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Properties of selectively starved Euglena
BIOCHIMICA ET BIOPHYSICA ACTA
195
BBA 95610
P R O P E R T I E S OF S E L E C T I V E L Y STARVED E U G L E N A
H T E P S T E I N AND E I A Z A B E T H A L L A W A Y
B*ology Department, Brandezs Unzverszty, Waltham, Mass (U S A ) (Received Novenaber 18th, 1966)
SUMMARY
It has been shown that starving Euglena for an essential nutrient causes at to reduce its nuclear and plastad complements by one-half If pressed b y extreme deprivation of the nutrient, the cell IS finally pushed to discard its plastid complement which is gratuitous in the heterotrophic medium ill which the cell is growing.
INTRODUCTION
In recent years it has been firmly established that some cellular organelles contain DNA. We have shared 1-4 in the demonstration that Euglena cells contain two DNA's in addition to the main, nuclear, DNA; these have been identified as being associated with the plastlds and the mltochondna. In suitable media Euglena can grow quite well either in the dark or after having had the chloroplasts entirely removed by various agents. Therefore, the plastlds and the plastid-assomated DNA are not at all indispensable to the cell. Accordingly it should be possible to alter the plastld DNA chemically or physically and study its subsequent properties without requiring its repair, this is presumably not true for the other DNA's, if damaged, since the cells would not be capable of reproduction. A study of such effect~ was initiated with a view towards ascertaining the relations, if any, between the nuclear and plastid DNA's. Selective starvation as a possible way of affecting the plastld DNA was chosen because of our interest in a second problem: that of obtaining specific labelling zn v,vo of plastld components. On light induction of chloroplast development several times as much non-plastid RNA and protein are synthesized as are plastld RNA and protein (BRAWERMAN, POGO AND CHARGAFF a) We thought it possible that during starvation for an essential nutrient, the plastlds might be preferentially discarded, and on replacing the nutrient just before complete plastid disappearance, plastid components would be preferentmlly synthesized Preliminary experiments of this type revealed some surprising properties of such selectively starved cells, and this paper reports on some of these propertms. B,och~m
Bzophys Acta, 142 (1967) 195-2o7
196
H. T
EPSTEIN,
E.
ALLAWAY
MATERIALS AND METHODS
Euglena gracklesvar. baclllarls was grown as p r e v i o u s l y described 6 in H u t n e r ' s p H 3.5 h e t e r o t r o p h l c m e d i u m in b a t c h cultures in e r l e n m e y e r flasks on a s h a k e r or in a c h e m o s t a t at room t e m p e r a t u r e u n d e r a b o u t 15o if-candles of illumination. P h o s p h a t e - f r e e m e d i u m was p r e p a r e d b y replacing the K H 2 P O a a n d (NH4)2HPO 4 of H u t n e r ' s m e d i u m with K H C Q a n d NH4HCO ~. N o r m a l m e d i u m , who~e p h o s p h a t e c o n c e n t r a t i o n is 5"1o a M, wa~ d i l u t e d m t o the p h o s p h a t e - f r e e m e d i u m a~ desired. F o r e x a m p l e , to p r e p a r e w h a t we designate P/2oo m e d m m , 5 ml of n o r m a l m e d i u m was d i l u t e d into IOOO ml of p h o s p h a t e - f r e e m e d m m . I n a similar way, ~ulfate-free m e d i u m was m a d e b y r e p l a c i n g the variou~ sulfates of n o r m a l m e d i u m (2.2-1o -3 M ,~ulfate) b y t h e i r chlorides U l t r a v i o l e t lrradlatlon~ were done as described p r e v i o u s l y s on cells in a m e d m m c o n t a m m g 0.05 M m a n m t o l a n d o o I M MgC12, which is the p h o s p h a t e - f r e e version of t h e resting m e d i u m p r e v i o u s l y used for o b t a i n i n g n o n - d l v l d m g cells. Low-phosp h a t e resting m e d i u m was o b t a m e d b y s t a t a b l e dilution of n o r m a l re~tmng m e d i u m into this p h o s p h a t e - f r e e resting m e d i u m Most e x p e r i m e n t s were done with cells grown in a c h e m o s t a t a d a p t e d from t h a t described b y ROTMAN7 a n d shown in F i g I F r e s h m e d i u m is c o n t m u o u s l y d r i p p e d in at the finely d r a w n tip, T, while cell suspension c o r r e s p o n d i n g l y drips out into t h e receiving flask, F. The d r i p r a t e was a d j u s t e d b y the Teflon stopcock, S, each m o r n i n g so t h a t the cell c o n c e n t r a t i o n s r e m a i n e d fairly constant. B y blowing
INPU
MEDIUML.~ HOSE
II
F i g I A c h e m o s t a t a d a p t e d f r o m ROTMAN ~ R a t e o f m e d m m i n p u t t o t h e g r o w t h c h a m b e r , G , is r e g u l a t e d b y t h e s t o p c o c k , S, so t h a t t h e cell c o n c e n t r a t i o n m t h e s a m e e v e r y m o r n i n g A f t e r the colnblned bubble-breaker and reservoir, R-B, m filled up to the height causing overflow, the v o l u m e ~s k e p t c o n s t a n t b y s ~ p h o n a c n o n i n t o t h e c o l l e c t i n g f l a s k , F B y b l o w i n g m t h r o u g h A, the mphon action can be stopped, and fluid accumulates m B-R until the overflow takes place. At any tune fluid can be siphoned off into F by sucking air out through A
Bwchzm
Bwphys
.4cta, 142 (1067) i()5 2o 7
PROPERTIES
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STARVED
EUGLENA
197
in through the air-hole, A, the chemostat action is stopped and cells accumulate in B-R, the combined bubble-breaker and reservoir. By sucking air out through A, the contents of the reservoir and growth chamber, G, can be caused to start flowing out into F. By using the reservoir in this way, large numbers of cells could be obtained In low-phosphate media, cell concentrations were about I.IOa-2'IoSper ml, m normal media they were 1.1o6 2.1o 6 per ml. Cell counts were made with a hemocytometer or a Coulter counter, depending on the sample volume available, these methods were crosschecked occasionally to ensure umformlty. After adding a pinch of MgCQ, chlorophyll was extracted with 85 °7o acetone and concentrations determined from absorbances at 663 and 645 mF. Otherwise extractions to measure RNA and DNA were carried out by the procedure of KEMPNER AND MILLER9 in which RNA was finally measured by the orclnol method and DNA by the Ceriottl method To make chromosome counts, cells were washed in a solution containing o.15 M NaC1 and O.lO M EDTA (dlsodium salt) (pH 8), treated with Viokase (I °/o, 15 ram), fixed with methanol-acetic acid (3.1, v/v), smeared on microscope slides, and stained with acetic acid-orcein (i °'o orcein in 50 ~o acetm acid). The Vlokase was made up in o 02 M potassium phosphate buffer (pH 8) for normal cells, in I o M mannitol brought to pH 8.5 for low-phosphate cells The procedure of PEARSEI° was used for Feulgen staining of the smears. Deoxyribonuclease pretreatment was at o.I mg/ml DNA in 3.1o -a M MgSO4 (pH 6.8) for 4 h at 36°. Control pretreatment was In the same solution without enzyme Electron micrographs were made as previously described n using glutaraldehyde fixation and OsO4 staining. Chloroplasts were observed in living cells by the red fluorescence of their chlorophyll and photographed as previously described 1~. Photomicrographs of the fluorescent cells were made using Polaroid infrared film which enabled us to obtain good results with exposures of only a few seconds.
EXPERIMENTS
AND
RESULTS
In the initial experiments to starve selectively for phosphate, o.I ml of cells growing in the hght in normal medium were diluted into 2o ml of phosphate-free medium (giving P/2oo), and cell growth proceeded until the stationary concentration of 4.IO5-5.IO5 cells per ml was reached. These cultures were noticeably less green than normal ones at the same cell concentrations, and observation with the fluorescence microscope revealed that this was due primarily to the cells containing fewer (and slightly smaller) chloroplasts per cell By the time stationary phase was reached, some cells had no traces of chlorophyll fluorescence, and when plated, many cells gave rise to entirely white colonies. When cells containing 3-5 chloroplasts per cell were subcultured into fresh P/2oo medium, they reverted toward normal amounts of chlorophyll and normal numbers (11-13) of chloroplasts. Only after several generations of growth did "degreening" begin again, and subcultures started at fairly high cell densities did not de-green noticeably. Some experiments have been carried out with cells having 3-5 chloroplasts per cell, and the results were similar to those to be described which used chemostat-grown cells. To avoid problems of reversion and to obtain a supply of Bzoch~m
Bzophys Zlcta, 142 ( 1 9 6 7 ) 195 2 0 7
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T. E P S T E I N ,
E. ALLAWAY
reproducible " l o w - p h o s p h a t e " cells, chemostat culture was b e g u n I n the experim e n t s to be described below, most work was done with cells grown an P/5oo m e d m m , a l t h o u g h some comparison experiments were done using P,"2oo, P/3oo, a n d P ' I o o o media.
I
The b,ochemwal contenls o~ P/5oo cells Table I gives some general d a t a for cells grown in n o r m a l a n d P/5oo media. As would be expected from the drop in R N A c o n t e n t b y a factor of a b o u t 3, the "l A I 3 L E i GENERAL
DAIA
ON
( H / ~ \ I O b T A , F-GROVVN
l e N A (pg p e r cell) D N A (pg p e r cell) C h l o r o p h y l l (pg p e r cell) C h l o r o p l a s t s p e r cell
EUGLENA
N o r m a l cells
P / 5 o o cells
3 ° 40 3 o 15 l I 13
lO-15 I 5 5 5 7
P/5oo cells are definitely smaller m d i a m e t e r t h a n n o r m a l cells. As was the case for n o n - c h e m o s t a t P/2oo cells, the r e d u c t m n in chlorophyll c o n t e n t is composed m a i n l y of a decrease m chloroplast n u m b e r b u t also p a r t l y of a decrease m chlorophyll per chloroplast Both these effects can be seen in the fluorescence pictures m Fig. 2, m which P a r t a is of n o r m a l cells a n d P a r t b is of P/5oo cells. The P/5oo cells c o n t a i n 5 7 chloroplasts, compared with the I I - I 3 in n o r m a l cells, a n d the~e are i n d i v i d u a l l y smaller t h a n those m the n o r m a l cells. We note p a r t i c u l a r l y t h a t chloroplast n u m b e r 1~ reduced b y the same factor as total cell DNA. A n i n t e r e s t i n g reference from these d a t a 1~ t h a t some nuclear a n d plastld ploidy ha~ been lost m the Er5oo ~ell~ We now t u r n to ~tudies designed to test this Inference
2 Nuclear plo~dy The most obvious way of i n t e r p r e t i n g the 50 °o decrease of D N A an P/5oo cells is t h a t of reduction of nuclear ploldy However, we have been dissatisfied with the v a r i a b i l i t y of the D N A m e a s u r e m e n t s which seems due to interfering substances associated with the presence of chloroplasts We have therefore estimated the n u m b e r of chromosomes in two ways. I n t e r p h a s e E u g l e n a chromosomes are in a partially-condensed or multig r a n u l a r state characteristic of m a n y protlsts. The n m n b e r of granules ~hould be related to the n u m b e r of chromosomes, a n d we have a t t e m p t e d to c o u n t t h e m using b o t h the electron microscope a n d the hght microscope Electron mmrographs of n o r m a l a n d P/5oo cells are shown m Fig. 3. The granuleb bhow up as dark blobs in the nuclei The granule n u m b e r s per mlcrograph sectmn were c o u n t e d a n d found to average a b o u t 45 for n o r m a l cells, a b o u t 22 for P/5oo cell~. The total granules per burat nucleus in acetic acid orcein p r e p a r a t i o n s were c o u n t e d xn the hght m~croscope, Bzoch~m
Bzopkr~
_4cla, 142 (1907) 195 207
~
Jql~~ap ~8
% n
~!
i~ii~~i~
:200
H, T. EPSTEIN, E
ALLAWAY
Fig 3 E l e c t r o n m m r o g r a p h s of s e c t i o n s (A) n o r m a l a n d (B) P / 5oo cells D a r k b l o b s are vi s i bl e m t h e nuclei, t h e r e b e i n g 4o-.5o m (X) a n d a b o u t 2o m (B) The bl obs are s e mi c o n d e n s e d or m u l t i granular mterphase chromosomes
lhock~m
l'~zopky,
.4cla, t 4 : 0 9 6 7 )
I05 207
PROPERTIES
OF SELECTIVELY
STARVED
EUGLENA
201
and similarly found to differ by a factor of 2, although the distributions were quite broad, as shown in Fig. 4, in which average granule numbers of 194 and 96 were found for the two types of cells. If we make plausible assumptions about granule size and section thickness of electron microscope preparations we can calculate that the numbers obtained from electron microscopy and light microscopy are in rough agreement. I0-
8J
-
-
60
120 180 240 GRANULE NUMBER
F i g 4 T h e d i s t r i b u t i o n o f g r a n u l e n u m b e r s m n o r m a l a n d P / 5 o o cells, a s d e t e r m m e d f r o m t o r e r o g r a p h s h k e t h o s e i n F i g 3 T h e c r o s s - h a t c h e d d l s t n b u t m n is t h a t o f t h e n o r m a l c e l l s T h e a v e r a g e numbers are about I9o for normal ceils and about 90 for P/5oo cells
Recently, improved techniques have yielded enough burst telophase nuclei with condensed mitotic chromosomes so that we can give estimates of chromosome numbers. Fig. 5 shows such preparations for both types of cells. The chromosome number for normal cells is 9 2 ± 2 and 4 ~ 3 for !ow-phosphate cells. The units being counted were found to be Feulgen-posltlve, and Feulgen staining was prevented b y deoxyribonuclease pretreatment. No deoxynbonuclease-sensmve staining was seen in the cytoplasm of either cell type. The variability of DNA measurements was at least partly associated with the different media and possibly slightly different light intensities in the various chemostats. To reduce these variables, cells were taken from a P/5oo chemostat and placed in an erlenmeyer flask with aeration. A small volume of I M phosphate was added to bring the medium up to normal phosphate concentration, and samples were taken daily. In this way cells could be studied in a fixed environment. The results of 4 such experiments are assembled in Fig. ~. Here it can be seen that the P/5oo amount of DNA is increased practically to normal after 24 h, and then remains at that value. Significant cell division began only after 24 h, but was roughly normal starting with 48 h Chlorophyll per cell was unchanged at 4 5 Pg per cell through 96 h.
3. Plashd plo~dy We have already presented evidence that the number of detectable plastlds has been reduced b y a factor of two in P/5oo cells. The same reduced number was found for chemostat cells grown in P[2oo and P[Iooo media. Given the reduction in chloroplast number, we were Interested in finding out if the plastid-associated DNA Bzoch*m. B*ophys. Acla, 142 ( 1 9 6 7 ) 1 9 5 - 2 o 7
202
H
3. L P S I L I N , L.
\LI..kW.\Y
Fig. 5 P h a s e - c o n t r a s t p h o t o m i c r o g r a p h s of c h r o m o s o m e c o m p l e m e n t s m b u r s t n u c l e i of (A) n o r m a l a n d (B) P / 5 o o ceils T h e r e are, r e s p e c n v e l y , a b o u t 9o a n d a b o u t 45 c h r o m o s o m e s
Bzochzm Bzophys .tcla, 142 (iq~7) t95 207
203
PROPERTIES OF SELECTIVELY STARVED EUGLENA
is also r e d u c e d or w h e t h e r it is r e t a i n e d in some form, p o s s i b l y m a s s o c l a t m n w i t h n o n - d e v e l o p i n g plastids. T h e m o s t direct w a y of d e t e r m i n i n g how m u c h p l a s t i d - D N A is p r e s e n t w o u l d be to m e a s u r e it. H o w e v e r , since it r e p r e s e n t s o n l y a b o u t 2 % of t h e t o t a l D N A , a c c u r a t e d e t e r m i n a t i o n of this a m o u n t is n o t a t p r e s e n t feasible. I n its m o s t d~rect
o
o STANDARD MEDIUM
/
~------{-t-°
i ,c
• ,.5oo MEDIUM
t~ Io
g ~o~
W ~,oo
g
~-
~o 10
C-O0
~. i
2
3
4
20
DAYS
4o
60
8o
ioo
DOSE IN SECONDS
which
Fig 6 The ceil concentration and DNA per cell in P15oo cells to enough phosphate was added to give the normal concentration The flags represent the range of the data for four experiments Fig 7 The effect of ultraviolet on green-colony forming ability of normal and P/5oo cells The lines are obtained from computer analyms of the data
form, the p r o b l e m is t h a t of m e a s u r i n g t h e a r e a u n d e r a p e a k in a d e n s i t o m e t e r t r a c i n g of CsCl-banded whole-cell D N A , w i t h t h e shoulder of t h e m a i n b a n d D N A t r a i l i n g in to the position of t h e c h l o r o p l a s t satellite D N A . A c c o r d i n g l y we h a v e used indirect methods. W e h a v e p r e v i o u s l y shown t h a t t h e n u m b e r of p r o p l a s t i d s seen b y fluorescence la per d a r k - g r o w n cell is similar to the 36 t a r g e t s 8 / o r u l t r a v i o l e t b l e a c h i n g ( i n a c t i v a t i o n of t h e g r e e n - c o l o n y - f o r m i n g a b i l i t y of t h e cells) curves. Since t h e action s p e c t r u m for bleaching is t h a t of nucleoprotein, we h a v e m a d e t h e r e a s o n a b l e a s s u m p t i o n t h a t bleaching is due to u l t r a v i o l e t d a m a g e to t h e D N A of the i n d i v i d u a l p r o p l a s t i d s . Therefore we should be able to m e a s u r e t h e n u m b e r of these n u c l e o p r o t e i n entities in l o w - p h o s p h a t e cells b y bleaching e x p e r i m e n t s . If t h e p l a s t i d D N A is r e a l l y reduced, t h e t a r g e t n u m b e r o b t a i n e d should equal t h e n u m b e r of p r o p l a s t i d s visualized b y fluorescence microscopy. U l t r a v i o l e t bleaching curves h a v e been o b t a i n e d for b o t h n o r m a l a n d P/5oo cells, a n d t h e results of 3 s e p a r a t e e x p e r i m e n t s are given in Fig. 7- B o t h curves look like m u l t i - t a r g e t curves. If we are to c o m p a r e t h e t a r g e t n u m b e r s of t h e two curves, t h e a s y m p t o t i c slopes m u s t be equal, i n d i c a t i n g t h e s a m e i n h e r e n t u l t r a v i o l e t s e n s i t i v i t y of the t a r g e t s a n d therefore t h a t the t a r g e t s are p r o b a b l y i d e n t m a l in the two t y p e s of cells. A c o m p u t e r was p r o g r a m m e d to d e t e r m i n e the a s y m p t o t i c slopes a n d t h e t a r g e t n u m b e r s from t h e s t r a i g h t line p o r t i o n s of t h e curves. T h e slopes were e x p e r i m e n t a l l y indistinguishable, being 11. 7 sec p e r hit for t h e n o r m a l cells a n d 11.8 sec per hit for the P/5oo cells. T h e respective t a r g e t n u m b e r s are 3 7 ± 2 Bzoch~m B,ophys
Acla, 142 (I967) 195-2o7
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and 18±2, from which we infer that the amount of plastld DNA in P/5oo cells is half that in normal cells. An accurate determination of the number of proplastlds was previously beyond our abfllty to measure because proplastlds were shown ~ to fuse by threes after 8-12 h after placing dark-grown cells in the light At those early times, some of the proplastlds contain so httle chlorophyll that they are too faint to he seen well. Of course, the number of proplastlds can also be estimated by multiplying the number of chloroplasts by three to give 30 for normal cells and 18 for P/'5oo cells In an attempt to measure the number of proplastids directly, we guessed that the fusion step might he inactivated by ultraviolet light, thereby permitting the proplastlds to develop individually to the point of being countable (note that bleachi n g - t h e formation of white colomes i~ not revolved because the color of the colonies formed tells only what the progeny of the lrradmted cells are like). Dark-grown P/5oo cells were given 2-3 lethal hits of ultraviolet light and placed in red (nonphotoreactlvating) hght for plastld deveh>pment. After 3 day~ many of the cells had developed apprecmbly, and contained 15 i~ small plastlds, as m the cell shown in Fig. 8, photographed by lt~ chlorophyll fluorescence It is easy to distinguish 14 plastlds and to see that at least two of them appear to be composed (>f two (fused)
Fig. 8. The plastld c o m p l e m e n t of a t y p m a l d a r k - g r o w n P/5oo cell given 3 lethal ultraviolet hits and t h e n exposed to red ( n o n - p h o t o r e a c t l v a t m g ) light for 3 days Fusion of m o s t proplastlds has n o t occurred There are 14 slngle-umt fluorescing plastlds, and three are seen to be c o m p o s e d of a t least two ( p r e s u m a b l y fused) plasttds
t3zochzm
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Acta, 142 (1907) I95 2o7
PROPERTIES OF SELECTIVELY STARVED EUGLENA
205
plastlds. A single such experiment with normal cells showed most developing cells with 30-40 such small plastids, but the increased number makes their accurate counting much more difficult.
~ IO tJ
§ O t)
5 o w g ~-oo u_
0
20
40 60 80 qO0 DOSEI~SECnNDS
Fig 9 The effect of ultraviolet h g h t on green-colony-forming ability of S / l o o o cells The hnes r e p r e s e n t the d a t a for n o r m a l and P/5oo cells as t a k e n f r o m Fig 7
Further evidence that the state of such selectively starved cells is a special and definite state of the cell is provided b y a few experiments on cells grown in low-sulfate medium S/Iooo cells also contain about 6 chloroplasts per cell, and as shown m Fig. 9, two ultraviolet-light bleaching experiments with such cells give results falling quite accurately on the curve obtained for the P/5oo cells, taken from Fig. 7.
DISCUSSION
The essential results of this paper are the values for DNA, chlorophyll, chloroplast numbers, and RNA In Euglena grown in our normal medium and in media in which the phosphate or sulfate concentrations have been markedly reduced. The accuracy of our DNA values can be estimated by a comparison with values for normal cells in the literature, although most such measurements are %1 other strains, frequently grown in different media and under different conditions of light intensity and temperature. Table I I presents a compilation of these data and shows that our value is generally in good agreement with those of other workers 5,9,15-18. In particular, for synchronously growing Z-strata cells, EDMUNDS15 found 2.2 pg DNA and 4 5 Pg DNA, respectively, for pre- and post-replication states. On the average, unsynchronized cells should have close to the mean of these two figures. 3.3 Pg DNA per cell, a value m good agreement with the 3.0 pg DNA per cell that we measured for our bacillans strain of Euglena. We tried several methods of extracting cells to obtain reproducible DNA values and tried both the Ceriotti and diphenylamine methods of estimating DNA. In our hands the dlphenylamine method was much more subject to variable interference from other substances present in the cell. The most reproducible extraction method was that of KEMPNER AND MILLER9. B*och*m
B,ophys
Acta, 142 (1967) I95-2o7
206
H
TABLE DNA
E
PER
T. E P S T E I N ,
E
ALLAWAY
II CELL
REPORTED
FOR
EUGLENA
grac*hs strain
Baclllans Mamx "IU'
"T" Z Z K l e b s Z, A T C C 1 2 7 1 6 Klebs, ATCC 12716 H e r e c a l l e d Z, l a t e r " u n l d e n t ~ f m d " Bacfllarls
pg D N 4 per cell
l?ef No
2 5 3 i 2 6 2 7 1 8* 3 2 2"*, 4 5 " " 3 4 3 7 3 o
i0 i6 16 16 ~7 5 i5 9 18 present
paper
* C a l c u l a t e d f r o m o 18 # g D N A - P p e r m g d r y w e i g h t , a s s u m i n g I lO 9 g d r y w e i g h t p e r c e l l (see r e f s 9, 15) *" T h e s e a r e t h e v a l u e s f o r s y n c h r o m z e d c e l l s b e f o r e a n d a f t e r D N A s y n t h e m s
The chromosome number found for normal cells, 92, was unexpected since LEEDALE19,20 estimated the related Klebs strata to have 45 chromosomes. This difference is not explained by our having looked at pre-mitotic nuclel because these were easily distinguished in our preparations by bemg uncountable as well as b y chromosome and cell morphology. However, his findings on a number of other members of the family Euglenlneae indicate that polyploidy is a regular feature ot this group. Therefore we consider it possible either that his strain differed from ours m ploldy or that it may have been in the same state as our low-phosphate cells whose chromosome number of 48 agrees with his number In fact, L E E D A L E ' S cultures were growing quite slowly in a blphasic soil-water system. As we have shown, the halfplold state may be brought about by sulfate as well as phosphate hmltation, and it is not unhkely that hmltatlons on other nutrients could effect similar reduction of ploidy. In nature, Euglena occurs m fresh waters and in moist soils. In the fresh waters it is very unlikely that phosphate and sulfate concentrations approach the molantms (respectively, 5 IO-a and 2.1o -3) used in our normal laboratory medium. Thus, what we have been calling reduction of ploldy might better be viewed a~ polyploidization m response to an unusually rich environment Otherwise, it could be expected that the cell would respond to lowered nutrient concentratlon~ simply by dividing slowly enough so that the accumulation of essential nutrients provided enough materials for its normal ploidy. The fact that half the chloroplasts (and proplastlds) are lost together with half the chromosomes may well imply some intrinsic nucleus-plastid relationship. If the plastids could reproduce independently of the nucleus, it could well have bee~ that the plastid ploldy would not have been reduced since maintenance would require only about 2 }~o of the phosphate needed by the nucleus. We are presently studying the response of cells to low-phosphate conditions in autotrophic medium to see if (and if so, how) the cells discard plastids or chromosome complements under conditions in which the photosynthetic activities of the plastlds are indispensable to the metabolic activities of the cell. B~oehzm
B~ophys
.4cta, i 4 2
(1967)
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PROPERTIES OF SELECTIVELY STARVED EUGLENA
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The reduction of ploidy makes other studies more feasible. For example, we are now studying the possibility of finding nuclear and plastld m u t a n t s whose presence m a y well have been masked in normal polyploid cells due to epistasls. If we can obtain such mutants, we plan to examine the posslbihty of finding sexual activity m half-ploid Euglena, whmh m a y well be more interested in such a c t l w t y since t h e y are less sated with chromosomes.
ACKNOWLEDGEMENTS
The authors are pleased to thank Miss J. JONES and Mr. E. LARSSON for their excellent assistance with many of the experiments, and Miss N. O'DoNOGHUE for her skilled electron microscopy. The work was supported by U.S. Public Health Servme grants GM-o6344-o 7 and GM-o6344-o8. REFERENCES i 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20
M EDELMAN, J A SCHIFF AND H T. EPSTEIN, J Mol B,ol, i i (1965) 769 M EDELMAN, H T EPSTEIN AND J A SCHIFF, J Mol B,ol, 17 (1966)463 G BRAWERMAN AND J EISENSTADT, Bzoch*m B2ophys Acta, 91 (1964) 477 D. S RAY AND P C HANAWALT, J Mol Bzol, i i (1965) 760 G BRAWERMAN, A O. POGO AND g CHARGAFF, B*ochzm B*ophys Acta, 55 (1962) 326 H. LYMAN, U T EPSTEIN AND J A SCHIFF, B*och*rn B*ophys Acta, 50 (1961) 3Ol B ROTMAN, J Bacterzol, 7° (1955) 485 H Z HILL, J A SCHIFF AND H. T. EPSTEIN, Bzophys. J , 6 (1966) 125 E S KEMPNER AND J H MILLER, B*och*m. B*ophys Acta, lO 4 (1965) I I A G E. PEARSE Hzstochermstry Theoretwal and Apphed, 2nd ed Little, Brown, and Co , Boston, 1961, p 822 Y BEN SHAUL, H. T EPSTEIN AND J a SCHIFF, Can J Botany, 43 (1965) 129. H T EPSTEIN, BOY DE LA TOUR AND J A. SCHIFF, Nature, 185 (196o) 825 H T EPSTEIN AND J A SCHIFF, J. Protozool, 8 (1961) 427 . Y BEN SHAUL, J A SCHIFF AND H T EPSTEIN, Plant Physzol , 39 (1964) 226 L N EDMONDS, J r , J Cellular Cutup Physzol, 62 (1964) 159 R NEFF, J Protozool, 7 (196°) 69 R M SMILLIE AND G KROTKOV, Can J Botany, 38 (196o)31 J R COOK, J Protozool, IO (1963) 436 G F LEEDALE, Nature, 18I (1958) 502 G. F LEEDALE, Bzol, Bull, 116 (1959) 162
B~ochzm B~ophys Acla, 142 (1967) 195-2o 7
195
BBA 95610
P R O P E R T I E S OF S E L E C T I V E L Y STARVED E U G L E N A
H T E P S T E I N AND E I A Z A B E T H A L L A W A Y
B*ology Department, Brandezs Unzverszty, Waltham, Mass (U S A ) (Received Novenaber 18th, 1966)
SUMMARY
It has been shown that starving Euglena for an essential nutrient causes at to reduce its nuclear and plastad complements by one-half If pressed b y extreme deprivation of the nutrient, the cell IS finally pushed to discard its plastid complement which is gratuitous in the heterotrophic medium ill which the cell is growing.
INTRODUCTION
In recent years it has been firmly established that some cellular organelles contain DNA. We have shared 1-4 in the demonstration that Euglena cells contain two DNA's in addition to the main, nuclear, DNA; these have been identified as being associated with the plastlds and the mltochondna. In suitable media Euglena can grow quite well either in the dark or after having had the chloroplasts entirely removed by various agents. Therefore, the plastlds and the plastid-assomated DNA are not at all indispensable to the cell. Accordingly it should be possible to alter the plastld DNA chemically or physically and study its subsequent properties without requiring its repair, this is presumably not true for the other DNA's, if damaged, since the cells would not be capable of reproduction. A study of such effect~ was initiated with a view towards ascertaining the relations, if any, between the nuclear and plastid DNA's. Selective starvation as a possible way of affecting the plastld DNA was chosen because of our interest in a second problem: that of obtaining specific labelling zn v,vo of plastld components. On light induction of chloroplast development several times as much non-plastid RNA and protein are synthesized as are plastld RNA and protein (BRAWERMAN, POGO AND CHARGAFF a) We thought it possible that during starvation for an essential nutrient, the plastlds might be preferentially discarded, and on replacing the nutrient just before complete plastid disappearance, plastid components would be preferentmlly synthesized Preliminary experiments of this type revealed some surprising properties of such selectively starved cells, and this paper reports on some of these propertms. B,och~m
Bzophys Acta, 142 (1967) 195-2o7
196
H. T
EPSTEIN,
E.
ALLAWAY
MATERIALS AND METHODS
Euglena gracklesvar. baclllarls was grown as p r e v i o u s l y described 6 in H u t n e r ' s p H 3.5 h e t e r o t r o p h l c m e d i u m in b a t c h cultures in e r l e n m e y e r flasks on a s h a k e r or in a c h e m o s t a t at room t e m p e r a t u r e u n d e r a b o u t 15o if-candles of illumination. P h o s p h a t e - f r e e m e d i u m was p r e p a r e d b y replacing the K H 2 P O a a n d (NH4)2HPO 4 of H u t n e r ' s m e d i u m with K H C Q a n d NH4HCO ~. N o r m a l m e d i u m , who~e p h o s p h a t e c o n c e n t r a t i o n is 5"1o a M, wa~ d i l u t e d m t o the p h o s p h a t e - f r e e m e d i u m a~ desired. F o r e x a m p l e , to p r e p a r e w h a t we designate P/2oo m e d m m , 5 ml of n o r m a l m e d i u m was d i l u t e d into IOOO ml of p h o s p h a t e - f r e e m e d m m . I n a similar way, ~ulfate-free m e d i u m was m a d e b y r e p l a c i n g the variou~ sulfates of n o r m a l m e d i u m (2.2-1o -3 M ,~ulfate) b y t h e i r chlorides U l t r a v i o l e t lrradlatlon~ were done as described p r e v i o u s l y s on cells in a m e d m m c o n t a m m g 0.05 M m a n m t o l a n d o o I M MgC12, which is the p h o s p h a t e - f r e e version of t h e resting m e d i u m p r e v i o u s l y used for o b t a i n i n g n o n - d l v l d m g cells. Low-phosp h a t e resting m e d i u m was o b t a m e d b y s t a t a b l e dilution of n o r m a l re~tmng m e d i u m into this p h o s p h a t e - f r e e resting m e d i u m Most e x p e r i m e n t s were done with cells grown in a c h e m o s t a t a d a p t e d from t h a t described b y ROTMAN7 a n d shown in F i g I F r e s h m e d i u m is c o n t m u o u s l y d r i p p e d in at the finely d r a w n tip, T, while cell suspension c o r r e s p o n d i n g l y drips out into t h e receiving flask, F. The d r i p r a t e was a d j u s t e d b y the Teflon stopcock, S, each m o r n i n g so t h a t the cell c o n c e n t r a t i o n s r e m a i n e d fairly constant. B y blowing
INPU
MEDIUML.~ HOSE
II
F i g I A c h e m o s t a t a d a p t e d f r o m ROTMAN ~ R a t e o f m e d m m i n p u t t o t h e g r o w t h c h a m b e r , G , is r e g u l a t e d b y t h e s t o p c o c k , S, so t h a t t h e cell c o n c e n t r a t i o n m t h e s a m e e v e r y m o r n i n g A f t e r the colnblned bubble-breaker and reservoir, R-B, m filled up to the height causing overflow, the v o l u m e ~s k e p t c o n s t a n t b y s ~ p h o n a c n o n i n t o t h e c o l l e c t i n g f l a s k , F B y b l o w i n g m t h r o u g h A, the mphon action can be stopped, and fluid accumulates m B-R until the overflow takes place. At any tune fluid can be siphoned off into F by sucking air out through A
Bwchzm
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.4cta, 142 (1067) i()5 2o 7
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in through the air-hole, A, the chemostat action is stopped and cells accumulate in B-R, the combined bubble-breaker and reservoir. By sucking air out through A, the contents of the reservoir and growth chamber, G, can be caused to start flowing out into F. By using the reservoir in this way, large numbers of cells could be obtained In low-phosphate media, cell concentrations were about I.IOa-2'IoSper ml, m normal media they were 1.1o6 2.1o 6 per ml. Cell counts were made with a hemocytometer or a Coulter counter, depending on the sample volume available, these methods were crosschecked occasionally to ensure umformlty. After adding a pinch of MgCQ, chlorophyll was extracted with 85 °7o acetone and concentrations determined from absorbances at 663 and 645 mF. Otherwise extractions to measure RNA and DNA were carried out by the procedure of KEMPNER AND MILLER9 in which RNA was finally measured by the orclnol method and DNA by the Ceriottl method To make chromosome counts, cells were washed in a solution containing o.15 M NaC1 and O.lO M EDTA (dlsodium salt) (pH 8), treated with Viokase (I °/o, 15 ram), fixed with methanol-acetic acid (3.1, v/v), smeared on microscope slides, and stained with acetic acid-orcein (i °'o orcein in 50 ~o acetm acid). The Vlokase was made up in o 02 M potassium phosphate buffer (pH 8) for normal cells, in I o M mannitol brought to pH 8.5 for low-phosphate cells The procedure of PEARSEI° was used for Feulgen staining of the smears. Deoxyribonuclease pretreatment was at o.I mg/ml DNA in 3.1o -a M MgSO4 (pH 6.8) for 4 h at 36°. Control pretreatment was In the same solution without enzyme Electron micrographs were made as previously described n using glutaraldehyde fixation and OsO4 staining. Chloroplasts were observed in living cells by the red fluorescence of their chlorophyll and photographed as previously described 1~. Photomicrographs of the fluorescent cells were made using Polaroid infrared film which enabled us to obtain good results with exposures of only a few seconds.
EXPERIMENTS
AND
RESULTS
In the initial experiments to starve selectively for phosphate, o.I ml of cells growing in the hght in normal medium were diluted into 2o ml of phosphate-free medium (giving P/2oo), and cell growth proceeded until the stationary concentration of 4.IO5-5.IO5 cells per ml was reached. These cultures were noticeably less green than normal ones at the same cell concentrations, and observation with the fluorescence microscope revealed that this was due primarily to the cells containing fewer (and slightly smaller) chloroplasts per cell By the time stationary phase was reached, some cells had no traces of chlorophyll fluorescence, and when plated, many cells gave rise to entirely white colonies. When cells containing 3-5 chloroplasts per cell were subcultured into fresh P/2oo medium, they reverted toward normal amounts of chlorophyll and normal numbers (11-13) of chloroplasts. Only after several generations of growth did "degreening" begin again, and subcultures started at fairly high cell densities did not de-green noticeably. Some experiments have been carried out with cells having 3-5 chloroplasts per cell, and the results were similar to those to be described which used chemostat-grown cells. To avoid problems of reversion and to obtain a supply of Bzoch~m
Bzophys Zlcta, 142 ( 1 9 6 7 ) 195 2 0 7
I98
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T. E P S T E I N ,
E. ALLAWAY
reproducible " l o w - p h o s p h a t e " cells, chemostat culture was b e g u n I n the experim e n t s to be described below, most work was done with cells grown an P/5oo m e d m m , a l t h o u g h some comparison experiments were done using P,"2oo, P/3oo, a n d P ' I o o o media.
I
The b,ochemwal contenls o~ P/5oo cells Table I gives some general d a t a for cells grown in n o r m a l a n d P/5oo media. As would be expected from the drop in R N A c o n t e n t b y a factor of a b o u t 3, the "l A I 3 L E i GENERAL
DAIA
ON
( H / ~ \ I O b T A , F-GROVVN
l e N A (pg p e r cell) D N A (pg p e r cell) C h l o r o p h y l l (pg p e r cell) C h l o r o p l a s t s p e r cell
EUGLENA
N o r m a l cells
P / 5 o o cells
3 ° 40 3 o 15 l I 13
lO-15 I 5 5 5 7
P/5oo cells are definitely smaller m d i a m e t e r t h a n n o r m a l cells. As was the case for n o n - c h e m o s t a t P/2oo cells, the r e d u c t m n in chlorophyll c o n t e n t is composed m a i n l y of a decrease m chloroplast n u m b e r b u t also p a r t l y of a decrease m chlorophyll per chloroplast Both these effects can be seen in the fluorescence pictures m Fig. 2, m which P a r t a is of n o r m a l cells a n d P a r t b is of P/5oo cells. The P/5oo cells c o n t a i n 5 7 chloroplasts, compared with the I I - I 3 in n o r m a l cells, a n d the~e are i n d i v i d u a l l y smaller t h a n those m the n o r m a l cells. We note p a r t i c u l a r l y t h a t chloroplast n u m b e r 1~ reduced b y the same factor as total cell DNA. A n i n t e r e s t i n g reference from these d a t a 1~ t h a t some nuclear a n d plastld ploidy ha~ been lost m the Er5oo ~ell~ We now t u r n to ~tudies designed to test this Inference
2 Nuclear plo~dy The most obvious way of i n t e r p r e t i n g the 50 °o decrease of D N A an P/5oo cells is t h a t of reduction of nuclear ploldy However, we have been dissatisfied with the v a r i a b i l i t y of the D N A m e a s u r e m e n t s which seems due to interfering substances associated with the presence of chloroplasts We have therefore estimated the n u m b e r of chromosomes in two ways. I n t e r p h a s e E u g l e n a chromosomes are in a partially-condensed or multig r a n u l a r state characteristic of m a n y protlsts. The n m n b e r of granules ~hould be related to the n u m b e r of chromosomes, a n d we have a t t e m p t e d to c o u n t t h e m using b o t h the electron microscope a n d the hght microscope Electron mmrographs of n o r m a l a n d P/5oo cells are shown m Fig. 3. The granuleb bhow up as dark blobs in the nuclei The granule n u m b e r s per mlcrograph sectmn were c o u n t e d a n d found to average a b o u t 45 for n o r m a l cells, a b o u t 22 for P/5oo cell~. The total granules per burat nucleus in acetic acid orcein p r e p a r a t i o n s were c o u n t e d xn the hght m~croscope, Bzoch~m
Bzopkr~
_4cla, 142 (1907) 195 207
~
Jql~~ap ~8
% n
~!
i~ii~~i~
:200
H, T. EPSTEIN, E
ALLAWAY
Fig 3 E l e c t r o n m m r o g r a p h s of s e c t i o n s (A) n o r m a l a n d (B) P / 5oo cells D a r k b l o b s are vi s i bl e m t h e nuclei, t h e r e b e i n g 4o-.5o m (X) a n d a b o u t 2o m (B) The bl obs are s e mi c o n d e n s e d or m u l t i granular mterphase chromosomes
lhock~m
l'~zopky,
.4cla, t 4 : 0 9 6 7 )
I05 207
PROPERTIES
OF SELECTIVELY
STARVED
EUGLENA
201
and similarly found to differ by a factor of 2, although the distributions were quite broad, as shown in Fig. 4, in which average granule numbers of 194 and 96 were found for the two types of cells. If we make plausible assumptions about granule size and section thickness of electron microscope preparations we can calculate that the numbers obtained from electron microscopy and light microscopy are in rough agreement. I0-
8J
-
-
60
120 180 240 GRANULE NUMBER
F i g 4 T h e d i s t r i b u t i o n o f g r a n u l e n u m b e r s m n o r m a l a n d P / 5 o o cells, a s d e t e r m m e d f r o m t o r e r o g r a p h s h k e t h o s e i n F i g 3 T h e c r o s s - h a t c h e d d l s t n b u t m n is t h a t o f t h e n o r m a l c e l l s T h e a v e r a g e numbers are about I9o for normal ceils and about 90 for P/5oo cells
Recently, improved techniques have yielded enough burst telophase nuclei with condensed mitotic chromosomes so that we can give estimates of chromosome numbers. Fig. 5 shows such preparations for both types of cells. The chromosome number for normal cells is 9 2 ± 2 and 4 ~ 3 for !ow-phosphate cells. The units being counted were found to be Feulgen-posltlve, and Feulgen staining was prevented b y deoxyribonuclease pretreatment. No deoxynbonuclease-sensmve staining was seen in the cytoplasm of either cell type. The variability of DNA measurements was at least partly associated with the different media and possibly slightly different light intensities in the various chemostats. To reduce these variables, cells were taken from a P/5oo chemostat and placed in an erlenmeyer flask with aeration. A small volume of I M phosphate was added to bring the medium up to normal phosphate concentration, and samples were taken daily. In this way cells could be studied in a fixed environment. The results of 4 such experiments are assembled in Fig. ~. Here it can be seen that the P/5oo amount of DNA is increased practically to normal after 24 h, and then remains at that value. Significant cell division began only after 24 h, but was roughly normal starting with 48 h Chlorophyll per cell was unchanged at 4 5 Pg per cell through 96 h.
3. Plashd plo~dy We have already presented evidence that the number of detectable plastlds has been reduced b y a factor of two in P/5oo cells. The same reduced number was found for chemostat cells grown in P[2oo and P[Iooo media. Given the reduction in chloroplast number, we were Interested in finding out if the plastid-associated DNA Bzoch*m. B*ophys. Acla, 142 ( 1 9 6 7 ) 1 9 5 - 2 o 7
202
H
3. L P S I L I N , L.
\LI..kW.\Y
Fig. 5 P h a s e - c o n t r a s t p h o t o m i c r o g r a p h s of c h r o m o s o m e c o m p l e m e n t s m b u r s t n u c l e i of (A) n o r m a l a n d (B) P / 5 o o ceils T h e r e are, r e s p e c n v e l y , a b o u t 9o a n d a b o u t 45 c h r o m o s o m e s
Bzochzm Bzophys .tcla, 142 (iq~7) t95 207
203
PROPERTIES OF SELECTIVELY STARVED EUGLENA
is also r e d u c e d or w h e t h e r it is r e t a i n e d in some form, p o s s i b l y m a s s o c l a t m n w i t h n o n - d e v e l o p i n g plastids. T h e m o s t direct w a y of d e t e r m i n i n g how m u c h p l a s t i d - D N A is p r e s e n t w o u l d be to m e a s u r e it. H o w e v e r , since it r e p r e s e n t s o n l y a b o u t 2 % of t h e t o t a l D N A , a c c u r a t e d e t e r m i n a t i o n of this a m o u n t is n o t a t p r e s e n t feasible. I n its m o s t d~rect
o
o STANDARD MEDIUM
/
~------{-t-°
i ,c
• ,.5oo MEDIUM
t~ Io
g ~o~
W ~,oo
g
~-
~o 10
C-O0
~. i
2
3
4
20
DAYS
4o
60
8o
ioo
DOSE IN SECONDS
which
Fig 6 The ceil concentration and DNA per cell in P15oo cells to enough phosphate was added to give the normal concentration The flags represent the range of the data for four experiments Fig 7 The effect of ultraviolet on green-colony forming ability of normal and P/5oo cells The lines are obtained from computer analyms of the data
form, the p r o b l e m is t h a t of m e a s u r i n g t h e a r e a u n d e r a p e a k in a d e n s i t o m e t e r t r a c i n g of CsCl-banded whole-cell D N A , w i t h t h e shoulder of t h e m a i n b a n d D N A t r a i l i n g in to the position of t h e c h l o r o p l a s t satellite D N A . A c c o r d i n g l y we h a v e used indirect methods. W e h a v e p r e v i o u s l y shown t h a t t h e n u m b e r of p r o p l a s t i d s seen b y fluorescence la per d a r k - g r o w n cell is similar to the 36 t a r g e t s 8 / o r u l t r a v i o l e t b l e a c h i n g ( i n a c t i v a t i o n of t h e g r e e n - c o l o n y - f o r m i n g a b i l i t y of t h e cells) curves. Since t h e action s p e c t r u m for bleaching is t h a t of nucleoprotein, we h a v e m a d e t h e r e a s o n a b l e a s s u m p t i o n t h a t bleaching is due to u l t r a v i o l e t d a m a g e to t h e D N A of the i n d i v i d u a l p r o p l a s t i d s . Therefore we should be able to m e a s u r e t h e n u m b e r of these n u c l e o p r o t e i n entities in l o w - p h o s p h a t e cells b y bleaching e x p e r i m e n t s . If t h e p l a s t i d D N A is r e a l l y reduced, t h e t a r g e t n u m b e r o b t a i n e d should equal t h e n u m b e r of p r o p l a s t i d s visualized b y fluorescence microscopy. U l t r a v i o l e t bleaching curves h a v e been o b t a i n e d for b o t h n o r m a l a n d P/5oo cells, a n d t h e results of 3 s e p a r a t e e x p e r i m e n t s are given in Fig. 7- B o t h curves look like m u l t i - t a r g e t curves. If we are to c o m p a r e t h e t a r g e t n u m b e r s of t h e two curves, t h e a s y m p t o t i c slopes m u s t be equal, i n d i c a t i n g t h e s a m e i n h e r e n t u l t r a v i o l e t s e n s i t i v i t y of the t a r g e t s a n d therefore t h a t the t a r g e t s are p r o b a b l y i d e n t m a l in the two t y p e s of cells. A c o m p u t e r was p r o g r a m m e d to d e t e r m i n e the a s y m p t o t i c slopes a n d t h e t a r g e t n u m b e r s from t h e s t r a i g h t line p o r t i o n s of t h e curves. T h e slopes were e x p e r i m e n t a l l y indistinguishable, being 11. 7 sec p e r hit for t h e n o r m a l cells a n d 11.8 sec per hit for the P/5oo cells. T h e respective t a r g e t n u m b e r s are 3 7 ± 2 Bzoch~m B,ophys
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ALLAWAY
and 18±2, from which we infer that the amount of plastld DNA in P/5oo cells is half that in normal cells. An accurate determination of the number of proplastlds was previously beyond our abfllty to measure because proplastlds were shown ~ to fuse by threes after 8-12 h after placing dark-grown cells in the light At those early times, some of the proplastlds contain so httle chlorophyll that they are too faint to he seen well. Of course, the number of proplastlds can also be estimated by multiplying the number of chloroplasts by three to give 30 for normal cells and 18 for P/'5oo cells In an attempt to measure the number of proplastids directly, we guessed that the fusion step might he inactivated by ultraviolet light, thereby permitting the proplastlds to develop individually to the point of being countable (note that bleachi n g - t h e formation of white colomes i~ not revolved because the color of the colonies formed tells only what the progeny of the lrradmted cells are like). Dark-grown P/5oo cells were given 2-3 lethal hits of ultraviolet light and placed in red (nonphotoreactlvating) hght for plastld deveh>pment. After 3 day~ many of the cells had developed apprecmbly, and contained 15 i~ small plastlds, as m the cell shown in Fig. 8, photographed by lt~ chlorophyll fluorescence It is easy to distinguish 14 plastlds and to see that at least two of them appear to be composed (>f two (fused)
Fig. 8. The plastld c o m p l e m e n t of a t y p m a l d a r k - g r o w n P/5oo cell given 3 lethal ultraviolet hits and t h e n exposed to red ( n o n - p h o t o r e a c t l v a t m g ) light for 3 days Fusion of m o s t proplastlds has n o t occurred There are 14 slngle-umt fluorescing plastlds, and three are seen to be c o m p o s e d of a t least two ( p r e s u m a b l y fused) plasttds
t3zochzm
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205
plastlds. A single such experiment with normal cells showed most developing cells with 30-40 such small plastids, but the increased number makes their accurate counting much more difficult.
~ IO tJ
§ O t)
5 o w g ~-oo u_
0
20
40 60 80 qO0 DOSEI~SECnNDS
Fig 9 The effect of ultraviolet h g h t on green-colony-forming ability of S / l o o o cells The hnes r e p r e s e n t the d a t a for n o r m a l and P/5oo cells as t a k e n f r o m Fig 7
Further evidence that the state of such selectively starved cells is a special and definite state of the cell is provided b y a few experiments on cells grown in low-sulfate medium S/Iooo cells also contain about 6 chloroplasts per cell, and as shown m Fig. 9, two ultraviolet-light bleaching experiments with such cells give results falling quite accurately on the curve obtained for the P/5oo cells, taken from Fig. 7.
DISCUSSION
The essential results of this paper are the values for DNA, chlorophyll, chloroplast numbers, and RNA In Euglena grown in our normal medium and in media in which the phosphate or sulfate concentrations have been markedly reduced. The accuracy of our DNA values can be estimated by a comparison with values for normal cells in the literature, although most such measurements are %1 other strains, frequently grown in different media and under different conditions of light intensity and temperature. Table I I presents a compilation of these data and shows that our value is generally in good agreement with those of other workers 5,9,15-18. In particular, for synchronously growing Z-strata cells, EDMUNDS15 found 2.2 pg DNA and 4 5 Pg DNA, respectively, for pre- and post-replication states. On the average, unsynchronized cells should have close to the mean of these two figures. 3.3 Pg DNA per cell, a value m good agreement with the 3.0 pg DNA per cell that we measured for our bacillans strain of Euglena. We tried several methods of extracting cells to obtain reproducible DNA values and tried both the Ceriotti and diphenylamine methods of estimating DNA. In our hands the dlphenylamine method was much more subject to variable interference from other substances present in the cell. The most reproducible extraction method was that of KEMPNER AND MILLER9. B*och*m
B,ophys
Acta, 142 (1967) I95-2o7
206
H
TABLE DNA
E
PER
T. E P S T E I N ,
E
ALLAWAY
II CELL
REPORTED
FOR
EUGLENA
grac*hs strain
Baclllans Mamx "IU'
"T" Z Z K l e b s Z, A T C C 1 2 7 1 6 Klebs, ATCC 12716 H e r e c a l l e d Z, l a t e r " u n l d e n t ~ f m d " Bacfllarls
pg D N 4 per cell
l?ef No
2 5 3 i 2 6 2 7 1 8* 3 2 2"*, 4 5 " " 3 4 3 7 3 o
i0 i6 16 16 ~7 5 i5 9 18 present
paper
* C a l c u l a t e d f r o m o 18 # g D N A - P p e r m g d r y w e i g h t , a s s u m i n g I lO 9 g d r y w e i g h t p e r c e l l (see r e f s 9, 15) *" T h e s e a r e t h e v a l u e s f o r s y n c h r o m z e d c e l l s b e f o r e a n d a f t e r D N A s y n t h e m s
The chromosome number found for normal cells, 92, was unexpected since LEEDALE19,20 estimated the related Klebs strata to have 45 chromosomes. This difference is not explained by our having looked at pre-mitotic nuclel because these were easily distinguished in our preparations by bemg uncountable as well as b y chromosome and cell morphology. However, his findings on a number of other members of the family Euglenlneae indicate that polyploidy is a regular feature ot this group. Therefore we consider it possible either that his strain differed from ours m ploldy or that it may have been in the same state as our low-phosphate cells whose chromosome number of 48 agrees with his number In fact, L E E D A L E ' S cultures were growing quite slowly in a blphasic soil-water system. As we have shown, the halfplold state may be brought about by sulfate as well as phosphate hmltation, and it is not unhkely that hmltatlons on other nutrients could effect similar reduction of ploidy. In nature, Euglena occurs m fresh waters and in moist soils. In the fresh waters it is very unlikely that phosphate and sulfate concentrations approach the molantms (respectively, 5 IO-a and 2.1o -3) used in our normal laboratory medium. Thus, what we have been calling reduction of ploldy might better be viewed a~ polyploidization m response to an unusually rich environment Otherwise, it could be expected that the cell would respond to lowered nutrient concentratlon~ simply by dividing slowly enough so that the accumulation of essential nutrients provided enough materials for its normal ploidy. The fact that half the chloroplasts (and proplastlds) are lost together with half the chromosomes may well imply some intrinsic nucleus-plastid relationship. If the plastids could reproduce independently of the nucleus, it could well have bee~ that the plastid ploldy would not have been reduced since maintenance would require only about 2 }~o of the phosphate needed by the nucleus. We are presently studying the response of cells to low-phosphate conditions in autotrophic medium to see if (and if so, how) the cells discard plastids or chromosome complements under conditions in which the photosynthetic activities of the plastlds are indispensable to the metabolic activities of the cell. B~oehzm
B~ophys
.4cta, i 4 2
(1967)
195-2o7
PROPERTIES OF SELECTIVELY STARVED EUGLENA
207
The reduction of ploidy makes other studies more feasible. For example, we are now studying the possibility of finding nuclear and plastld m u t a n t s whose presence m a y well have been masked in normal polyploid cells due to epistasls. If we can obtain such mutants, we plan to examine the posslbihty of finding sexual activity m half-ploid Euglena, whmh m a y well be more interested in such a c t l w t y since t h e y are less sated with chromosomes.
ACKNOWLEDGEMENTS
The authors are pleased to thank Miss J. JONES and Mr. E. LARSSON for their excellent assistance with many of the experiments, and Miss N. O'DoNOGHUE for her skilled electron microscopy. The work was supported by U.S. Public Health Servme grants GM-o6344-o 7 and GM-o6344-o8. REFERENCES i 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20
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B~ochzm B~ophys Acla, 142 (1967) 195-2o 7