EFFECT OF LIGHT ON THE CHLOROPHYLL FORMATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. Previous studies have shown that when Chlorella protothecoidesis grown in a medium rich in glucose and poor in nitrogen source(urea), apparently chlorophyll-less cells with profoundly degeneratedplastids—referred to as "glucose-bleached cells—areproduced either in the light or in darkness. When the glucose-bleachedcells are incubated in a medium enriched with the nitrogen sourcebut without added glucose, an active formation of chlorophylloccurs after a certain lag period under illumination, whilein darkness a very small amount of chlorophyll is formed atabout the same time as in the light. The stimulating effectof light on the chlorophyll formation is not appreciably affectedwhen the photosynthetic CO₂-fixation of greening algal cellsis blocked by the addition of CMU. In the present study, itwas further found that the light-enhanced chlorophyll formationproceeds, although at a somewhat lower rate, under aerationof CO₂-free air. All the experiments in this work were doneunder these non-photosynthetic conditions to exclude any influenceof photosynthates.
2. The effect of light (from daylight fluorescentlamps) on thechlorophyll formation in the glucose-bleachedalgal cells wassaturating at about 1,000 lux. Blue light wasfound to be mosteffective; yellow, green and red light followingin the orderof decreasing effectiveness.
3. When the bleachedalgal cells were illuminated for a short periodin the lag phaseof chlorophyll formation and subsequently incubatedin darkness,there occurred an appreciable enhancement of chlorophyllformationin the dark. When the short illumination was appliedat differenttimes of the lag phase, the enhancement was inducedto almostthe same extent. But the longer the duration of theilluminationduring the lag phase, the greater was the enhancementof chlorophyllformation in the subsequent dark incubation.In such experimentsblue light was most effective and red lightleast, as it wasthe case in the experiments of continuous illumination.An intervenientillumination of the bleached cells at lowertemperatures orunder the atmosphere of N2 produced little orno enhancementof the chlorophyll formation in the subsequentdark incubation.
4. Based on these results, it was concluded that the light enhancementof chlorophyll formation in the glucose-bleached algal cellsis mediated by a non-chlorophyllous photoreceptor(s), absorbingmaximally blue and yellow light, and that a light-induced changeof the photoreceptor is immediately followed by a certain dark(temperaturedependent and aerobic) process(es) which is connected,directly or indirectly, to the chlorophyll synthesis.

(Received August 10, 1967; )     

DEVELOPMENT OF PHOTOSYNTHETIC ACTIVITIES DURING THE PROCESS OF CHLOROPLAST FORMATION IN CHLORELLA PROTOTHECOIDES

1. By growing Chlorella protothecoides in a medium rich in glucoseand poor in nitrogen source (urea), entirely chlorophyll-lesscells, called "glucose-bleached’ cells, were obtained.These cells were found to have neither discernible plastid structuresnor photosynthetic activities. When these cells were incubatedin a nitrogenenriched mineral medium without added glucose,a remarkable formation of fully organized chloroplasts occurredin the light and only partially organized chloroplasts weredeveloped in darkness.
2. In the dark-incubated algal cells asmall but appreciable amountof chlorophyll was formed, beingaccompanied by developmentof significant activities for thePMS- and FMN-catalyzed photophosphorylationsand the HILL reaction.The development of the capacity for performingphotosyntheticCO₂-fixation, however, was negligible.
3. During the processof "re-generation" of chloroplasts in thelight, there occurredactive formation of chlorophyll followedby development of allthe photic activities mentioned above.Chlorophyll formationas well as development of the photic activitiesproceeded firstin a manner of autocatalytic reaction and laterin the formof the first-order reaction. It was inferred thatthe light-absorbingagent which mediates the chlorophyll synthesisis chlorophyllitself.
4. The activities for the PMS- and FMN-photophosphorylations,theHILL reaction and photosynthetic CO₂-fixation were recognizedalready in the algal cells at an early stage of greening inthe light, in which the "discs" were developed but no completelamellar structure was observed. Further processes of increaseof these photosynthetic and related activities—as measuredat a high and a lower light intensities—were studied inrelation to the chlorophyll formation under continuous illuminationand under light-dark conditions. It was found that the PMS-photophosphorylationactivity was developed always in parallel with the chlorophyllformation under these different light conditions. Developmentof the activities for the other photic reactions, however, lagged,to different extents, behind the formation of chlorophyll inthe later phase of greening of algal cells under these conditions.
5. Based on these results the modes of formation of the componentsinvolved in these photic reactions were surmised.

(Received September 15, 1965; )     

CHANGES IN CONTENTS OF CARBOHYDRATE AND FATTY ACID IN THE CELLS OF CHLORELLA PROTOTHECOIDES DURING THE PROCESSES OF DE- AND RE-GENERATION OF CHLOROPLASTS

1. Previous work has demonstrated that when cells of Chlorellaprotothecoides are grown mixotrophically under illuminationin a medium rich in nitrogen source (urea) and poor in glucose,normal green cells are obtained, while in a medium rich in glucoseand poor in the nitrogen source, strongly bleached cells containingapparently no discernible chloroplast structures — called"glucose-bleached" cells — are produced either in thelight or in darkness. When the green cells are incubated ina glucose-enriched mineral medium without added nitrogen source,they are fairly rapidly bleached with concomitant degenerationof chloroplast structures (" bleaching "). When, on the otherhand, the "glucose-bleached" cells are transferred in a nitrogen-enrichedmedium without added glucose under illumination, they turn greenwith regeneration of chloroplasts (" greening "). In the presentstudy changes in contents of carbohydrate and fatty acid inalgal cells were followed during these processes of "bleaching"and "greening.".
2. During the process of "bleaching", the quantityof glucose existingin the insoluble carbohydrate fraction ofalgal cells increasedrapidly and markedly. A considerable increasewas also observedin the contents of cells in oleic, linoleicand palmitic acids.It was noted, however, that linolenic aciddecreased in quantityduring the most active phase of cell bleaching.
3. During the process of "greening", the glucose in the insolublecarbohydrate fraction rapidly decreased, suggesting that itis utilized, as carbon and energy sources, for the chloroplastregeneration. Linolenic acid was found to be synthesized inparallel with formation of chlorophyll. A peculiar pattern ofchange in contents was observed with oleic and palmitic acids,which was interpreted as being related with the process of cellulardivision occurring incidentally during the process of greening.

(Received September 24, 1966; )     

"GIGANTISM" OF CHLORELLA VULGARIS I. MECHANISM OF INDUCTION OF CIGANTISM

1. Based on the microscopic observations, two stages, "giant cellstage" and the subsequent "palmelloid body stage", were distinguishedin the process of formation of giant Chlorella induced by theaddition of sugars. The "giant cell" is much larger in sizethan the control cell, but the other morphological featuresare the same as those of the latter. The "palmelloid body" isa form composed of many conjoined autospores.
2. When a highconcentration of glucose was maintained in the medium,gigantismwas also maintained. Under this condition, the algashows acyclic transformation between "giant cell" and "palmelloidbody"without returning to the small single cells.
3. Large amountsof carbohydrate composed of hexose were foundto be accumulatedin the giant algal cells, and it was inferredthat this carbohydrateaccumulation causes greater enlargementof cell volume as comparedwith control cells.
4. Uronic acids, which were found to be absentin the control cells,were formed and lost in the cells culturedin the glucose mediumin parallel with the appearance and disappearanceof gigantism.
5. Pectic substances, from which uronic acids areconsidered tobe derived during the extraction procedure, werefound to bepresent only in giant Chlorella.
6. The conjoinedautospores in giant Chlorella (at the palmelloidbody stage)were separated to some extent by the addition ofEDTA, and theresulting cells were similar to control Chlorellacells.
7. Basedon these results it was inferred that inductive formationofthe pectic substances is causally related with the appearanceof "palmelloid body".

¹ Present address: Department of Chemistry, College of GeneralEducation, Osaka University, Toyonaka, Osaka.     

DEGRADATION AND FORMATION OF SULFOLIPID OCCURRING CONCURRENTLY WITH DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES

1. It has been demonstrated that when the cells of Chlorella protothecoidesare grown mixotrophically under illumination in a medium richin nitrogen source (urea) and poor in glucose, the normal greencells are obtained, while in a medium rich in glucose and poorin the nitrogen source, entirely chlorophyll-less cells withprofoundly degenerated plastids ("glucose-bleached" cells) areproduced, irrespective of whether in the light or in darkness.The "glucose-bleached" cells turn green with regeneration offully organized chloroplasts when incubated in a nitrogen-enrichedmedium in the light ("light-greening"), while in the dark theybecome pale green with formation of only partially organizedchloroplasts ("dark-greening"). When, on the other hand, thegreen cells are transferred into a medium enriched with glucose,they are bleached fairly rapidly with degeneration of chloro-plastsin the light as well as in darkness ("bleaching"). Using ³⁵Sas a tracer, investigations were made on the changes of contentsof the algal cells in sulfolipid and other sulfur compoundsduring the processes of the greening and bleaching.
2. By determiningthe radioactivities of chromatographically separatedsulfur-containingcompounds of the uniformly ³⁵S-labeled green("G") and "glucose-bleached"("W") cells, it was found thatthe concentration of a speciesof sulfolipid (discovered byBENSON et al.) as well as thoseof glutathione, sulfotriosesand most of the other sulfur-containingcompounds were at least5 times higher in the "G" cells thanin the "W" cells, whilesulfoquinovosyl glycerol was presentin approximately equalamounts in the two types of cells.
3. Phospholipidcontents and compositions in the two types of algalcells werefound to be practically identical.
4. The sulfolipid contentof algal cells increased and decreasedalmost in parallel withthe processes of greening and bleaching,respectively.
5. Studyingthe mode of incorporation of radiosulfate into varioussulfurcompounds of algal cells during the processes of "light-anddark-greening" and "bleaching" (lasting about 70 hr), itwasfound that active ³⁵S-incorporation into sulfolipid occurredthroughout the process of "light-greening," while in the "dark-greening"and "bleaching" the active incorporation abruptly ceased afterthe initial 24 hr period of experiments. It was suggested thatthe biosynthesis of the sulfolipid is closely related to theformation of photosynthetic apparatus in chloroplast.
6. Whenthe ³⁵S-labeled green cells were bleached in a medium containingno radiosulfate, the ³⁵S-sulfolipid and most of other ³⁵S-sulfurcompounds decreased markedly but the ³⁵S-sulfoquinovosyl glycerolincreased considerably. It was inferred that the deacylationof the sulfolipid, a surfactant lipid, with formation of watersoluble sulfoquinovosyl glycerol may be a cardinal event ofbleaching process, causing a disintegration of the intact architechtureof photosynthetic apparatus.
7. Based on these observations itwas concluded that the sulfolipidis an integral component ofphotosynthetic structure.

¹This work was partly reported at the Symposium on Biochemistryof Lipids, sponsored by the Agricultural Chemical Society ofJapan, Sapporo, July, 1964.     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: V. DEGENERATION OF CHLOROPLASTS INDUCED BY DIFFERENT CARBON SOURCES, AND EFFECTS OF SOME ANTIMETABOLITES UPON THE PROCESS INDUCED BY GLUCOSE

1. The green cells of Chlorella protothecoides were bleached todifferent extents when incubated (in the dark) in the nitrogen-freemedia containing, besides basal mineral nutrients, glucose,fructose, galactose, glycerol or acetate. Glucose and fructosewere found to have the strongest bleaching effect. Additionof a nitrogen source (urea) caused a considerable reductionof the bleaching. It was assumed that from the different carbonsources a certain common intermediate(s) causing the bleachingis formed, and that in the presence of the nitrogen source thesubstance is removed by reacting with it.
2. Using glucose asbleach-inducing agent, the effects of someantimetabolites uponthe processes of bleaching, division andgrowth of green algalcells were investigated, and it was demonstratedthat the processof bleaching occurs without being accompaniedby growth anddivision of the algal cells.
3. It was found that during theprocess of bleaching no net increasesin RNA and protein tookplace.

(Received March 11, 1965; )     

Effect of cycloheximide on the process of "glucose-bleaching" in Chlorella protothecoides

The process of bleaching of Chlorella protothecoides inducedby the addition of glucose was strongly inhibited by cycloheximide,an inhibitor of protein synthesis, whereas it was suppressedonly weakly by chloramphenicol, puromycin and ethionine. Whencycloheximide was added simultaneously with glucose at the beginningof die bleaching experiment, no bleaching of algal cells occurredduring the subsequent incubation. When it was added after glucose,the bleaching of algal cells proceeded for a period of timeas actively as in the control, then gradually ceased. Cycloheximidewas found to suppress the uptake of glucose by algal cells,and to severely inhibit the assimilation of glucose into lipidswhen added at the beginning of the bleaching experiment. Theseinhibitory effects of cycloheximide are discussed in relationto the induction of "glucose-bleaching" in algal cells. (Received December 16, 1968; )     

Further studies on the metabolism of glucose in the process of "glucose-bleaching" of Chlorella protothecoides

Previous studies have demonstrated that when cells of Chlorellaprotothecoides are incubated in a medium containing glucosebut no nitrogen source, they are profoundly bleached with degenerationof chloroplast structure and photosynthetic activity. When anitrogen source (urea) is added to the glucose medium, bleachingof algal cells is greatly suppressed. In this work the metabolismof glucose in the process of glucose-induced bleaching was studiedusing ¹⁴C-glucose as tracer. Changes in algal cell activityfor ¹⁴CO₂-evolution and ¹⁴C-incorporation into various cellularsubstances from ¹⁴C-glucose were followed. Most conspicuouswere increases in cellular activities for assimilating ¹⁴C-glucoseinto lipids (fatty acids) and glucose polymer. When urea wasadded to the glucose medium, the incorporation of ¹⁴C by algalcells into fatty acids was greatly reduced, while the assimilationof ¹⁴C into glucose polymer was increased. These and previous observations suggest that the formation oflarge amounts of lipids (fatty acids) probably is causally relatedto the induction of algal cell bleaching. (Received March 5, 1969; )     

CHANGES IN EMISSION SPECTRA OF PHOTOSYNTHETIC PIGMENTS IN VIVO

1. The effects of 3-(4'-chlorophenyl)-1, 1-dimethylurea (CMU)onthe fluorescence of photosynthetic pigments in vivo wereinvestigatedin blue-green, red and brown algae and in isolatedspinach chloroplasts.CMU caused an increase in steady statelevel of fluorescenceof chlorophyll a, but did not influencethe fluorescence ofphycobilins. The spectrum of the fluorescenceincrement hada peak at 685 m/µ and a shoulder at 730–740mµ.These two bands probably arise from chlorophyll a(C_f684) belongingto pigment system II.
2. On excitation of chlorophylla in a red alga, Porphyra yezoensis,a fluorescence band witha peak at 720 mµ was observedbesides a shoulder at 685mµ. The 720 m band is inferredto arise from chlorophylla (probably, C_f-1) in pigment systemI.
3. On addition of CMUto the algal cells, the induction of fluorescencewas modifiedto take a simple time course. The induction wasobserved onlywith respect to the fluorescence of chlorophylla, but not inthe fluorescence of phycobilins. The spectrumof the "transient"fluorescence showed two emission bands ofchlorophyll a at 685mµ and 740 mµ, and was quitesimilar in form tothe spectrum of the CMU-caused increase insteady state fluorescence.
4. These facts were interpreted in terms of the correlation offluorescence of chlorophyll a and the photochemical reactionsof photosynthesis

(Received July 20, 1967; )     

Acetate metabolism in the process of "acetate-bleaching" of Chlorella protothecoides

Green cells of Chlorella protothecoides when incubated in amedium containing acetate but no nitrogen source, have beenshown to be bleached as strongly as in glucose-induced bleaching.Using U-¹⁴C-acetate as tracer, the acetate metabolism of algalcells during the process of acetate-induced bleaching was investigated.Changes in algal cell activities for respiration and assimilationof added ¹⁴C-acetate were followed during bleaching processesin "acetate-adapted" and "non-adapted" green cells. As in glucose-inducedbleaching of algal cells, algal cell activity for incorporating¹⁴C into lipids showed the most characteristic change, suggestingthat lipogenesis is causally related to the occurrence of bleachingin algal cells. (Received March 5, 1969; )     

THE ROLE OF RESPIRATION AND PHOTOSYNTHESIS IN THE CHLOROPLAST REGENERATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. As previously demonstrated, entirely chlorophyll-less cellsof Chlorella protothecoides are obtained when the alga is grownin a medium rich in glucose and poor in nitrogen source (urea).These cells, which are referred to as "glucose-bleached" cells,have neither discernible chloroplast structures nor photosyntheticactivity. When the "glucose-bleached" cells are incubated, inthe light, in a nitrogen-enriched mineral medium without addedglucose, they turn green, after an induction period, with regenerationof chloroplasts and development of the capacity for performingnormal photosynthesis. In the present study, changes in respiratoryactivity of algal cells during the process of greening (chloroplastregeneration) were followed, and the effects of various inhibitorsof respiration and photosynthesis on the greening process wereexamined. 2. The glucose-bleached cells showed a very low activity ofrespiration, and the activity increased markedly during an earlyphase of chloroplast regeneration, showing, however, a decreaseduring the subsequent phase of greening. 3. Some antimetabolites which inhibited the cell respiration,were found to suppress also the greening of cells. 2,4-Dinitrophenoland azide, potent inhibitors of oxidative phosphorylation, acceleratedconsiderably both the respiration and greening of algal cells.CMU inhibited completely photosynthesis of the greening cells,but suppressed only slightly the greening process. 4. Based on these results it was concluded that the primaryrole of respiration in the chloroplast regeneration in the glucose-bleachedcells is to produce oxidized carbon compounds (and perhaps reducedforms of NAD and NADP) for various biosynthetic reactions. Itwas further suggested that ATP may be supplied for the chloroplastregeneration by a certain means different from the oxidativephosphorylation or photophosphorylation. The activities of photosyntheticphosphorylation and CO₂-fixation developing in the greeningcells do not appear to play any essential role in the chloroplastregeneration. (Received December 27, 1965; )     

EFFECT OF CHROMATIC LIGHTS ON PHYCOBILIN FORMATION IN A BLUE-GREEN ALGA, TOLYPOTHRIX TENUIS

1. The formation of phycobilin pigments in a blue-green alga Tolypothrixtenuis was investigated with special reference to the effectsof preillumination with colored lights.
2. It was discoveredthat the algal cells are capable of formingphycobilin pigmentsin the dark, if they have been previouslyilluminated for severalhours in the presence of CO₂.
3. The color of light applied inthe later period of preillumination(chromatic illumination)was found to affect the ratio of phycoerythrinto phycocyaninformed in the subsequent dark period. A greenlight acceleratesthe dark-formation of phycoerythrin, a redlight that of phycocyanin,and the two lights counteractingwith each other in their effects.
4. These directive effects of the "chromatic illumination" canbe accomplished within a very short period, for instance, in3 minutes if it is preceded by sufficient "preillumination"with an incandescent or day light fluorescent light. The reactionsoccurring during the period of chromatic illumination does notrequire the presence of CO₂ and the aerobic condition.
5. Thealga can be grown heterotrophically when supplied with casaminoacids and glucose. Under such a condition the alga forms phycocyanintogether with chlorophyll and carotenoids, but not phycoerythrin.
6. On the basis of the results obtained, a tentative scheme forthe biosynthesis of phycobilin pigments in the alga was proposed,assuming the light-induced formation of unknown precursors whichare converted into phycocyanin and phycoerythrin in the subsequentdark period.

(Received July 4, 1960; )     

EFFECTS OF LIGHT ON THE DEOXYRIBONUCLEIC ACID FORMATION AND CELLULAR DIVISION IN CHLORELLA PROTOTHECOIDES

1. It has been demonstrated previously that when Chlorella protothecoidesis grown in a medium rich in glucose and poor in nitrogen source(urea), chlorophyll-less cells with markedly degenerated plastids—called "glucose-bleached" cells—are produced eitherin the light or in darkness. When the glucose-bleached cellsare incubated in a medium enriched with the nitrogen sourcebut without added glucose, normal green cells with fully organizedchloroplasts are obtained in the light, and pale green cellswith partially organized chloroplasts in darkness. During theseprocesses of chloroplast development in the glucose-bleachedcells, there occurs, after a certain lag period, an active DNAformation followed by a more or less synchronous cellular division.In the present study the effects of light on the DNA formationand cellular division were investigated in the presence of CMUor under aeration of CO²-free air to exclude the interveninginfluence of photosynthetic process.
2. It was revealed thatlight severely suppresses the DNA formationand cellular divisionof the glucose-bleached cells while enhancingremarkably theirgreening. The suppression was saturated atthe light intensityof about 1,000 lux. Blue light was mosteffective, being followedby green, yellow and red light inthe order of decreasing effectiveness.
3. Further experiments unveiled that light exerts two apparentlyopposing effects on the DNA formation depending upon the timeof application during the incubation of algal cells. When thealgal cells were illuminated only during the lag period beforethe active DNA synthesis, there occurred an enhancement of theDNA synthesis occurring during the subsequent dark incubation.When, on the other hand, the cells were transferred to the lightfrom darkness at or after the start of the DNA synthesis, itcaused an almost complete abolition of the subsequent synthesisof DNA in the algal cells. No such effects of light were observedwith RNA and protein (total)
4. These findings were discussedin relation to the process ofchlorophyll formation occurringconcurrently in the algal cells.

(Received August 10, 1967; )     

SYNCHRONOUS CULTURE OF CHLORELLA: I. KINETIC ANALYSIS OF THE LIFE CYCLE OF CHLORELLA ELLIPSOIDEA AS AFFECTED BY CHANGES OF TEMPERATURE AND LIGHT INTENSITY

1. Using the technique of synchronous culture, investigationsweremade of the effects of temperature and light-intensityon cellularlife cycle of Chlorella ellipsoidea. Some improvementsin theculture technique for obtaining a good synchrony of algalgrowthwere described.
2. By following the changes of averagecell volume and cell numberoccurring during culturing, therates of the following processesof life cycle were determined:(i) "growth" (or the increasein cell mass) occurring from thestage of smaller cells (D_a)to the stage of ripened cell (L₃),(ii) "ripening" (or processofformation of "nuclear substances"as estimated from the averagenumber of daughter cells formedfrom single mother cell), and(iii) " maturing and division" which leads to the full maturationof mother cells (L-cells)and their division into separate daughtercells (D-cells).
3. "Growth"and "ripening" were found to be dependent in light,"maturingand division" light-independent. The time requiredfor "growth"and "ripening" (C) is dependent on temperaturebut independentof light intensity, the onset of "maturing anddivision" occurringat the same time (D) of culturing undervaried light intensities.The average cell volume at this stage(L₃),however, was foundto be markedly modified by light intensity;larger with highertemperatures (see Fig. 4).
4. Changes in incubation temperature(under the condition of saturatinglight intensities) were foundto affect the life cycle in thefollowing way: (i) The timeof onset of "maturing and division"(D), varies markedly withculturing temperature; earlier athigher temperatures, (ii)The average cell volume at this stagealso depends on temperature; smaller at higher temperatures.
5. The average number of daughtercells (n) emerging from singlemother cells, was found to beuninfluenced by culturing temperature;(4.0–4.1 underthe conditions of the present study). Itwas found that thedivision number n is remarkably varied bychanging the lightintensity in the "growth" and "ripening"phases; 2.0 at 1 kilolux,3.7 at 5 kilolux, 4.2 at saturatinglight intensities (10 and25 kilolux). This finding was explainedby assuming a light-dependentformation of "nuclear substances"during the "growth" and "ripening"phases, the quantity of thesubstances in the cell at L₃ stagedeterminig the division number.
6. The experimental data wereanalyzed reaction kinetically, therate constants and othercharacteristics of the reactions constitutingthe processesof life cycle were determined, and values forthe apparent activationenergy for each reaction were computed.The reactions were discussedwith special reference to theirrelationship with photosyntheticprocess was discussed.

(Received November 7, 1959; )     

EFFECT OF ULTRAVIOLET LIGHT UPON VARIOUS PHYSIOLOGICAL ACTIVITIES OF CHLORELLA CELLS AT DIFFERENT STAGES IN THEIR LIFE CYCLE

1. Using Chlorella ellipsoidea as material, investigations weremade of the effects of ultraviolet irradiation upon variousactivities of cells at different developmental stages in theirlife cycle. Cell activities investigated were photosynthesis,respira tion, over-all growth, modes of synchronous growth andcell division as well as the formation of nucleic acids. Theu. v.- light applied was 30 µµW/cm²in intensityand 2537 Å in wavelength.
2. The most u. v.-sensitive wasthe over-all growth activity, andin this respect the irradiationapplied at the L₂-stage wasmore inhibitive than that givenat the D-stage. The next mostvulnerable was the photosyntheticactivity, the sensitivitybeing the same in the D- and L-cells.The most resistant towardu.v. was the endogenous respirationof D-cells followed by theirrespiration using exogenous glucoseas substrate. The L₂-cellsappeared to be unable to use exogenousglucose as substrateof respiration, but their endogenous respirationwas considerablystronger than that of D-cells, and its u. v.-sensitivitywasthe same as that of glucose respiration of D-cells.
3. WhenD-cells were u. v. irradiated immediately before the startofsynchronous culture, growth and cell division as well astheformation of DNA and RNA were retarded in proportion totheu. v.-dose applied. The division number (n) was normal (around4) at lower u.v.-doses (1-2 minute irradiation), but was reducedto a half (about 2) at a higher dose.
4. When, during the synchronousculture, 1-minute u.v.- irradiationwas applied at various stagesof the ripening phase, the divisionwas retarded, but the cells,after attaining an abnormally largesize, divided into about8. If the irradiation was given atthe L₄-stage, the divisionnumber was practically unmodified(n=4.5), although the divisionwas somewhat retarded comparedwith that of the control culture.When a 1-minute irradiationwas given at the L₂-stage, thereoccurred an apparent stimulationof DNA- and RNA-formation,a phenomenon which corresponds tothe production of a largernumber of daughter cells than itwas the case in control cultures.
5. Thus the cells which were moderately u.v.-irradiated at differentstages of synchronous culture were able to complete their lifecycle, but later a certain portion of irradiated cells becameunable to grow normally.

¹Present address: Department of Biochemistry, Dartmouth MedicalSchool, Hanover, New Hampshire, U.S.A. (Received March 6, 1961; )     

Photosynthetic metabolism of 14CO2 in the process of the "glucose-bleaching" of Chlorella protothecoides

Changes in photosynthetic carbon metabolism during the glucosebleaching of Chlorella protothecoides cells were investigatedusing NaH¹⁴CO₃ as tracer. Several hours after incubating thegreen algal cells in the glucose medium in the dark, the ratesof ¹⁴C-incorporation into glucose polymers and sucrose decreasedand the incorporation into the lipid fraction (fatty acids)greatly increased. At this stage, the rate of photosynthetic¹⁴CO₂ fixation and the chlorophyll content were practicallythe same as in the starting green cells. Afterwards, the photosyntheticcapacity and chlorophyll content continued to decrease throughoutthe experimental period. In contrast, when photosynthetic ¹⁴CO₂fixation of green cells was carried out in the medium containingglucose, the rate of ¹⁴C-incorporation into glucose polymersincreased, though there was no change in the incorporationsinto sucrose and the lipid fraction. ¹Part of this investigation was reported at the Conference "ComparativeBiochemistry and Biophysics of Photosynthesis" (Japan-U.S. CooperativeScience Program) held at Hakone, Japan in 1967. ²Present address: Faculty of Agriculture, Tamagawa University,Machida-shi, Tokyo, Japan. (Received June 10, 1974; )     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: IV. EFFECTS OF 5-FLUOROURACIL, DIHYDROSTREPTOMYCIN, CHLORAMPHENICOL AND ACRIDINE ORANGE ON THE PROCESSES OF GREENING AND DIVISION OF "GLUCOSE-BLEACHED" ALGAL CELLS

1. The "glucose-bleached" cells of Chlorella protothecoides, whichwere obtained by the method described previously, were transferredto a glucose-free medium containing basal mineral nutrientsalone in the dark, and after a certain period of time, the cellsuspension was supplied with urea and light to induce the greeningof cells. At different times before and after the provisionof urea and light, the inhibitors were applied to the cultureto test their effects upon the process of greening.
2. Markedgreening of the glucose-bleached cells occurred aftera lagperiod in the control culture. 5-Fluorouracil inhibitedthecell greening strongly when it was applied at differenttimesbefore the provision of urea and light. When applied aftertheprovision of urea and light, the suppressive effect of 5-fluorouracilgradually decreased with the delay of its application. No inhibitiveeffect was observed when the uracil analogue was added laterthan the 12th hr after the provision of urea and light, thetime around which the chlorophyll formation started in the controlculture. On the other hand, the cell division was much morestrongly affected by 5-fluorouracil. Even when it was appliedat the 18th hr after the provision of urea and light, the celldivision was completely halted, indicating that the greeningand division of the glucose-bleached cells are separate processes.Different mechanisms of action of the uracil analogue towardsthese two processes were suggested.
3. Dihydrostreptomycin showedits strongest suppressive effectwhen added at the beginningof the dark incubation of algalcells in the glucose-free medium,and with the delay of application,its effect was progressivelyreduced, even during the periodof the dark incubation. Thesuppression, however, was stillmarked when it was applied atthe 15th hr.
4. Chloramphenicol was found to inhibit stronglythe chlorophyllformation and protein synthesis, but, to a muchlesser extent,RNA synthesis. Acridine orange suppressed thecell greeningand division at such a low concentration as 1.5µg/ml.
5. Based on these observations it was concludedthat synthesesof nucleic acid and protein are essential processesfor thegreening of the glucose-bleached algal cells. Successiveeventsoccurring in the greening process were discussed.

(Received March 9, 1965; )     

SYNCHRONOUS CULTURE OF CHLORELLA: II. CHANGES IN CONTENT OF VARIOUS VITAMINS DURING THE COURSE OF THE ALGAL LIFE CYCLE

1. Chlorella ellipsoidea was grown synchronously and the changesin content of various vitamins during the algal life cycle werefollowed either by chemical or microbiological assay methods.
2. In terms of µg per gram of cell dry weight, the contentof some vitamins (niacin, biotin, inositol and choline) remainedalmost constant throughout the algal life cycle, while thatof others (vitamin B₆-complex, pantothenic acid, folic acid,thiamine and riboflavin) was found to decrease more or lessmarkedly during the "growing phase" and increase at later phasesof "ripening". The content of p-aminobenzoic acid increasedonly at an early stage of "ripening", and that of ascorbic acidincreased only at the stages in which photosynthesis occurredmost actively.
3. These results were discussed in an attemptto interprete theirrelationship with the previously reportedobservations pertainingto the physiological and biochemicalevents occurring in thelife cycle of the alga.

(Received November 7, 1959; )     

STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM III. POSTOXIDATIVE FIXATION OF CARBON DIOXIDE

1. The cells of Thiobacillus thiooxidans, which had been in contactwith sulfur or sulfide in air (or CO₂-free air), could fix addedCOa very rapidly after replacing air with nitrogen. This fixationis designated as the postoxidative fixation.
2. "Preoxidation"of the sulfur compounds is mandatory for theoccurrence of thepostoxidative fixation.
3. The cells which had preliminarilyoxidized sulfide could notshow the CO₂-fixation, when theywere placed under an anaerobiccondition in the absence of thesulfur compound.
4. These results indicate that sulfur compoundsmay have an importantrole as the electron donor for the reductionof CO₂, besidestheir role as the substrate of respiration tosecure energyfor the fixation of CO₂

(Received March 6, 1962; )     

"GIGANTISM" OF CHLORELLA VULGARIS I. RELATION OF GIGANTISM TO CELL GROWTH AND DIVISION

1. The sugars which induced gigantism of Chlorella cells wereglucose,fructose, galactose, mannose, xylose and arabinose.These sugarswere utilized as respiratory substrates by thealgal cells.
2. The cellular division of Chlorella was stimulatedby glucoseand galactose, but suppressed by fructose, mannose,xylose andarabinose, while all these sugars evoked gigantism.No correlationwas found between cellular division and gigantism,
3. The photosynthetic activity of giant Chlorella varied withthesorts of sugars added. It was decreased by glucose, fructoseand mannose, but was unaffected by other sugars such as galactose,xylose and arabinose.
4. The respiratory activity of giant Chlorellacells as much higherthan that of control cells.
5. The amountsof protein-N and dry weight per unit volume of giantChlorellawere much less than those of control cells.

¹ Present address: Department of Chemistry, College of GeneralEducation, Osaka University, Toyonaka, Osaka.