Differential Uptake of Tritiated Thymidine into Hetero- and Euchromatin in Melanoplus and Secale

Grasshoppers of the species Melanoplus differentialis were injected with tritium-labelled thymidine. At intervals thereafter autoradiographic stripping film was applied over Feulgen squashes and sections. In this species during early prophase of meiosis the sex chromosome forms a heterochromatic block large enough to be resolved in tritium autoradiographs. A study of the squash preparations reveals that the sex chromosome is synthesizing DNA at a different period of time from the euchromatic autosomes. Since there is a developmental sequence of spermatocyte cysts along the testicular tubes it is possible from the sections to show that the heterochromatin synthesizes DNA later than does the euchromatin. To find out whether the results obtained in Melanoplus were characteristic of heterochromatin in general, young seedlings of rye were grown in a tritiated thymidine solution and Feulgen squashes were made as for Melanoplus. In rye leaf nuclei there is a large block of heterochromatin constituted by the proximal regions of the chromosomes and a euchromatic one formed by the median and distal regions of the same chromosomes. Here also the heterochromatin synthesizes DNA at a different period of time from the euchromatin. It is concluded that in rye the asynchrony of synthesis occurs within each chromosome. Counts of silver grains over the two types of chromatin in nuclei of Melanoplus and Secale disclosed that the number of grains per unit area was two to three times higher over the heterochromatin. To check the DNA content, Feulgen photometric measurements were made of Melanoplus nuclei at the same stage. The Feulgen and grain counts agree in showing that the heterochromatin contains two to three times more DNA per unit area than the euchromatin.     

Nuclease accessibility of chromatin from a heterotic hybrid and from parental inbreds

The DNAase II, Mg²⁺ procedure was used to fractionate the seedling chromatin of a heterotic maize hybrid and its parental inbreds FRM017 and FRN28. The hybrid and the more vigorous maize inbred FRN28 have 14 and 11 percent soluble chromatin (euchromatin) respectively, while the less vigorous FRM017 contains 30 percent. The unfractionated chromatin of the hybrid contains less protein than either inbred. The RNA contents of the unfractionated chromatin of the hybrid and of FRN28 are similar and are one-half that of less vigorous FRM017. Hybrid euchromatin contains relatively more protein and RNA than DNA as well as higher proportions of protein and RNA than heterochromatin, unfractionated chromatin, or inbred euchromatin; this suggests a more “efficient” type of activity, as reflected by the low amount of euchromatin and a high proportion of RNA and chromosomal proteins.     

Differential DNA synthesis in heterochromatic and euchromatic chromosome sets of Planococcus citri

In males of the mealy bug Planococcus citri, Nur (1966) counted five heterochromatic (H) and about 5, 10, 20, 40, or 80 euchromatic (E) chromosomes in testis sheath nuclei which were undergoing endomitosis. He suggested that the H chromosomes were not replicating and that the nuclei were becoming polyploid as a result of successive cycles of replication of only the E chromosomes. This hypothesis was tested using autoradiography with H³-thymidine to detect DNA synthesis and microspectrophotometric measurements of the Feulgen reaction in nuclei to detect quantitative changes in DNA. — The integrated absorbance of the whole nucleus and of the isolated clump of heterochromatic chromosomes (H body) in polyploid testis sheath nuclei were measured using the mechanical scanner of the CYDAC system. The absorbance of the H body was similar in all testis sheath nuclei examined and was not significantly different from the absorbance of a haploid set of H chromosomes measured after meiosis. The absorbance of the euchromatic component varied in different sheath nuclei, the values closely corresponding to the terms of the series 2c, 4c, 8c. This series is expected if the DNA in the E chromosomes is exactly doubled at each cycle of replication. — Autoradiographs showed that most labeled sheath nuclei had silver grains localized exclusively over euchromatin. With one exception, the remainder of the labeled nuclei had silver grains over both euchromatin and the H body. The observation that euchromatin was much more heavily labeled than the H body and that labeled H bodies occurred at a low frequency and only in the presence of labeled euchromatin suggests that the H body did not incorporate the label and that the silver grains over the H body were the result of -particles which originated in proximal euchromatin.     

Sub-nuclear fractionation. II. Intranuclear compartmentation of transcription in vivo and in vitro

DNA-dependent RNA polymerase activities were measured in subnuclear fractions obtained from rat liver by the procedure described in the preceding paper [14]. Most of the total nuclear enzyme was recovered in a form bound to chromatin with only small amounts as free enzyme in the nucleoplasm. The multiple eukaryotic RNA polymerases were resolved according to the endogenous template to which they were bound and which they continue to transcribe in vitro. The A and B forms of the enzyme were distinguished from each other by their differential sensitivities to α-amanitin, exogenous native and denatured DNA, thermal denaturation at 45 °, Mg²⁺ and Mn² ions, high ionic strength and by the binding of ${}^{14}\text{C-methyl}\text{-γ-}\text{amanitin}$. RNA polymerase B (α-amanitin-sensitive) was exclusively recovered in the nucleoplasmic and euchromatin fractions. RNA polymerase A was recovered in the dispersed nucleolar as well as in heterochromatin. By assaying in the presence of α-amanitin subnuclear fractions that had been pre-incubated at 45 °C a third enzyme (form C) was located exclusively in heterochromatin fractions. Only the euchromatin associated RNA polymerase B was capable of initiating the synthesis of new RNA chains in vitro on endogenous template at low ionic strength. Raising the ionic strength abolished initiation but accelerated chain elongation by this form of enzyme.When nuclear RNA was labelled in vivo, newly made RNA turned over rapidly in the nucleoplasm but accumulated in the euchromatin + membrane fraction. RNA in the nucleolar fraction accumulated gradually after a lag period, whereas a significant amount of rapidly-labelled nuclear RNA was recovered in the heterochromatin fractions. The distribution of RNA labelled in vivo compared with that of RNA polymerase activities suggested that RNA synthesized in vivo is rapidly translocated from its site of synthesis to some other sites within the nucleus.     

Sub-nuclear fractionation. I. Procedure and characterization of fractions

A procedure for fractionation of nuclei from rat liver, Xenopus liver and Xenopus erythrocytes is described. It is based on mild sonication of isolated nuclei for 7–12 sec in a nearly isotonic medium, separation of nuclear sap and centrifugation on a discontinuous sucrose density gradient containing Na and K citrate. Nuclei are thus separated in a single operation into 8 fractions representing nucleoplasm, euchromatin, nucleoli, heterochromatin and nuclear membranes. The sub-nuclear fractions were characterized by chemical composition (DNA, protein, RNA and phospholipid), electron microscopy, thermal denaturation properties of chromatin, relative binding of ${}^3\text{H-actinomycin D}$, polyacrylamide gel electrophoresis of nuclear proteins and titration of membranes against Triton X-100. Approx. 10% of total DNA was recovered as heterochromatin associated with membranes but the bulk of nuclear membranes co-sedimented with the major euchromatin zones. Subnuclear fractions prepared in this way retain virtually all the RNA polymerase activity bound to chromatin [41].     

Ultrastructural Studies of H-1 Parvovirus Replication VI. Simultaneous Autoradiographic and Immunochemical Intranuclear Localization of Viral DNA Synthesis and Protein Accumulation

The localization of H-1 viral replicative-form double-stranded DNA and progeny single-stranded DNA replication in parasynchronously infected, simian virus 40-transformed newborn human kidney cells was studied with high-resolution electron microscope autoradiography (80-nm silver grains). We analyzed wild-type H-1 and ts1 H-1 (a conditional mutant defective in progeny single-stranded DNA synthesis). The proportion of the total DNA synthesis that was viral was estimated to be >90% by comparing the amount of [(3)H]thymidine uptake in cultures infected with wild-type H-1 versus ts14 (an H-1 mutant defective in DNA replication). Simultaneous staining with cytochrome c-conjugated anti-H-1 immunoglobulin G was performed to ensure that cells incorporating [(3)H]thymidine (2- to 60-min pulses) were H-1 infected. The sites of H-1 replicative-form (in ts1-infected cells) and progeny (in wild-type-infected cells) DNA synthesis were identical. Immunospecifically labeled nuclei at the earliest stages of infection exhibited dense clusters of silver grains over material extruded from nucleolar fibrillar centers. These foci became larger with increasing cellular damage, forming a limited number of H-1 DNA synthetic centers in the euchromatin. Each island-like focus was surrounded by tufts of heterochromatin containing high concentrations of unassembled H-1 capsid proteins. In late phases of infection, the heterochromatin became completely marginated, and the nucleoplasm contained only euchromatin that exhibited randomly distributed sites of H-1 DNA replication. This indicates that H-1 DNA synthesis begins at localized euchromatic or nucleolar sites and then spreads outward. Immunostained heterochromatin and nucleolar chromatin never incorporated [(3)H]thymidine. Our results suggest that H-1 proteins and cellular cofactors associated with the fibrillar component of the nucleolus and the euchromatin may play a role in the regulation of H-1 DNA synthesis.     

Interphase chromosomal deoxyribonucleoprotein isolated as a discrete structure from cultured cells

A new procedure is described for the preparation of interphase chromatin from cultured mouse cells (line P815). The primary objective of this procedure was to eliminate exchanges of histones between deoxynucleoprotein molecules; this objective is shown experimentally to have been attained. The chromatin is released from cells by the non-ionic detergent Nonidet P40 in medium of low ionic strength (0.1 mM-KNa₂PO₄), and may then be sedimented as a structure which conserves the general form and ultrastructural characteristics of chromatin within the cell. The nuclear envelope cannot be detected in these structures by electron microscopy, and their content of choline-containing phospholipids is less than 10% of that of nuclei. The maintenance of form in this structure must thus depend on properties of the chromatin itself, and possibly on the more compact peripheral chromatin.Soluble DNP² prepared by shearing these structures has the same relative contents of DNA, histones, non-histone proteins and RNA as DNP prepared by standard methods. Analyses by electrophoresis on polyacrylamide gels of the non-histone proteins reveals certain differences from the pattern of these proteins in DNP prepared by a salt precipitation method. The template activity for RNA synthesis, in the presence of Escherichia coli RNA polymerase of sheared, soluble DNP prepared by this procedure, is comparable to that of DNP prepared by other methods. However, in the absence of exogenous RNA polymerase the rate of RNA synthesis by structured (unsheared) chromatin is about ten times higher than the rate using sheared DNP.The rapid removal of the nuclear envelope in this lysis procedure allowed experimental examination of the origin of the histones and non-histone proteins of DNP. When DNP was prepared from a mixture of two populations of cells, one containing DNA distinguishable by a density label and the other containing radioactively labelled proteins, radioactive proteins were found exclusively in DNP of normal density, and not in dense DNP and vice versa. It is concluded that the proteins of DNP prepared in this way are not acquired during the preparation procedure but were already associated with DNA in vivo, and that other proteins are not bound non-specifically to DNA during the preparation of DNP. When a mixture of DNP molecules prepared, in this way is precipitated in 150 mm-NaCl and redissolved, some radioactively labelled histones migrate onto dense DNA molecules.This procedure is suitable for routine, quantitative isolation of chromosomal DNP from small numbers of cells; it is also applicable to cells of other cultured lines.     

The de novo and salvage pathways for the synthesis of pyrimidine residues of RNA predominate in different locations within the mouse doudenal epithelium

Summary RNA synthesis was examined by radioautography in mouse doudenal epithelium using ³H-uridine as a tracer of the salvage pathway and ³H-orotic acid as a tracer of the de novo pathway. The incorporation of the two precursors was estimated by counting silver grains in light-microscopic and electron-microscopic radioautographs at successive levels of crypt and villus. With both precursors, silver grains were found over all epithelial nuclei, but in numbers varying by location. Thus, after ³H-uridine injection, the number of grains was high over nucleolus and nucleoplasm in the base of the crypt, declined gradually in the middle and top of the crypt, and was low along the villus. After ³H-orotic acid, the number of grains was fairly low throughout, but peaked over the nucleoplasm in lower villus cells. The ³H-uridine reaction over nucleolus and nucleoplasm in crypt cells was interpreted as synthesis by the salvage pathway of ribosomal RNA and heterogeneous RNA, respectively, whereas the ³H-orotic acid reaction over the nucleoplasm of some villus cells indicated that these cells synthesized heterogeneous RNA by the de novo pathway.     

CHANGES IN CHROMATIN TEMPLATE ACTIVITY AND THEIR RELATIONSHIP TO DNA SYNTHESIS IN MOUSE PAROTID GLANDS STIMULATED BY ISOPROTERENOL

Chromatin template activity of mouse parotid glands increases after a single injection of isoproterenol (IPR), a procedure that causes, after a lag period of 20 hr, a marked stimulation of DNA synthesis and cell division in salivary glands of rodents. The increase in chromatin template activity occurs as early as 1 hr and peaks between 6 and 10 hr after IPR, paralleling previously reported changes in the incorporation of uridine-³H into total cellular RNA of mouse parotids. Template activity was measured in vitro in a system in which parotid gland chromatin was incubated with an exogenous RNA polymerase isolated from Escherichia coli. Similar results were obtained when template activity of parotid gland chromatin was assayed using an homologous RNA polymerase from mouse liver. Chromatin template activity in mouse parotids was also studied after the administration of drugs capable of inducing in salivary glands both DNA synthesis and secretion or secretion alone. The results indicate that the increased chromatin template activity occurring 6 hr after IPR is related to the subsequent onset of DNA synthesis. Furthermore, the increased chromatin template activity caused by IPR is inhibited by the previous administration of puromycin, an inhibitor of IPR-stimulated DNA synthesis.     

Template activity of chromatin during stimulation of cellular proliferation in human diploid fibroblasts

1. Contact-inhibited confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to divide by replacing the medium with fresh medium containing 30% foetal calf serum. 2. Of the cells 40–75% are stimulated to divide with a peak DNA synthesis between 15 and 21h and a peak mitotic index between 28 and 30h after stimulation. 3. In the first 12h before the initiation of DNA synthesis there is a biphasic increase in the incorporation of [³H]uridine into RNA of whole cells. 4. This is paralleled by a similar biphasic stimulation of chromatin template activity measured in vitro in a system in which purified cell chromatin is incubated with an exogenous RNA polymerase isolated from Escherichia coli. 5. The changes in chromatin template activity are believed to represent activation of the genome, with more sites available for RNA synthesis, and to account almost entirely for the changes in RNA synthesis occurring in the whole cell.     

Chromatin- and nuclei-directed ribonucleic Acid synthesis in sugar beet root

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg²⁺ or Mn²⁺) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.     

Changing rates of DNA and RNA synthesis in Drosophila embryos

Rates of DNA and RNA synthesis during Drosophila embryogenesis were measured by labeling octane-treated embryos with [¹⁴C]thymidine and [³H]uridine. Radioactivity incorporated per hour was converted to rates of synthesis using measurements of the pool-specific activity during the labeling periods. The rate of DNA synthesis during early embryogenesis increases to a maximum at 6 hr after oviposition and then decreases sharply. Measured rates of DNA synthesis were used to calculate that the total amount of DNA per embryo doubles every 18 min at blastoderm, every 70–80 min during gastrulation, and less than once every 7 hr at later stages. The rate of RNA accumulation per embryo increases continuously during the first 14 hr of embryogenesis. The rate of nuclear RNA synthesis per diploid amount of DNA, however, decreases fivefold between blastoderm and primary organogenesis. The cytoplasmic poly(A)⁺ RNA synthesized by blastoderm embryos associates rapidly with polysomes. The relatively high rate of synthesis of polysomal poly(A)⁺ RNA per nucleus at blastoderm allows the small number of nuclei present at blastoderm to make a significant quantitative contribution to the informational RNA active in the early embryo. At the end of blastoderm, approximately 14% of the mRNA being translated in the embryo has been synthesized after fertilization.     

Purification and properties of tyrosinases from Vibrio tyrosinaticus

Rat liver chromatin which has been briefly sonicated is fractionated by treatment with low concentrations of magnesium ion. At 1.5 mm Mg²⁺, where approximately 20–25% of the chromatin remains soluble after low-speed centrifugation, chemical and physical analysis of the Mg-soluble and Mg-insoluble chromatin fractions show that the fractions possess markedly different properties. The Mg-soluble chromatin has more protein and RNA than the Mg-insoluble chromatin. The histone composition of the two fractions as shown by electrophoretic analysis is similar, but many of the acidic proteins are qualitatively and quantitatively different. The molecular weight of the Mg-soluble chromatin is less than that of the insoluble chromatin based on sedimentation behavior and gel filtration experiments. The soluble chromatin has nearly twice the template activity for RNA synthesis in vitro with added RNA polymerase as the Mg-insoluble chromatin and contains approximately 80% of the in vivo rapidly labeled RNA found in the total chromatin preparation. In addition the Mg-soluble chromatin has a significantly greater amount of “accessible” DNA (62%) as measured by polylysine binding than Mg-insoluble chromatin (48%). The data suggest that (a) fractionation of chromatin preparations can be achieved by titration with Mg²⁺, and (b) chromatin soluble in low concentrations of Mg²⁺ may be enriched in actively transcribed portions of the genome.     

RNA Stimulated by Indole Acetic Acid

THE stimulation of RNA synthesis in plant cells by auxins^1,2 may be due, in part, to gene derepression, since chromatin isolated from hormone-treated cells is a better template. Mathysse and Phillips³ demonstrated that an acceptor protein for auxin can interact with the chromatin to derepress the genome. In their system the hormone and protein do not affect the rate of RNA synthesis if pure DNA. is used as a template. However, the actual mechanism of auxin on RNA synthesis by isolated RNA polymerase system has yet to be elucidated.