Jerome A. Schiff

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.

Iron-containing proteins in euglena: I. Detection and characterization

Abstract Iron-containing proteins present in Euglena have been examined. The properties of pure samples of two of these—cytochrome c (552, Euglena) and cytochrome c-type (558, Euglena)-have been determined in some detail. These include spectrochemical characteristics, reactivity, stability, amino acid composition, isoelectric point, midpoint potential and nature of haem prosthetic group, in addition to molecular weight and haem content. A third haem protein, particle bound, and associated with the chloroplast fraction, is characterized as a cytochrome b6. Evidence for the existence of an a-type cytochrome and ferrodoxin is also presented.

Events surrounding the early development of Euglena chloroplasts: Photoregulation of the transcription of chloroplastic and cytoplasmic ribosomal RNAs

Abstract During light-induced chloroplast development in dark-grown resting cells of wild-type Euglena gracilis Klebs var. bacillaris Pringsheim, plastid ribosomal RNA (pl-rRNA) increases from about 2 to 25% of the total cellular RNA; concomitantly, 32 PO 4 3− is incorporated linearly from 0 to 48 h. The net amount of cytoplasmic ribosomal RNA (cytrRNA) does not change during light-induced chloroplast development in wild type, but 32 PO 4 3− is incorporated with a peak rate at 24–36 h, indicating that turnover of cyt-rRNA takes place during light-induced plastid development. The extent of this turnover is about 60% by 36 h of development. Such light-induced turnover of cyt-rRNA also occurs in resting cells of dark-grown W3BUL, a mutant of Euglena which lacks detectable plastid DNA and protochlorophyll(ide), with similar kinetics but to one-third the extent of wild-type cells. Labeling of cyt-rRNA at 3 h of development is primarily in the polysome fraction compared with the monosomes, indicating that newly synthesized ribosomes are immediately active in protein synthesis. Labeling of pl-rRNA continues at the same rate in darkness after a brief light induction while cyt-rRNA labeling decreases 50% every 3 h in darkness after a brief light induction. Labeling of cyt-rRNA in wild type and W3BUL is induced most effectively by blue light, green and red light being relatively ineffective. Labeling of pl-rRNA in wild-type cells is induced most effectively by blue light and red light, and the ratio of effectiveness of blue to red to green is the same as the ratios of absorption in these regions of the protochlorophyll(ide) holochrome from beans. These results are consistent with other data which indicate that the nonplastid aspects of plastid development are under control of a nonchloroplast blue light receptor while the plastid events are under the control of the plastid-localized protochlorophyll(ide) system.

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.

[12] Isolation of mutants from Euglena gracilis

Publisher Summary This chapter discusses the isolation of mutants from Euglena gracilis . Most mutations observed in Euglena are nonchromosomal, leading to a consideration of the mitochondrial and plastid genomes. Euglena is an obligate aerobe and cannot grow by fermentative processes; therefore, all mitochondrial mutations, which affect function, are lethal in the dark. There are more than 500 mitochondria per cell, and the probability of segregation of a mutation present in any one mitochondrion is very small. Although mutation of the mitochondrial DNA occurs (aberrant mitochondria are occasionally seen in electron micrographs of mutagenized cells), it is unlikely that any of these could be recovered as mutant cell lines. These arguments also apply to any mutations in the chromosomal or mitochondrial genomes that produce gene products affecting the chloroplast. Experimental evidence suggests that mutations of the chloroplast genome can be detected. Mutant strains might represent clones derived from cells in which all but one of the plastid genomes were prevented from replicating by the mutagenic treatment, and a mutation was induced in the one surviving genome. This mutated genome could then have replicated several times to produce a cell in which all of the chloroplast genomes carried the mutation. It seems likely that the mutants expressed as plastid phenotypes in Euglena are mutations or deletions of the plastid DNA. This is consistent with the observation that the plastid of Euglena is more sensitive than the viability of the cell to alteration by most environmental agents.

The Development, Inheritance, and Origin of the Plastid in Euglena

Publisher Summary This chapter describes the development, inheritance, and origin of the plastid in Euglena . The nucleus, chloroplast, and mitochondrion are cellular organelles having double membranes and the ability to divide. A significant proportion of the energy and small molecules required for plastid development comes from outside the chloroplast and some of it from the mitochondrion. The information for constructing plastid proteins originates in the plastid and nuclear DNAs. The translation of nuclear information occurs on cytoplasmic ribosomes while the translation of plastid information occurs on plastid ribosomes. Cytoplasmic contributions to plastid formation begin earlier than plastid contributions during light-induced chloroplast formation, and the lag period during the first 12 hours is devoted to cytoplasmic events preceding the developmental changes in the plastid. The plastid in Euglena is prokaryotic in structure and composition, and the plastid originated from the endosymbiotic invasion of an organism that is similar to a primitive blue-green alga or the contemporary proplastid.

Blue-light-induced absorbance changes associated with carotenoids in Euglena

When treated with blue light, intact cells of Euglena gracilis Klebs var. bacillaris Cori, bleached strain W3BUL, show a series of positive peaks at 384, 411, and 440 nm in the blue-light-minus-dark difference spectrum; bleached strain 1224-5/24 shows a series of positive peaks at 386, 417, and 448 nm under the same conditions. The same changes are observed in a 27,000xg supernatant from darkgrown W3. The absorption change appears to be a consequence of shifts in the absorption of carotenoids; it is not seen in cells of W3BUL grown on SAN 9789 (4-chloro-5-(methylamino)-2-(α,α, α-trifluoro-m-tolyl)-3(2H)pyridazinone) to deplete the carotenoids or in cells of W10BSmL, a mutant lacking carotenoids. Inhibitors of flavin-mediated reactions, reductants and valinomycin had no effect on the activity of the system. The activity in the 27,000xg supernatant was associated with material of a molecular weight more than 2.5×106 and was insensitive to heating for 2 min at 100° C but was reduced or eliminated on longer heat treatment or addition of Triton X-100, indicating a possible association with membrane material. Photoactivity is enriched in the lower density fractions of a flotation gradient, and correlates with the ζ-carotene content in all fractions. Similar spectral changes can be obtained by comparing the iodine catalyzed cis-to-trans isomerization of ζ-carotene in a CS2-CHCl3 solvent. The action spectrum for the absorbance change shows effectiveness peaks in the 370–390 and 420–448-nm regions, with no marked effectiveness past 500 nm. Thus the photosensitizer may not be a carotenoid (at least not a normally-occurring C40 carotenoid). These blue-lightinduced absorption changes and their action spectra are discussed in relation to such blue-light-mediated responses as carotenogenesis, chloroplast development and phototaxis.

Events surrounding the early development of Euglena chloroplasts: cellular origins of chloroplast enzymes in euglena.

SUMMARY: During chloroplast development, the large increases in ribulose diphosphate carboxylase (RUDPCase) activity and cytochrome 552 concentration follow the patter of chlorophyll synthesis, in that the formation of these two enzymes is inhibited by streptomycin (Sm) and by chloramphenicol (Cm) beyond 12 h of development. Neither enzyme can be detected in W3BUL, a mutant of Euglena in which chloroplasts and chloroplast DNA are undetectable. In contrast, the NADP-linked triose phosphate dehydrogenase (NADP-TPDase), another plastid-localized enzyme, increases in activity without the 12 h lag normally observed for chlorophyll synthesis; this increase in activity is not inhibited by Sm and Cm, but is inhibited by cycloheximide, an antibiotic which acts on 87 S cytoplasmic ribosomes. NADP-TPDase activity is present at the same level in W3BUL as in the dark-grown wild-type organisms. These data are interpreted to mean that NADP-TPDase is coded in the nuclear DNA, and is translated on 87S cytoplasmic ribosomes. The sensitivity of the increase in cytochrome 552 and RUDPCase activities to Sm and Cm indicates that they are translated, at least in part, on the 68 S ribosomes of the chloroplast. Thus, chloroplast differentiation in Euglena is dependent upon information and synthetic machinery from both the plastid and the rest of the cell. Since total cellular protein does not change significantly during chloroplast development in resting cells, we conclude that protein turnover probably occurs.

Studies of sulfate utilization by algae

Summary1.Incubation of high specific activity adenosine-5′-phosphosulfate (AP35S) with crude enzyme fractions or purified APS-sulfotransferase from wildtype and from a mutant which does not grow on sulfate (Sat2-) leads to binding of label to protein as judged by separation on Sephadex G-25. Crude extracts of mutants Sat1-, Sat6-, and Sat7-R1 lacking APS-sulfotransferase show 10 fold lower binding, indicating that APS-sulfotransferase activity is required.2.Protein-bound radioactivity is not released by mild acid and exchanges with sulfite excluding bound APS; this suggests a linkage of the organic thiosulfate type (R-S-SO3-). Labeled protein releases radioactive SO32- and GSSO3- with gluthathione (GS-); with BAL or S2-, SSO32- is released.3.The reaction of APS with APS-sulfotransferase is heat labile, but once bound, the radioactivity is still released by thiols from the heated protein.4.Aged purified APS-sulfotransferase loses binding activity which is restored by adding the supernatant from heated fresh extract, indicating the participation of a cofactor, consistent with the view that a low molecular weight carrier participates in the main pathway of reduction.