Uptake of glycine-N15 by components of cell nuclei

1. The uptake of glycine-N¹⁵ by components of cell nuclei was studied. The nuclear components were derived both from tissues with high metabolic rates-mammalian liver, kidney, and pancreas-and from cells with relatively low rates of metabolism-avian erythrocytes and echinoderm sperm. N¹⁵ uptake by nuclear components of liver, kidney, and pancreas was far more rapid than by those of erythrocytes and sperm. 2. The nuclear components of liver, kidney, and pancreas for which measurements were made were DNA, histone, and residual protein of chromatin. Uptake into DNA was low, into histone higher, and into residual protein much higher still, being comparable with that into mixed cytoplasmic protein. 3. A comparison of the uptake of N¹⁵ by the chromosomal components, histone and DNA of liver, pancreas, and kidney showed that chromosomal "activity" varies in different cells and also in the same cell depending upon its over-all activity.     

The incorporation of N15 glycine by avian erythrocytes and reticulocytes in vitro

A study of the incorporation of glycine-N¹⁵ by chicken red cells in vitro has shown that: 1. There is no detectable nitrogen turnover in the histone or desoxyribonucleic acid of erythrocytes or reticulocytes. 2. Hemoglobin synthesis in the nucleated reticulocyte proceeds at 2 to 3 times the rate observed in the mature erythrocyte. 3. The uptake of glycine-N¹⁵ by heme is 9 to 14 times the corresponding uptake into hemoglobin, and 12 to 20 times the calculated uptake into globin. 4. Maturation of the red cell results in a decline in the rate of synthesis of both heme and globin, but the deceleration is much more marked in globin. synthesis. 5. No significant differences could be detected in the low N¹⁵ incorporations of nuclear and cytoplasmic hemoglobins.     

Synthesis of protein in the pancreas. III. Uptake of glycine-N15 by the trypsinogen and chymotrypsinogen of mouse pancreas

The uptake of glycine-N¹⁵ into the trypsinogen and chymotrypsinogen of mouse pancreas is much higher than that into any ribonucleoprotein component of the pancreas that has so far been investigated.     

METHODS FOR THE PURIFICATION OF THYMUS NUCLEI AND THEIR APPLICATION TO STUDIES OF NUCLEAR PROTEIN SYNTHESIS

Procedures are described for the purification of calf thymus nuclei using mild hypotonit shock to break intact cells, and layering techniques to remove cytoplasmic debris. Ficolc (a high polymer of sucrose) was dissolved in isotonic sucrose to give dense solutions suitable for gradient centrifugation. The method yields nuclei which can incorporate amino acids in vitro. Thymus nuclei isolated under isotonic conditions were incubated with C¹⁴-amino acids and later purified by centrifugation through dense sucrose solutions. The distribution of radioactivity in different nuclear proteins was measured and it was found that isotopic amino acids are actively incorporated into characteristically chromosomal proteins, such as the arginine-rich and lysine-rich histones. Protein synthesis in the nucleus is markedly inhibited by puromycin and by agents, such as 2,4-dinitrophenol, which inhibit ATP synthesis. The synthesis of histones is also inhibited by puromycin, but the uptake of several amino acids into the lysine-rich histone fraction seems less sensitive to puromycin inhibition than is uptake into the arginine-rich histones or other proteins of the nucleus. High resolution autoradiography using tritiated leucine and observing grain distribution over thin sections of isolated nuclei and whole cells shows that amino acid incorporation occurs within the nucleus and is not due to cytoplasmic contamination.     

Alterations in turnover of labelled precursors incorporated into rat liver nuclear macromolecules during azo dye feeding

Continual feeding of either 4-dimethylaminoazobenzene (DAB) or 2-methyl-4-dimethylaminoazobenzene (2-MeDAB) to rats resulted in an increase in the uptake but a decrease in the turnover of [³H]lysine in all the nuclear proteins of rat liver. The pattern of lysine turnover in acidic nuclear proteins from DAB-fed animals was more similar to that of normal acidic nuclear proteins than that from the 2-MeDAB-fed animals. The histone fractions showed an increase in uptake after dye feeding which was greater in the lysine-rich fractions than in the arginine-rich fractions. During DAB feeding both the uptake and rate of turnover of [³H]thymidine were greatly increased, but with the noncarcinogenic 2-MeDAB the uptake of the precursor was lower and the rate of turnover slower than in normal animals. These differences in metabolism in response to azo dye feeding are discussed in relation to azo dye carcinogenesis.     

Some aspects of the desoxyribonuclease activities of animal tissues

It has been found that many animal tissues contain "acid" desoxyribonucleases with pH optima near 5.2. A chemical method for the determination of this activity is described. The pancreatic desoxyribonuclease crystallized by Kunitz and shown to have a neutral pH optimum occurs in the pancreas together with the "acid" enzyme, but only the "neutral" enzyme occurs in the pancreatic juice. The ratio of "neutral" to "acid" DNAase activities in the pancreas is greater than 200, but in all other tissues examined there is no appreciable concentration of the neutral enzyme. It is concluded that neutral DNAase, like trypsin or lipase, has a digestive function. Some problems in the activation of the secretory enzyme in neutral pancreatic extracts are described. This activation can be interpreted in terms of a specific inhibitor or an inactive form of the enzyme. A comparison of the "acid" DNAase activities of different organs of the calf, horse, chicken, mouse, and rat indicates a possible connection between the DNAase concentration of a tissue and its capacity for proliferation or regeneration. However, the comparative DNAase activities of fetal and adult tissues do not support the view that DNAase function is limited to some simple role in the mechanics of cell division. Studies on the incorporation of glycine-N¹⁵ into the desoxypentose nucleic acids of avian red cells, and mouse liver, pancreas, and kidney show that the N¹⁵ uptake into the DNA of the chromosome is most rapid in tissues with high DNAase concentrations. No N¹⁵ incorporation is observed in the DNA of avian red cells, which have negligible concentrations of the enzyme. The analyses of tissues and nuclei isolated in non-aqueous media show that the bulk of the enzyme occurs in the cytoplasm of the cell, and that nuclear concentrations vary from tissue to tissue. A theory relating the DNAase activity of the cell to its over-all desoxypentose nucleotide metabolism is discussed. No evidence has been found for the presence of inhibitors of the "acid" DNAase in animal tissues.     

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.     

NUCLEIC ACID AND PROTEIN METABOLISM DURING THE MITOTIC CYCLE IN VICIA FABA

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C¹⁴ and uridine-H³ were used as ribonucleic acid (RNA) precursors, thymidine-H³ as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C¹⁴ as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA.     

A comparative study of the nonhistone proteins of rat liver euchromatin and heterochromatin

Rat liver was fractionated into template-active (euchromatin) and template-inactive (heterochromatin) fractions by controlled shearing and glycerol gradient centrifugation. The histone and nonhistone proteins associated with each fraction were compared. No qualitative differences in histone content were observed, but heterochromatin contained 1.5 times more histone protein than did euchromatin. The nonhistone proteins of each chromatin fraction were fractionated on the basis of salt solubility into loosely bound (those extracted by 0.35 m NaCl), tightly bound (those extracted by 2.0 m NaCl), and residual nonhistone proteins (those not extracted by 2.0 m NaCl). Euchromatin contained 3.7 times more loosely bound nonhistone proteins than did heterochromatin, while the latter contained twice as much residual nonhistone protein. Euchromatin was devoid of tightly bound nonhistone protein, a component of heterochromatin. Electrophoretic analysis of these nonhistone protein fractions revealed marked heterogeneity, with a number of bands unique to either eu- or heterochromatin.     

Rat liver chromatin non-histone proteins and glucocorticoid binding

We have previously characterized a specific corticosterone binding protein in chromosomal non histone proteins (NHP) from rat liver. In this paper, we present evidence that a relationship exists between this protein and the cytoplasmic glucocorticoid receptor. The binding capacity of NHP is reduced by 40 p. cent when this fraction is isolated from adrenalectomized animals. Incubation of isolated nuclei with the glucocorticoid hormone receptor complex results in a decrease in the specific radioactivity of the cytoplasmic proteins and simultaneously in a rapid uptake of the isotope by the nucleus; radioactive hormone was extracted along with the NHP. Evidence is presented that the NHP component binding the hormone is closely related or identical to the cytoplasmic receptor-proteins. Progesterone and corticosterone compete similarly for the binding of dexamethasone to nuclear and cytoplasmic forms of the receptor. However the nuclear form of the receptor has a higher affinity for corticosterone (K_a : 6 × 10⁹ M⁻¹) than for dexamethasone (K_A : 10⁸ M⁻¹) in vitro.A mixture of rat liver NHP and cytosol was shown to bind specifically more corticosterone than when the two proteins were incubated separately with the hormone. The Scatchard analysis shows that the enhancement of binding is due to an interaction of nuclear and cytoplasmic proteins leading to the appearance of a stable protein-protein complex which has a high affinity for the hormone (K_a : 2 × 10⁸ M⁻¹). KCl prevented this interaction. Complex formation does not require the presence of the hormone. The experiments presented here favor the hypothesis of the existence of a regulatory protein in the nucleus. This protein associated with the binding protein to reveal or enhance the active form of the receptor.     

Studies on the mode of action of calciferol. XVIII. Evidence for a specific high affinity binding protein for 1,25 dihydroxyvitamin D3 in chick kidney and pancreas.

Cytosol fractions prepared from rachitic chick kidney and pancreas were analyzed for binding of vitamin D₃ metabolites by sucrose density gradient centrifugation. Both cytosol fractions were found to contain a 3.6S macromolecule which specifically binds 1,25-dihydroxy[³H] vitamin D₃ and in addition a 5 to 6S macromolecule which binds 25-hydroxy[³H]vitamin D₃. Sucrose gradient analysis of a KCl extract prepared from kidney or pancreas chromatin resulted in a peak (3.6S) of bound 1,25-dihydroxyvitamin D₃ which could not be distinguished from the cytoplasmic binding component. The interaction of 1,25-dihydroxy[³H]vitamin D₃ with the cytoplasmic binding component of both tissues occurred at low concentrations of hormone with high affinity.     

Evidence for Two Metabolically Distinct Types of Ribonucleic Acid in Chromatin and Nucleoli

Patterns of radioisotope incorporation are useful characteristics in describing cellular RNA fractions, and have indicated a distinctive "nuclear" RNA. In order to characterize the RNA fractions of the two nuclear components, nucleoli and chromatin, and to determine thereby the precise localization of the RNA typical of isolated nuclei, time-courses of P³² incorporation into nucleolar, chromosomal, and cytoplasmic RNA of Drosophila salivary glands have been determined from autoradiograms. Two experiments are reported which cover 12 and 18 hour periods, including an initial 2 hour feeding on P³². Concentrations of RNA-P³² (identified by ribonuclease digestion) were determined by grain counts. After 1 hour only the nucleolar RNA is labelled. Activity is detectible in chromosomal and cytoplasmic RNA after the 2nd hour. The nucleolar fraction reaches its maximum activity shortly after transfer of the larvae to non-radioactive food, the other fractions several hours later. Maximum activities persist in the chromosomal and cytoplasmic fractions; nucleolar activity decreases after the 9th hour. The observed differences in times at which incorporation begins and maximum activities are reached, and in maintenance of maximum activities indicate that chromosomal and nucleolar RNA are distinct fractions. The metabolic characteristics which have been ascribed to "nuclear" RNA apply only to the nucleolar fraction.     

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.     

Acetylation of histones of rat testis.

When [1-¹⁴C]acetate was injected into rats intratesticularly in the presence of cycloheximide to inhibit protein synthesis, the label was incorporated into histone fractions F2a1 and F3 and into non-histone chromosomal proteins of each of the following stages of spermatogenesis: spermatogonia-preleptotene spermatocytes, leptotene-zygotene-pachytene-diplotene primary spermatocytes, and spermatids. Acetylation of histones was particularly active in the spermatid stages. There was no significant incorporation of acetate into the lysine-rich histone fractions F1 and X₁.In early periods of in vivo incorporation of [³H]amino acids into histones the acetylated histone F2a1 fractions had higher specific activities than the main band of F2a1, but with the passage of time the label moved into the principal band to the extent that specific activities in the acetylated and principal bands were approximately equal at 6 days. However, at 24–36 days the specific activities were again higher in the acetylated bands than in the principal band of F2a1. These data support the conclusions of Candido, Louie, and Dixon, from experiments with trout testis, that acetylation of histone F2a1 may be important in the process of combination of this protein with DNA in chromatin at the spermatogonia-primary spermatocyte stage and also in the subsequent removal of this histone for replacement by protamines at the spermatid stage.[³H]Amino acids were incorporated into histone fractions X₁ and F1 at approximately equal rates, and there was no evidence that one of these fractions was a precursor of the other.Chromatin of the seminiferous epithelial cells of rat testis has a firmly bound acetylase which catalyzes the in vitro acetylation of histones F3 and F2a1 by acetyl CoA.     

Cellular compartmentalization of protein kinase activity during the cell cycle

The quantities and types of protein kinases found in the cytoplasmic and nuclear or chromosomal compartments of interphase and mitotic human culture cells were compared. Using histone as substrate, the total quantity of kinases recovered from cytoplasmic and chromosomal fractions of mitotic cells was several times greater than from cytoplasmic and nuclear fractions of interphase cells. In both mitotic and interphase cells, more activity was recovered from cytoplasmic fractions than from chromosomal or nuclear fractions, respectively. When activity against various substrates was examined, mitotic chromosomal extracts were found to display the greatest preference for the H1 fraction of histones. Neither cytoplasmic nor chromosomal fractions from mitotic cells exhibited enhanced activity in the presence of cAMP, whereas the activity of both cytoplasmic and nuclear fractions of interphase cells was enhanced. Protein kinases, previously identified by nondenaturing polyacrylamide gel electrophoresis as present in the cytoplasmic fraction of mitotic but not interphase cells, were also present in chromosomal fractions of mitotic cells; only one of these kinases may be present in nuclear extracts of interphase cells. In addition, the profiles of nuclear extracts of interphase cells differ from their cytoplasmic fractions. These results indicate that there are protein kinases which are restricted to the mitotic phase of the cell cycle and that they apparently partition between the cytoplasmic and chromosomal compartments of cells in mitosis.     

Buoyant density centrifugation of sea urchin embryo chromatin on sucrose-glucose gradient

Unsheard chromatin isolated from sea urchin embryos was submitted to buoyant density centrifugation in sucrose-glucose gradients. The main peak of blastula chromatin was at a density position of 1.299±0.028±0.009 g ml^-1 whereas at gastrula stage a shift to a lower buoyant density position of (1.276±0.021±0.007 g ml^-1) was observed. Besides the main peak, a small band with a density of 1.18 g ml^-1 was noticed. The lighter fraction differed from the heavy one in a higher histone to DNA ratio, a lower proportion of the F-1 histone, and a lower nonhistone to DNA ratio. The most pronounced developmental alterations of proteins were observed at the level of nonhistone protein patterns of the light fractions.     

Chromosomal proteins of Drosophila melanogaster and an approach for their localisation on polytene chromosomes

Chromosomal proteins have been prepared from embryos of Drosophila melanogaster and separated into histone and nonhistone fractions by a procedure which completely avoids exposure to extremes of pH. These fractions have been characterised by amino acid analysis and gel electrophoresis. Antisera have been prepared against whole chromatin and against the two chromosomal protein fractions. — A new method is described for the preparation of Drosophila salivary chromosomes. This method employs microdissection techniques and completely avoids the use of acid fixatives. Preservation of fine structure in these preparations is comparable to, if not better than, that in classical acid-fixed preparations. Antisera against embryo chromatin and chromosomal protein fractions react with the salivary chromosome preparations. These reactions exhibit selectivity with different chromosomal structures. Evidence is presented suggesting a specific distribution of protein antigens along the chromosome.     

Properties of Reconstituted Chromatin and Nucleohistone Complexes

WE have prepared complexes of calf thymus DNA with whole histone, with individual histone fractions and with whole histone and acidic chromosomal proteins from the same tissue by taking up the components in 5 M urea, 2 M NaCl and gradually removing the urea and salt¹. These nudeoproteins were examined in a “Philips EM 300” electron microscope after phosphotungstate-uranyl acetate staining²; their template activities for RNA synthesis by Escherichia coli DNA-dependent RNA polymerase were compared; their protein³ and DNA⁴ contents were measured and the fraction of total DNA phosphate groups free to bind basic dye⁵ was estimated.