the parent wild-type strain and containing approximately three times the usual number of chloroplasts. Further subculturing has resulted in cells which have reverted to a normalsize but have kept their additional number of chloroplasts. To determine whether the mutation consisted of interference with either chloroplast development or chloroplast replication, we extracted DNA from both the wild-type strain and mutant strain, WiBVL (79), by the Marmur method (20) as modified for Euglena by Leff et al. (21). The preparation was analyzed for chloroplast DNA by density-gradient centrifugation carried out in a Spinco Model E ultracentrifuge for 40 hours at 44,770 rev/min. So that we mightsee the satellite bands, we overloaded the centrifuge cells with DNA, 160 to 170 yg being added to each cell. A sample of SP-8 phage DNA was used as a density marker (22), and the DNA buoyant densities were cal- culated according to the procedure of Schildkraut et al. (23). Figure 2 repreSents the tracing of patterns obtained directly from the ultracentrifuge using a photoelectric scanner attachment (24), As in previous studies of DNA extracted from either wild-type E. gracilis satellite bands are observed in addition to the main band of nuclear DNA. These satellite bands appear at buoyant densities (p) of 1.693 g/cmand 1.689 g/cm? and represent mitochondrial DNA and chloroplast DNA, respectively (Fig. 2A) (25), The DNA of the mutantstrain contains only a single satellite band (p = 1.693 g/cm‘) repreThis type of pattern has been reported for various bleached mutant strains of ABSORBANCE E. gracilis and is considered to represent those cells which can no longer produce chloroplasts by virtue of having lost their chloroplast DNA. In these experiments it appears that ! a me os t 1.693 1.689 visible light, acting through the endogenous chlorophyll pigment, is responsible for the disappearance of DNA associated with chloroplasts and consequently for the disappearance of all J ' 1 plastid structures from the mutantcells. The mechanism is unknown, but T 1.743 1.712 DENSITY (g/cm?) Fig. 2. Tracings of ultraviolet absorbing material separated by CsCl density-gradient centrifugation of DNA from Euglena gracilis var. bacillaris. The samples were centrifuged at 44,770 rev/min at 20°C for 40 hours. The band at p = 1.743 g/cm} is a marker DNA of known density from phage SP-8. As both samples were overloaded, only the ascending and descending limbs of the nuclear DNA band can be observed at p = 1.712 g/cm. (A) represents DNA extracted from wild-type E. gracilis var. bacillaris and is similar to previously reported patterns with the nuclear DNA band at p = 1.712 g/cm? and a double satellite representing both chloroplast DNA (9 = 1.689 g/cm?) and mitochondrial DNA (o = 1.693 g/cm’). (B) is the tracing obtained from the DNA of the mutant strain WiBVL, which has only a single satellite band at » = 1.693 g/cm’ representing mitochondrial DNA and lacks any trace of a chloroplast DNA satellite. 1334 , these organisms (27). or certain colored mutant strains, two senting mitochondrial DNA (Fig. 2B). _— velopment of aplastidic colonies is made possible by the use of a heterotrophic medium which provides the cells with nutrients usually made available through photosynthesis. In an autotrophic medium this type of mutation would appear as an increase in cell death inasmuch as survival of the cells depends on their ability to photosynthesize. We therefore conclude that in the case of microorganisms containing chlorophyll, sunlight may act as a natural mutagenic agent and thus serve as an important factor in the evolution of could involve photosensitized oxidation of DNA directly or photooxidation of some of the crucial enzymes involved in DNAreplication. The mutagenic mechanism does not appear to be due to localized heating brought about by the absorption of intense visible radiation, for the induction of white mutant colonies by heat requires active cell division over several generations (26). At the high light intensities we used, there was no cell division during the 6-hourirradiation period. Thus, visible light, in the absence of an exogenous photosensitizing pigment can produce viable and stable muta- tions. Our ability to observe this mutation instead of the killing effect reported previously for visible light, is due to the fact that E. gracilis can live equally well on a heterotrophic or auto- trophic medium. The growth and de- J. LEFF N. I. KRINSKY Department of Pharmacology, Tufts University School of Medicine, Boston, Massachusetts 02111 References and Notes 1.H, F. Blum, Photo-dynamic Action and Diseases Caused by Light (Reinhold, New York, 1941). 2. R. W. Kaplan, Nature 163, 573 (1949); Arch. Mikrobiol. 24, 60 (1956). 3. B. A. Kihlman, Nature 183, 976 (1959); R. B. Uretz, Radiat. Res. 22, 245 (1964); M. M. Roth, J. Bacteriol. $3, 506 (1967). 4. M. M. Mathews, J. Bacteriol, 85, 322 (1963). 5. R. W. Kaplan and C. Kaplan, Exp. Cell Res. 11, 378 (1956). 6. M. R. Zelle and A. Hollaender, in Radiation Biology, A. Hollaender, Ed. (McGrawHill, New York, 1955), vol. 2, p. 365. . H. E. Kubitschek, Science 155, 1545 (1967). .N. I. Krinsky, in Photophysiology, A. C. Giese, Ed. (Academic Press, New York, in press), vol, 3, ° 9. C. L. Greenblatt and J. A. Schiff, J. Protozool. 6, 23 (1959). 16. H. Lyman, H. T. Epstein, J. A. Schiff, Biochim. Biophys. Acta 50, 301 (1961). 11. C. Sironvai and O, Kandler, ibid. 29, 359 (1958). 12. W. R. Sistrom, M. Griffiths, R. Y¥. Stanier, J, Cell. Comp. Physiol. 48, 473 (1956). 13. N. I. Krinsky, in Biochemistry of Chloroplasts, T. W. Goodwin, Ed. (Academic Press, New York, 1966) vol. 1, p. 423. 14. M. S. Bamji and N. I. Krinsky, J. Biol Chem. 240, 467 (1965). 15. N. IL. Krinsky and A. Gordon, Fed. Proc. 24, 232 (1965). 16. W. A. Maxwell, J. D. MacMillan, C. O. Chichester, Photochem. Photobiol. 5, 567 (1966). 17, J. D. MacMillan, W. A. Maxwell, C. O. Chichester, ibid., p, 555. 18. J. A. Schiff and H. T. Epstein, in Reproduction: Molecular, Subceilular and Cellular, M. Locke, Ed. (Academic Press, New York, 2-4 phyll. With one exception these white colonies have not reverted to green cells. The exception was observed in a stationary-phase liquid culture of one of the white colonies, where green cells started to appear. These were abnormal Euglena, about five times the size of 1965), p. 131. 19. The system used for identifying mutant Strains of E. gracilis has been borrowed from Drs. J. A. Schiff and H. T. Epstein, Department of Biology, Brandeis University; W indicates white, B indicates bacillaris strain, and VL represents the mutagenic agent, that is, visible light. 20. J. Marmur, J. Mol. Biol. 3, 208 (1961). 21. J. Leff, M. Mandel, H. T. Epstein. J. A. Schiff, Biochem. Biophys. Res. Commun. 13. 126 (1963); M. Edelman, thesis, Brandeis University (1965). 22. We thank Drs. H. V. Aposhian and M. Nishihara of the Department of Microbiology, Tufts University School of Medicine for the sample of highly purified SP-8 phage DNA. 23. C. L. Schildkraut, J. Marmur, P. Doty, J. Mol, Biol. 4, 430 (1962). 24 We thank Dr. R. H. Haschemeyer of the Department of Biochemistry, Tufts University School of Medicine for assistance in carrying out the analytical ultracentrifuge analyses. SCTENCE, VOL. 158