Correlation between synthesis and methylation of DNA in HeLa cells

For the whole cell cycle the methylation of DNA was studied in synchronized $\text{HeLa}$ cells and in nuclei isolated from them. In the intact cells the methylation of DNA cytosine runs parallel to DNA synthesis. The pattern of DNA cytosine methylation by the isolated nuclei is almost identical to that obtained with the whole cells. Since the isolated nuclei do not synthesize DNA, it is shown that DNA methylation continues for at least 30 min after DNA synthesis is over. No DNA minor thymine is found in the isolated nuclei.     

Activation of the template activity of isolated rat liver nuclei for DNA synthesis and its inhibition by NAD

The template activity of isolated rat liver nuclei for DNA synthesis assayed with $\text{E.}$$\text{coli}$ DNA polymerase was found to be dependent upon the presence of Ca²⁺ or Mg²⁺ in the incubation medium. DNA was prepared from isolated nuclei subjected to conditions which activated the template and centrifuged in an alkaline sucrose gradient. The distribution profile showed that smaller fragments were formed, suggesting enhancement of endonucleolytic activity. When isolated nuclei were incubated with NAD to induce poly(adenosine diphosphate ribose) formation and were subjected to the activation conditions, the template for DNA synthesis remained unchanged. The distribution profile in an alkaline sucrose gradient of DNA prepared from these nuclei and control nuclei was identical. The present findings suggest that the template-activating system for DNA synthesis was blocked when isolated nuclei were treated with NAD $\text{in}$$\text{vitro}$.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

Unscheduled DNA synthesis in isolated hepatic nuclei after treatment of rats with methylnitrosourea in vivo

Administration of the carcinogenic methylating agent, methylnitrosourea, to rats caused a significant increase in endogenous DNA synthesis assayed subsequently in isolated hepatic nuclei $\text{in}\text{vitro}$. DNA synthesis was related directly to the dose of carcinogen and inversely to the interval between treatment and isolation of nuclei. This synthesis appears to represent the continuation $\text{in}\text{vitro}$ of unscheduled, reparative DNA synthesis initiated in damaged cells $\text{in}\text{vivo}$.     

An improved method for the isolation of yeast nuclei active in RNA synthesis in vitro

An improved method has been developed for the isolation of nuclei from $\text{Saccharomyces cerevisiae}$ for the study of RNA synthesis $\text{in vitro}$. Utilization of Ficoll in the isolation procedure greatly increases the activity of RNA polymerase in isolated nuclei. Nuclei prepared by this procedure are essentially free of mitochondrial DNA.     

Inhibition of DNA synthesis in embryonic mouse retina as a result of gene interaction.

It has been shown by autoradiography using ³H-thymidine that 11-day mouse embryos doubly homozygous for the autosomal recessive genes fidget (gene symbol fi) and ocular retardation (or), have three to five times fewer labelled nuclei in their retina anlages as do normal (genotype +/+ +/+) embryos singly homozygous for fidget (+/+ $\text{fi}\text{fi}$) or ocular retardation (+/+ $\text{or}\text{or}$). In 11-day embryos of +/+ +/+, +/+ $\text{fi}\text{fi}$ and +/+ $\text{or}\text{or}$ genotypes the labelled nuclei are localized mainly in the inner zone of the retina anlage. However, in double homozygotes the indices of labelled nuclei were not significantly different in the inner and outer zones of the retina anlage. The retina anlage of 12-day double homozygote, $\text{fi}\text{fi}\text{or}\text{or}$, has practically no nuclei synthesizing DNA. Consequently, the mutant genes fi and or which prolong the G₁ period of the cell cycle in single homozygotes, act synergetically to stop DNA synthesis in the retina anlage cells of 12-day $\text{fi}\text{fi}\text{or}\text{or}$ embryos.     

The in vivo equivalence of a cell-free system for RNA processing and transport

The equivalence of messenger RNA released (transported) from isolated rat liver nuclei to three selected media, with messenger RNA normally released to liver cytoplasm $\text{in vivo}$, has been evaluated by competitive DNA: RNA hybridization. Near normal nuclear restriction was exhibited by nuclei in media fortified with ATP, salts, spermidine and dialyzed cytosol. The RNA transport in the latter system was markedly inhibited by colchicine as was also the transport of RNA $\text{in vivo}$. Both nuclear restriction and sensitivity of the RNA transport to colchicine in media lacking spermidine and cytosol deviated significantly from the $\text{in vivo}$ norm. The results emphasize the importance of establishing the $\text{in vivo}$ equivalence in cell-free systems designed to study RNA synthesis, processing and transport.     

Chromatographic resolution of two DNA polymerase activities from bloodstream forms of Trypanosomabrucei: Differential responses to exogenous polyamine addition

A single peak of DNA polymerase activity from extracts of $\text{T.}\text{brucei}$, obtained by DEAE-cellulose and phosphocellulose ion-exchange chromatography, was resolved into two peaks differing in KCl concentration necessary to elute them from a DNA-agarose column. Peak I (eluting at 0.2 M KCl) and Peak II (eluting at 0.4 M KCl), differed in response to increasing KCl concentrations, although both functioned optimally with Mg²⁺ as divalent cation when DNA synthesis was directed either by activated DNA or poly (dC)·(dG)_12–18. Due to the potential significance of polyamines in the metabolism of $\text{T.}\text{brucei}$, the effect of exogenous polyamine on rates of DNA synthesis by the peak I and II enzymes was compared with that of murine DNA polymerase alpha. Only the peak I enzyme was significantly stimulated (up to 4-fold) by the biologically active polyamines spermine and spermidine at physiological concentrations. The response of the peak I enzyme resembled that of the alpha polymerase. This result suggests a possible functional difference between peak I and II enzymes, as well as a potential target site for trypanocidal drug development.     

Chromatin replication in isolated nuclei from bovine lymphocytes

DNA replication in isolated nuclei from Concanavalin A-stimulated and resting bovine lymphocytes has been studied. Nuclei from S phase lymphocytes incorporate 4–7 times more (³H)dTTP than nuclei from resting cells. The DNA synthesis was dependent on ATP, Mg²⁺ and all four deoxynucleoside triphosphates and was linear for about 60 min. The newly synthesized DNA is nuclear and DNase-sensitive and is the product of discontinuous and semiconservative replication. After limited digestion with micrococcal nuclease the $\text{in vitro}$ replicated DNA was found to occur in nucleosomes prior to joining of primary DNA pieces. Addition of a protein extract from replicating cells stimulated the DNA synthesizing capacity of nuclei from resting lymphocytes. A preliminary characterization of this extract is given.     

Preparation of metaphase chromatin of Physarumpolycephalum without the loss of repressed RNA synthesis

A novel method for the preparation of intact chromatin from the slime mold $\text{Physarum}\text{polycephalum>}$ which retains the $\text{in}\text{vivo}$ property of RNA synthesis is described. Preparations from G₂-cells were highly active, while those from metaphase-cells were inactive. The plasmodial cells were disrupted by gentle homogenization on a polyethylene sieve in a neutral isotonic sucrose medium containing Mg⁺⁺, deoxycholate and EGTA, a Ca⁺⁺-chelating agent. The nuclei were lysed in a hypotonic buffer without use of EDTA and chromatin was precipitated by centrifugation after addition of Mg⁺⁺.     

Translational control of the expression of the β subunit gene of E., coli RNA polymerase

Using the plasmid pNF1337 as template, a mRNA preparation has been obtained that directs the $\text{in}\text{,}\text{vitro}$ synthesis of fMet-Val, the N-terminal dipeptide of the β subunit of RNA polymerase. RNA polymerase holoenzyme specifically inhibits the mRNA-directed synthesis of fMet-Val showing that the autoregulation by RNA polymerase of β,β′ synthesis is at the level of translation. L factor ($\text{nusA}$ gene product) stimulates the synthesis of fMet-Val from a DNA template but not from mRNA. Rifampicin has no effect on the mRNA-directed synthesis of fMet-Val or the ability of RNA polymerase to inhibit fMet-Val synthesis.     

Replication of repeated DNA in human cells

The replication pattern of the repeated sequence families of human DNA has been studied by means of DNA reassociation curves. Early- and late-replicating DNA fractions were obtained from synchronized cultures of KB cells by labeling cells with bromodeoxyuridine (BUdR) early or late in the DNA synthesis period and isolating the BUdR-containing DNA by CsCl density-gradient centrifugation. Highly repeated and moderately repeated sequence classes labeled with ${}^{14}\text{C-deoxycytidine}$ either early or late in the DNA synthesis period were also prepared. The effect of the isolated early- or late-replicating BUdR-DNA on the rate of reassociation of the ${}^{14}\text{C-labeled}$ repeated sequences was then tested. Increasing concentrations of early- or late-replicating BUdR-DNA were added to a constant amount of either ${}^{14}\text{C-labeled}$ early- or late-replicating repeated sequences, and the fraction of label in double-stranded DNA was determined. Analysis of the DNA reassociation curves so obtained indicates that some repeated sequence families are replicated throughout the DNA synthesis period whereas others are replicated primarily in the second half. This is true for both the highly-repeated and moderately-repeated sequence classes.     

Induction of single-strand regions in nuclear DNA by adriamycin.

Incubation of adriamycin with isolated nuclei converts nuclear DNA to a form which is susceptible to hydrolysis by $\text{Neurospora}$$\text{crassa}$ nuclease an enzyme highly specific for the cleavage of single-stranded DNA. The effect of adriamycin on nuclear DNA incubated in the presence of the nuclease can be determined by measuring the release of acid-soluble nucleotides or by analyzing the DNA after centrifugation in neutral sucrose gradients. Similar changes in chromatin structure are not observed during incubation of nuclei with adriamycin alone. In addition to adriamycin, daunomycin and ethidium bromide are also active in inducing the formation of DNA structures which are susceptible to the $\text{Neurospora}$$\text{crassa}$ nuclease. The results suggest that certain antitumor agents can induce the formation of single-strand regions in nuclear DNA and that these sites probably occur as a result of a DNA strand separating event.     

Excision of thymine dimers by proteolytic and amber fragments of E. coli DNA polymerase I

Excision of thymine dimers from specifically incised ultraviolet irradiated DNA by $\text{E. coli}$ DNA polymerase I is stimulated by concurrent DNA synthesis. The 36,000 molecular-weight “small fragment” obtained by limited proteolysis of DNA polymerase I, which retains only the 5′ → 3′ exonuclease activity, also excises thymine dimers, but at one-tenth the rate of the intact enzyme. However, the rate of excision is increased by addition of the “large” 76,000-molecular weight fragment. With the further addition of the 4 deoxynucleoside triphosphates, permitting DNA synthesis to occur, excision approaches rates observed with the intact enzyme. The same result was obtained with a fragment of DNA polymerase I with 5′ → 3′ exonuclease activity that is present uniquely in polymerase I $\text{amber}$ mutants.     

Phosphorylation of nuclear matrix proteins in isolated regenerating rat liver nuclei

The phosphorylation of nuclear matrix proteins from normal and regenerating rat liver nuclei was examined using an $\text{in vitro}$ system of isolated nuclei and γ-³²P-ATP. Phosphorylation of the nuclear matrix proteins was 2–3 fold higher than that of the total nuclear proteins in normal nuclei. The level of phosphorylation of the matrix proteins was enhanced an additional three fold at a period in liver regeneration (12 hours) just preceding the onset of DNA synthesis.     

Expression and subcellular distribution of mouse cytochrome P1-450 mRNA as determined by molecular hybridization with cloned P1-450 DNA

Cytochrome P₁-450 (P₁-450) is defined as that cytochrome most closely associated with 3-methylcholanthrene (MC)-induced aryl hydrocarbon hydroxylase (AHH) activity. Recently a cloned DNA sequence (clone 46) was shown to represent a portion of the P₁-450 structural gene [Negishi $\text{et}\text{al.}\text{,}\text{Proc. Nat. Acad. Sci. U.S.A.}\text{78}$: 800–804 (1981)]. Poly(A⁺)-enriched RNA was isolated from total liver homogenate, membrane-bound polysomes and from free polysomes at various times after MC treatment of $\text{Ah}$-responsive $\text{C57BL}\text{6N}$ (B6) and $\text{Ah}$-nonresponsive $\text{DBA}\text{2N}$ (D2) inbred mice. The poly(A⁺)-enriched RNA was separated by methylmercury-agarose gel electrophoresis and hybridized to nick-translated [³²P]DNA from clone 46. By means of this RNA-DNA hybridization, only 6% of total polysomal P₁-450 mRNA exists in free polysomes after 24 h of MC treatment. The data indicate that the endoplasmic reticulum is the principal site of synthesis for this integral microsomal protein.Studies of induction kinetics following MC treatment provided the evidence of the rapid increase of total liver and membrane bound P₁-450 mRNA preceding the synthesis of apo-P₁-450 and the increase of AHH activity.     

The effects of different concentrations of glycerol and dimethylsulfoxide on the metabolic activities of kidney slices

Kidney slices either were exposed to the cryoprotectants for 1 hr at room temperature and subsequently washed and incubated in fresh KR buffer containing only the radioactive metabolic tracers, or were immediately incubated for 2 hr at 37 °C in KR buffer containing the cryoprotectant and the tracers. Exposure to glycerol by incubation of kidney slices in Krebs-Ringer bicarbonate buffer containing varying concentrations of glycerol from 0 to 70% ($\text{v}\text{v}$) resulted in a pronounced inhibitory effect on the protein synthesizing activity, while thymidine incorporation into DNA and the α-aminoisobutyric acid uptake through the cell membranes were less affected. Exposure of the tissue to buffer containing dimethylsulfoxide (Me₂SO) in concentrations of 10 to 20% ($\text{v}\text{v}$) resulted in a stimulatory effect on metabolism. At higher concentrations, Me₂SO was toxic resulting in damaging effects on protein and DNA synthesis as well as on membrane integrity. The stimulatory effects of exposure to low concentrations of Me₂SO on protein and DNA synthesis in kidney slices were concluded to be the result of an increased transport of precursors through the cell membranes.