H.T. Epstein

Studies of chloroplast development in Euglena: XII. Two types of satellite DNA

Three species of DNA from wild-type cells of Euglena gracilis var. bacillaris are described. Main band DNA (density = 1·707 g/cm3) most probably is assignable to the nucleus and is found in all strains examined. The satellite region (density = 1·688 g/cm3) previously described ( Leff, Mandel, Epstein & Schiff, 1963 ) has now been resolved into two DNA satellites, Sc (density = 1·686 g/cm3) and Sx (density = 1·691 g/cm3), both of which are present in wild-type cells, Sc is found in enriched quantities in the chloroplast fraction of green cells and is found in light-grown and dark-grown wild-type cells and in all mutants capable of forming at least a partial chloroplast, but is absent from all mutants incapable of plastid formation. Conversion of strains capable of chloroplast formation to strains incapable of this by means of ultraviolet treatment brings about a loss of Sc. This is consistent with ScDNA being a constituent of the chloroplasts. Sx is found in wild-type cells and in all mutants examined, whether capable or incapable of forming chloroplasts. On cell fractionation, Sx is absent from the chloroplast fraction and is found in the small-particle fraction of the cells along with mitochondrial cytochromes. From density calculations, main band DNA contains 48% guanine+cytosine : 52% adenine+thymine; Sx contains 31% guanine+cytosine: 69% adenine+thymine; and Sc contains 26% guanine+cytosine : 74% adenine+thymine. All three DNA species appear to be double-stranded.

Studies of chloroplast development in Euglena I. Inactivation of green colony formation by U.V. light

Abstract Irradiation of Euglena gracilis with non-lethal doses of u.v. light prevents the formation of chloroplasts in the progeny. This inactivation is photoreactivable with efficiencies approaching 100%. The albino cells produced through irradiation form colorless clones which never revert to green, indicating that this is a heritable effect. The action spectrum for u.v. inactivation of chloroplast formation shows peaks at 260 and 280 mμ.implicating nucleoprotein as the probable site of u.v. action. Evidence obtained from the inactivation kinetics of dark-grown and light-grown cells, the presence of sectored colonies after u.v., the lack of lethality, and the multiplicity of inactivation sites suggest to us that the u.v. sensitive site is cytoplasmic. The evidence leads us, then, to the hypothesis that there are self-reproducing cytoplasmic entities in Euglena which contain nucleoprotein and which are responsible for chloroplast formation.

Studies of chloroplast development in Euglena. II. Photoreversal of the u.v. inhibition of green colony formation.

Abstract The inhibition of chloroplast formation in u.v. irradiated progeny of Euglena gracilis can be reversed by visible light with efficiencies approaching 100%. If the cells are permitted to divide after u.v. treatment, photoreactivability is rapidly lost. The kinetics of this loss are fairly consistent with the hypothesis that entities responsible for chloroplast formation are prevented from being replicated by u.v. light and are diluted out among the progeny. There is no decay of photoreactivability in non-dividing cells. The action spectrum for photoreactivation of chloroplast formation shows a single broad peak in the near-u.v. region of the spectrum clearly separating this phenomenon from the light induction of chloroplast formation which has effectiveness peaks in the blue and red regions of the spectrum. The evidence presented is consistent with the hypothesis of cytoplasmic entities which are replicated at cell division and which control chloroplast formation.

Isolation and characterization of DNA from the mitochondrial fraction of Euglena.

DNA purified from streptomycin-bleached cells of Euglena gracilis var. bacillaris exhibits a main band (1·707 g/cm3) and a satellite band (1·691 g/cm3) upon ultra-centrifugation in cesium chloride. Using recently established procedures, a purified mitochondrial fraction was prepared which consists almost exclusively of intact mitochondria on the basis of its electron microscopic appearance. DNA extracted from this fraction exhibits a three- to fourfold enrichment of the satellite band. However, treatment of the mitochondrial fraction with DNase prior to DNA extraction eliminates adhering main-band material and yields a cesium chloride density profile consisting entirely of satellite DNA. This DNA is double stranded and is estimated to be present per Euglena mitochondrion in an amount equal to 9 to 22 × 106 daltons.

Studies of chloroplast development in Euglena: III. Experimental separation of chloroplast development and chloroplast replication

Abstract Since red light induces chloroplast formation in Euglena and is inactive in photoreactivation, it has been possible to separate the effect of ultraviolet light on chloroplast development from its effect on chloroplast replication. Cells which had received an ultraviolet dose sufficient to yield 100% albino colonies when plated under non-photoreactivating conditions were exposed to red ligth (which does not photoreactivate) and the synthesis of chlorophyll and chloroplasts was followed with time after irradiation under resting conditions. The conversion of protochlorophyll to chlorophyll was found to be unaffected by ultraviolet light. Chloroplast development (followed by fluorescence microscopy) was found to be entirely normal in ultraviolet irradiated cells. When these cells were plated under non-photoreactivating conditions, 100% albino colonies resulted. It is concluded, therefore, that ultraviolet light does not affect the development of precursor bodies into chloroplasts in the ultraviolet irradiated cells but acts specifically to prevent the replication of chloroplasts or the structures which manufacture them, at cell division.

Properties of selectively starved euglena.

Abstract It has been shown that starving Euglena for an essential nutrient causes it to reduce its nuclear and plastid complements by one-half. If pressed by extreme deprivation of the nutrient, the cell is finally pushed to discard its plastid complement which is gratuitous in the heterotrophic medium in which the cell is growing.

Studies of Chloroplast Development in Euglena: XIII. Variation of Ultraviolet Sensitivity with Extent of Chloroplast Development

Ultraviolet (UV) inactivation of green colony-forming ability of several different types of Euglena gracilis var. bacillaris was studied. The observed target numbers are not widely different, while the doses required to produce a single inactivation event (D(o)) vary with the type of cell used. In dark-grown cells adapting to the light in resting medium and in an X-ray-induced mutant, D(o) is proportional to the chlorophyll content of the cells. However, in hyperdeveloped cells which contain abnormally high amounts of chlorophyll, the correlation does not hold, suggesting that it is not chlorophyll per se which is responsible for the differences observed. D(o)s of colony-forming ability (viability) of light-grown and dark-grown cells are found to differ by the same factor as those of green colony-forming ability. Stationary phase and exponential phase cells show a small difference in D(o) with no obvious difference in target multiplicity. The multiplicity of the various target curves has been re-evaluated by computer and found to be between 30 and 40.

Studies of Chloroplast Development in Euglena: XIV. Sequential Interactions of Ultraviolet Light and Photoreactivating Light in Green Colony Formation

Photoreactivation (PR) of green colony-forming ability in Euglena is pH-insensitive from pH 6.0 to 8.0 and temperature-sensitive with a maximum rate at 35 degrees C. There is no PR at 0 degrees C. The rate of PR varies with the growth stage of the cells; PR of exponential phase cells is slower than that of stationary phase cells. The reciprocity rule holds for PR over a 6-fold range of intensity. The shape of PR curves is a function of the UV dose; there appears to be a progressive increase in multiplicity until a limiting multiplicity is reached as indicated by the fact that curves for high doses are superposable. Dark-grown and light-grown cells give the same PR response for comparable UV doses. UV inactivation of cells which have been treated with UV and then with PR light shows that, if the PR dose is sufficiently large, the same UV-inactivation curve is obtained as for nonpretreated control cells. Doses of PR lower than the saturating dose produce UV-inactivation curves, the ultimate slopes of which are parallel to the slope of the control curve, but which show reduced multiplicity. The multiplicity of these curves increases with increasing PR dose. The UV inactivation of green colony-forming ability in Euglena is completely photoreactivable at the doses studied, in contrast with the UV inactivation of colony-forming ability, which occurs at considerably higher UV doses and behaves like most other photoreactivable systems, showing a photoreactivable sector of 0.32.

Studies of Chloroplast Development in Euglena: XV. Factors Influencing the Decay of Photoreactivability of Green Colony Formation

When UV-treated cells of Euglena gracilis var. bacillaris are incubated in the dark in a nutrient medium which permits cell division, they lose the ability to be photoreactivated. The rate of this loss increases with the UV dose. For any given UV dose, the rate of decay increases with increasing growth rate. The same phenomena are observed in light-grown and in dark-grown cells, although the sensitivity to UV of the light-grown cells is smaller by a factor of 1.7. The kinetics of photoreactivation (PR) change during the decay of photoreactivability only if the cells are incubated in growth medium. A UV-inactivation curve for cells photoreactivated only after appreciable PR shows the same slope as that for untreated cells (number of UV-sensitive targets). These results are discussed from the point of view of possible models.