The Metabolism of Glucose and Acetate in Aspergillus niger

1. The metabolism of a citric-acid-forming strain of A. nigerwhengrown on a glucose-acetate medium has been investigated.
2. Acetate greatly accelerated the rate of utilization of glucose.
3. Citric acid production from glucose was much increased bythepresence of acetate.
4. The formation of oxalate from glucose-acetatecultures was muchless than from acetate alone.
5. In some cultureslarge amounts of glucose and acetate were consumedbut no acidicproducts were formed.

     

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; )     

Studies on the destruction of indole-3-acetic acid by a species of Arthrobacter I. On the induction of the enzyme responsible for the destruction

1. The induction of an IAA-destroying enzyme in Arthrobacter sp.that can utilize IAA as its sole source of carbon and nitrogenwas investigated.
2. 1. The enzyme was most effectively inducedby 10–³ to2x10–³ M IAA, at pH 6.5.
3. 2. All testedIAA analogs were unable to induce the enzyme.Analogs otherthan indole-3-lactic acid were rather inhibitoryon the inductionwith IAA.
4. 3. The induction period was shortened with the ageof culturein both polypeptone and acetate media.
5. 4. Pretreatmentof the bacterium with IAA caused a shorteningof the inductionperiod.
6. 5. The induction was inhibited by various antibiotics,aminoacid analogs and nucleobase analogs.
7. 6. The inductionwas less remarkable in actively proliferatingcells than itwas in slowly proliferating ones.

(Received July 1, 1967; )     

PARTIAL PHOTO-REPRESSION OF THE GLYOXYLATE BY-PASS IN EUGLENA

1. Addition of exogenous acetate or ethanol to autotrophic culturesof Euglena gracilis strain Z induces formation of the glyoxylateby-pass.
2. Visible light decreases the activity of malate synthasein greenEuglena by about 50%. No such effect was found in apermanentlybleached mutant.
3. Aconitase activity parallelsthat of malate synthase, but isocitricdehydrogenase activityis constant under all conditions examined.
4. Oxygen consumptionis proportional to the activities of malatesynthase and aconitase,but not to that of isocitric dehydrogenase.
5. The results ofsimilar studies with other growth substrates(pyruvate, malate,succinate) suggest that some of the oxygenconsumed by C₂-grownEuglena may not be associated with energyproduction.

(Received March 25, 1966; )     

The mechanism of phosphate permeation in purified bean mitochondria

The permeability properties and mechanism of Pi transport wereinvestigated in purified bean mitochondria.

1. Purified bean mitochondria are impermeable to small moleculesand ions. However, Pi, arsenate, acetate and formate can enterthe osmotically active space of bean mitochondria.
2. Nigericinor the association of valinomycin and FCCP cause mitochondrialswelling in isoosmotic potassium phosphate.
3. The SH-blockingreagents mersalyl, pHMB and NEM inhibit variousmitochondrialfunctions dependent on the translocation of Piand arsenateacross the membrane. These include the respirationstimulatedby ADP, Ca²⁺+Pi, and K⁺+valinomycin +Pi; the swellingin ammoniumphosphate medium and, in the presence of nigericin,in potassiumphosphate medium; the energy-linked yalinomycin-inducedswellingand the subsequent CICCP-induced shrinking. The uncoupler-stimulatedrespiration, as well as the other processes when acetate issubstituted for Pi, are not influenced by SH reagents.
4. Mersalyland pHMB cause complete inhibition at about 20 nmoles/mgprotein,whereas, NEM is effective at about 1 µmole/mgprotein.The inhibition by mersalyl and pHMB, but not that byNEM, issigmoidal and reversed by 2-mercaptoethanol. Non-inhibitoryamounts of mersalyl protect the Pi transport from irreversibleinhibition by NEM.
5. We concluded that a carrier-mediated transportsystem for Piis present in bean mitochondria, and that someof its propertiesare similar to the Pi carrier of animal mitochondria.

(Received June 5, 1975; )     

Salt-induced alterations of the fluorescence yield and of emission spectra in Chlorella pyrenoidosa

1. The addition of salts to the suspending medium induces a decreasein the yield of chlorophyll a fluorescence in normal and DCMU-poisonedintact algal cells of Chlorella pyrenoidosa. Potassium and sodiumacetate cause a pronounced lowering of the fluorescence at relativelylow concentrations (0.01–0.1 M). MgCl₂ and KCl cause asimilar lowering of fluorescence but at much higher concentrations(0.1–0.4 M). In contrast to sodium acetate, ammonium acetatedoes not cause any significant change in the fluorescence transient.
2. Unlike the case in isolated chloroplasts, MgCl₂ decreasestheratio of short wavelength (mainly system 2) to long wavelength(mainly system 1) emission bands in both DCMU poisoned and normalcells. Since these salt-induced changes do not appear to berelated to the redox reactions of photosynthesis, the saltsmight have caused a decrease in the mutual distance betweenthe two photosystems by changing the microstructure of the chloroplastsin vivo thereby facilitating the spillover of excitation energyfrom strongly fluorescent system 2 to weakly fluorescent system1.
3. The light induced turbidity changes in intact algal cells,asmeasured by the increase in optical density at 540 nm, isreducedin the presence of these salts. However, MgCl₂ producesthegreatest reduction while Na acetate the least, even thoughbothof these salts (at the concentrations used) cause largesuppressionof the fluorescence transient. Moreover, the lightinduced turbiditychanges were, essentially irreversible.
4. Ashigh concentrations of salts increase the osmotic potentialof the bathing medium, it seems that the osmotic changes aswell as the ionic changes in the intact algal cells are responsiblefor the fluorescence quenching and changes in the mode of excitationtransfer observed in this study. In the case of low concentrationsof salts (e.g., 0.1 M Na or K acetate) the effects are predominantlyionic, and in the case of very high concentrations of MgCl₂(0.4 M), the osmotic effects play a much larger role.

(Received July 30, 1973; )     

SPORULATION OF THE YEAST, HANSENULA SATURNUS: II. INFLUENCE OF CARBON-NITROGEN RATIO OF CULTURE MEDIUM

1. Several factors affecting sporulation of a wild yeast, Hansenulasaturnus, especially carbon sources and the carbon-nitrogenratio of sporulation medium were studied.
2. The sporulationis stimulated at a certain definite C/N ratioof glucose medium.
3. Several carbon sources such as ethanol, acetate, lactate,glycerol,succinate, glucose, gluconate and citrate are utilizedby theorganism both for growth and sporulation.
4. The numberof spores in an ascus depends on the C/N ratio ofthe medium.An increase in the ratio stimulates the yield of2-and 3-sporedasci, especially of the former. One-spored ascibecome abundantas this ratio decreases.
5. Lysine promotes sporulation in anacetate medium, and its presencein a large amount in glucosemedium also stimulates sporulation,while a small amount isinhibitory. When lysine was employedas the sole nitrogen source,most of the asci were 1-spored.
6. It is discussed that sporulationof yeast is induced by a balanceof metabolism, rather thanby one definite "sporulation substrate".

¹ Present address: Laboratory of Microbiology, Department ofAgriculture, T{circumflex}hoku University, Sendai. (Received May 23, 1961; )     

STUDIES ON PROTEIN SYNTHESIS IN TOMATO COTYLEDONS AND LEAVES. II. INTERMEDIATE STAGES OF PROTEIN SYNTHESIS

1. The activities of RNase, ATPase and proteolytic enzymes inextractsof tomato leaves have been investigated with regardto detrimentaleffects on protein synthesis. The use of coppersulphate asan inhibitor of RNase is described.
2. The effectof microbial contamination on amino acid uptake hasbeen studiedin relationship with cell-free systems, and thehigh activitynoted underlines the necessity for aseptic procedures.
3. Methodsfor the preparation of cell-free systems capable ofamino acidactivation and incorporation into protein in an asepticenvironmentare discussed.

¹Present address: Dept. of Horticultural Science, Universityof Wisconsin, Madison, Wisconsin, U.S.A.     

STUDIES ON PROTEIN SYNTHESIS IN TOMATO COTYLEDONS AND LEAVES. I. PROTEIN SYNTHESIS AND TURNOVER IN INTACT COTYLEDONS AND LEAVES

1. Details of aseptic culture of virus-free tomato seedlings usedin comparative in vivo and in vitro studies on protein synthesisare described.
2. Developmental changes in the levels of DNA,RNA, protein andchlorophyll content of seedling cotyledonsand leaves were recorded,and are related to protein synthesis.
3. Incorporation of isotopically labeled carbon into proteinwasfollowed both by photosynthetic uptake of ¹⁴CO₂ and by theuptakeof ¹⁴C-amino acids through the roots.
4. A marked stimulationby light of ¹⁴C uptake was observed, andthe higher rate of¹⁴C incorporation from ¹⁴CO₂ than from ¹⁴C-aminoacids intothe protein fraction is discussed in relation tothe pathwaysof protein synthesis in tomato leaves, and alsowith regardto protein turnover.

¹Present address: Dept. of Horticultural Science, Universityof Wisconsin, U.S.A.     

The Development of Cells in the Growing Zones of the Root

1. Data are presented which show the change in volume, dry weight,respiration, and protein content of the cell during developmentfrom the meristematic to the mature fully extended state.
2. Thedata have been obtained by making serial sections of knownthicknessalong the root, making the relevant observations onthe sections,and relating the values obtained to the totalnumber of cellsin the sections. An apparatus is described forobtaining serialsections from a group of roots.
3. It is shown that the weightof the wall, the protein content,and respiration all increaseduring extension, and the increasesduring active growth withrespect to respiration and proteincontent occur in two phases.In the first the increases arelarge per unit increment in volume;in the second they are relativelysmaller. When extension ceasesa third phase in the developmentof the cell sets in in whichrespiration and protein contentdecrease slightly.
4. The datashow that the protein content and the respiration inthe meristematiccell are considerably lower than they are infully vacuolatedcell.

     

CHANGES IN THE RESPONSE OF CARROT ROOT CALLUSES TO THE ROOT EXTRACT OCCURRING DURING THEIR SUCCESSIVE CULTURES

1. Alcohol extract of carrot root promoted the growth of the carrotroot callus which had been succesively cultured for more than18 months (CCL) on the medium containing WHITE'S inorganic salts,sucrose, yeast extract and 2, 4-D, but only a weak promotionwas observed for the growth of the carrot root callus whichhad been cultured for less than 14 months (CCS).
2. The activesubstances were fractionated by Amberlite IR-120and AmberliteIRA-400 into four fractions; C, D, E, and F. Eachfraction seemedto act synergistically to produce the effectof the whole carrotroot extract on the growth of CCL.
3. Fraction F of the carrotroot extract, which was adsorbed byAmberlite IRA-400 but notby Amberlite IR-120, promoted thegrowth of CCL in the presenceof other fractions, but had noeffect on the growth of CCS.So the different responses to thealcohol extract of the carrotroot calluses having differentlengths of successive cultureperiod seemed to depend mainlyon the ability of respondingto fraction F.
4. Using four strains of carrot root callusesof different origin,it was ascertained that different responsesof carrot root callusesto fraction F depended on the lengthof their culture and noton their strain-specific characters.
5. The substances active for the growth of CCL in the carrotrootextract passed through a dialysis membrane. These substanceswere little affected by autoclaving and remained in the aqueouslayer when shaken with several organic solvents: n-butanol,ethyl acetate, chloroform, benzene, ethyl ether and carbon tetrachloride.
6. Alcohol extract of carrot root also promoted the growth ofcarrotroot explant, tobacco stem callus and sunflower crowngall tissue.

(Received December 24, 1964; )     

Studies in the Nitrogen Metabolism of Apple Fruits: THE CLIMACTERIC RISE IN RESPIRATION IN RELATION TO CHANGES IN THE EQUILIBRIUM BETWEEN PROTEIN SYNTHESIS AND BREAKDOWN

1. A close parallelism in the drift of the rate of respirationand the protein-N/ non-protein-N ratio is shown to occur bothin apple fruits attached to the tree and when detached fromthe tree at various stages of development and stored for severalmonths at 12 C.
2. In detached fruits the fall in respirationwhich occurs immediately(during the first 48 hours) after pickingis only accompaniedby a concomitant fall in net protein invery young fruits inwhich active cell division is taking place.Subsequently, infruit of all ages when a climacteric rise inrespiration occursit is accompanied by a net increase in protein.
3. It is argued that the climacteric rise in respiration is aresultof increase in the level of protein which will be expectedtoreduce the ATP/ADP ratio.
4. Over the climacteric period,although rate of respiration andnet protein content both rise,R rises more rapidly than proteinand, subsequently, falls ata faster rate than P. It is suggestedthat this may be due tothe ‘new’ protein containinga higher proportionof enzyme(s) directly involved in respirationand leading, forexample, to a reduction in the ATP/ADP ratio.

     

FLAVOPROTEIN IN CHLORELLA ELLIPSOIDEA CATALYZING TPNH-LINKED REDUCTION OF PLASTOCYANIN

1. An enzyme possessing a capacity of catalyzing reduction of thecopper protein, plastocyanin, with reduced pyridine nucleotides(TPNH-plastocyanin reductase) was isolated from Chlorella ellipsoidea.The procedures of purification and the properties of the purifiedenzyme are described.
2. From the results of chromatographicand enzymic tests, the prostheticgroup of the enzyme was identifiedas FAD. No evidence was obtainedto indicate the participationof metal ions in the reaction.
3. The enzyme utilizes both TPNHand DPNH as electron donors, butthe reaction is about 12 timesfaster with TPNH than with DPNH.
4. The enzyme, with TPNH aselectron donor, catalyzes the reductionof Chlorella plastocyanin,cytochrome c, 2,6-dichlorophenolindophenol and oxygen in adecreasing order of reaction rate.The reaction with oxygenas electron acceptor was found to bemuch more strongly acceleratedby the addition of higher concentrationsof flavins as comparedwith the reaction with other acceptors.FAD and FMN added tothe reaction mixture are not appreciablyreduced.
5. The propertiesof the enzyme are compared with those of alliedchloroplastenzymes reported by various investigators and thepossible roleof the new enzyme in photosynthesis is discussed.

(Received January 18, 1961; )     

The Absorption of Ions by Excised Root Systems: II. OBSERVATIONS ON ROOTS OF BARLEY GROWN IN SOLUTIONS DEFICIENT IN PHOSPHORUS, NITROGEN, OR POTASSIUM

1. Barley plants were grown in complete culture solution and indeficiencies of phosphorus, nitrogen, or potassium for a periodof about 6 weeks. Excised roots of these plants were treatedwith a complete, aerated culture solution at 25? C. for varyingperiods of time, and the changes in respiration rate, phosphorus,nitrogen, potassium, sugars, and starch contents measured.
2. Therewere changes in fresh weight and dry weight of the excisedrootsduring treatment. The dry weight decreased with time butthewater-content changes were variable. There was a gain orlossof water by the roots according to the treatment.
3. In all casesthe deficient roots increased in content of theelement in whichthey were originally deficient. The roots ofthe plants suppliedwith full nutrient usually decreased incontent of phosphorus,nitrogen, and potassium, but exceptionsoccurred and the reasonsare discussed.
4. In most of the experiments described simultaneousloss of oneion and gain of another occurred.
5. Nitrogen-deficientroots accumulated nitrate when exposed toa complete nutrientsolution, and some of this was assimilatedwith formation ofprotein. Under similar conditions nitrogen-richroots decreasedin nitrogen content and proteolysis took place.
6. There wasa rapid fall in sucrose and reducing sugar contentof the excise'roots. The starch content was initially verysmall and showedlittle change with time.
7. The respiration rate declined withtime in all treatments exceptwhere a nitrogen deficiency existed.Here the respiration rateincreased to a maximum value at about8 hours and then fell.This increase in rate is attributed toprotein synthesis. Noevidence of a ‘salt respiration’was observed evenwhen active uptake of phosphorus or potassiumwas occurring.
8. In most instances the carbon dioxide evolvedin respirationgreatly exceeded the carbon dioxide equivalentof the sugarconsumed in the same period. Exceptions were foundwith thenitrogen-deficient roots where less carbon dioxidewas evolvedthan the equivalent of sugar consumed. It is probablethat apart, at least, of the sugar unaccounted for was usedin proteinsynthesis.
9. Where the carbon dioxide of respirationwas in excess of theequivalent of sugar consumed, protein oramino-acid is the mostprobable substrate. Respiration rateis found to be relatedboth to nitrogen and sugar content.

     

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURES

1. The longer the period of stock culture, the more remarkableis the growth inhibition by 8-azaguanine in callus.
2. Chloramphenicol,5-methyltryptophane and mitomycin C exert greaterinhibitionon growth in CCL than in CCS.
3. Bud formation is inhibited bysome concentrations of chloramphenicolwithout accompanyinginhibition of the growth.
4. Cell size and the contents of RNA,DNA, protein and lipid percell of CCL are greater than thoseof CCS, respectively. Thecontents per cell of RNA and lipidin "mitochondrial fraction"are higher in CCL than in CCS.
5. Incorporationof guanine-8-¹⁴C into RNA of CCS occurs rapidlyin the first12 hr and slows down thereafter, but that in CCL-RNAincreasessteadily for 16 hr. This difference in rate of theincorporationafter 12 hr between CCS and CCL is principallydue to the differencein rate of the incorporation into RNAof nuclear, mitochondrialand soluble fractions.

1. The rate of RNA breakdown in CCL wasnot so great as the rateof synthesis.
2. 8-azaguanine (10^–3and 10^–4M) inhibits incorporationof guanine-8.¹⁴C intoRNA of both CCS and CCL during 14 hr,but thereafter (up to25 hr) it inhibits the incorporation intoCCL-RNA alone leavingthat into CCS-RNA unaffected.

1. In CCL 510^–5M 8.azaguaninedoes not affect total radioactivityincorporated into bulk RNA,but inhibits incorporation intoRNA of "mitochondrial fraction".

(Received December 23, 1964; )     

Purification and properties of the photoactive particle corresponding to photosystem II

1. 1) Purification of the photoactive particle corresponding tophotosystem II (particle II) was achieved using a combinationof procedures including digitonin- and Triton X-100-treatments,sonication, and differential- and density-gradient centrifugations.
2. 2) The "purified" particle II preparation showed only oxygenevolution activity and showed no NADP⁺ photoreduction activityeven when an electron donor couple was added.
3. 3) The chemicalcompositions of this particle and of the particlecorrespondingto photosystem I (particle I) were compared withrespect tothe composition and contents of their chlorophylls,carotenoids,cytochromes, plastoquinones, protein and lipid.Marked differenceswere found.
4. 4) Using the two photoactive particles I and IIlacking therespective counterpart activity, a partial successwas attainedin reconstituting a system, in which photoinducedelectron flowfrom water to NADP⁺ was observed, provided thatsuitable intermediateelectron carriers, e. g. plastoquinoneand plastocyanin, wereadded.
5. 5) The nature of the two photoactiveparticles obtained andtheir relationship to particles so farreported are discussed.

¹This study was aided by grants from the Ministry of Education(407160-1965, 91402-1966, 1967). Financial support from SanyoBroadcasting Scientific Foundation is also acknowledged withcordial thanks. (Received October 23, 1968; )     

Further Studies on the Acid Metabolism of Aspergillus niger: THE FORMATION AND UTILIZATION OF OXALIC ACID

1. Some recent works on the formation of oxalic acid by variousfungi are critically considered.
2. The present work deals withthe role of oxalic acid in the metabolismof Aspergillus niger.
3. When glucose solutions were supplied to preformed mats ofthefungus oxalic acid accumulated, attaining an equilibriumlevelwhich was not exceeded despite the presence of a considerableconcentration of glucose.
4. When the glucose supplies were depletedthe oxalic acid concentrationfell steeply to a low level.
5. Theconcentration of oxalic acid was dependent on the glucoseconcentration.In three separate series of experiments it wasshown that theoxalic acid concentration diminished with increasingglucoseconcentration.
6. Similar results were obtained when the cultureswere rearedfrom spores on culture solutions with the normalamounts ofnutrient salts but different glucose concentrations.
7. In all cases the CO₂ output increased with the glucose concentration.
8. When cultures were supplied with glucose+oxalic acid, theconcentrationof the latter fell steeply to the equilibriumlevel attainedon glucose only. In a culture receiving glucose+oxalicacid,with the oxalic acid concentration somewhat below thenormalequilibrium concentration, the formation of oxalic acidfromthe glucose ceased as soon as the equilibrium level hadbeenattained.
9. When 1 per cent. oxalic acid only was suppliedto the fungusthe concentration gradually diminished to a lowlevel. When3 per cent. oxalic acid was supplied the rate ofacid utilizationsoon fell to low value.
10. In several experimentsit was shown that the rate of CO₂ outputwas higher from culturessupplied with glucose+excess oxalicacid than from culturessupplied with glucose only.
11. The rate of oxalic acid carbonloss was always below that ofthe CO₂ carbon output both incultures supplied with oxalicacid only and in cultures receivingglucose+oxalic acid.
12. The cultures were incapable of utilizingneutral sodium oxalateand the presence of this substance hadno effecft on the ofCO₂ output.
13. The results indicate thatthe utilization of oxalic acid isassociated with the liberationof at least an equivalent amountof CO₂.
14. It is suggested thatthe utilization of oxalic acid is promotedby the presence ofglucose, thus accounting for the lower oxalicacid concentrationsand higher rates of CO₂ output of cultureswith higher glucoseconcentrations.

     

VARIATION OF WATER CONTENT IN HELIX ASPERSA MULLER IN A NATURAL ENVIRONMENT

1. Daily and seasonal variations occur in the water content ofHelix aspersa Müller.
2. The water content is not constantthroughout 24 hours but fluctuatesby 2 to 10%. There is noapparent daily rhythm.
3. Humidity has an effect on the watercontent. Falls in relativehumidity to 60% or less, triggera decrease of the snails' hydration,over a range of temperatures,as well as a brief pause in activity.
4. Only external climaticconditions produce noticeable fluctuationsof water content,and they more particularly affect juvenileswhich undergo greaterevaporo-transpiration.
5. A seasonal rhythm in water contentexists in Helix aspersa,with maxima during spring and autumnand minima in June andJanuary.
6. The water content of juvenilesis always superior to that ofthe adults.
7. The water contentaffects the duration of the snail's activephase.

(Received 22 September 1988; accepted 5 February 1989)     

A Comparison of the Aerobic and Anaerobic Respiration of Apples

1. The loss of carbohydrate, acid, and cell-wall material, andthe production of carbon dioxide and alcohol, have been studiedin apples under aerobic and anaerobic conditions.
2. The changesin all these metabolites, except the cell-wall material,proceedin a regular manner.
3. The presence of oxygen has a conservingeffect on the loss ofcarbohydrate.
4. The presence or absenceof oxygen is without effect on the rateof loss of acid.
5. Therate of loss of acid is proportional to the logarithm ofitsconcentration and is constant for a given variety of apple.
6. The loss of carbohydrate plus acid accounts quantitativelyforthe production of carbon dioxide and alcohol, both in airorin nitrogen.
7. The anaerobic respiration of carbohydrateby apples is identical,so far as the nature and quantity ofthe end products are concerned,with the alcohol fermentationof yeast.
8. The carbon dioxide which is produced in nitrogen,over and abovethat produced in fermentation, is equivalentto that which wouldbe produced by oxidation of the acid.

     

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; )