Germination-induced Changes in Chromosomal Proteins of Spring and Winter Wheat Embryos

The template activity of chromatin from winter wheat embryos gradually increased during germination and was regulated with some nonhistone proteins different from the two major ones, molecular weight 39k and 59k polypeptides, previously reported.     

Onset of deoxyribonucleic Acid synthesis in germinating wheat embryos

Germinating wheat embryos (Triticum vulgare var. Florence) synthesize proteins before the onset of DNA synthesis. The onset of DNA replication occurs at about 15 hours of germination and was shown to depend on proteins synthesized before 9 hours of germination with the use of blasticidin S, a specific inhibitor of protein synthesis. A 10-fold increase in the activity of DNA-dependent DNA polymerase was found in extracts derived from germinated embryos, as compared to the activity found in extracts from ungerminated embryos.     

Protein kinase associated with chromatin and changes in substrate-specificity during germination of wheat seeds

Protein kinase activity was associated with chromatin in wheat ( Triticum aestivum L. cv. Mukakomugi) embryos. The kinase activity did not change significantly during germination, whereas the activity of poly (ADP-ribose) synthetase decreased significantly. The protein kinase activity in chromatin was inhibited by NAD, NADH, and ADP-ribose, and was enhanced by treatment of the chromatin with snake venom phosphodiesterase or soybean trypsin inhibitor. The activity in chromatin was not stimulated by cyclic AMP. Different subfractions of the histones, H1 and H2, were mainly phosphorylated in germ and 3 day-germinated seedling chromatins. The histones, H3 and H4, seemed unable to accept phosphate from ATP in the in vitro reaction system. Different acidic non-histone chromosomal proteins were phosphorylated in germ and 3-day-germinated seedling chromations, and germ-specific and seedling-specific acidic non-histone chromosomal proteins seemed unable to accept phosphate from ATP.     

Properties of the genome in normal and SV-40 transformed WI-38 human diploid fibroblasts. III. Turnover of nonhisone chromosomal proteins and their phosphate groups.

The turnover of nonhistone chromosomal proteins and their phosphate groups was compared in normal and in SV-40 virus transformed WI-38 human diploid fibroblasts. Cells were pulse labelled with tryptophan-³H and ³²P for 30 minutes and the specific activities of tryptophan-³H and ³²P in the various molecular weight classes of nonhistone chromosomal proteins were determined during the first four hours following termination of labelling. While a rapid turnover of high molecular weight nonhistone polypeptides (142, 000 to 200, 000 Daltons) is evident after one hour in SV_40 transformed cells, the specific activities of these nonhistone chromosomal polypeptides are not significantly decreased in normal cells. In contrast, a rapid turnover of low molecular weight (30, 000 to 51, 000 Daltons) nonhistone chromosomal proteins occurs during the first hour in normal WI-38 cells with no corresponding decrease in the specific activities of these proteins in SV-40 transformed cells. There is no apparent net turnover of phosphate groups on nonhistone chromosomal proteins in either normal or SV-40 transformed cells four hours following pulse labelling. Rather, during the first four hours significant fluctuations are observed in the ³²P specific activities of defined molecular weight fractions. Taken together with previous reports of differences in the composition, synthesis and phosphorylation of nonhistone chromosomal proteins in normal and SV-40 transformed human diploid cells the present results further indicate the complex nature of the alterations in these proteins which accompany viral transformation.     

The effect of 20-hydroxyecdysone on synthesis of chromosomal and cytosol proteins in imaginal discs

Over 600 cytosol and 300 nonhistone chromosomal proteins of mass-isolated imaginal discs of Drosophila melanogaster have been resolved by two-dimensional electrophoresis. More than half of the nonhistone chromosomal proteins fall into families with effectively constant apparent molecular weight but varying isoelectric points. At least six chromosomal proteins differ distinctly in proportions between embryos and imaginal discs. The synthesis of six cytosol proteins is increased, and one decreased with incubation of the discs in vitro with 20-hydroxyecdysone. Two disc acidic chromosomal proteins are specifically synthesized in the presence of 20-hydroxyecdysone. Their isoelectric points and molecular weights are similar to those of the subunits of vertebrate steroid hormone receptor proteins. However, although ecdysteroid receptor activity is associated with purified chromatin, no ecdysteroid-dependent increase in receptor activity is detected during in vitro culture of discs.     

Nuclear protein modification and chromatin substructure. 3. Relationship between poly(adenosine diphosphate) ribosylation and different functional forms of chromatin.

The relationship between poly(adenosine diphosphate) ribosylation of nuclear proteins and functionally different forms of chromatin from mid-S-phase HeLa nuclei was investigated. The major observations emerging from this study were that unique nonhistone proteins were modified in mid-S-phase HeLa nuclei. The major acceptor for poly(adenosine diphosphate-ribose) [poly(ADP-Rib)] was an internucleosomal nonhistone protein (protein C; 125 000 molecular weight). Histones H3, H1, H2b, and H2a but not H4 were ADP-ribosylated in S-phase nuclei. Chromatin fragments preferentially released by micrococcal nuclease were enriched in nonhistone proteins, poly(ADP)-ribosylated nuclear proteins, poly(ADP-Rib) polymerase activity and nascent DNA from the DNA replicating fork. In extended forms of chromatin, contiguous to the DNA replicating fork, poly(ADP-Rib) polymerase was maximally active. However, in chromatin distal to the replicating fork (i.e., more condensed structures), nucleosomal histones and histone H1 were not significantly ADP-ribosylated, and poly(ADP-Rib) polymerase activity was depressed two- to threefold. The data suggest that a subset of nucleosomes in extended regions of chromatin is subject to extensive ADP ribosylation.     

De Novo Biosynthesis of Deoxyribonucleic Acid Polymerase during Wheat Embryo Germination

An 8-fold enhancement in the activity of a DNA-dependent DNA polymerase was found in extracts from germinating wheat (Triticum vulgare var. Florence) embryos, as compared to the activity found in extracts from ungerminated embryos. The enhancement of this activity during the first hours of germination is concomitant to the increase of a Dnase activity. The two activities could be separated and the increased level of the DNA polymerase upon germination was observed in an enzymatic fraction which contains very low DNase activity. Addition of the protein synthesis inhibitor, blasticidin S, to germinating wheat embryos, reduced the increase in DNA polymerase. Incorporation of radioactive amino acids into a phosphocellulose preparation, which contains the DNA polymerase starts during the first 6 hours of germination. The amount of radioactivity incorporated is doubled in the next 6 hours, and the incorporation is continued between 12 and 18 hours of germination.     

Changes of Template Activity and Proteins of Chromatin during Wheat Germination

Relationships between changes in template activity and composition of chromatin during germination of wheat embyros (Triticum aestivum L.) were investigated. The template activity of chromatin was determined with exogenous DNA-dependent RNA polymerase II (EC 2.7.7.6) prepared from wheat embryos. It was essentially constant for 18 hours of germination, corresponding to 2.5% of that of a native calf thymus DNA. Thereafter, the activity increased 2-fold and 5-fold at 24 and 60 hours of germination, respectively.     

Chromosomal proteins of conifers

During imbibition and germination of jack pine seeds, the composition of the total extractable chromatin varied. Relative to DNA, the histone levels decreased as the nonhistone chromosomal proteins (NHCP) increased. New chromosomal proteins were synthesized after 2 days of imbibition as judged by recovery of ¹⁴C-amino acids from the major protein fractions. Phosphorylation of histones from ³²P-phosphoric acid was detected before the incorporation of ¹⁴C-amino acids. In the seed the synthesis and relative changes of chromatin coincided with a fall in total soluble protein and free arginine N. By contrast, adenylate energy charge, free glutamine N and in vitro template activity of chromatin increased during chromatin protein synthesis. When seeds had germinated for 4 days after the start of imbibition more radioactivity, derived from free ¹⁴C-amino acids, was recovered from the NHCP than from the histones. The percentage amino acid composition of most histone fractions remained stable, whereas the composition of NHCP changed more with time. The phosphorylation of NHCP was 8- to 41-fold greater than that of the histones. Phosphorylation of histone H4 was not detected at any stage of germination. Correlations between recovery of radioactivity (³²P and ¹⁴C) from chromosomal proteins and higher adenylate energy charge were positive.     

Fractionation, isolation and characterization of nonhistone chromosomal protein from calf thymus.

1) A method is described for the separation and fractionation of nonhistone chromosomal proteins from salt-urea dissociated calf thymus chromatin. After precipitating DNA in the dissociated chromatin solution with LaCl3, the chromosomal proteins in the supernatant were fractionated by SP-Sephadex C-25 column chromatography using a combination of NaCl stepwise and linear gradient elutions. Much care was taken to prevent proteolytic degradation of the chromosomal proteins during the preparation. 2) Among the protein fractions separated by this chromatography, twenty subfractions were found to be homogeneous on SDS-polyacrylamide gel electrophoresis. These purified proteins account for about 18% of the whole chromosomal protein. Eleven subfractions of these purified nonhistone proteins had ratios of acidic to basic amino acids above 1.0 and the nine remaining subfractions had ratios below 1.0, corresponding to nonhistone proteins of basic character. 3) The molecular weights of the purified nonhistone proteins ranged from 7,400 to 19,000.     

Factors affecting the onset of deoxyribonucleic acid synthesis during wheat embryo germination. Study of the changes in DNA polymerases A, B and C and the pool of DNA precursors.

DNA synthesis starts about 12 h after water imbibition in wheat embryos. We have determined that noticeable amounts of labelled thymidine are found inside the embryo only after 6 hr of germination. DNA polymerase C from ungerminated wheat embryos decreased markedly in activity during the first hours of germination, whereas the activities of DNA polymerases A and B increased, having a maximum at about 15 h or germination. Serological evidence has suggested a clear antigenic relationship between DNA polymerases A and C. Although the pool of ATP increases rapidly after water imbibition, the increase in the pool of dNTP species was much slower.     

Isolation and characterization of a membrane-bound proteinase from rat liver.

The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 m phosphate buffer. After a 1330-fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 ± 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone H1, which was split into a high molecular weight fragment (M_r 13,000). The highly phosphorylated histone H1 isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as H1 from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl-l-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl-l-tyrosine ethyl ester is 5.0 × 10^?3m. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, l-1-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.     

Effect of the conditions of isolation and incubation of the nuclei on digestion of DNA chromatin by calcium- and magnesium-dependent endonuclease. Change in the spectrum of chromosomal proteins and possible role of DNA-protein interactions]

Digesting of chromosomal DNA of interphase rat liver nuclei by Ca, Mg-dependent endonuclease in situ in the presence of chelating agents results in the appearance of the soluble DNP--up to 30% of the total DNA. In addition, 50% of the chromatin is solubilised after mild ultrasonication. In the absence of the chelating agents the degree of fragmentation is considerably increased. The process is accompanied by a loss of some histone and nonhistone chromosomal proteins; the nonhistone proteins are lost selectively. The preliminary removal of the nuclear membrane and significant part of the proteins by tritone X-100 promotes the chromatin degradation and the appearance of low molecular weight fragments. The DNA-fragments of solubilised chromatin are similar to the DNA-fragments of residual chromatin, but in the presence of the chelating agents the latter does not contain monomeric fragments.     

The seed proteins of white spruce and their mobilization following germination

Buffer-soluble proteins that have subunit molecular weights, in the presence of sodium dodecyl sulphate (SDS), of 47, 31 and 27 kilodaltons (kDa) form the major storage proteins in the mature white spruce [ Picea glauca (Moench) Voss] seed. These proteins were found mainly in the megagametophyte. but smaller amounts were identified in the embryo. Following the completion of germination, this reserve was rapidly hydrolyzed in both tissues and probably plays a major nutritional role in the germinated seed. Buffer-insoluble proteins were also found in megagametophytes and embryos from the mature seed. These proteins were soluble in buffer only if SDS was present. Predominant in this class of proteins were several that have a subunit molecular weight and structure that is characteristic of seed crystalloid storage proteins; the subunits were shown to be heterodimers with polypeptide molecular weights in the 33 kDa to 37 kDa and 23.5 kDa to 25 kDa ranges. This reserve was rapidly hydrolyzed in the germinated seed. Storage protein hydrolysis was accompanied by a significant increase in the soluble amino acid pool in both megagametophytes and embryos. Cell-free extracts of mature seed megagametophytes and embryos contained leucine-naphthylamidase (leuNAase) activity. Following germination. this activity was maintained at a constant level in megagametophytes but increased substantially in embryos.     

Effect of 5-iodo-2′-deoxyuridine on physical properties and nonhistone chromosomal proteins of chromatin from Burkitt somatic cell hybrids

A Burkitt somatic cell hybrid line, D98/HR-1, contains latent Epstein-Barr virus genome which can be induced to express by 5-iodo-2′-deoxyuridine (IdUrd). Chromatins were isolated from confluent monolayers of D98/HR-1 grown in the presence and absence of IdUrd and its effect on the chromatin was studied by circular dichroism, ethidium bromide binding capacity, and template activity. IdUrd-Treated chromatin consistently exhibits a 20 to 25% decrease in positive ellipticity of circular dichroism at 275 nm. Scatchard plots of spectrophotometric titration of chromatin with ethidium bromide indicate a 25% reduction of the number of primary binding sites in IdUrd-treated chromatin which corresponds with a 25% reduction in template activity of the same chromatin. These effects are reversible when IdUrd-treated cells were released into IdUrd-free medium for 3 h. However, the differences in circular dichroism spectra and dye binding capacity are not observed between DNA isolated from IdUrd-treated and nontreated cells. These results suggest that chromosomal proteins or modification of protein-DNA interactions are responsible for the alterations. Polyacrylamide gel electrophoretic analysis of the chromosomal proteins reveals that a class of nonhistone chromosomal protein in the high molecular weight region (above 130K) is lacking in IdUrd-treated cells.     

Selective interaction of 5'-bromodeoxyuridine substituted DNA with different chromosomal proteins.

Chromosomal proteins selectively interact with 5'-bromodeoxyuridine (BrdUrd) substituted DNA relative to unsubstituted DNA. The relative affinities of chromosomal proteins for BrdUrd-DNA and unsubstituted DNA were measured by both thermal chromatography on hydroxylapatite and selective retention on nitrocellulose filters. Certain chromosomal proteins have a high affinity for hydroxylapatite; thus, during thermal chromatography of chromatin, the single-stranded DNA component percolates across a bed of adsorbed proteins as it elutes. We have measured the relative affinities of Brd-Urd-DNA and normal DNA for chromosomal proteins by chromatographing appropriate mixtures on hydroxylapatite. The results show that, under these conditions, the histone components, rather than the nonhistone chromatin proteins, retard the BrdUrd-substituted DNA. In addition, the individual histones vary in the degree of their affinity for BrdUrd-DNA in the order H3 greater than H4 greater than H2A greater than H2B greater than H1. We have used the property that protein-DNA complexes have a preferential affinity for nitrocellulose filters over naked DNA to measure the selective binding of BrdUrd-DNA and unsubstituted DNA's to both histone and nonhistone chromosomal proteins at low temperatures. The histones selectively retained BrdUrd-DNA on filters in the order H4 greater than H2A greater than H3 greater than H2B greater than H1. Using this assay, the nonhistones displayed greater selectivity toward BrdUrd-DNA than the histone fraction. We interpret these results to mean BrdUrd-containing DNA has a specific affinity for certain chromosomal proteins with BrdUrd-DNA may be the basis for selective inhibition of cytodifferentiation by the thymidine analogue, BrdUrd.