Plant Physiol. (1975) 56, 600-604

Ribulose 1, 5-Diphosphate Carboxylase Synthesis in EuglenaII. EFFECT OF INHIBITORS ON ENZYME SYNTHESIS DURING REGREENING AND SUBSEQUENT

TRANSFER TO DARKNESS

Received for publication April 28, 1975

J. MICHAEL LORD, TERENCE L. ARMITAGE, AND MICHAEL J. MERRETTPostgraduate School of Studies in Biological Sciences, University of Bradford, Bradford, Yorkshire, BD7 lDPEngland

ABSTRACT

Dark-grown Euglena gracilis Klebs strain Z Pringsheimcells, which have been partially regreened in the light, showa striking, continued synthesis of the chloroplast enzymeribulose 1,5-diphosphate carboxylase on transfer back intodarkness. This dark synthesis of the enzyme was completelyprevented by the addition of 15 ,g/ml of cycloheximide tothe culture medium but was unaffected, for at least 8 hours,by the addition of 1 mg/ml of D-threo-chloramphenicol.The addition of either cycloheximide or D-threo-chloram-phenicol to dark-grown cultures at the onset of illumina-tion completely inhibited the light-induced synthesis ofribulose 1, 5-diphosphate carboxylase. When cells which hadbeen illuminated in the presence of D-threo-chlorampheni-col, and hence were unable to synthesize ribulose 1,5-di-phosphate carboxylase, were transferred to darkness in theabsence of this inhibitor, synthesis of the carboxylase thenoccurred. Dark-grown cells which had been illuminated inthe presence of cycloheximide failed to synthesize the en-zyme when placed in the dark in the absence of cyclohexi-mide. The addition of 5-fluorouracil to regreening culturesto prevent light-induced transcriptional steps completelyblocked the synthesis of ribulose 1,5-diphosphate carboxyl-ase.

The illumination of dark-grown, bleached Euglena graciliscells initiates the synthesis of the chloroplast enzyme ribulose1 ,5-diphosphate carboxylasel (23). The activity of this enzymeincreases, along with the cellular level of chlorophyll and otherenzymic and structural components of the developing chloro-plast, for 3 to 4 days, by which time the cells are fully regreenedand photosynthetically competent (4, 7). In a previous publica-tion, we reported that if at anytime during the regreening processthe cells are transferred back into darkness, there is a strikingcontinued synthesis of RuDPCase in the dark (21). These cellsgrew rapidly in darkness, but the increase in RuDPCase was notrelated to cell-division; cells regreening in the resting mannitolmedium (26) and placed in the dark likewise synthesized RuDPC-ase, although cell division did not occur (unpublished obser-vation).The present study involved a further examination of this phe-

I Abbreviation: RuDPCase: ribulose 1, 5-diphosphate carboxylase.

nomenon using inhibitors that specifically prevent genetictranscription or translation in either the chloroplast or nucleusand cytoplasm. The implications of these results are discussed inrelation to the contributions of the chloroplast and nucleargenomes during the synthesis of the two subunit types whichconstitute the multimeric chloroplast RuDPCase.

MATERIALS AND METHODS

Growth of Alga. Dark-grown cells were obtained by growingEuglena gracilis Klebs strain Z at 25 C, with stirring, in themedium of Hutner et al. (15) in the dark for 4 days. The cellswere harvested by centrifugation and were transferred to freshgrowth medium from which all carbon sources other than vita-mins had been omitted. Cultures were maintained in the dark for48 hr at 25 C to deplete reserve carbohydrates. Resting cells wereregreened by exposure to continuous white light (6000 lux) afterthe addition of D-glucose to give a final concentration of 22.5 g/l.Regreening cultures were transferred back into darkness afterharvesting the cells by centrifugation and resuspending them inan equivalent volume of Hutner's medium (15).

Inhibitors were dissolved in the appropriate culture mediumand sterilized by Seitz filtration before being added to cultures attimes indicated under "Results."

Inhibitors. The D-threo-isomer of chloramphenicol was usedexclusively throughout this work (8). The final concentration ofinhibitors used was D-threo-chloramphenicol, 1 mg/ml; cyclo-hemixide, 15 ,g/ml; 5-fluorouracil, 5 mm; rifampicin, 250 ,ug/ml.All inhibitors were obtained from Sigma (U.K.)

Preparation of Extracts. Euglena cells were harvested by cen-trifugation, washed with 20 mnim tris-HCl, pH 8, containing 10mnm MgCl2 and 1 mm dithiothreitol, and then resuspended in asmall volume of this buffer. The cells were disrupted by three15-sec bursts of ultrasonic waves (M.S.E. ultrasonic disintegrator,1.5 amp). Microscopic examination revealed that this treatmentresulted in virtually 100% cell breakage. Cell debris was removedby centrifuging at 20,000g and 4 C for 10 min, and the super-natant was used immediately.RuDPCase Assay. The RuDP-dependent incorporation of

NaH'4C03 into acid-stable product was determined exactly asdescribed previously (21).

RESULTS AND DISCUSSION

Euglena RuDPCase has been recently purified and charac-terized (22). This enzyme has the high mol wt and distinctivequaternary structure which is typical for this enzyme isolatedfrom higher plants and green algae (11, 16, 18, 20, 27). In thepresence of sodium dodecyl sulfate, the enzyme dissociates intotwo nonidentical subunits of mol wt 55,000 and 15,000; eight

600

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RuDP CARBOXYLASE SYNTHESIS IN EUGLENA

large and eight small subunits are thought to constitute themultimeric chloroplast enzyme (22). In keeping with current con-cepts regarding the origin of synthesis of this enzyme in eukary-otic cells (3, 6, 12, 13, 19), it is assumed that the larger subunit ofEuglena RuDPCase is coded for by chloroplast DNA and syn-thesized on 68S chloroplast ribosome, while the small subunit iscoded for by nuclear DNA and synthesized on 87S cytoplasmicribosomes.

D-Threo-chloramphenicol and streptomycin are effective in-hibitors of protein synthesis in bacteria, binding to the 30S sub-unit of the 70S bacterial ribosome (10, 17). These inhibitors alsoprevent protein synthesis on the 70S type ribosomes isolated fromeither Euglena chloroplasts or mitochondria but they do notaffect protein synthesis on the larger cytoplasmic ribosomes (2).In contrast, cycloheximide specifically blocks protein synthesisby cytoplasmic ribosomes (2). When chloramphenicol at a con-centration of 1 mg/ml is added to the suspending medium ofEuglena cells, it effectively blocks protein synthesis on 68S chloro-plast ribosomes; RuDPCase synthesis during the regreening ofdark grown cells was completely inhibited (5, 7). This concentra-tion of chloramphenicol has little effect on cell division orviability in Euglena, suggesting that it does not block protein syn-thesis on cytoplasmic ribosomes. This ineffectiveness has beenconfirmed by Bovarnick et al. (4, 5) who reported that chloram-phenicol, even at 2 mg/ml, did not prevent the synthesis ofNADP-linked triose phosphate dehydrogenase in regreeningEuglena. This enzyme is apparently translated on 87S cytoplasmicribosomes since its synthesis was completely prevented by 15,ug/ml of cycloheximide (5). The failure of chloramphenicol toinhibit cell division in Euglena suggests that protein synthesis onmitochondrial ribosomes is not prevented in vivo, possibly be-cause mitochondria are impermeable to this inhibitor (5).

In the present work we have used cycloheximide at a concen-tration of 15 Ag/ml. This concentration effectively blocks proteinsynthesis on cytoplasmic ribosomes in Euglena (2) and preventscell division and Chl synthesis when dark-grown cells are placedin the light (7, 25). When this inhibitor is used at lower concentra-tions the situation is more complex; 2 to 5 Ag/ml actually stimu-lates chloroplast development (25) although the reasons for thisare unclear. It is assumed here that 1 mg/ml of chloramphenicolspecifically blocks protein synthesis on 68S chloroplast ribosomesincluding the synthesis of the large subunit of RuDPCase, whereas15 Ag/ml of cycloheximide specifically blocks protein synthesison 87S cytoplasmic ribosomes including the synthesis of the smallsubunit of RuDPCase.Dark-grown Euglena cells which had been regreening for 24 hr

in the light were harvested and resuspended in an equivalent vol-ume of fresh Hutner's medium. Transferring such cultures todarkness for a further 24 hr resulted in a linear increase inRuDPCase activity during this period (Fig. 1A). When the re-greening cells were suspended in Hutner's medium containingchloramphenicol, and were placed in the dark, RuDPCaseactivity again increased (Fig. 1B), the rate of increase during thefirst 8 hr in darkness being the same as that observed in controlcultures (Fig. 1A). The presence of cycloheximide, however,completely prevented the dark increase in RuDPCase activity(Fig. 1C).RuDPCase activity increased when expressed on a protein

basis in both the control culture (Fig. 2A) and in the culture con-taining chloramphenicol (Fig. 2B) during the first 8 hr aftertransfer to darkness. On a per cell basis, this increase was lesspronounced and was completely inhibited by cycloheximide(Fig. 2C).i The effect of chloramphenicol and cycloheximide on the darksynthesis of RuDPCase was the same regardless of what stageduring regreening the cells were transferred to the dark. As shownin Figure 3, chloramphenicol permitted the synthesis of RuDPC-

0 8 16 24Hrs in dark

FIG. 1. Effect of inhibitors on the increase in RuDPCase activityafter transferring regreening Euglena cultures to darkness. A: Controlculture; B: culture containing 1 mg/ml of chloramphenicol; C: culturecontaining 15 jug/mI of cycloheximide.

ase during a 24-hr dark period with cells which had been re-greened for 24, 48, or 72 hr in the light, whereas cycloheximideresulted in complete inhibition.These data (Figs. 1-3) indicate that the increase in activity of

RuDPCase observed when regreening Euglena cultures are trans-ferred to darkness is dependent upon protein synthesis on 87Scytoplasmic ribosomes but not synthesis on 68S chloroplastribosomes. The failure of chloramphenicol to prevent the increasein activity may have resulted from the synthesis of an excess oflarge subunit, with respect to the small subunit of the enzyme, onchloroplast ribosomes in the light before transfer to darkness.The inhibition of RuDPCase synthesis in the dark in the presenceof cycloheximide indicates that the continued synthesis of thesmall subunit as a translational product of cytoplasmic ribosomesis prevented and, if this is the case, that such synthesis is necessaryfor the increase in RuDPCase activity.The addition of either chloramphenicol or cycloheximide to

regreening cultures in the light prevented the synthesis ofRuDPC-ase during a 24-hr period (Fig. 4). One interpretation of theseresults is that chloramphenicol and cycloheximide inhibit the syn-thesis of the large subunit and the small subunit of RuDPCase,respectively, and that the synthesis of both subunits is required forthe assembly of the functional multimeric carboxylase. Recentwork has shown that cycloheximide completely blocks the light-dependent synthesis of Euglena chloroplast aminoacyl-tRNA

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LORD, ARMITAGE, ANDMERRETP.

0-6

0*4

A

0-2-

0

06

.:

-0 0-420Q.C)

E

D

0

C

0

0-2-

0 2 4 6 8Hrs indark

0-2

activation of amino acids by cytoplasmically-synthesized chloro-plastic aminoacyl-tRNA synthetases, or the transcription of themRNA for the small subunit from nuclear DNA and its transla-tion on cytoplasmic ribosomes. When cells were transferred todarkness in the absence of chloramphenicol, the translation oflarge subunit mRNA accumulated in the light and the continuedsynthesis of the small subunit would permit the assembly of themultimeric carboxylase.

0-1

03r

0-2

-ii

100-1 o-

I...cD .G-

u

E000-2

0-2

0-1

FIG. 2. Changes in the activity of RuDPCase in relation to the pro-tein content of extracts (a) or the cell number of the cultures (0) aftertransferring regreening Euglena cultures to darkness. A: Control cul-ture; B: culture containing 1 mg/ml of chloramphenicol; C: culturecontaining 15 ,/g/ml of cycloheximide.

synthetases during the regreening of dark-grown cells (14). Thesechloroplast synthetases are apparently transcribed from nucleargenes and are translational products of cytoplasmic ribosomesbefore being compartmentalized within the chloroplasts. Thus,by inhibiting the light-induced synthesis of chloroplast amino-acyl-tRNA synthetases on cytoplasmic ribosomes, cycloheximidemight indirectly inhibit chloroplast protein synthesis, particularlythe translation on chloroplast ribosomes of the mRNA for thelarge subunit of RuDPCase. The possibility that the synthesis ofone subunit type is dependent on or regulated by the concurrentsynthesis of the other subunit type cannot be discounted.

Cells which had been exposed to light for 24 hr in the presenceof the inhibitors were harvested, washed, and resuspended infresh Hutner's medium in the absence of inhibitors, and wereplaced in the dark for 24 hr. The cells which had been exposed tocycloheximide during the light period showed no increase inRuDPCase activity during the subsequent dark period. This lackof increase may be the result of the inhibition of large subunitsynthesis in the light via the effect of cycloheximide on chloro-plast aminoacyl-tRNA synthetases as discussed above; or, per-haps more likely, this lack of effect occurs because the in-hibitor once bound to the cytoplasmic ribosomes is not readilydisplaced, cannot be completely removed by washing the cellsand hence continues to inhibit the synthesis of the small subunit.On the other hand, cells which had been regreened in the presenceof chloramphenicol showed a marked increase in RuDPCase ac-tivity in the dark in the absence of this inhibitor, although nosynthesis had occurred in the previous light period (Fig. 4).Chloramphenicol inhibits the translational step on chloroplastribosomes, so it should not prevent the light-induced transcriptionof the mRNA for the large subunit within the chloroplast, the

II

I 1 I

-A -

---A

0 20 40 60 80 100Hrs in light

FIG. 3. Effect of inhibitors on RuDPCase synthesis during a 24-hrdark period after different periods of regreening in the light. Regreeningcells were transferred (at the times indicated by the arrows) to darknessfor 24 hr in the presence of 1 mg/ml of chloramphenicol (@) or 15 ,g/ml of cycloheximide (A); cells continuously illuminated (0).

0-1

0-08

'D 0-06E0

0

0-02

00 24 48

Hours

FIG. 4. Effect of inhibitors on RuDPCase synthesis in the light andthe dark. No inhibitor in light, chloramphenicol present in dark (0);no inhibitor in light, cycloheximide present in dark (0); chlorampheni-col present in light, no inhibitor in dark (A); cycloheximide present inlight, no inhibitor in dark (A).

Light I Dark

2

3

. ,'

602 Plant Physiol. Vol. 56, 1975

B

1-

4

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RuDP CARBOXYLASE Sl

Table I. Effect ofLight Intensity during a 24-hr Regreening Periodon Synthesis of Enzyme

RuDPCase synthesis was blocked by the addition of t mg/ml ofchloramphenicol during a 24-hr regreening period and activity wasdetermined after a subsequent 24-hr period in darkness in the ab-sence of the inhibitor.

RuDPCase Activity

Light Intensity After 24 hr in After further 24 hrlight plus in dark without Increase

chloramphenicol inhibitor

lux X 103 ugnits/1OO ml culture fold

2 0.009 0.047 5.24 0.011 0.053 4.86 0.009 0.048 5.38 0.010 0.050 5.010 0.010 0.049 4.9

Chloramphenicol completely inhibited RuDPCase synthesisduring regreening over a range of light intensities. The removalof chloramphenicol resulted in a marked increase in RuDPCaseactivity during a subsequent 24-hr dark period; an approximately5-fold increase in the level of carboxylase activity was observeddpring this period, regardless of the light intensity during re-greening (Table I).The increase in RuDPCase activity, observed in the dark with

cells which had been regreened in the presence of chlorampheni-col and then had been resuspended in fresh Hutner's medium,was completely prevented by the addition, at the time of transferto darkness, of either chloramphenicol itself or cycloheximide(Fig. 5). This indicates that cells in which protein synthesis onchloroplast ribosomes in the light had been blocked by the pres-ence of chloramphenicol were dependent upon translational stepson both chloroplast and cytoplasmic ribosomes during the darkperiod for enzyme synthesis. This is readily explained in the caseof cells treated with chloramphenicol during both the light anddark periods; synthesis of the catalytically-active, large subunit onchloroplast ribosomes would be blocked at all stages. The failureof cells, treated with chloramphenicol in the light, to synthesizeRuDPCase in the dark in the presence of cycloheximide is lesseasily explained. Such cells are able to perform translational stepson chloroplast ribosomes in the absence of inhibitors, and if theeffect of cycloheximide is simply to block protein synthesis oncytoplasmic ribosomes, the data indicates that an excess of smallsubunit has not been accumulated in the light. This may indicatethat the synthesis of the large chloroplast subunit in the light, inthis experiment blocked by chloramphenicol, is necessary for con-comitant synthesis ofthe small subunit on cytoplasmic ribosomes.Alternatively, since the synthesis of the large subunit in the lighthas been blocked, the data could also be interpreted as suggestingthat the synthesis of the small subunit on cytoplasmic ribosomesblocked in the dark in the presence of cycloheximide, is requiredfor the synthesis of the large chloroplast subunit. Recent work onthe effect of inhibitors on RuDPCase synthesis during the green-ing of detached etiolated pea shoots has provided some evidencethat may indicate that the small subunit is a positive factor re-

quired for either the transcription ofmRNA for the large subunitfrom chloroplast DNA or its translation on chloroplast ribosomes(9). The inhibitor used was 2-(4-methyl-2,6-dinitroanilino)-N-methyl-propionamide, which inhibits protein synthesis on cyto-plasmic ribosomes but not on chloroplast ribosomes in plants;furthermore, it does not inhibit the synthesis of the large subunitof RuDPCase by isolated chloroplasts (9). When this inhibitorwas supplied to greening, detached pea shoots, it was expected toinhibit selectively the synthesis of the small subunit, with the pos-sibility that the large subunit would accumulate in the tissue.

eNTHESIS IN EUGLENA 603.

Measurement of the large subunit content of the tissue by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis showed thatthis was not the case, and led Ellis (9) to suggest that the presenceof the small subunit may be necessary for, and may control, thesynthesis of the large subunit. This interpretation does not includethe possibility that inhibitors of protein synthesis on cytoplasmicribosomes might block the light-induced synthesis on such ribo-

008

' 0-06E0

0

*A 0-04

0-02

00 24 48

Hours

FIG. 5. Effect of inhibitors on RuDPCase synthesis in the dark bycells in which light-dependent enzyme synthesis had been blocked bythe addition of chloramphenicol. Chloramphenicol present in light, noinhibitor in dark (A); chloramphenicol present in both the light anddark periods (0); chloramphenicol present in light, cycloheximidepresent in dark (-).

Light Dark

015 -

E 01

0'~~~~~.CD)

005 -

0 24 48Hours

FIG. 6. Effect of 5-fluorouracil on RuDPCase synthesis in the lightand the dark. Control culture (0); culture containing 5 mM 5-fluoroura-cil (0)-

Light | Dark |

I' />- _ > = S 3~~~~~~~~~~~~~~~~~~~~

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LORD, ARMITAGE, AND MERRETT

somes of components required for protein synthesis within thechloroplast, e.g. chloroplast aminoacyl-tRNA synthetases (14).The data reported in the present paper do not allow us to differ-entiate between this hypothesis or alternative explanations forthe control of the synthesis of RuDPCase.The synthesis of Euglena RuDPCase during a 24-hr regreening

period and a subsequent 24-hr dark period was prevented by theaddition of 5-fluorouracil at concentrations known to inhibitRNA synthesis in Euglena (Fig. 6, and ref. 24). When this in-hibitor was removed at the time of transfer to darkness, no syn-thesis was observed during the dark period. It is possible that theinhibitor was not effectively removed; nevertheless, this resultclearly indicates that the synthesis of RuDPCase in either thelight or the dark period requires light-dependent transcriptionalsteps.The chloroplast-specific inhibitor ofRNA polymerase, rifampi-

cin (28), added at a concentration of 250 ,g/ml, had no effect onRuDPCase synthesis during regreening or the subsequent darkphase (data not shown). This concentration of rifampicin haslikewise been reported to be without effect on RuDPCase synthe-sis in synchronous cultures of the green alga, Chlamydomonasreinhardtii (1). Assuming a specific site of action for rifampicin,these results suggest that chloroplast DNA transcription is notrequired for the synthesis of RuDPCase and, in particular, thatthe large subunit of the enzyme is a transcriptional product ofnuclear DNA, although translated on chloroplast ribosomes (1).It seems possible that the concentration of rifampicin used was toolow for effective inhibition; the work of Chan and Wildman hasshown that the large subunit of tobacco RDPCase is a transcrip-tional product of chloroplast genes (6).The results obtained in the present study suggest that the in-

crease in RuDPCase observed after Euglena cells which havebeen regreening in the light are transferred to darkness (21) maybe attributable to an excess of large subunit synthesized onchloroplast ribosomes in the light. If this synthesis is blocked bythe addition of chloramphenicol, the increase in RuDPCase ac-tivity, which ensues when such cells are placed in the dark, indi-cates an accumulation of large subunit mRNA and activatedamino acids in the light, which allows translation of this peptidein the dark. The inhibition of the dark increase in RuDPCaseactivity by cycloheximide suggests the continuing synthesis ofthe small subunit on cytoplasmic ribosomes in the dark. After a

period of regreening, RuDPCase synthesis proceeds faster duringa subsequent 24-hr dark period than it does in cells maintainedin continuous light (Fig. 3, and ref. 21). This could be attribut-able to regulation of enzyme synthesis by light, which possiblycontrols the rate of synthesis of the small subunit.

Acknowledgment-The authors thank Professor B. A. 'McFadden, WashingtonState University, for critically reading the manuscript.

LITERATURE CITED

1. ARMSTRONG, J. J., S. J. SURZYCKI, B. 'MOLL, AND LEVINE, R. P. 1971. Genetic

transcription and translation specifying chloroplast components in Chlamydom-onas reinhardi. Biochemistry 10: 692-701.

2. AVADHANI, N. G. AND D. E. BUETOW. 1972. Isolation of active polyribosomes fromthe cytoplasm, mitochondria, and chloroplasts of Euglenza gracilis Biochem. J.128: 353-365.

3. BLAIR, G. E AND R. J. ELLIS. 1973. Protein synthesis in chloroplasts. 1. Light-driven synthesis of the large subunit of fraction I protein by isolated pea chloro-plasts. Biochim. Biophys. Acta 319: 223-234.

4. BOVARNICK, J. G., S. W. CHANG, J. A. SCHIFF, AND S. D. SCHWARTZBACH. 1974.Events surrounding the early development of Euglena chloroplasts: experi-ments with streptomycin in non-dividing cells: J. Gen. Microbiol. 83: 51-62.

5. BOVARNICKC, J. G., J. A. SCHIFF, Z. FREEDMAN, AND J. M. EGAN. 1974. Eventssurrounding the early development of Euglena chloroplasts: cellular origins ofchloroplast enzymes in Euglena. J. Gen. Microbiol. 83: 63-71.

6. CHAN, P. AND S. G. WILDMAN. 1972. Chloroplast DNA codes for the primarystructure of the large subunit of fraction I protein. Biochim. Biophys. Acta277: 677-680.

7. DAVIS, B. AND M. J. MERRETT. 1975. The glycolate pathway and photosyntheticcompetence in Euglena. Plant Physiol. 55: 30-34.

8. ELLIS, R. J. 1969. Chloroplast ribosomes: stereo-specificity of inhibition by chlor-amphenicol. Science 163: 477-478.

9. ELLIS, R. J. 1975. Inhibition of chloroplast protein synthesis by lincomycin and2-(4-methyl-2, 6-dinitroanilino)-N-methylpropionamide. Phytochemistry 14:89-93.

10. FLAKS, J. G., E. C. COX, ANDJ. R. WHITE. 1962. Inhibition of polypeptide synthe-sis by streptomycin. Biochem. Biophys. Res. Commun. 7: 385-389.

11. GIVAN, A. L. AND R. S. CRIDDLE. 1972. Ribulose diphosphate carboxylase from

Chlamydomonas reinhardii: purification, properties, and its mode of synthesisin the cell. Arch. Biochem. Biophys. 149: 153-163.

12. GOODING, L. R., H. RoY, AND A. T. JAGENDORF. 1973. Immunological identifica-tion of nascent subunits of wheat ribulose diphosphate carboxylase on ribosomesof both chloroplast and cytoplasmic origin. Arch. Biochem. Biophys. 159: 324-335.

13. GRAY,J. C. AND R. G. 0. KEKWICK. 1974. An immunological investigation of thestructure and function of ribulose 1 ,5-diphosphate carboxylase. Eur.J. Biochem.44: 481-489.

14. HECKER, L. I., J. EGAN, R. J. REYNOLDS, C. E. Nix, J. A. SCHIFF, AND W. E.BARNETT. 1974. The sites of transcription and translation for Euglena chloro-plastic aminoacyl-tRNA synthetases. Proc. Nat. Acad. Sci. U.S.A. 71: 1910-1914.

15. HUTNER, S. H., M. K. BACH, AND G. I. M. Ross. 1956. A sugar-containing basalmedium for vitamin B1, assay with Euglena. J. Protozool. 3: 101-112.

16. IWANIJ, V., N. H. CHUA, AND P. SIEKEVITZ. 1974. The purification and some

properties of ribulose diphosphate carboxylase and of its subunits from thegreen alga Chlamydomonas reinhardtii. Biochim. Biophys. Acta 358: 329-390.

17. KAJI, H. AND Y. TANAKA. 1968. Binding of dihydrostreptomycin to ribosomalsubunits. J. Mol. Biol. 32: 221-230.

18. KAWASHIMA, N. AND S. G. WILDMAN. 1970. Fraction I protein. Annu. Rev. PlantPhysiol. 21: 325-358.

19. KAWASHIMA, N. AND S. G. WILDMAN. 1972. Studies on fraction I protein. IV.Mode of inheritance of primary structure in relation to whether chloroplast or

nuclear DNA contains the code for a chloroplast protein. Biochim. Biophys.Acta 262: 42-49.

20. LORD,J. M. AND R. H. BROWN. 1975. Purification and some properties of Chlorellafusca ribulose 1 5-diphosphate carboxylase. Plant Physiol. 55: 360-364.

21. LORD, J. M. AND M. J. MgERRETT. 1975. Ribulose diphosphate carboxylase syn-thesis in Euglena. Increased enzyme activity after transferring regreening cellsto darkness. Plant Physiol. 55: 890-892.

22. McFADDEN, B. A., J. M. LORD, A. RoWE, AND S. DILKS. 1975. Composition,quaternary structure and catalytic properties of D-ribulose-1,5-diphosphatecarboxylase from Euglena gracilis. Eur. J. Biochem. 54: 195-206.

23. SCHIFF, J. A. 1973. The development, inheritance, and orgin of the plastid inEuglena. Adv. Morphog. 10: 265-312.

24. SMILLIE, R. M. 1963. Formation and function of soluble proteins in chloroplasts.Can. J. Bot. 44: 123-154.

25. SMILLIE, R. M., D. G. BISHOP, G. C. GIBBONS, D. GRAHAM, A. M. GRIEVE,J. K. RAISON, AND B. J. REGER. 1971. Determination of the sites of synthesis ofproteins and lipids of the chloroplast using chloramphenicol and cyclohexi-mide. In: N. K. Boardman, A. W. Linnane, and R. M. Smillie, eds., Autonomyand Biogenesis of Mitochondria and Chloroplast. North Holland, Amsterdam.pp. 422-433.

26. STERN, A. I., J. A. SCHIFF, AND H. P. EPSTEIN. 1964. Studies of chloroplast de-velopment in Euglena. 5. Pigment biosynthesis, photosynthetic oxygen evolu-tion, and carbon dioxide fixation during chloroplast development. Plant Physiol.39: 220-226.

27. SUGIYAMA, T., I. ToMoEo, AND T. AKAZAWA. 1971. Subunit structure of ribulose

1, 5-diphosphate carboxylase from Chlorella ellipsoidea. Biochemistry 10: 3406-3411.

28. SURZYCKI, S. J. 1969. Genetic functions of the chloroplast of Chlamydomona.sreinhardi: effect of rifampicin on chloroplast DNA-dependent RNA polymerase.Proc. Nat. Acad. Sci. U.S.A. 63: 1327.

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