627COGGINS & LEWIs-K GIBBERELLATE & REGREENING OF ORANGE
Literature Cited
1. BLASS, U., J. M. ANDERSON, & M. CALVIN. 1959.Biosynthesis & possible functional relationshipsamong the carotenoids, & between chlorophyll a &chlorophyll b. Plant Physiol. 34: 329-333.
2. CAPRIO, J. M. 1956. An analysis of the relationbetween regreening of Valencia oranges & meanmonthly temperatures in southern California.Proc. Am. Soc. Hort. Sci. 67: 222-235.
3. COGGINS, C. W., Jr., H. Z. HIELD, & M. J. GARBER.1960. The influence of potassium gibberellate onValencia orange trees & fruit. Proc. Am. Soc.Hort. Sci. 76: 193-198.
4. ERICKSON, L. C. 1960. Color development in Va-lencia oranges. Proc. Am. Soc. Hort. Sci. 75:257-261.
5. JONES, W. W. & T. W. EMBLETON. 1959. Thevisual effect of nitrogen nutrition on fruit qualityof Valencia oranges. Proc. Am. Soc. Hort. Sci.73: 234-236.
6. MEYER, B. S. & D. B. ANDERSON. 1952. PlantPhysiology. D. Van Nostrand Company, Inc.,New York.
7. SMITH, J. H. C. & A. BENITEZ. 1955. Chloro-phylls: analysis in plant materials. In: ModernMethods of Plant Analysis, K. Paech & M. V.Tracey, eds. Springer-Verlag, Berlin 4: 142-196.
Ribonucleic Acid-Polyphosphate From AlgaeI. Isolation & Physiology"2
David L. Correll 3 & N. E. TolbertDepartment of Biochemistry, Michigan State University, East Lansing
An RNA-polyphosphate complex has been isolatedfrom Mycobacteria by Winder and Denneny (31) andfrom Aspergillus by Belosersky and Kulaev (3).Both groups of investigators postulated that chemicalbonds existed between the RNA and the polyphos-phate. P32-phosphate was rapidly incorporated intoacid-insoluble RNA-polyphosphates in Aspergillus(22) and the specific activity of the polyphosphatewas shown to be higher than the specific activity ofthe RNA. In the case of Azotobacter, Zaetseva, andBelozersky (32) showed a similar pattern of incor-poration of p32 into the acid-insoluble polyphosphatesand the only phosphate fraction labeled more rapidlywas ATP. These properties indicate that a metabolicimportance should be associated with the RNA-poly-phosphate fraction from microorganisms.
No careful purification or detailed characteriza-tion of an RNA-polyphosphate has been reported.Also no convincing evidence for a metabolic role forthese complexes has been demonstrated. However,all reports indicate that the RNA-polyphosphates aremajor components of the phosphorus and nucleic acid
1 Received March 5, 1962.2 Journal article No. 2908 of the Michigan Agricultural
Experiment Station.3 Present address: Division of Radiation and Organ-
isms, The Smithsonian Institution, Washington 25, D. C.
fractions from microorganisms. These reports alsoagree that there is a rapid turnover of the polyphos-phate in these complexes. Consequently, this paperpresents results of a more detailed study of the isola-tion and properties of RNA-polyphosphate with somespeculations about its possible metabolic role.
Many reports have established that polyphosphatesare normal constituents of algae (1, 9, 13, 18, 25, 27,28, 29). In Acetabularia the polyphosphates have beenfound in spheres in the cytoplasm (29), while inZygnemataceae the polyphosphates were found in thechloroplasts along with a high concentration of RNA(18). Polyphosphates have been reported in nuclearequivalents which contain RNA and DNA, in addi-tion, and which have also been referred to as pseudo-vacuoles ( 13, 21 ). In all these previous reports frac-tions containing only polyphosphates and those con-taining both the polyphosphate and RNA have notbeen differentiated.
Materials & Methods- Anabaena Culture. An inoculum of the blue-
green alga, Anabaena variabilis Kiitz, was obtainedfrom the Algal Culture Laboratory, Botany Depart-ment, University of Indiana. This alga was masscultured in 6-liter flasks with medium C. as describ-ed by Kratz and Myers (20). The cultures at 30 C
were aerated with 1 % CO, in air, and illuminatedwith 500 ft-c of continuous light from incandescentbulbs. Details of the mechanics of the culture systemare reported elsewhere (7). For harvest, the culturemedium was centrifugedl in a refrigerated centrifuge,the cells resuspended in cold distilled water and cen-
trifuged for 6 minutes at 1.,770 X g to establish theirpacked-cell volume, p.c.v.
The correlation between age of culture, p.c.v., andpercentage cells in a state of division is shown infigure 1. After about ten (lays conditions becamegrowth limiting, resulting in a high proportion ofcells in a state of division. The increase in packedcell volumle in cultures over 9 or 10 days of age was
largely caused by the production of an excessiveamount of sheath-material encasing the filaments.
- Chlorella Culture. A culture of Chlorella Pvre-noidosa V.N. 2.2.1 and suggested conditions for syn-chronous growth were obtained from R. R. Schmidt,Departmlenit of Biochemistry and Nutrition. VirginiaPolytechinic Institute. The apparatus for milass syn-
chronization of Chlorella was dlesigned and construct-ed from plexiglass so that 20 liters of culture could bemaintained at 25 C under light saturations conditionsfrom (layliglht-type fluorescent bulbs and aerated with
5 C CO., in air (7). Chlorella synchronization was
induce(l by raising a 600 nml subculture in a separate
apparatus to a density of about one milliliter p.c.v.
per 100 ml of mediuml which was sufficient to keep at
w
w
-J
(n-> Z:0
Z()
)5:VJ a-Ji
I
zU1J
w
0-
z
w
CHz
w
-i
*s.
,I:
1 3 5 7 9 11
CULTURE AGE (DAYS)Fig. 1. Increases in packed cell volume and per-
centage dividing cells in a typical Anabaena populationduring growth.
YOUNG 6 HR.DAUGHTER CELLS}
so
9 HR.& HOURS IN 3HR.
/ RDARKNESS
O HR. 18 HR.
HOURS IN MOTHER CELL
1500 FOOT-CANDLES WITH 4
AUTOSPORES
3 HR. 0 15 HR.
GROWING ~jN CLEAR
GWCELLS DIVISION
6 HR. * 0 12 HR.
9 HR.
Fig. 2. Synchronization cycle of Chlorella pyrenzoi-dosa (Van Niel 2.2.1) at 25 C.
least 80 % of the cells at the young daughter-cellstage. These cells were removed by centrifugationiancl resuspended in 20 liters of freslh medium in thesynchronization apparatus. The (lilute algal suspen-
sion was first subjected to 800 ft-c of liglht for 18hours and then to 12 hours of darkniess. Thereafter,the culture was maintained on a 30-hour cycle consist-ing of 18 hours of 1500 ft-c of light followed by 12hours of darkness. After four light cvcles the cellsere considered adequately synchronized and liar-
vests were begun. Usually 15 or 16 liters of culturewere harvested and the remainder was dlilutedl to 20liters with fresh me(lium for continuation of the cul-ture in its synchronous state. Harvesting of theChlorella was carrie(d out witlh a Sharples continuouscentrifuge with a large jet. The packed algae were
transferred to 100 ml tubes and centrifuged in a
refrigerated centrifuge, wvashed once with distille(l
water, andI finally' centrifuged at 2,120 X g for 10minutes to establish their p.c.v. They were storedlfrozen until usedl for analyses.
Chlorella cells were harveste(d an(d RNA-polv-
phosphate isolations were performed for every 3hours in the life cycle of 30 hours. The type ofChlorella cells M lhich predomlinated at each of theharvest periods (luring the synchronization cycle isshown in figure 2. These harvests could not be nmadeconisecutively dluring one life cycle of the culture, forit was necessary to obtain about 15 to 30 ml of p.c.v.
each time for analyses. Therefore, each harvest atthe selected stage of growth was mla(le after at leastthree to four conpl)lete cycles of growth. In one
case a culture was mlaintained for 21 hours in lightbefore harvesting. In another case 40 ppm of clhlor-amphenical was addled to the culture after 9 hoursof light and the algae were harvested at the 12-hourlight stage.
1 Fractionation Procedures for Phosphorus Com-ponents. In figure 3 the methods used to fractionatethe phosphorus components of Anabaena are outlined.This procedure, up to the crude preparation for thepolyphosphate-RNA, was a slight modification of theprocedures reported by Kulaev and Belozersky (22)
and Juni et al. (17). The phenol extraction hasbeen described by Gierer and Schramm (11) and thefinal material has been designated as the phenolpreparation.
The procedure developed for isolating the RNA-polyphosphate from Chlorella was also based upon
Harvested, Washed Cellsextract 4 times withEtOH: ether (3:1)
phenol extractions. A frozeln samiiple of algae wasthawe(d in 300 ml of (listille(l water wvhich was ad-
juste(l (at intervals) to pH 11.5 with KOH. Thisinitial treatnment solublizedl but di(d not hydrolyze thepolyphosphate-RNA material. After 1 hour at roomtemperature the pH wvas adjusted to 7.4 with IICland(I 300 ml of cold, water-saturated phenol was add-e(l. The mixture was shakeni for 5 miiinutes an(d thencentrifuged for 8 minutes at 5 C. The upper (aque-ous) phase was savedl. 300 mil of coldl water-saturatedplienol was added, an(d the extraction repeated. Thecombine(d aqueous phase wNas then extracted fourtimles with ether andl evaporated un(ler redluced pres-sure for a short time to remlove the residual ether.Einough1 sodiunm acetate wvas adde(d to make a 1 %solution, and one drop of 2 N HCl and four volumiiesof alcohol were then adde(l. Additional sodiunm ace-tate was a(ddedl slowvly with stirring until a flocculentprecipitate formed which containe(I the RNA-poly-phosphates. The solution was then put in a freezerfor 1 hour with occasional stirring. The precipitatewx-as removed by centrifugation at 0 C, washed withalcohol, and (Iried at reduced pressure.
- DEAE-Cellulose Chromlatography. The poly-phosplhate-RNA obtained fronm the solvent fraction-ation procedures was furthier separated on a diethyl-aminoethylcellulose (DEAE-cellulose) column.Prior to use the DEAE-cellulose was ashedl in a
large column first with a saturated NaCl solution,then with 0.1 N NaOH, andl finally with a large vol-ume of 0.01 \t tris-lydr-oxvmnetlyl-aninlo-nmetlhane(tris) buffer, pH 7.6, until the pH of the eluate was
7.6. Columns 20 cnm long an(d either 2 cim in diameterfor material from Anabaena or 1 cim in (liameter formaterial from Chlorella wrere packedl writh only verygentle pressure and wx-ashe(d with a small volume ofthe tris buffer. The precipitate fromi a fractionationprocedure wvith Chlorella was (dissolved in 30 ml ofthe tris buffer, appliedI to the colunin, anid ashedwith 20 ml of the buffer. The columns were connect-e(l to a 500 ml imlixinlg flask which w\as filled with thetris buffer and the mlixing flask was in turn connect-ed to a reservoir of a salt solution. For separationof material from Anabaenca, 2 at NaCl was addedl tothe reservoir, and 10 ml fractions were collected fromthe colunmn. In the work \vith Chlorella, a series ofNaCl solutions was added in sequence to the reservoir:200 ml of 1 -t NaCI, 200 ml of 2 Mr NaCI, 200 ml of3 alr NaCl, andl 200 nml of 4 Mt NaCl. Fractions were
collecte(d (5 mil) froml the columns.1 Analyses. Total phosphorus was determined bvthe technique of King (19). Acid-labile phosphate-phosphorus was (letermine(l by lhydrolyzing samplesfor 7 nminutes witlh 1 N HCI in a boiling water bathand then determininig orthophosphate by the methodof Fiske and Subbarow (10). For the RNA-poly-phosphate fractions the nonacid-labile phosphoruswvas obtainedl by the (lifference between these twoanalyrses (difference-P).
Ribose was determined by the orcinol reaction(6) using a 40-minute hy(droly sis with 6 N HCl in aboiling water bath. A solution of adenylic acid wasused as a standard. Deoxyribose was tested for bythe diphenylamine reaction (5). Total nucleic acidlwas dletermined by the metlhodl of Webb (30). Pro-tein was determined by both the method of Lowryet al. (23) using bovine seruml albuiminl as a stand-ard, and by the ninhydrin reaction.
Optical (lensities in the ultraviolet region weremeasured with a Beckmlaln, nmo(lel DU, spectrophloto-meter witlh 1 cm quartz cells. An OD unit of RNAwas equivalent to the amlounit of material in 1 mlwhich had an absorbance of 1.0 at 26-0 mA. TheRNA in the fractions was hydrolzed to the free nu-cleotides by incubation for 18 hours with 0.5 N KOHat 40 C. Subsequent neutralization \ith coldl 36 'HClO4 resulted in the precipitation of KCIO1 w\hichwas filtered off. The filtrate was subjectedl to col-umn chromatography by the method of Hurlbert et al.(14). The separatedl nucleotide solutions were thenilyophilized to renmove the formic aci(l. Ultravioletspectra were determline(d at both aciclic and basic pHto establish identities. The miajor nucleotidles werealso identified by subjecting the RNA in the fractionisto 1 hour of hydrolysis with 1 N- HCI in sealedltubes ina boiling water bath. The hydrolysates wvere thenichromatographed on Whlatmiian No. 1 paper by the (le-scending technique with anl isobutyric acid-NH10H-water (66/1/33) solvent (26).
tion of phosphorus in the fractions obtained from theisolation procedure (fig 3) are shown in table I fortwo cultures of Anabaenia of (lifferent ages as rep-resented bv their p.c.v. In both cases the comiibined
Table IDistribution of Phosphorus in Anabaena
Harvested at 2 Culture Densities
Phosphorusfractions
P-lipidsAcid soluble
organic-PResidueOrtho-POligopoly-PRNA-poly-P
(crude prep.)
Total P
Culture No. 1(density = 5.6ml p.c.v.*/l)
Culture No. 2(density = 12ml p.c.v. ,/1)
,ug P/ml %0 jug P/ml <
p.c.v. p.c.v.
2 0.3 8 1.468 10.2 5 0.9
29224130215
4.3 7733.5 17519.5 15332.5 168
(134) ** (20.2) **668 100 586
13.129.926.128.7
100
* p.c.v. = packed cell volume.** Figures in parenthesis are A,-P values and represetit
the labile polyphosphate portioni of this fraction.
oligo- and RNA-polyphosphates constituted about 40to 50 % of the total phosphorus and the bulk of the re-maining phosphorus was orthophosphate.
Phenol preparations of the RNA-polyphosphatecomponent from a number of Anabaena cultures wereanalyzed (table II). Protein was either absent oronly present in trace amounts. The total phosphorus(parameter A) and labile phosphorus (B) variedamong the Anabaena cultures, but there was alwaysmore labile phosphorus than stable phosphorus inthese fractions as illustrated by the ratio of 7-minuteacid-labile phosphorus to difference phosphorus (D).The amount of stable RNA-phosphorus was esti-mated by the difference between the total and acid-labile phosphorus (C) or by multiplying the amountof nucleic acid (E) by the gravimetric factor, 1/10.3.The amount of nucleic acid phosphorus was of thesame magnitude by either method of calculation (C &F). This approximate correspondence indicated thatmajor amounts of other phosphorus compounds werenot present as contaminants. Also a correspondencewas found between ribose as determined with the or-cinol reaction (G) and ribose as calculated (H) bymultiplying the stable phosphorus by the appropriategravimetric factor. For different experiments, con-siderable variability among the parameters examinedreflected large biological variations in the algae.Subsequent data with Chlorella has indicated thatthese differences were probably caused by variationin the stage of growth of the Anabaena at the momentof harvest.
No significant contamination by DNA existed,since no deoxyribose was found in crude preparationsof RNA-polyphosphate from Anabaena. When analiquot of a phenol preparation of RNA-polyphos-phate was subjected to alkaline hydrolysis and thenucleotides were resolved by ion-exchange chroma-tography, the following mole percentages were ob-tained: CMP, 22.8; AMP, 20.9; GMP, 31.6, andUMP, 24.7. Acid hydrolysates of Anabaena RNA-polyphosphate were also prepared and separated bypaper chromatography. Adenine, guanine, cytidylicacid, and uridylic acid were identified, but nothymidylic acid was detected.
- DEAE-Cellulose Chromatography of RNA-poly-phosphate From Anabaena. The RNA-polyphos-phate fractions from seven Anabaena cultures of in-creasing density or p.c.v. were isolated as phenolpreparations (fig 3). These preparations werefurther fractionated on DEAE-cellulose columns andthe elution patterns as measured by the optical den-sities at 260 mju and total phosphorus are shown forthree representative cultures in figure 4. Theamount of polyphosphate from the young culture (4
cr
wa-
E0(0N\
I-
0i
30 40
TUBE NUMBER
II
cn
:
U,
0xa-C,,0Ia.-J
0A-
Fig. 4. Elution patterns of RNA-polyphosphate ob-tained from Anabaena cultures of various densities uponDEAE-cellulose chromatography.
Fig. 5. Total acid-insoluble RNA and phosphorus per
10 ml p.c.v. in Anabaena cultures of different densities.One RNA OD unit is equivalenit to 1 ml of solution withan absorbance of 1.0 at 260 m,t.
ml p.c.v.) was much greater than from the old culture(30 ml p.c.v.). From the old culture there was a
large RNA peak., which apparently contained very
little polyphosphate.The total phosphorus and total OD units of RNA
for all the DEAE-cellulose fractionations from eachAnabaena culture were plotted against culture den-sity, representing culture age, on a scale selected forcomparative purposes (fig 5). This relationship ofthe total RNA to total phosphorus, which was pre-
dominantly acid-labile polyphosphate, could be divid-ed into three parts. In the younger cultures (about5 ml p.c.v. per liter nutrient) there was an excessiveamount of polyphosphate, even though there was alsoa relatively large amount of RNA. A second condi-tion was represented by rapidly growing cultures inwhich the ratio of OD units of RNA to mg of totalphosphate was fairly constant. Finally in the very
old cultures of about 30 ml p.c.v. per liter the amountof RNA increased without a corresponding increasein polyphosphate.
Large amounts of amino acids and peptides have
been found covalently bonded to RNA as usuiallyisolated (12, 15). However, the Anabaena RNAfractions separated in this study on DEAE-cellulosecolumns gave no detectable ninhydrin reaction beforeor after a 1-hour hydrolysis in 2 N HCI at 100 C.1 RNA-Polyphosphate Complexes From Synchron-ous Chlorella. Because of wide variability in theRNA-polyphosphate content from Anabaena of dif-ferent ages, a synchronous culture of Chlorella was
used as a source of material. AMost of the Chlorellacells at one harvest time were of the samiie age, buteach harvest time at 3-hour intervals represented adifferent stage in the life cycle of this alga (fig 2).The RNA-polyphosphate fractions were initially ex-tracted by the phenol procedure described in themethods section. Afterwards, they were chroma-tographed on DEAE-cellulose columns and the sunmsof the RNA and phosphorus from each chroma-tographic fractionation are shown in figure 6. Al-
ba:
z
0
z
0
>
CL0a.
Hj
0 3 6 9 12 15 18 3 6 9 12
HOURS IN LIGHT <-f--HOURS IN DARK-H
TYPE OF SYNCHRONOUS CHLORELLA CULTURE
Fig. 6. Total RNA and phosphorus per 10 ml p.c.v.
of Chlorella during synchronous growth. In one case
( *) chloramphenicol was added at nine hours of light.One RNA OD unit is equivalent to 1 ml of solution withan absorbance of 1.0 at 260 m,t.
though the data in figure 6 are on the basis of p.c.v.,
during one life cycle the p.c.v. increased two- or three-fold (16), so that total RNA or total phosphorus per
culture would have shown even greater increases (lur-
ing the first 9 hours of growth. There was a close
correlation between changes in amount of total phos-phorus and amount of total RNA in these fractionson the basis of the algal p.c.v. at each stage in theChlorella life cycle. A three-fold increase in the
amount of RNA and polyphosphate per mlilliliterp.c.v. occurred during the first 9 hours in the light,when the cells grew in size but not in number. Be-tween 9 and 12 hours in the light, a large decrease inthe RNA and polyphosphate took place, when the cellsshould have been preparing for nuclear division.
DEAE-cellulose elution patterns from eight rep-
resentative stages in the Chlorella life cycle were
determined by optical densities at 260 m,u and totalphosphorus (fig 7 & 8). For each fractionation, sixelution areas of RNA and polyphosphate, labeled Aand F on the figures, were selected for analysis, sincethey were eluted at about the same place in each ex-
periment and since the amounts of each showed sys-
tematic rather than erratic changes. Changes inRNA in these six areas (A-F) duriiig the life cycle
10 ml p.c.v. of Chlorella from synchronous cultures ofdesignate state in their life cycle (fig 2). DEAE-Cellu-lose fractions A to F are designated above each elutionpattern.
a:
0
0%
:2
-j
CL)
0
0.
0
I
a.
0
0O 20 40 60 80 100
TUBE NUMBER
Fig. 8. Elution patterns of RNA-polyphosphate per10 ml p.c.v. of Chlorella from synchronous cultures.DEAE-cellulose fractions A to F are designated aboveeach elution pattern.
of Chlorella are illustrated in figure 9. All the RNAcomponents increased in total amount during 0 to 9hours of light and areas D, E, and F decreased sharplybetween 9 and 12 hours of light. Areas B, D, and Eagain increased during the first 6 hours in the dark.Area F, which was probably of highest molecularweight, because it required the highest salt concen-tration for elution, showed the greatest changes.Treatment with chloramphenicol from the 9- to the12-hour light period prevented a rapid decrease inthe RNA content of areas D and E which normallyoccurred during this period. In fact, the RNA inareas D and E continued to increase during this 3-hour treatment at a rate about equivalent to the pre-vious 9-hour cell growth phase. However, the chlor-amphenicol had little influence upon the rapid disap-pearance of the large RNA fraction in area F after
.30> ~~B
U20 A
I0
0
s100 ,_ \ '
cr 80 _ ,
roo
TYPE OF SYNCHRONOUS CHLORELLA CULTURE
Fig. 9. Changes in Chlorella RNA during synchron-ous growth. In one case (@*) chloramphenicol wasadded at nine hours of light for 3 hours before harvest.DEAE-Cellulose fractions labeled A through F werefrom figures 7 and 8.
the 9-hour light stage. In the dark part of the lifecycle there was a build-up of RNA in the first 6hours, and in the last 6 hours of darkness, the RNAcontent returned to a low level. In order to main-tain a viable culture a dark period or one of lowlight intensity was necessary. During this darkperiod changes did occur in total RNA and in quali-tative distribution among the different chromato-graphic fractions.
Changes in the amount of total phosphorus ineach of the six elution areas from the DEAE-cellulosecolumns are shown in figure 10. As with RNA,phosphorus, mainly as polyphosphate, increased in allareas during the first 9 hours in the light and thenall but area A decreased rapidly between 9 and 12hours in the light. The amounts of polyphosphate inareas D and E were by far the largest, whereas thelargest RNA fraction was F. There was, however,a significant amount of polyphosphate in all areasexcept F which contained little phosphorus after 12hours of light. Chloramphenicol treatment at the www.plantphysiol.orgon May 15, 2018 - Published by Downloaded from
HOURS IN LIGHT *|* HOURS IN DARK-HTYPE OF SYNCHRONOUS CHLORELLA CULTURE
Fig. 10. Changes in total phosphorus of ChlorellaRNA-polyphosphate fractions. DEAE-Cellulose frac-tions are labeled A to F.
9-hour light period did not influence the loss ofpolyphosphates from areas D, E, and F although itdid prevent RNA loss from areas D and E.0 Ratios of RNA to Polyphosphates. In table IIIthe amounts of total RNA, total phosphorus, and acid-labile phosphorus are summarized from nmany Ana-baena and Chlorella phenol preparations which hadbeen fractionated on DEAE-cellulose columns. Thevalues are the sum of the combined areas of the totalelution pattern. The ratio of 20 RNA optical den-
sity units per milligram of total phosphorus in thetotal RNA-polyphosphate present in the middle partof the Anabaena growth curve (fig 5) was about thesame as in synchronous Chlorella in the light growthphase of their life cycle. This similarity seems sig-nificant, although it may be a coincidence of ourlimlitedl data. The low ratio observed in Chlorellakept in continuous high light was caused by a (le-crease in total RNA rather than an increase in totalphosphorus.
Each combine(d eluate area from a DEAE-cellulosecolumn fractionationi of the RNA-polyphosphatesfrom a Chlorella culture was analyzed for ribose anid(leoxyribose. No dleoxyrilbose could be found. Ri-bose was found in each eluted fraction. From areasD and E the Ag ribose per RNA OD unit calculatedlas 13 from area D and 17 yg from area El an(d avalue of 15 iAg or 0.1 /uimole ribose per RNA OD unitwas arbitrarily taken for approximate calculationis.Since only purine nucleotides were (letermiiined in theorcinol reaction, this value slhould be approximatelydoubled (0.2 Atmoles total ribose per RNA OD unit).These calculations wvere carrie(d out in order to at-tach a physical meaning to the ratio of 20 RNA ODunits per mg total phosphorus (table III). There-fore, one RNA OD unit was equixalent to 50 Ag ol1.6 umole of phosphorus. Thus for every mlicro-mole of ribose in the total system of complexes therewere about eight micromoles of phosplhorus of whiclh7 micromoles would be polyphosphate-phosphorns.This calculate(d ratio of RNA phosphorus to polyphos-phate-phosphorus mzay be in consi(lerable error, but
Table IIIInfluence of Light Upon Ratio of Total RNA OD Units to mg Total Phosphorus*
Culture conditions RNA units/ mg total-P/ mg97-P/10 ml p.c.v. 10 ml p.c.v. 10 ml p.c.v.
A7-P RNA units/Diff.-P mg total-P
3.6 ml p.c.v./l7.2 ml p.c.v./l8.0 ml p.c.v./l
11.2 ml p.c.v./l13.0 ml p.c.v./l25.0 ml p.c.v./l29.6 ml p.c.v./l
it serves to emphasize that there was much more poly-phosphate than RNA-phosphorus present.
The ratio of 7-minute acid-labile phosphorus todifference-phosphorus (tables II & III) varied fromabout 1 to 16 and was usually 8 or less. Acid hy-drolysis should have resulted in the release of allphosphate which was bound only by anhydride link-ages.
DiscussionMaterial isolated from Anabaena and Chlorella
by solvent extractions and then DEAE-cellulose ionexchange column chromatography contained bothRNA and polyphosphate. This fraction containedbetween 25 to 35 % of the total phosphorus in Ana-baena and a major portion of the alga's RNA. NoDNA, amino acids, protein or significant amounts ofother phosphorus compounds were detectable. RNAwas identified by alkaline hydrolysis to the free nu-cleotides which were isolated by column chroma-tography and identified by their ultraviolet spectra.Adenine, guanine, cytidylic acid, and uridylic acidwere also identified by paper chromatography of acidhydrolysates. Polyphosphate was identified by suchclassical characteristics as alkali-stability, acid-lability, precipitation with barium ion at pH 4.0, andstrong metachromatic reaction with toluidine blue.Thus, RNA and polyphosphate were the only twocomponents identified in the isolated material.
When RNA-polyphosphate isolated from Chlorellawas chromatographed on DEAE-cellulose columns,the material separated into numerous fractions. Sixelution areas were selected because of consistencyin chromatographic position and systematic changesduring synchronous growth of the algae. Each areacontained both the RNA and polyphosphate compo-nents. However, the relative amount of material ineach area depended upon the stage of algae growth.Further, the relative distribution between RNA andpolyphosphate was not the same in the different frac-tions. Area F, which eluted at the highest salt con-centration, contained a small amount of polyphosphaterelative to its RNA content. Areas D and E, incontrast, always contained a large amount of polyphos-phate. During the first 9 hours of light both RNAand polyphosphate synthesis were rapid. During thefirst 6 hours the amount of RNA in area F increasedmost rapidly (fig 9), but during the period of 6 to 9hours RNA in area F did not increase significantly,while RNA components in D and E continued to in-crease. These data suggest that the RNA portion ofthe fractions was synthesized first and was isolatedin area F. The polyphosphate for area D and E maythen have been elaborated by an enzymatic process in-volving ATP with a consequential modification of thechromatographic properties of the RNA. The result-ing RNA-polyphosphate was then found in areas Dand E. This hypothesis is supported by polyphos-phate synthesis which was more rapid from 6 to 9hours of light than between 0 and 6 hours. This hy-pothesis is consistent with speculations by other in-
vestigators (2, 4, 8), that polyphosphates are syn-thesized on the surface of RNA.
A large synthesis of Chlorella RNA was noted dur-ing the first 6 hours of darkness without a corres-ponding synthesis of polyphosphate. One wondersabout the function of this RNA, since it was syn-thesized and lost during a period of dark starvationand suspended growth of the cells. A similar situa-tion occurred in a large synthesis of RNA by anauxophilic mutant of Escherichia coli when methio-nine was withheld (24). When methionine wasadded to that deficient medium, no protein synthesisoccurred for some time suggesting that the accumu-lated RNA was nonfunctional.
A ratio of about 1 mole of nucleotides to 7 molesof polyphosphate-phosphorus was consistently foundin the total RNA-polyphosphate fractions from bothChlorella and Anabaena providing the algae cultureswere in a state of rapid growth and not under theinfluence of abnormal environments. These calcula-tions were based upon total RNA analysis by ODand ribose as compared to total phosphorus. Varia-tions from the ratio of 7 polyphosphate-phosphorusper nucleotide were noted for Chlorella at threestages of growth. In early light stages polyphosphatesynthesis lagged behind RNA synthesis as was re-flected by a reduction of the ratio to 5. This ratiodropped to 3 polyphosphate-phosphorus per nucleo-tide when the algae were treated with chloramphenicolbecause of a failure of normal RNA utilization. Inthe dark the algae accumulated RNA without a build-up of polyphosphate and the ratio was about 4 after6 hours of darkness.
The complex changes in the RNA-polyphosphatefractions cannot now be precisely correlated withstages of cellular growth and division. However,the increase in RNA-polyphosphate in the first 9hours of light could simply involve a preparation forcertain energy requiring syntheses which take placebetween 9 and 12 hours of light when the RNA-poly-phosphate was rapidly utilized. This utilization justpreceded nuclear division at 14 hours of light. Thecorrelation between RNA-polyphosphate content andthe stages of Chlorella growth seems very significant,but biological variations must be considered sinceeach 3-hour harvest was taken 3 or 4 days apart (lur-ing several weeks in order to obtain sufficient ma-terial for the analyses.
SummaryAlgae, Anabaena, and Chlorella, were mass cul-
tured for the isolation of RNA-polyphosphate frac-tions. In actively growing Anabaena cells total poly-phosphates constituted about 40 to 50 % of the totalphosphorus, and the polyphosphate in the RNA-polv-phosphate fraction amounted to about 30 %. Theisolation consisted of solvent extraction proceduresfollowed by DEAE-cellulose chromatographic frac-tionation into a spectrum of RNA-polyphosphatetypes. Six areas from the column chromatogramswere then analyzed for amounts of RNA and polv-phosphate. No other components were detected.
All of the chromatographic areas contained bothRNA and polyphosphate. Within the total RNA-polphosphate complex there was about one nucleotideto seven polyphosphate phosphorus from both Chlorel-la and Anabaena providing the algae cultures werein a state of rapid growth.
The relative amount of RNA-polyphosphate ineach chromatographic area depended upon the stageof synchronous Chlorella growth. During the first9 hours of light, RNA-polyphosphate synthesis wasrapid and accumulatedl in the three areas that elutedwith the highest salt concentration. During the next3 hours these fractions decrease(l while the cells werepreparing for nuclear division. Chloramphenicolprev-ented the decrease in RNA at that time withoutaffecting polyphosphate utilization. Significantchanges also occurred during the rest of the Chlorellalife cycle.
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