Biochemical studies on the induction of chloroplast development in Euglena gracilis

BIOCHIMICA ET BIOPHYSICA ACTA

475

BBA 95638

BIOCHEMICAL VELOPMENT

STUDIES

ON T H E

IN E U G L E N A

INDUCTION

OF CHLOROPLAST

DE-

GRACILIS

I. N U C L E I C A C I D M E T A B O L I S M D U R I N G INDUCTION*,** A. GNANAM*** AND J. S. KAHN Departments ot Biochemistry and Botany, North Carolina State University, Raleigh, N. C. (U.S.A.) (Received November Ist, 1966)

SUMMARY W h e n h e t e r o t r o p h i c Euglena gracilis, either d a r k - g r o w n or e x p o n e n t i a l l y growing, is t r a n s f e r r e d to a u t o t r o p h i c conditions, t h e chloroplasts are p r e f e r e n t i a l l y i n d u c e d to develop while cell division is delayed. The nucleic acid e x t r a c t s from purified isolated chloroplasts were f o u n d to c o n t a i n all t h e m a j o r molecular species such as soluble R N A , D N A , a n d r i b o s o m a l R N A . D u r i n g t h e e a r l y hours of chlor o p l a s t i n d u c t i o n u n d e r a u t o t r o p h i c conditions, a chloroplast-specific messenger R N A ( m R N A ) , which is n o t f o u n d u n d e r c o n t i n u e d h e t e r o t r o p h i c conditions, is formed. T r e a t m e n t w i t h s t r e p t o m y c i n a n d 5-fluorouracil, which are k n o w n to inh i b i t chloroplast d e v e l o p m e n t , also i n h i b i t t h e f o r m a t i o n of this m R N A . The induction a p p e a r s to be v e r y similar to t h e inducible s y s t e m s described in b a c t e r i a a n d t h e evidence indicates t h a t c h l o r o p l a s t D N A is t h e p r i m e r for t h e n e w l y f o r m e d m R N A . S t r e p t o m y c i n a p p e a r s to block t h e chloroplast d e v e l o p m e n t before t r a n s c r i p t i o n at D N A level, while 5-fluorouracil blocks t h r o u g h a general interference w i t h R N A m e t a b o l i s m including t h a t of m R N A .

INTRODUCTION I n a d d i t i o n to being t h e site of p h o t o s y n t h e s i s , chloroplasts also r e p r e s e n t a self-reproducing u n i t of t h e c y t o p l a s m , like the nucleus. I t is k n o w n t h a t chloroplasts in Euglena gracilis are inducible s t r u c t u r e s a n d e x h i b i t p a r t i a l a u t o n o m y from t h e Abbreviations: MAK column, mettlylated albumin-coated kieselguhr column; tRNA, transfer RNA; rRNA, ribosomal RNA; mRNA, messenger RNA. * Contribution from the Biochemistry and Botany Departments, North Carolina Agricultural Experiment Station, Raleigh, N. C., U.S.A. Published with the approval of the Director of Research as Paper No. 2256 of the Journal Series. "* This report constitutes a portion of a thesis submitted by A. G. in partial fulfilment of the requirements for the degree of Doctor of Philosophy. *** Current address: Department of Genetics, Development and Physiology, Plant Science Building, Cornell University, Ithaca, N. Y., U.S.A.

Biochim. Biophys. Acta, 142 (i967) 475-485

470

A. GNANAM, J . S . KAHN

remainder of the cell. The formation of the photosynthetic apparatus requires the synthesis of new enzymes and structural proteins; consequently, cells containing developing chloroplasts maintain a high rate of protein synthesis, most of which is found within the chloroplasts 1. Histochemical studies coupled with electron microscopy have indicated the presence of both ribo- and deoxyribonucleic acids within the chloroplasts. The involvement of nucleic acids in chloroplast development has been indirectly demonstrated with nucleotide analogues like 5-fluorouracil 2. RNA was found in the form of ribosomes and their aggregation as polysomes a and the presence of DNA was confirmed with the aid of analytical density-gradient centrifugation techniques 4. Although frequent reference is made to the similarity between chloroplast induction and the inducible enzyme systems of bacteria and other organisms, no direct evidence to substantiate this analogy is available; although RNA fractions with some template activity have been extracted from mature chloroplasts 5, their actual involvement in chloroplast development has not been demonstrated. The study reported here was initiated with the purpose of elucidating the general pattern of nucleic acid metabolism in the early hours of chloroplast induction by the use of pulse-labeling technique, and investigating the pattern of nucleic acid metabolism associated with chloroplast development.

MATERIALS AND METHODS

Euglena gracilis, strain z, was axenically grown in both heterotrophic and autotrophic medium at room temperature (23-25 °) in a modified Hutner's medium s. 2-1 erlenmeyer flasks, each containing I 1 of medium, were incubated at room temperature on a rotary shaker under IOOO if-candles of fluorescent Gro-Lux lights. 5 % COo in air was circulated over the autotrophic cultures. After desired periods of growth, the cells were harvested by centrifugation and washed with o.oi M NaC1 and distilled water. Chloroplasts were isolated as described b y CARELL AND KAHN7. Chlorophyll was determined b y the method of ARNONs and protein by the method of LowRY et al 2. Pulse labeling o/ nucleic acids with a2p. The washed cells (5 g fresh weight) were suspended in IOO ml of phosphate-free autotrophic or heterotrophic medium to which 500 ooo counts/rain per ml carrier-free orthophosphate (E. R. Squibb and Sons, New York, New Brunswick, N. J.) had been added. Special precautions were taken to sterilize the NaC1 solution, distilled water and centrifuge tubes whenever long-term labeling or short-term incubation in heterotrophic medium was involved. Such precautions were not necessary for short-term labeling experiments in autotrophic medium since it contained no organic carbon source and also because of the low pH. After required periods of incubation, cells were collected b y centrifugation and washed with 0.05 M phosphate buffer (pH 6.7) and distilled water. Extraction o/nucleic acid and/ractionation on methylated albumin-coated kieselguhr (MAK) column. The nucleic acids were extracted with phenol and fractionated over MAK columns according to the procedure of MANDELL AND HERSHEY 1°. 2 mg of nucleic acid were added to the column in 3 ° m l of starting buffer and were fracBiochim. Biophys. Acta, I42 (i967) 475-485

NUCLEIC ACIDS IN CHLOROPLAST INDUCTION

477

tionated with a linear gradient from o. 4 M NaC1 to 1.2 M NaC1 in o.o5 M phosphate buffer (pH 6.9). Fractions of approx. 5 ml were collected, and the elution pattern of nucleic acids was monitored with an ultraviolet absorption meter using a 265 m/~ filter. Whenever isotopic labeling studies were carried out, I-ml aliquots were plated for determination of 3~p incorporation into nucleic acids.

RESULTS

The complications involved in the metabolic processes of cell division should be minimized in any study on chloroplast development in order to distinguish the changes directly attributable to the process of chloroplast formation from other, more general, alterations attendant upon cell division. The transfer of dark-grown or exponentially growing heterotrophic cells of Euglena provides an excellent opportunity for such exclusive chloroplast developmental studies. As seen in Fig. i, cells

I

Hetero

×

c o

Auto 21

0

0

. . . . .

--o .

.

.

.

.

.

.

.

.

.

.

.

-o

.

I 16

.

.

.

.

I 32

.

.

.

.

.

.

.

.

.

-o

I 48

Time(h)

Fig. i. The r a t e of g r o w t h of E u g l e n a cells u n d e r a u t o t r o p h i c and h e t e r o t r o p h i c conditions.

transferred to autotrophic conditions do not divide for at least 48 h, whereas cells under heterotrophic medium multiply at an exponential rate after a short intial lag. This was found to be true of all autotrophically growing cells regardless of whether the culture was grown heterotrophically in light or dark before inoculation. However, during this period, the chloroplasts developed rapidly. It appeared that under autotrophic conditions cell division was suppressed until the chloroplasts had fully developed. This was in contrast to the heterotrophic conditions under which cell division was accelerated while the chloroplast development was suppressed, as shown in Table I, which is based on time studies on chloroplast development in dark-grown cells. The carotene and chlorophyll content of autotrophic cells were, in general, far greater t h a n those of heterotrophic cells. Their photosynthetic capacity, as measured b y the rate of 02 evolution, also followed the same pattern, although the difference is not of Biochim. Biophys. Acta, 142 (1967) 475-485

47 b

A. GNANAM, J. S. KAHN

TABLE I PIGMENT COMPOSITION, PHIC AND AUTOTROPHIC

Type o/ cells

Darkbleached Heterotrophic (24 h) Heterotrophic (12o h) Autotrophic (120

h)

PHOTOSYNTHESIS AND C E L L S O F E . gracilis

PHOTOPHOSPHORYLATION

STUDIES

ON HETEROTRO-

Chlorophyll (/zg/Io~ cells) -a b

a/b

None

None

None

217.5

23-1

9.4

lO.64

96.8

None

1i72.2

63.5

i8. 4

22.61

i2o.2

3.42

2162.5

204.2

lO-5

45.49

17o.3

1.78

Ratio

Carotene Photo(ktg/zo 6 cells) synthesis 0 3 evolution (/zmoles/ io 6 cells) o.z ,14 Na2COa

--

Photophosphorylation* (tzmoles o/ A T P /ormed per mg protein per h)

None

* Photophosphorylation was found to be quite variable. Data represent typical experimental results.

t he same m a g n i t u d e . H o w e v e r , the rate of p h o t o p h o s p h o r y l a t i o n was lower in a u t o t r o p h i e cells t h a n h e t e r o t r o p h i c ones. Also, u n d e r a u t o t r o p h i c conditions, t h e chloroplasts d ev el o p e d r a p i d l y and reached their m a x i m u m size and n u m b e r within 24 h. D u r i n g this period, however, the t o t a l p r o tei n c o n t e n t per cell did n o t change. T h e effects of different m e t a b o l i c inhibitors of the chloroplast d e v e l o p m e n t (as m e a s u r e d b y chlorophyll formation) are given in Tables I I and I I I .

TABLE I I THE EFFECT FORMATION

OF STREPTOMYCIN, p-FLUOROPHENYLALANINE IN GREENING EUGLENA

AND 5-FLUOROURACIL

Treatment

A m o u n t o/ chlorophyll developed in 2 4 h (l~g/ 2 o 6 cells)

Control Streptomycin (15o/*g/inl) p-Fluorophenylalanine (2/,moles/nil) 5-Fluorouracil (2/*moles/ml)

io26.7 563.9 586.5 244. 7

ON CtlLOROPHYLL

Inhibition (%)

46 44 73

TABLE III THE EFFECT OF 5-FLUOROURACIL OF EUGLENA

CONCENTRATION

ON CHLOROPHYLL

Treatment

A m o u n t o/ chlorophyll developed in 24 h (izg/zo 8 cells)

Autotrophic medium (control) plus o.I/,mole/ml fluorouracil plus i /,mole/ml fluorouracil plus 2 #moles/ml fluorouracil Heterotrophic medium

891.I 638. 4 404.3 244. 7 231.9

Bioehim. Biophys. Acta, 142 (1967) 475-485

FORMATION

Inhibition (%)

28 55 73 74

IN CHLOROPLASTS

NUCLEIC ACIDS IN CHLOROPLASTINDUCTION

479

Nucleic acids ol Euglena. The nucleic acids of Euglena ceils were extracted in the presence of 1 % Dupanol. Separation of nucleic acids b y chromatography on a MAK column with a linear gradient of 0. 4 M to 1.2 M NaC1 in Na2HPO 4 buffer (pH 6.9) resulted in elution of oligonucleotides, followed b y transfer RNA (tRNA) peak, DNA and D N A - R N A complex, and ribosomal RNA (rRNA) and messenger RNA (mRNA). The distribution of these fractions in the eluent peaks is shown in Fig. 3 and Fig. 6, and is typical of all separations. The identification of eluted fractions is operational and based on work with bacteria, plants, and algae n,12. The identity of the t R N A fraction from Euglena cells was confirmed as follows: [14ClaminoacylRNA's were prepared from the lO5 ooo x g supernatant fractions according to the method reported b y SUEOKA AND YAMANE 1~, and they were then mixed with the total nucleic acid extracts and fractionated on MAK columns. The radioactive peak coincided with the t R N A peak. Similarly, the rRNA peak was identified by using the RNA extracted from the ribosomal fraction of Euglena cells (fraction between 2I ooo x g and lO5 o o o x g ) . The rRNA peak was never resolved into light and heavy fractions as reported in studies on bacteria and higher plants. RNA extracts from heterotrophic and autotrophic cells, compared b y fractionation of the MAK column, showed no appreciable difference except for a very small difference in the size of the D N A - R N A complex peak. RNA extracts from apochlorotic cells, reversibly bleached b y growing them in the presence of 15o ffg/ml streptomycin for short periods, also contained the fractions that were common to normal green Euglena cells except for a quantitative difference in the D N A - R N A complex fraction as seen by the relative size of the peak in Fig. 2. Similarly, phenolic

0.3 0.2

~

~

¢a

%

2o

40

60

~o

Fraction No.

Fig. 2. Fractionation of nucleic acid from streptomycin bleached (reversibly) Euglena cells. extracts of RNA prepared from isolated, purified chloroplasts in the presence of I o/ /O Dupanol, on passing through a MAK column separated into tRNA, D N A - R N A complex, and rRNA. This was the same as for extracts of whole cells, except for the occurrence of a proportionately large amount of D N A - R N A complex. It appears that chloroplasts contain the same complement of nucleic acids as does the whole cytoplasm (Fig. 3)-

Studies on m R N A /ormation with a2p Time course studies. In order to find the optimal time for incubation with 32p, Biochim. Biophys. Acta, 142 (1967) 475-485

480

A. GNANAM, J. S. KAHN

DNA-RNA 0.3(

A Oligonucleotldes

I

rRNA \

/'zX X

mRNA

O.10 0.02 _ _ 1 0

I 20

I

40

I

60 Fraction No,

I

80

100

Fig. 3. Fractionation of nucleic acids from isolated chloroplasts of Euglena on MAK column. preliminary pulse-labeling experiments at different periods of incubation were conducted. Nucleic acids from cells incubated for 5 and IO rain with 3sp showed rapid incorporation of the label into oligonucleotides and t R N A , but no incorporation into the remaining fractions. The R N A extracted from the cells t h a t were incubated with 32p for 4 ° min showed even a larger a m o u n t of radioactivity in the first major peak but, in addition, the D N A - R N A complex fractions also showed a significant a m o u n t of asp incorporation. The radioactive peak did not coincide, however, with the absorbance peak of the D N A - R N A complex fraction. A slight shift to the left was noticeable, corresponding to one or two fractions. There was no radioactivity incorporated into the r R N A fractions. On incubation with a2p for I h, it was observed t h a t in addition to the t R N A and D N A - R N A complex fraction the m R N A part of the third peak showed appreciable incorporation of asp. Very little incorporation could be seen in the ribosomal fractions (Fig. 4). On incubation with asp for 2 h, however, 4000 '

I 1

Oligonucleotides

,

~

" 0,2

~

J'~I O.1

3000

,

mRNA

'~,

DNA-RNA ,

rRNA,,'"

2000

Z

'~

,

-

1OOO

c

C

% '

#o

4'0

6b

do

IO~o

U

Fraction No.

Fig. 4. Fractionation of nucleic acid from "pulse-study" ceils under autotrophic conditions. --, absorbance; - - - , radioactivity. an overall increase of radioactivity was noted in all the fractions, including the ribosomes. As indicated previously, Euglena cells, when transferred to autotrophic conditions, do not divide for at least 48 to 56 h. On this basis, I-h incubation is equivalent to I 2 % of its life cycle and thus would be a reasonable equivalent to the time employed in pulse-labeling bacteria and other microorganisms. t~iochim. Biophys. Acta, 142 (1967) 475-485

481

N U C L E I C ACIDS IN CHLOROPLAST I N D U C T I O N

3sP-Labeling studies under autotrophic conditions. To determine whether or not m R N A was produced during chloroplast induction, Euglena cells were transferred to autotrophic medium with carrier-free ~2p and incubated for I h in the light. At the end of the incubation period, the cells were harvested and washed. One half of the cell mass was used for RNA extraction and the other half was suspended in a normal autotrophic medium with unlabeled phosphate. After further incubation for 2 h, the cells were collected and nucleic acid was extracted as before. Henceforth, the former half will be designated as "pulse-study" cells and the latter as "chase-study" ceils. The nucleic acid extract from the pulse-study cells, upon separation on the MAK column, showed radioactive peaks corresponding to the oligonucleotides and t R N A fractions, to the left (ascending half) of D N A - R N A complex peak and to the far right (descending slope) of rRNA (Fig. 4). When the chase-study extracts were separated in the same way the high radioactivity in m R N A disappeared and, in general, the radioactivity coincided with absorbance peaks (Fig. 5). To determine

b

0.3

i

t

:

!

GO00

f,,

0.2

~

c

3000

O.1

q

TI & o: "~ o

J-__

I 20

i

I

40

t

60

Fraction

80

No.

o 100

F i g . 5. F r a e t i o n a t i o n of n u c l e i c a c i d s o n M A K c o l u m n f r o m " c h a s e - s t u d y " conditions.----, absorbance; ---, radioactivity.

cells under autotrophic

whether or not the fraction identified as m R N A under autotrophic conditions was directly involved in chloroplast development, the formation of this peak was studied under continued heterotrophic conditions, where chloroplast development is known to be suppressed (Fig. 6). The formation of m R N A was found to be greatly curtailed. t

'

0.3

J

Ii

i

300O I

0.2

'

2ooo t

~ 0.1 oJ

~

.

~

.g

.

1000 E m

"

o ; - - J-/

i 20

, 40 60 Fraction No.

, 60

F i g . 6. F r a c t i o n a t i o n of n u c l e i c a c i d s f r o m " p u l s e - s t u d y " - - , absorbance; ---, radioactivity.

10~o

o

o

cells under heterotrophic

conditions.

Biochim. Biophys. Acta, 142 (1967) 4 7 5 - 4 8 5

482

A. GNANAM, J. S. KAHN

This was further confirmed by pulse labeling the cells under autotrophic conditions in the presence of known inhibitors of chloroplast development like streptomycin (Fig. 7) and 5-fluorouracil (Fig. 8). I

ii 1

0.3

3000L

0.2

2000 O_

%

2;

40

60

80

o

Fraction No.

Fig. 7. F r a c t i o n a t i o n of nucleic acids from " p u l s e - s t u d y " cells u n d e r a u t o t r o p h i c c o n d i t i o n s in t h e p r e s e n c e of 1 5 o / , g / m l s t r e p t o m y c i n . - - , a b s o r b a n c e ; - - -, r a d i o a c t i v i t y .

7 i ~000

Q3

-)000 .if_

r

o.1

1000

;

0--, 0

20

40

60 F r a c t i o n No.

80

0 I(30

i 8

Fig. 8. F r a c t i o n a t i o n of nucleic acids f r o m " p u l s e - s t u d y " cells u n d e r a u t o t r o p h i c c o n d i t i o n s in t h e p r e s e n c e of 2 # m o l e s / m l 5-fluorouracil. - - - , a b s o r b a n c e ; - - -, r a d i o a c t i v i t y .

DISCUSSION

The development of chloroplasts in Euglena cells on exposure to light or on growth under autotrophic conditions constitute an inducible system quite comparable to the initiation of specific enzyme formation by specific inducers and the extensive changes in cellular metabolism upon phage infection 14. This induction process involves the formation of complex structures which contain a considerable amount of protein of the cell and the appearance of enzymatic activities associated with photosynthesis. If the above analogy is correct, the formation of a distinct species of RNA with a high metabolic turnover and with base compositions resembling that of DNA should be detected. It should thus be possible to isolate this fraction from the RNA extracts of pulse-labeled cells under conditions of chloroplast induction. The results of the pulse-labeling experiments reported here confirmed the formation of mRNA Biochim. Biophys. Acta, 142 (I967) 475-485

NUCLEIC ACIDS IN CHLOROPLAST INDUCTION

483

during the induction period. In a subsequent 2-h "chase-study" period, this fraction disappeared, indicating its short half life (Fig. 7). The association of this m R N A with chloroplast formation was established by comparison with cells grown under heterotrophic conditions where development of chloroplasts was suppressed and also b y using known inhibitors of chloroplast development such as streptomycin and 5fluorouracil. According to the current hypothesis, transfer of information from DNA to the sites of protein synthesis involves a mechanism whereby single-stranded DNA serves as the template for polymerization of complementary ribopolynucleotide. If continued formation of m R N A is a necessary concomitant, it should be possible to find the native D N A - R N A hybrids in cells actively engaged in chloroplast development. Indirect evidence for the existence of this hybrid was obtained in the second major nucleic acid fraction observed through MAK column analysis (Fig. 6). Composition of this second major fraction is rather complex. In an extensive study on this fraction, CHERRYn had found that it consists of 25 % RNA, 25 % rapidly metabolized DNA and 5o o/ /o non-metabolized DNA. SAMPSON et al. 15 have shown that the growing regions of root and leaf tissue contains as much as 2o % of their DNA in a low molecular weight form which is metabolically labile. This latter form of DNA is eluted from the MAK column with a lower NaC1 concentration than that with which higher molecular weight DNA is eluted. Although they do not report the presence of RNA in their DNA fraction, it was shown that only 8o °/o of the DNA is composed of deoxyribonucleotides. Even though the presence of RNA in the DNA fraction does not unequivocally prove that it is in the form of a D N A - R N A complex, the RNA and the rapidly metabolized DNA appear to be chemically associated n. A similar pattern of rapid labeling in the left half of the DNA peak together with the association of RNA has been reported from extracts of Chlorella as welP 2. Apparently, the DNA that is found associated with this peak represents only a fraction of the total cellular DNA and has a different nucleotide composition from the total cellular DNA 16. It appears to be very labile and can be extracted along with RNA. These facts are indicative of the possibility that this fraction represents the portion of nuclear DNA that is active in transcription at the time of extraction, possibly in a single-strand form and free from histone complexing. On the other hand, it m a y be the more labile extrachromosomal DNA that has been reported from various cytoplasmic fractions. Under our experimental conditions in Euglena, it is conceivable that DNA from the chloroplasts m a y be induced to transcribe its message and, hence, this peak m a y represent the native D N A - R N A hybrids together with the rapidly labeled RNA. Evidence for this possibility can be seen from Table IV; the comparative amount of the D N A - R N A complex fraction appears to depend upon the level of chloroplast development in the cells. This compares favorably with the findings of L E ~ et al. 4 that an enrichment of the preparation for chloroplasts of Euglena also enriched for the satellite band in cesium chloride density-gradient analysis of DNA. A more direct evidence for the relationship of this fraction with m R N A formation can be seen from time-course studies using ~2p (Fig. 4). The appearance of the m R N A fraction is always preceded b y a radioactive peak in the left half of the D N A RNA complex fraction which corresponds to the "hybrid". Similarly under conditiom inhibitory to the formation of mRNA, there is a concomitant suppression of the Biochim. Biophys. Acta, 142 (1967) 475-485

484

A. GNANAM, J . S . KAHN

T A B L E IV EFFECT OF STREPTOMYCIN TREATMENT ON THE D N A - R N A COMPLEX FRACTION OF NUCLEIC ACIDS EXTRACTED FROM VARIOUS EXPERIMENTAL CELLS P e r c e n t a g e d i s t r i b u t i o n of a b s o r b a n c e u n d e r v a r i o u s f r a c t i o n s as c a l c u l a t e d f r o m t h e a r e a c o v e r e d by different fractions.

Treatment

Heterotroph (mature) S t r e p t o m y c i n (I h) S t r e p t o m y c i n (3 h) S t r e p t o m y c i n b l e a c h e d ceUs Isolated chloroplasts

Composition (%) Oligonucleotides and t R N A

DNA and DNA-RNA

14.72 14.64 15.99 27.50 16.16

14.39 13.36 lO.12 5.5 ° 27.67

complex

r R N A and mRNA 52.o 7 68.78 70.04 57.20 43.39

radioactive peak in the D N A - R N A complex fraction, probably indicating that m R N A is in fact formed from the D N A - R N A complex fraction. In the absence of such direct evidence, CHERRY11 denotes this m R N A fraction as D-RNA indicating its base composition is analogous to the DNA fraction. Although both streptomycin and 5-fluorouracil were found to inhibit primarily m R N A synthesis, their mechanisms of action differed markedly. With streptomycin treatment, there appeared to be no interference with ribosomal RNA metabolism as seen from the rapid synthesis of ribosomes. This is represented by the radioactive peak corresponding to the light rRNA section (Fig. 7). Since no m R N A was formed in the presence of streptomycin, the site of action of the latter must be prior to the formation of mRNA, which would then implicate DNA. Its effects on metabolically active or labile DNA can be seen from Table IV. Though the data given in Table IV are semi-quantitative, there appears to be a progressive decrease in the D N A - R N A complex fraction, which represents the labile DNA, with increasing periods of streptomycin treatment. In this connection, it m a y be recalled that prolonged treatment with streptomycin leads to an irreversible loss of chloroplasts in Euglena. This can best be explained on the basis of its interference with DNA rather than RNA. However, the possibility of its secondary effects upon other processes, such as protein synthesis 17, is not excluded. On the other hand, as compared to streptomycin-treated and normal cells, 5-fluorouracil does not interfere with DNA as evidenced b y the size of the D N A - R N A complex peak in Fig. 8. However, there is an overall reduction in 32p incorporation in all the RNA fractions. Because this agent is a base analogue specific to RNA, its mode of operation appears to be an interference with overall metabolism of ribonucleic acids, including that of mRNA. To date, there is no report of a permanent bleaching in 5-fluorouracil treated cells. The observed general effect of ribonucleic acid metabolism is in agreement with earlier findings with Staphylococcus is and soybean seedlings 1~. In conclusion, when chloroplasts are induced to develop under autotrophic conditions, a specific m R N A is formed and is inhibited b y heterotrophic conditions, streptomycin and 5-fluorouracil, which are also known to inhibit chloroplast development. The induction appears to be very similar to the inducible systems deBiochim. Biophys. Acta, 142 (1967) 475-485

NUCLEIC ACIDS IN CHLOROPLASTINDUCTION

485

scribed in bacteria, and the evidence indicates that chloroplast D N A is the primer for the newly formed m R N A .

ACKNOWLEDGEMENT

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