Were Probablycytoplasmic, Since Multiplicities of Approximately Thirty Were Ob- Damage to Chloroplast Replication Was Fully Phot

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STUDIES OF CHLOROPLAST DEVELOPMENT IN EUGLENA, VIII. CHLOROPLAST-ASSOCIATED DNA* BY MARVIN EDELMAN, CHARLES A. COWAN, H. T. EPSTEIN, AND JEROME A. SCHIFF

DEPARTMENT OF BIOLOGY, BRANDEIS UNIVERSITY, WALTHAM, MASSACHUSETTS Communicated by David R. Goddard, September 8, 1964 Previous studies in this laboratory1-3 of ultraviolet (UV) inactivation of chloroplast replication in Euglena established the following. (1) The UV-sensitive sites were probably cytoplasmic, since multiplicities of approximately thirty were observed in the inactivation curves which coincided with estimates of the numbers of proplastids in dark-grown cells. This was further strengthened by the finding that doses of UV which completely inactivated chloroplast replication had no effect on cell viability. The cytoplasmic localization of these sites was later confirmed by Gibor and Granick4 using an ultraviolet microbeam technique. (2) The ultraviolet damage to chloroplast replication was fully photoreactivable2 and the action spectrum for this was the same as for other known DNA repair systems such as T2 bacteriophage in Escherichia coli. (:3) The action spectrum for UV inactivation of chloroplast replication showed peaks at 260 and 280 mt,' suggesting the participa- tion of a nucleoprotein as the photoreceptor. Taken together, the data suggested a series of cytoplasmic nucleoprotein entities in Euglena cells which are responsible for chloroplast replication and raised the possibility of the existence of a plastid- localized species of DNA. We were subsequently able to demonstrate5 the presence of two types of DNA in light-grown, chloroplast-containing cells of Euglena. The major component had a density of 1.707 gm cm-3 ("main band"). A minor component of density 1.685 gm cm-3 ("satellite band") was also present in density gradient separations of whole cell DNA. Both bands were composed of double-stranded molecules, since heat denaturation resulted in the usual shifts of both bands to higher densities. Since the satellite band was undetectable in a mutant of Euglena (W3BUL) in- capable of forming chloroplasts although the main band was present, the satellite DNA was tentatively assigned to the chloroplasts. The work to be reported here confirms the validity of this assignment and describes some properties of the two types of DNA. Reports of plastid-associated DNA in other organisms have also appeared.6-10 Materials and Methods.-Cell growth and chloroplast isolation: Euglena gracilis var. bacillaris was cultivated as described previously1 on Hutner's medium pH 3.5.11 Cells in the exponential phase of growth were used in all experiments. Chloroplasts were isolated from these cells by the method of Eisenstadt and Brawerman,12 which they developed for autotrophically grown cells of the Z strain: their method is a great step forward in obtaining relatively clean, nonleaky chloroplasts from Euglena. DNA isolation: DNA was isolated by the method of Marmur" with minor modifications., It proved essential to keep all volumes minimal (approximately one tenth those recommended by Marmur). The isopropanol step in Marmur's procedure was found to be unnecessary for these studies and was generally omitted. Density gradient centrifugation: Analytical density gradient centrifugation was carried out in a Spinco Model E ultracentrifuge as described by Meselson, Stahl, and Vinograd14 and by Schild- kraut et al.'5 Routinely, between 6 and 12 1Ag of DNA were introduced into the centrifuge cells 1214 Downloaded by guest on September 23, 2021 VOL. 52, 1964 BOTANY: EDELMAN ET AL. 1215

to permit the detection of minor components. Centrifugation was carried out at 44,700 rpm and 250C for 20 hr. Ultraviolet-absorption photographs were taken with Kodak commercial film -nd tracings made with a Joyce-Loebl double-beam recording microdensitometer. Two methods were used for preparative separation of the two types of DNA: (1) DNA obtained as described above was mixed with a cesium chloride solution to yield a final density of 1.700 in a total volume of 2 ml. This solution was placed in a 5-ml cellulose nitrate centrifuge tube and subjected to centrifugation at 33,000 rpm (100,000 X g) for 72 hr in a SW39 rotor in a Spinco model L ultracentrifuge with a chamber temperature of 4°C. This method gave good separation of main band and satellite band only at total DNA concentrations below 50 ,&g per tube. Separation of the two bands could not be achieved at higher concentrations. (2) Contin- uous preformed cesium chloride gradients were prepared in 5-ml cellulose nitrate centrifuge tubes with the aid of a small, double-chamber mixing device. The DNA to be separated was either floated on top of the gradient or was introduced at a point near its buoyant density. These tubes were then centrifuged as described in method (1) above. With this modification, good separation of main band from satellite was obtained with total concentrations of DNA greater than 200 ,g per tube. In both cases, the following methods were used to isolate the separated DNA bands. After centrifugation, a hole was punctured in the bottom of the centrifuge tube, and fractions were col- lected dropwise (two drops/fraction, approximately 30 fractions/ml) in order of decreasing density. Each fraction was then diluted in 1 ml of SSC (NaCl, 0.15 M plus sodium citrate, 0.015 M, pH 7.0).13 Absorption at 258 m1A was measured in a Cary model 14 spectrophotometer. Determination of thermal denaturation curves of purified DNA's: Purified DNA fractions from several preparative separations were pooled, concentrated by air evaporation through a dialysis membrane, and exhaustively dialyzed against SSC. The method of Marmur and Doty'6 was em- ployed to obtain the denaturation curves and to calculate the denaturation mid-point (Tm). Results and Discussion.-From our earlier data,5 any Euglena cells capable of forming chloroplasts should contain the satellite band. We therefore examined the DNA from dark-grown cells of Euglena which contain proplastids and which form chloroplasts on light induction."7 18 Figure 1 shows that the satellite DNA is indeed present in these cells. If the satellite DNA is indeed plastid-associated, it should be possible to demonstrate an enrichment of the satellite material in the chloroplast fraction from light- grown cells during cell fractionation. Figure 2 shows a greater than 15-fold enrichment of the satellite band in DNA extracted from chloroplasts prepared from Euglena by the method of Eisenstadt and Brawerman.'2 Enrichments as great as 30-fold have been obtained in some experiments. This evidence supports the idea that this is indeed chloroplast DNA. Main-band DNA is presumably nuclear in origin, since it is present in all cells of

1.707 DARK GROWN CELLS

FIG. 1.-DNA from dark-grown Euglena. W l A densitometer tracing of a UV-absorption z l photograph of dark-grown Euglena DNA taken after 20 hr at 44,770 rpm and 250C in l the analytical ultracentrifuge. The band of 1-763 density 1.763 is fully deuterated Pseudomonas aeruginosa used as a density standard. Bands of density 1.707 and 1.688, respectively, represent main band and satellite band Euglena DNA. 1688

DENSITY Downloaded by guest on September 23, 2021 1216 BOTANY: EDELMAN ET AL. PROC. N. A. S.

706 LIGHT GROWN CELLS

1742

1685

> 1706 D CHLOROPLASTS

1685

1742

DENSITY FIG. 2.-Enrichment of satellite-band DNA in the chloroplast fraction. Densitometer tracings made from UV-absorption photographs of light-grown Euglena cellular DNA and chloroplast DNA taken after 20 hr at 44,770 rpm and 250C in the analytical ultracentrifuge. The band of density 1.742 is SP-8 (subtiis phage) DNA which was used as a density standard. The band of density 1.706 is Euglena main-band DNA, while the band of density 1.685 is the chloroplast satellite.

Euglena thus far examined.5 Its presence in the chloroplast fraction is readily accounted for by a very slight contamination of this fraction with small, whole Euglena cells. It is calculated that one whole cell among 325 chloroplasts would yield a banding profile of 50 per cent main band and 50 per cent satellite band. Since this degree of contamination was indeed present as determined by light and electron microscopy, it appears most likely that the main band in the "purified" chloroplast fraction is due to whole cell contamination and is not endogenous to the chloroplast. In order to determine some of the physical properties of the two types of DNA from Euglena, preparative cesium chloride gradients were devised to separate measurable amounts of highly purified main-band and satellite-band DNA. Downloaded by guest on September 23, 2021 VOL. 52, 1964 BOTANY: EDELMAN ET AL. 1217

FRACTION NUMBER 17 19 21 23 25 27 0.12-

0.10-

_08-

C\ 06]

0 04

.02-

0.00- FRACTION NO 19 FRACTION NO. 24 1,707 1685

LU z

U)0

> ~~~~~~~~~1742

742

DENSITY FIG. 3.-Separation of the two types of Euglena DNA. Preparative density gradient centrifugation (upper fig.) was carried out as in Materials and Methods with DNA extracted from the chloroplast fraction of light-grown Euglena cells. Peak drops were rerun (lower figs.) in the analytical ultracentrifuge, photographed, and traced as in Fig. 2. Figure 3 shows the results of one such separation using method (1) described in Materials and Methods. Two major bands are separated from the DNA of isolated chloroplasts, one centering at fraction 19, the other at fraction 24 as dripped from the centrifuge tube. When these two fractions were separately recentrifuged in a cesium chloride density gradient in the analytical ultracentrifuge, fraction 19 was found to contain pure main-band DNA while fraction 24 contained pure satellite- band DNA. In order to determine the temperature denaturation curves for Euglena DNA, pure main band and satellite band, prepared as above, were subjected to heating and measurement of UV absorption as described by Marmur and Doty."6 Both the density of DNA and the denaturation temperature (Tm) are functions of the base composition of the nucleic acid involved.'5 16 Figure 4 shows the corrected Downloaded by guest on September 23, 2021 1218 BOTANY: EDELMAN ET AL. PROC. N. A. S.

'15- E o FIG. 4.-Determination of the Tm values for LO isolated main-band and satellite-band DNA. Euglena DNA, suspended in SSC, was heated as Zl 3- Z t described by Marmur and Doty." Absorb- <' ance at the elevated temperatures relative to a:o A/ ; that of the native material at 250C was plotted a function of the temperatures to which the .asDNA was exposed. The T. value is the mid- LO o CHLOROPLAST SATELLITE point of the hyperchromic increase of the indi- > / it BAND Tm=81 5 C vidual DNA absorbance-temperature profiles. aS * MAIN BAND Tm=92°C Relative absorbance measurements are cor- z f rected for thermal expansion of the solutions. cc:W 70 80 90 100 TEMPERATURE `C

optical density increases observed and the calculated Tm values of 81.50C for the satellite band and 920C for the main band. Table 1 compares the base-pair compositions calculated from the measured densities and Tm values with the aid of the published relationships."5 16 The main band is approximately 50 per cent adenine plus thymine, and 50 per cent guanine plus cytosine. The satellite is quite different, showing about 72 per cent adenine plus thymine, and 28 per cent guanine plus cytosine. These values do not include the small amounts of methyl cytosine known to be present in Euglena DNA to the extent of about 2.3 per cent.'9

TABLE 1 Euglena DNA BASE-PAIR COMPOSITIONS FROM DENSITY10 AND Tm1" MEASUREMENTS -Per cent Adenine + Thymine- -Per cent Guanine + Cytosine- From density From Tm From density From Tm (gm/cm3) (0C) (gm/cm,) (0C) Main band 52 45 48 55 Chloroplast satellite band 74 70 26 30 Estimates from several experiments indicate that the satellite DNA represents about 3 per cent of the total cell DNA, the remainder being main-band DNA. A calculation can thus be made as to the DNA content of an individual chloroplast. Euglena cells contain approximately 12 X 10-10 ug of DNA per chloroplast, and thus approximately 2.4 X 106 nucleotide units. Assuming all these units to be linked up in one molecule, we arrive at a molecular weight of 7.2 X 10.8 This value is minimal, since it is calculated on the basis of extracted DNA. Cairns20 has demonstrated that the nucleic acid of E. coli is of the order of 1 X 109 molecular weight and probably consists of one molecule. Thus, the amounts of DNA in the chloroplast and in a bacterial cell appear to be similar. Summary.-The previous work cited in the introduction suggested that a plastid- localized species of DNA exists in Euglena cells. Data presented here of;the enrichment of this DNA species in the chloroplast fraction supports this suggestion. The plastid-localized DNA is shown to be quite different in base-pair composition from the main DNA component, which is presumably nuclear. We estimate an amount of DNA close to that found in bacterial cells to be present in the Euglena chloroplast. It remains to be shown that this DNA is indeed informational for the formation of chloroplast structures. Downloaded by guest on September 23, 2021 VOL. 52, 1964 BIOCHEMISTRY: LOEB AND GELBOIN 1219

The authors are indebted to Drs. J. Marmur and M. Mandel for the deuterated Ps. aeruginosa and SP-8 DNA standards, respectively. * Supported by a grant from the National Institutes of Health (RG-6344). 1 Lyman, H., H. T. Epstein, and J. A. Schiff, Biochim. Biophys. Acta, 50, 301 (1961). 2 Schiff, J. A., H. Lyman, and H. T. Epstein, Biochim. Biophys. Acta, 50, 310 (1961). 3Ibid., 51, 340 (1961). 4Gibor, A., and S. Granick, J. Cell Biol., 15, 599 (1962). 5 Leff, J., M. Mandel, H. T. Epstein, and J. A. Schiff, Biochem. Biophys. Res. Commun., 13, 126 (1963). 6Chun, E. H. L., M. H. Vaughan, Jr., and A. Rich, J. Mol. Biol., 7, 130 (1963). 7Sager, R., and M. Ishida, these PROCEEDINGS, 50, 725 (1963). 8 Kirk, J. T. O., Biochim. Biophys. Acta, 76, 417 (1963). 9 Baltus, E., and J. Brachet, Biochim. Biophys. Acta, 76, 490 (1963). 10 Gibor, A., and M. Izawa, these PROCEEDINGS, 50, 1164 (1963). 11 Greenblatt, C. L., and J. A. Schiff, J. Protozool., 6, 23 (1959). 12Eisenstadt, J., and G. Brawerman, Biochim. Biophys. Acta, 76, 319 (1963). 13 Marmur, J., J. Mol. Biol., 3, 208 (1961). 14Meselson, M., F. W. Stahl, and J. Vinograd, these PROCEEDINGS, 43, 581 (1957). 15 Schildkraut, C. L., J. Marmur, and P. Doty, J. Mol. Biol., 4, 430 (1962). 16Marmur, J., and P. Doty, J. Mol. Biol., 5, 109 (1962). 17Epstein, H. T., and J. A. Schiff, J. Protozool., 8, 427 (1961). 18Ben-Shaul, Y., J. A. Schiff, and H. T. Epstein, Plant Physiol., 39, 231 (1964). 19 Brawerman, G., D. A. Hufnagel, and E. Chargaff, Biochim. Biophys. Acta, 61, 340 (1962). 20 Cairns, J., J. Mol. Biol., 4, 407 (1962).

METHYLCHOLANTHRENE-IND UCED CHANGES IN RAT LIVER NUCLEAR RNA* BY LAWRENCE A. LOEBt AND HARRY V. GELBOIN CARCINOGENESIS STUDIES BRANCH, NATIONAL CANCER INSTITUTE, BETHESDA, MARYLAND Communicated by Seymour S. Kety, August 26, 1964 A single in vivo administration of 3-methylcholanthrene (MC) into rats increases the activities of certain liver microsomal enzyme systems.' 2 The MC stimulation of microsomal enzyme activity may be due to an increased rate of enzyme synthesis. Thus, microsomes isolated from MC-treated rats have a greater rate of amino acid incorporation into protein when either free amino acids2 3 or amino- acyl sRNA3 are used as precursors. The MC stimulation of microsomal benzpyrene hydroxylase4 and aminoazo dye demethylase5 is prevented by the in vivo administration of puromycin, an inhibitor of protein synthesis. Furthermore, the administration of actinomycin D, an inhibitor of DNA-dependent RNA synthesis,6 prevents the MC stimulation of both microsomal amino acid incorporation and benzpyrene hydroxylase activity,7 suggesting that these effects of MC are mediated by DNA-directed RNA synthesis. If the stimulation of specific microsomal en- zymes is the result of changes in the "gene-action system" ("the whole series of biochemical processes which lead from a gene to the phenotypic character by which it is recognized"8), then one might observe MC-induced alterations in the metab- olism of nuclear RNA, the amount of RNA present in liver cell nuclei, and the bio- logical activity of the isolated RNA. Downloaded by guest on September 23, 2021