MODE OF NUCLEAR DIVISION IN SYNCHRONOUS CULTURES OF CHLORELLA: COMPARISON OF VARIOUS METHODS OF SYNCHRONIZATION

1. Chlorella ellipsoidea was grown synchronously using variouspossible techniques and the mode of nuclear division in eachcase was followed by staining the nuclei according to FEULGEN.
2. A satisfactory synchrony in respect to nuclear and cellulardivision was obtained by starting the culture from a homogeneouspopulation of young and small cells and by discontinuing theillumination at the stage which was called the L₃-stage. Thestarting young cells were invariably mononuclear and the L₃-cellswere either dinuclear or tetranuclear. When the L₃ were incubatedin the dark, they ripened further, and after passing througha tetranuclear stage (referred to as the L₄) divided into fourmononuclear daughter cells which have been called the D_n-cells.The most clear-cut and repetitive synchronous culture was obtainedwhen the culture (in the light) was started from the D_n-cellsand the illumination was discontinued at the L₃-stage untilthe fully ripened cells divided into four each of D_n-cells.
3. An apparently "synchronous" culture was also obtained by themethod of programmed light-and-dark regimen, in which a randomculture is subjected to a regular alternation of light and darkperiods of adequate durations. In this case, however, the cellsat different stages of culture showed irregular nuclear patterns,and the average "division number" of mother cells was not constant,being subject to change between 4.0 and 4.9.

(Received May 25, 1961; )     

DISTRIBUTION AND TURNOVER OF PHOSPHATE COMPOUNDS IN GROWING CHLORELLA CELLS

1. Using the Chlorella cells which had been uniformly labeled with³²P, the distribution of phosphorus in various fractions ofcell material was investigated. Uniformly ³²P-labeled Chlorellawas further grown in a P-free medium or in a standard "cold"medium, and the change of distribution of ³²P (as well as theuptake of exogenous P) in various cell fractions was followed.
2. Analysis of the ³²P-labeled algal cells showed that the highestin P-content was the fraction of RNA followed by those of polyphosphates,lipid, nucleotidic labile phosphate compounds, DNA and protein(in decreasing order). ATP and ADP were found to be only minorfractions of the total labile phosphates.
3. On incubating the³P-labeled alga in a P-free medium, the P.contentsin the fractionsof DNA, protein, lipid and ATP increased, thosein polyphosphatesand ADP decreased, and that in RNA remainedalmost unchanged.When the ³²P-labeled alga was further grownin the normal "cold"medium, DNA and protein increased withthe expenditure of endogenous³²P, but with practically no incorporationof external P. Inthe meantime the P in polyphosphates decreasedconsiderably,and the RNA fraction incorporated a large amountof externalP but only a little of endogenous³²P.
4. It was inferred that,under the experimental conditions of thepresent study, thephosphorus used in the syntheses of DNA andprotein was primarilytaken from polyphosphates, while thatused in the synthesesof RNA, phospholipid and polyphosphateswas, for the most part,taken from the extracellular P-source.

¹A part of this paper was read at the Vth International Congressof Biochemistry, Moscow, August 10–16, 1961. (Received June 4, 1961; )     

SYNCHRONOUS MASS-CULTURE OF CHLORELLA

A new culture apparatus was constructed to obtain large quantitiesof synchronized cells of Chlorella ellipsoidea. Two flat culturechambers made of lucid acrylate resin (each 20 liters in capacity)were placed in a water thermostat together with a bank of 17daylight fluorescent lamps, and the culture was run by the methodstarting from a homogeneous population of D_s-cells accordingto TAMIYA et al. It was demonstrated that with this apparatusone can obtain as much as 200–400 mg (dry weight) eachof algal material at 8 or 9 different stages in one cell cycle.Completely synchronized cell cycles could be repeated as manyas 10 times in one series of experiment, indicating that theapparatus can produce 2–4g (dry weight) each of algalcells of different developmental stages. (Received April 2, 1964; )     

ROLE OF SULFUR IN THE CELL DIVISION OF CHLORELLA, WITH SPECIAL REFERENCE TO THE SULFUR COMPOUNDS APPEARING DURING THE PROCESS OF CELL DIVISION. I.

1. The role of sulfur in the cell division of Chlorella was studiedby following the fate of the sulfur supplied to the sulfur-deficientcells using ³⁵S as a tracer.
2. The sulfur-deficient cells whichwere unable to perform celldivision were made capable of divisionby the provision of ³⁶S-labeledsulfate under non-photosynthesizingconditions. Soon after theprovision of sulfate the labeledsulfur went rapidly into thecold perchloric acid (PCA)-solublefraction of algal cells,almost entirely in the form of sulfateand/or some other inorganicsulfur substance (s). With the lapseof time, more or less remarkablechanges occurred in the patternof ³⁵S-distribution in differentfractions of cell material.It was noticed that, at the onsetof cell division, a sulfur-containingpeptide-nucleotide compound(s)(SPN), which has been reportedearlier, appeared in a largequantity in the cold PCA-solublefraction, and that its quantitydecreased gradually during thesubsequent process of cell division,suggesting that the compoundwas transformed into some othersubstance (s), presumably withits nucleotide moiety going intonucleic acids and the peptidemoiety going into some essentialproteins.
3. Another noteworthyphenomenon observed during the process ofcell division wasthe incorporation of ³⁶S in a group of hotPCA-soluble substances.These sulfur substances were revealedto be sulfur-containingnucleotidic compounds, which might possiblybe some essentialcomponents of, or substances in close relationto, deoxypentosenucleic acid (DNA).

(Received March 1, 1960; )     

PRECISE MEASUREMENT OF THE CHANGE OF STATISTICAL DISTRIBUTION OF CELL SIZE OCCURRING DURING THE SYNCHRONOUS CULTURE OF CHLORELLA

Change of statistical distribution of cell size occurring inthe synchronous culture of Chlorella ellipsoidea was followedby using a COULTER counter. The culture was started using apopulation of D-cells which showed a sharp distribution of cellsize. During the growth phase, there occurred characteristicchanges in the height and shape of the distribution curve; namely,the height first decreased with broadening of the distributionand then increased to the level even higher than the originalpeak. The broadening of the curves, which indicates the loweringof degree of homogeneity of population, occurred during theperiod of most active growth, and the homogeneity was restoredat later stages of ripening where the growth became sluggish.When the ripened cells (L₃) were transferred to the darknesstheir volume decreased to some extent before the occurrenceof cellular division. It was assumed that the shrinkage of cellsobserved may be partly due to exudation of water from the cellsand partly to the consumption of fuel material caused by enhancedrespiration, both having been shown to occur at the stage ofcell maturation. (Received June 12, 1964; )     

ROLE OF SULFUR IN THE CELL DIVISION OF CHLORELLA, WITH SPECIAL REFERENCE TO THE SULFUR COMPOUNDS APPEARING DURING THE PROCESS OF CELL DIVISION II.

1. Chlorella cells, which had been grown synchronously undersulfur-deficient conditions and thus rendered unable to performcell division, were made capable of nuclear and cellular divisionby being supplied with ³⁵S-labeled sulfate and nitrate underphotosynthesizing conditions, and the fate of sulfur duringthese recovery processes was followed. 2. When the S-starved cells were provided with sulfate aloneunder photosynthesizing conditions, cells grew appreciably inmass performing nuclear division but remaining incapable ofcellular division. During these processes most of the ³⁵S wasfound to be incorporated into the protein fraction of algalcells. 3. When the cells which had been stalemated at the above-mentionedstage were supplied with nitrate, they grew further in massand eventually performed cellular division. During this periodthe ³⁵S was found to be distributed not only in the proteinfraction, but also in an appreciable amount in the cold andhot acid-soluble fractions. 4. By paper-electrophoretic experiments it was found that thenature of the sulfur substances appearing in the hot acid-solublefraction changed strikingly during the process of cellular division.Zone electrophoresis and an anion-exchange chromatography ofthese substance isolated from the cells at the completion ofcellular division, disclosed that they were most probably deoxypentosepolynucleotides containing sulfur in some form yet unidentified. 5. It was demonstrated that there exist some antagonistic relationsbetween the protein synthesis and the formation of these sulfur-containingdeoxypentose polynucleotides, and that the former predominatesunder photosynthesizing conditions while the latter outweighsunder nonphotosynthesizing conditions. (Received August 9, 1960; )