Incorporation of deoxythymidine triphosphate into DNA of fractions of mouse liver in vitro

236

J. Klose & R. A. Flickinger

between 5 to 10 %. Sufficient nuclei, however, can be obtained from 50 mg pollen to carry out a base anlysis of the nuclear RNA. About 45 000 vegetative nuclei and 75 000 generative nuclei are required for a base analysis (a total of about 1 000 cpm 32P). Nuclei were isolated from pollen grown for 25 min in 32P-containing medium. The RNA was extracted and its base composition determined (table 1). These figures are the means (&SE.) of 3 separate experiments. Although the adenylic acid content of the RNAs made by the two nuclei are similar and high, there are significant differences in the other nucleotides indicating that at least some of the species of RNA made by the two nuclei are different. It is also found that more radioactivity is present in the RNA isolated from vegetative nuclei. They synthesize almost twice the amount of RNA synthesized by the generative nuclei. The conclusions regarding differences in types and amounts of RNA synthesized by the two nuclei would only be valid if the assumption made, that there are no differences in the pool sizes of the different nucleotides in the two nuclei, is correct. Further studies on improvement of yields of nuclei and the detailed characterization of the RNA and proteins associated with and synthesized by the two nuclei are in progress. The assistance of Linda Rueckert and Robert Kulikowski in the obtaining of the pictures is gratefully acknowledged. This work was supported by NIH (grant GM02014) and Research Foundation of State University of New York.

References 1. LaFountain, K L & Mascarenhas, J P, Plant physiol 47, suppl. (1971) 39 (Abstract). 2. Mascarenhas, J P, Am j bot 53 (1966) 563. 3. Mascarenhas, J P & Bell, E, Biochim biophys acta 179 (1969) 199. 4. Mascarenhas, J P & Bell, E, Dev biol21(1970) 475. 5. Penman, S, Smith, I, Holtzman, E & Greenberg, H, Nat1 cancer inst monograph 23 (1966) 489. Received January 7, 1972 Revised version received March 27, 1972 Exptl Cell Res 73 (1972)

Incorporation of deoxythymidine triphosphate into DNA of fractions of mouseliver in vitro J. KLOSE and R. A. FLICKINGER, Department of Biology, State University of New York at Buffalo, Buffalo, N. Y. 14214, USA

Summary The heterochromatin fraction isolated from mouse liver nuclei incorporates more labeled dTTP into DNA than does the euchromatin fraction in an in vitro system for DNA synthesis. Saturation of these fractions with microbial DNA nolvmerase. as well as the use of isolated mouse sat&& and main peak DNA, suggests that the heterochromatin DNA is not a better template than euchromatin DNA. The addition of exogenous DNA in the in vitro systems shows that heterochromatin has a higher level of dTTP incorporation than an equivalent amount of euchromatin DNA suggesting that a higher DNA polymerase activity is the primary cause of the higher level of incorporation of precursor into heterochromatin DNA in vitro.

Several autoradiographic studies of mammalian cell DNA synthesis have shown that the DNA of heterochromatin is not only latereplicating but also has a higher rate of DNA synthesis [l, 5, 7, 81. The relation between the heterochromatic state and a higher rate of DNA synthesis is demonstrated by the two X-chromosomes in the female mammal, one of which is heterochromatic and has a higher rate of DNA synthesis than the euchromatic X-chromosome [7, 81. In the present study levels of incorporation of deoxythymidine triphosphate (dTTP) into the DNA of heterochromatin and euchromatin fractions of mouse liver nuclei were determined using an in vitro system under endogenous conditions or when exogenous DNA or DNA-polymerase was added. The purpose of this study was to determine whether isolated heterochromatin has a higher level of incorporation of precursor into DNA than euchromatin and, if so, to what degree it is attributable to superior template properties or to greater enzyme activity. Material and Methods Nuclei of mouse livers were isolated, sonicated and separated into heterochromatin and euchromatin

dTTP incorporation fractions [lo]; the euchromatin fraction was recovered by high speed centrifugation (150000 g for 2 h) instead of precipitation with ethanol. Portions of the heterochromatin or euchromatin preparations containing 50 pg DNA were used in an in vitro system for DNA synthesis. The reaction mixture contained 40 ,umoles Tris-HCl (pH 7.8), 4 pmoles MgCl?, 1 pmole KCI, 0.5 pmoles sodium versenate, 0.5 pcmoles mercaptoethanol, 0.1 pmoles each of dATP, dGTP, dCTP. 0.8 nmoles dTTP and 2.5 &i 3H-TTP (snec. act. 11.1 Ci/mmole) in a total volume of 0.5-ml. Exogenous activity was determined by incubating 100 pg of native DNA (calf thymus DNA; Worthington Biochem. Corp.) with the above system. The reaction was carried out at 30°C and terminated by the addition of 2 ml of cold 10 % TCA containing 1 % sodium pyrophosphate. After exhaustive washing by centrifugation with 5 % TCA plus 1 % sodium pyrophosphate the precipitates were collected by filtration on 934 AM glass filters (4 filters/precipitate) and counted in a liquid scintillation counter in a toluene PPOPOPOP solution. Omission of dATP, dGTP and dCTP was found to reduce the incorporation of labeled dTTP into DNA by 85 %. To insure that labeled dTTP entered DNA, DNA was isolated [4] from a reaction mixture. This DNA was radioactive and incubation for 8 h at 37°C with 500 ,ug of electrophoretically pure DNase I (Worthington Biochem. Corp.) in 1 ml of 0.01 M Tris-HCl, pH 7.5, 2 mM MgCl, converted all this radioactivity to an acid-soluble form.

The results show that the isolated heterochromatin fraction had a higher endogenous level of incorporation of labeled dTTP into DNA in vitro than did the euchromatin fraction Table 1. Incorporation of dTTP into DNA of mouse heterochromatin and euchromatinjractiona expressedas pmoles dTTP incorporated/ minlrx. mixture& S.D.

Mouse chromatin fraction (50 pg DNA)

Endogenous system (no DNA added)

Heterochromatin fraction Euchromatin fraction

65.5 x 1OV 14.9 x 10-g 30.3 x 10-S & 8.3 x lO-9

a The system contained pmoles each of unlabeled 0.8 nmoles of dTTP. The four separate experiments cate.

Exogenous system (native calf thymus; ~dy$wo PE9

203.2 k25.2 128.3 ?c20.6

x 1O-9 x 1O-9 x 1O-9 x lo-$

3H-dTTP (2.5 ,uCi), 0.1 dATP, dGTP, dCTP, and values are the average of each performed in dupli-

into heterochromatin

231

and euchromatin

Table 2. Template activity of mousesatellite and main band DNA in the presenceof frog embryo nucleiapmoles 3H-dTTP incorporated/ minlrx. mixture* S.D. Mouse DNA fraction Satellite DNA Main band DNA No exogenous DNA

Amount added 15l-a 15 pg

Gastrula nuclei (30 pg DNA)

Tailbud nuclei (30 Pug DNA)

131.6 x lO-9 316.7 x 1O-9 161.9 x 1O-8 i 8.9 ): lo+’

63.3 i- 3.4 73.3 k3.1

36.1 x lo+ il.5 x 10-S

10.1 y 10-S +0.96 x lo+’

x x x x

lo+ 10-S 10-g 10-g

a The system contained 3H-dTTP (2.5 ,Li), 0.1 Fcmoles each of unlabeled dATP. dGTP. dGTP and 0.8 nmoles TTP. These values are the’average of four separate experiments each performed in duplicate.

(table 1). The addition of saturating amounts of calf thymus DNA as template for the endogenous enzyme revealed that heterochromatin had a higher exogenous activity than had euchromatin (table 1). Doubling the amount of heterochromatin or euchromatin in the incubations approximately doubled the incorporation of labeled precursor, suggesting that the enzyme involved is operating under first order kinetics. DNA of the heterochromatin fraction isolated from mouse liver consists primarily of satellite DNA (at least 70%) and the euchromatin fraction is almost free of satellite DNA [lo]. A similar percentage of satellite DNA was found to be present in our heterochromatin preparations. These two DNA fractions were prepared in order to ascertain their template activity for dTTP incorporation into DNA. DNA was isolated from mouse liver nuclei and purified [4]. DNA was separated into satellite and main band DNA fractions by CsCl density gradient centrifugation [3]. To compare the template activity of these Exptl

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Res 73 (1972)

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J. Klose & R. A. Flickinger

Table 3. Template activity of mouse heterochromatin and euchromatin fractions in the presence of E. coli DNA polymerase” Mouse chromatin fractions (50 /-cz DNA)

ymoles $H-dTTP incorporated/min/rx.

Heterochromatin Euchromatin

5 470 x 10-s* 166.5 x 10-S 8 519 x 1O-9& 50.1 x10-Q

mixture

a Forty units of E. coli DNA polymerase were present in each incubation mixture together with SHdTTP (2.5 ,&i), 0.1 ,umoles each of unlabeled dATP, dGTP, dCTP and 0.8 nmoles TTP. These values are the average of four separate experiments each performed in duplicate.

DNA fractions the same reaction mixture was used as described previously, but an exogenous source of enzyme was added instead of the mouse chromatin fractions. Isolated and sonicated nuclei of frog embryos (early gastrulae, stage 10 of Shumway [9] and tailbuds, stage 18) have a considerable DNApolymerase activity when exogenous DNA is added~ (Klose & Flickinger, unpublished data), and these nuclei were utilized in this experiment as a source of enzyme. Satellite and main peak mouse DNA (15 pg) were added to frog nuclei containing 30 pg of DNA in 0.5 ml of the reaction mixture. The satellite DNA was a slightly poorer template for the frog embryo enzyme than was the main peak DNA (table 2). Experiments were carried out, using mouse heterochromatin or euchromatin fractions, containing 50 ,ug DNA as template, with saturating amounts of purified E. coli DNApolymerase (40 units/O.5 ml reaction mixture; spec. act. 5 000 unitsjmg Biopolymers Inc.). Although the reaction between the bacterial enzyme and the chromatin had not been completely characterized and the bacterial enzyme may be primarily a repair enzyme [2], the use of this enzyme did allow a general comparison of the template activity of the Exptl CelI Res 73 (1972)

heterochromatin and euchromatin fractions. When E. coli DNA polymerase was added to the systems the template activity of the heterochromatin fraction was lower than that of the euchromatin fraction (table 3). This suggests that the properties of the template DNA do not account for the higher activity of the heterochromatin fraction compared to euchromatin in the endogenous system. The heterochromatin fraction has a higher level of incorporation of dTTP into DNA than an equivalent amount of euchromatin (table l), while heterochromatin (table 3) or its DNA (table 2) is a slightly poorer template for added enzyme. Therefore it seems likely that greater enzymatic activity accounts in part for the higher level of incorporation of precursor into heterochromatin DNA in vitro. The fact that the incorporation of dTTP is inhibited by 85 % when three of the deoxynucleoside triphosphates are omitted suggests that this enzyme is DNA polymerase involved in replicative activity. The observed results could be due to more rapid degradation of newly synthesized DNA in the euchromatin incubation. However, the addition of 3H-labeled frog DNA (2 000 cpm) to the euchromatin incubations showed a similar level of acid soluble radioactivity at time zero and at the-end of the incubation. Hence little or no DNase activity is present. Synchronized HeLa cells have a higher rate of DNA synthesis in late S phase of the cell cycle due to a faster rate of DNA replicon growth and an increase in the number of active replicons [6]. If our results reflect to some extent the situation in vivo, the higher rate of DNA synthesis of mouse heterochromatin could also be explained by a higher activity of DNA polymerase associated with this fraction. This research was supported by a grant from NIH.

Heterochromatic

Material

References 1. 2. 3. 4. 5. 6. I. 8. 9.

10.

segment of number 9 chromosome

239

and Methods

Chromosomes were obtained from whole blood shortComings, D E, Chromosoma 29 (1970) 434. delucia, P & Cairns,J, Nature 224(1969)1146. term cultures in Eagle minimum essential medium Flamm,W G, Bond, H E & Burr, H E, Biochim (spinner modified) for 65 to 72 h using Wellcome biophys acta 129 (1966) 310. PHA. Cells were harvested by treatment with diluted Greene, R F & Flickinger,R A, Biochimbiophys calf serum in water (1 :7), fixed in methanol/glacial acetic acid (3 : 1) for 15 to 30 min and subsequently acta217(1970)447. suspended in 45 9/o glacial acetic acid for 3-4 min Hsu, T C, J cell biol 23(1964)53. Painter, R B & Schaefer,A W, J mol biol 58 before warm air blow drying. Using a modification of a technique described earlier [9] slides were (1971)289. Schmid, W & Leppert, M F, Cytogenetics8 stained for 5 min with a 2 9/,Giemsa solution (Harleco, (1969)125. Azure Blend type) in 0.1 % NazHP04-12 H,O buffer Schneider, L K & Rieke, W 0, J cell biol 33 adjusted to pH 11.6 with NaOH. They were then washed in running tap water for l-2 min, dried and (1967) 497. Shumway, W, Anat ret 78 (1940) 139. mounted. Photographs were taken on Agfa isopan II and Yasmineh, W G & Yunis, T J, Exptl cell res 59 IFR films with a ZeissPhotomicroscope prints were made on Agfa BEHI photographic paper. (1970) 69.

Received January 10, 1972 Revised version received March 16, 1972

With this procedure, cells and metaphases appear pale blue, but two chromosomes show a large, red-purple, juxtacentromeric

segment. In well preserved metaphases, these chromosomes were identified as C, (fig. 1). Slpecific cytological recognition of the heteroIn cultured interphase lymphocytes, two redchromatic segment of number 9 chromosome purple bodies were observed corresponding in man to these chromosomal segments (fig. 2). The R. GAGNG and C. LABERGE, Human Genetics proportion of nuclei and metaphases Research Quebec-IO,

Centre, Lava1 University Quebec, Canada

Medical

Centre,

Heterochromatic segmentsare recognized on most juxta-centromeric regions of human chromosomes with denaturation-renatura-

tion Giemsa staining procedures [I]; bands also appear on human chromosomes following various treatments [lo]. Such techniques have shown differences in the staining intensity of the heterochromatic regions, more specifically the one on chromosome C, which is often less intensely stained than those on chromosomes A, and E,, which show heavier staining [5]. A simple cytological technique is presented which reveals specifically the heterochromatic segment on chromosome C, in metaphase as well as in interphase. This technique suggests different kinds of heterochromatic segmentsand the possibility

that

the constitutive

heterochromatin

of

chromosome C, represent a special satellite DNA.

showing these chromatin segments was evaluated at 25 to 30 %, although this pH induces cellular damage which could explain

the results. This technique has been tried on routine cases for 2 months, and results have been

quite reproducible. Sometimes the centromeres of D and G chromosome appeared red and bands were noted on chromosomes, but staining of the heterochromatic areas on A, and E,, chromosomes, which are known to be as large as those on C, chromosomes, were never observed. The crucial points of this technique are the pH and the freshness of the staining solution. These two heterochromatic segments are visible from pH 11.3 to 11.9 with a maximum at pH 11.6, the specificity for chromosome C, being directly

correlated

with increasing pH. A new solution must be prepared for each batch of slides. The last step in 45 Y0 glacial acetic acid also appears Exptl

Cell Res 73 (1972)