresis. They are most evident between the non-histone chromosomal proteins obtained from stromal and parenchymal nuclei. However, some differences are also found for the parenchymal nuclei, between the diploid parenchymal and the tetraploid parenchymal, and between them and the nuclei involved in the synthesis of DNA respectively. 3. Drastic alterations in the nuclear proteins are found after the administration of thioacetamide. The changes observed are complex and not uniform. They vary with the age of the animal and the type of nucleus. In general an increase in the soluble proteins and non-histone chromosomal proteins and a decrease in the residual proteins is observed. There is a decrease in the specific radioactivity of soluble and residual proteins. 4. Electrophoretic analysis of the non-histone chromosomal proteins showed that specific changes occurred after administration of thioacetamide, which are different in adolescent and young adult rats.     

Chromatin changes related to dedifferentiation and differentiation in tobacco tissue culture (Nicotiana tabacum L.)

Non-histone chromosomal proteins (NHP) were isolated from different stages of Nicotiana tabacum L. pith dedifferentiation to callus and callus redifferentiation. The NHP were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis on slab gels and analyzed by densitometry. Simultaneous histological changes are reported. In both processes, some high molecular weight protein (HMWP) bands increase drastically in an induction period, previous to cell proliferation, and decrease when cell division declines. Some low molecular weight protein bands, intense in pith tissue, decrease early when callus is forming and increase when cells differentiate. chromatin template activity is high when cells proliferate, coinciding with maximum HMWP-bands intensity.Abbreviations HMWP high molecular weight proteins - IAA indole-3-acetic acid - LMWP low molecular weight proteins - NHP non-histone proteins - TA template activity