Ribonucleic acids from the mitochondria of bleached Euglena gracilis z

132

BIOCHIMICAET BIOPHYSICAACTA

B B A 96572

R I B O N U C L E I C ACIDS FROM T H E MITOCHONDRIA OF B L E A C H E D EUGLENA

GRACILIS z

II C H A R A C T E R I Z A T I O N OF H I G H L Y POLYMERIC R I B O N U C L E I C ACIDS STEVE~I KRAWIEC* AND JEROME M. EISENSTADT Department o/Mwrobzology, Yale Umverszty School ol Medw*ne, New Haven, Corm o65zo (U. S.A )

(Received March i2tb, I97o)

SUMMARY I. The presence of 0.2 M NaC1 reduces nuclease activity during phenol extraction of cytoplasmic and mltochondrial nbonucleic acids from bleached Euglena gracilis z.

2 Sedimentation analyses reveal that the cytoplasm contains 25- and I9-S RNA's and that the mitochondna contain 14- and II-S RNA's. 3 Thermally induced hyperchrommity spectra demonstrate that mitochondrlal RNA's contain predominantly AU pairs whereas cytoplasmic RNA's contain large numbers of GC pairs. 4. Descending paper chromatography indicates that the mole% composition of mltochondrial RNA is 39.5 % adenine, 33.2 % uracil, 14. 9 % guanine, and 12 5 % cytosine. The composition of cytoplasmic RNA is 23.4 % adenine, 22 3 % uracil, 31.o % guanine, and 23.4 % cytosine. 5. The compositions, secondary structures, and sedimentation characteristics of bleached E. grackles mitochondrial and cytoplasmic RNA's are different. Although distinct from bacterial RNA, highly polymeric mltochondrial RNA appears more similar to procaryotic than eucaryotlc RNA

INTRODUCTION During the past several years, mitochondrial RNA from fungi l-s, algae 9 protozoa l°,n, angiosperms 1, and vertebrates lza3 has been extracted and characterized This report describes some physical and chemical characteristics of mitochondrla and cytoplasmic RNA's from bleached E. gracdzs z For the isolahon of native mitochondrial RNA's, an extraction procedure which yields native RNA's from whole cells was applied to purified mltochondria. The extent of degradation of cytoplasmic RNA's was gauged by the sedimentation pattern of the nucleic acids In sucrose gradients A typical sedimentation profile for highly polymeric (ribosomal) RNA's exhibits two major components 14-1~, the more rapidly sedlmentlng "~" component containing approximately twice the amount of RNA as the less rapidly sedimentmg "~" component 16 " Address after July ISt, 197° Department of Biology, Lehigh Umvermty, Bethlehem, Pa 18Ol5, U S A B*ochzm Bzophys Aela. 217 (197° ) 132-141

EUGLENA MITOCHONDRIALRNA. II

133

The isolation of native RNA molecules from Escherwhia col, and the mitochondria and cytoplasm of E. grac,lis allows analyses of the composition, secondary structure, and sedimentation characteristics of the macromolecules. Comparisons of such results Indicate that mitochondnal RNA is different from both procaryotic and eucaryotm cytoplasmic RNA but more similar to the former than the latter. An abstract of this information has appeared previously TM.

METHODS AND MATERIALS

Orgamsms The preparahon of streptomycin-bleached (SB3) and heat-bleached (HB4) mutants of E. grac,lis z has been described previously 9. E. coli AB 3000 was obtained from Dr. E. A. Adelberg.

Growth Mutants of E. gracilis were grown under conditions reported previously 9 on Medium A described by GREENBLATTAND SCHIFF19. E coli was grown on Medium B described by SHIH et al. ~° supplemented with I/zg vitamin B 1 per ml. For the preparation of labeled ribosomes, 25 #g uracil per ml and 50 #C [3Hluridine were also added to the medium

Isolation o] m#ochondria The harvesting and disruption of cells, the subcellular fractionation of the slurry, and the purification of mitochondria have been described 9.

Extraction o / R N A RNA was extracted as previously described 9 with one modification: during nuclem acid extraction or storage, aqueous solutions contained the concentrations of NaC1 specified in the text.

Rad,oact, ve mon,tormg Radioactive samples not exceeding o.I ml were absorbed on 2.4-cm W h a t m a n 3 MM paper discs The discs were washed as described by MANS AND NOVELL121 with two modifications: hydrolysis at 9°0 in 5 % trichloracetic acid was omitted, and incubation in ethanol-ether was performed at room temperatures.

Sed,mentatwn analys,s Small volumes of RNA solutions were layered on the surface of hnear, continuous, 5-ml or i2-ml, 5-20 % sucrose gradients 22 containing o.oi M Tris-HC1 (pH 7.4) and NaC1 (see figure legends for concentrations). The 5-ml gradients were centrifuged at 2 ° at 48 000-50 ooo rev./min for 3.5-4 h without braking m a Beckman SW-5o rotor. The I2-ml gradients were centrifuged at 2 ° at 41 ooo rev./min for 5.58 h without braking in a Beckman SW-4I rotor. In gradients that had 15o-3oo/zg RNA, the bottom of the mtrocellulose centrifuge tube was pierced and Io-drop samples (approx. 0.2 ml) were collected. The samples were diluted with 0.6 or 0.8 ml water and absorbance at 260 m# was measured. Bzochzm. B~ophys. Acta, 217 (197 o) 132-141

134

s . K R A W I E C , J. M. E I S E N S T A D T

In gradients that contained 5o-15o/~g RNA, a fine-bore needle was lowered through the gradient to the bottom of the nitrocellulose tube. The contents were pumped through an L K B Uvicord scanner equipped with a flow cell and absorbance at 258 m F was recorded with a Sargent SRL recorder.

Thermally ,nduced hyperchrom,czty The absorbance of RNA dissolved in o.15 M NaCl-o.oI 5 M sodium citrate was measured at 5-m# intervals from 230 through 31o nap at ambient temperature (25-29 °) and three other temperatures: 41.5-43.5 °, 57.5-60.5 °, and 71.5-73.5 ° (see refs. 23, 24). The absorbance was measured with a Zeiss PMQ-II spectrophotometer equipped with a thermostated circulating pump, and the temperatures were measured with a thermocouple. The hyperchromlcity was calculated after the absorbance at 31o m F was subtracted from other absorbance measurements made at the same temperature 25.

Chromatography Chemical base analysis was performed by a method of descending paper chrom a t o g r a p h y described b y AAMODT AND EISENSTADT2e. A 95 % confidence interval for the mole% of each base was established with Student's t test 27. RESULTS

Sedimeniat, on analysis Extraction of E. gracil,s cytoplasmic RNA's with no precautions to minimize nuclease activity yields degraded RNA's :7,2s,~9. In sedimentation profiles, such de-

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F i g I S e d i m e n t a t i o n p r o f i l e s of b l e a c h e d E. grac*l,.s c y t o p l a s m i c R N A ' s e x t r a c t e d i n t h e p r e s e n c e a n d a b s e n c e of o.2 M NaC1. S a m p l e s of i M N a C l - l n s o l u b ! e c y t o p l a s m i c m a t e r i a l p r e p a r e d w l t h (- - -) a n d w i t h o u t ( ) o 2 M NaC1 w e r e l a y e r e d o n t h e s u r f a c e of t 2 - m l 5 - 2 0 % s u c r o s e g r a d i e n t s c o n t a i n i n g o . o i M T r l s - H C 1 ( p H 7.4) a n d 5 m M NaC1 a n d c e n t r i f u g e d a t 41 ooo r e v . / m m for 7 5 h a t 4 ° A b s o r b a n c e a t 258 m / , w a s m e a s u r e d w l t h a n L K B U v m o r d E coh r R N A s e r v e d as a standard.

B*och*m B,ophys Acta, 217 ( I 9 7 o) 132-141

EUGLENA MITOCHONDRIALR N A II.

135

graded R N A ' s exhibit a m a j o r I9-S c o m p o n e n t a n d a n u m b e r of m i n o r species 3°,31. TAKANAM132 d e m o n s t r a t e d t h a t the yield of different species of R N A from r a b b i t liver ribosomes varied with the c o n c e n t r a t i o n of NaC1 present d u r i n g extraction. Accordingly, samples of E. gracilis cell-free homogenate were extracted with 80 °/o phenol solutions c o n t a i n i n g various c o n c e n t r a t i o n s of NaCI. I n Fig. I, the sediment a t i o n profiles of I M NaCl-insoluble R N A ' s prepared in the presence a n d absence of o 2 M NaC1 are superimposed. I n the presence of salt, the yield of R N A is approx. 2-fold greater a n d the s e d i m e n t a t i o n profile of the R N A ' s exhibits p r o m i n e n t 25- a n d I9-S c o m p o n e n t s (c/. refs. 17, 28, 29) R N A ' s prepared in the absence of salt r e t a i n the I9-S c o m p o n e n t b u t the 25-S c o m p o n e n t disappears a n d the a m o u n t s of 17- a n d Io-S R N A ' s Increase. The a m o u n t of R N A in the 25-S "o~" peak is 1.25 times the a m o u n t of R N A in the I9-S "fl" peak suggesting t h a t d e g r a d a t i o n of the "~" c o m p o n e n t is n o t completely p r e v e n t e d b y o 2 M NaC1. The possibility t h a t the more r a p i d l y s e d i m e n t i n g c o m p o n e n t was a n aggregate of more slowly s e d i m e n t i n g R N A ' s was excluded b y preparing cytoplasmic R N A ' s in the presence of i mM NaC1 a n d t h e n dialyzing the p r o d u c t against 0.2 M NaC1. S e d i m e n t a t i o n analysis indicates t h a t exposure of the degraded R N A ' s to 0.2 M NaC1 does n o t produce a 25-S c o m p o n e n t (Fig. 2). S e d i m e n t a t i o n profiles of R N A ' s

A

B

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z

=< p.

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I

f B ($B3)

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Fig 2. The effect of 0.2 M NaCI on the aggregation and degradation of bleached E. grackles cytoplasmic RNA's. The curves illustrate the sedimentation profiles of I M NaCl-msoluble RNA's (A) extracted m the presence of o 2 M NaC1 and then (B) dialyzed against water, or (C) extracted in the presence of I mM NaC1 and then (D) dzalyzed against 0.2 M NaCI. Centrzfugatlon conditions and the standard were the same as those presented in the legend of Fig. I. Fig. 3- Sedimentation profiles of RNA's extracted from the nntochondrla of heat-bleached (A) and streptomycin-bleached (B) E. grac*l*s. Condzt]ons were the same as those presented in the legend of Fzg. I except that the centrzfugataon time was 7 h.

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s. KRAWIEC, J. M. EISENSTADT

e x t r a c t e d in t h e p r e s e n c e of o.2 M NaC1 a n d d i a l y z e d a g a i n s t w a t e r r e v e a l a d i m i n u t i o n of 25-S R N A s u g g e s t i n g a g r e a t e r l a b i l i t y of t h e m o r e r a p i d l y s e d l m e n t m g c o m p o n e n t 1v,33,34. T o isolate n a t i v e m l t o c h o n d r i a l R N A ' s , p u r i f i e d m i t o c h o n d r l a f r o m s t r e p t o m y c i n - a n d h e a t - b l e a c h e d E . g r a c , lzs w e r e e x t r a c t e d w i t h a p h e n o l - o 2 M NaC1 (4:1, w / v ) e m u l s i o n . T h e s e d i m e n t a t i o n profiles of t h e p r o d u c t s (Fig. 3) r e v e a l 14- a n d I I - S R N A ' s If 0.2 M NaC1 is o m i t t e d d u r i n g e x t r a c t i o n , t h e a m o u n t of I 4 - S R N A d i m i n i s h e s a n d a n 8 - 9 - S c o m p o n e n t a p p e a r s . T h e y i e l d of I M NaC1i n s o l u b l e R N A f r o m p u r i f i e d m i t o c h o n d n a was 2.0 # g / r a g p r o t e i n . T h e s e d i m e n t a t i o n r a t e s of m i t o c h o n d r l a l R N A ' s are g r e a t l y i n f l u e n c e d b y Mg ~÷ (refs. 3, 4). If I m M MgC12 is p r e s e n t in all s o l u t i o n s u s e d in t h e e x t r a c t m n , s t o r a g e , a n d a s s a y of E . g r a c i l i s m i t o c h o n d r i a l R N A , t h e 14- a n d I I - S c o m p o n e n t s s e d i m e n t as 17. 7- a n d I 4 - S c o m p o n e n t s , r e s p e c t i v e l y (Table I). T h e s e results i n d i c a t e t h a t c o n s i d e r a b l e m o d i f i c a t i o n of t h e s e c o n d a r y s t r u c t u r e of m l t o c h o n d r i a l R N A c a n O c c u r 35.

TABLE I T H E E F F E C T OF m g 2+ ON T H E S E D I M E N T A T I O N OF

Mltochondnal Mltochondrlal E colt rRNA E. colt rRNA

RNA "~" component RNA "fl" component "~z" component "fl" component

E grac~lzs

M I T O CH O N D RI A L R ~ A

Sedtmented tn gradzents contaznzng 5 m M NaC1

Sedtmented *n gradtents contazntng J: rnM M e C l 2

Ratw o/sedimentation rates tn I m M M g C l 2 and 5 m M N a C l

14 iI 23"* 17 7**

17 7* i4" 23"* 17 7"**

i i i i

26 27 o o

* All solutions used m the extraction, storage, and assay of these mltochondrlal RNA's contained i mM MgC1z. "* E coh, rRNA was extracted in the absence of salts *** Ahquots of E colt RNA were eqmhbrated with I mM MgC12 solutions for 48 h. Sedimentation coefficients were calculated by the relation %3/Dza -- sx/D ~, where s~3 = 23 and Dza equals the distance the 23-S component moved m a sucrose gradmnt containing 5 mM NaCI sx and D~ equal the sedimentation coeffmmnt ar)d distance traveled, respectively, of the RNA being characterized RNA extracted in the absence of Mg 2+ were sedlmented at 2 ° in 5-ml, linear, continuous, 5-20 °/o sucrose gradients containing o Ol M TrIs-HC1 (pH 7 4) and 5 mM NaC1 at 48 500 rev./ mm for 3 5 h RNA's equilibrated or extracted with i mM MgC1a were sedlmented under slmdar conditions except that t m M MgCI z was substituted for 5 mM NaC1 in the gradients

14- a n d I I - S R N A ' s c a n be i s o l a t e d f r o m s u b - m i t o c h o n d r l a l f r a c t i o n s p r e p a r e d b y l y s l n g p u r i f i e d m l t o c h o n d r l a w i t h 0 5 % d e s o x y c h o l a t e or T r i t o n X - I o o , c e n t r i f u g i n g t h e l y s a t e a t 105 000 × g for 2 - 3 h, a n d t h e n e x t r a c t i n g a t 4 ° tile clear, colorless p e l l e t w i t h 80 °/o p h e n o l c o n t a i n i n g 0 2 M NaC1. R l b o n u c l e o p r o t e i n p a r t i c l e s w i t h t h e s e d i m e n t a t i o n c h a r a c t e r i s t i c s of r i b o s o m e s or r i b o s o m a l s u b u n i t s w e r e n o t a p p a r e n t in t h e r e s u s p e n d e d 105 000 x g p e l l e t (c/. ref. 13). F u r t h e r m o r e , a p p l i c a t i o n of t h e lyric t e c h n i q u e s a n d s e d i m e n t a t i o n a n a l y s e s of RIFKIN e t a l . 3 or KUNTZEL AND NOLL* to p u r i f i e d m l t o c h o n d r l a d i d n o t r e v e a l r i b o s o m e s or s u b u n i t s Colorimetnc and spectrophotometrlc analyses indicate that the extraction proc e d u r e y i e l d s c y t o p l a s m i c a n d m l t o c h o n d r i a l R N A ' s free f r o m d e t e c t a b l e p r o t e i n a n d B t o c h t m B w p h y s Acta, 217 (197 ° ) I32-I4I

E U G L E N A MITOCHONDRIAL R N A .

II

137

DNA 9. In addition, exposure of the phenol extraction products to 2 mg ribonuclease per ml for 30 min at room temperature destroys all rapidly sedimenting components in the gradients. Simultaneous extraction of E. coli ribosomes labeled with [3H~uridine and E. gracd, s mitochondna or 5000 ×g supernatant demonstrates that 0.2 M NaC1 inhibits E. grac,lis nuclease(s) If 0.2 M NaC1 was present during nucleic acid extraction (Figs. 4 C and 4D), the sedimentation profile of radioactive material was nearly eqmvalent to that of undegraded E. col, rRNA (Fig. 4A). If salt was absent (Fig. 4B), the 23-S component of the radioactive RNA was partly degraded, a condition consistent with the greater susceptibility to nucleases of the more rapidly sedimenting component of rRNA ~3,~. These results suggest that E. gracdis nuclease activity is reduced when 0.2 M NaC1 is present. Such an inhibition of E. grac,l,s ribonuclease m a y be comparable to the inhibition of a rat liver ribonuclease b y 0.2 M Na + (ref. 36). Furthermore, the observation t h a t equivalent amounts of 25-S RNA can be extracted from fresh or 8-h-old cell-free homogenates suggests that the cytoplasmic RNA's are not degraded prior to phenol extraction. Nevertheless, the possibility that mitochondrial RNA's are hydrolyzed during the isolation and purification of the organelles has not been excluded.

1 25

72*

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B

C

D

E col/ + Eugleno grottos

E col/ + Eugleno ~mo/hs cytoplasm

E col/ + Eugleno grac/I/$ rnztochondrlo

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Fig 4 Inhlbztmn of endogenous nuclease a c t i v i t y w i t h o 2 M NaCI The curves illustrate the s e d i m e n t a t i o n profiles of radioactive R N A extracted from 3H-labeled E col, ribosomes (A) mixed w i t h s t r e p t o m y c i n - b l e a c h e d E. grac~hs c y t o p l a s m (]3) containing o 2 M NaC1 (C), or mixed with m l t o c h o n d r l a (purified lOOO-5OOO× g pellet) containing o 2 M NaC1 (D) The samples were layered on t h e surface of 5-ml 5-2o % sucrose gradzents containing o.oi M Trls-HC1 (pH 7.4) and 5 mM NaC1. The gradients were centrifuged at 2 ° at 5 ° ooo r e v / m m for 3.5 h (A, B, C) or 4 h (D) In an SW-5o rotor. A b s o r b a n c e at 258 rot, was m e a s u r e d w i t h an L K B Uvtcord e q m p p e d with a flow cell. 7-drop samples were collected for radioactive monitoring. Fig 5- T h e r m a l l y reduced h y p e r c h r o m i c l t y spectra of bleached E. gracd*s cytoplasmic (A) and mltochondrlal (B) I M NaCl-msoluble R N A ' s and E. coI,~ r R N A (C). F o r details of experiment, see t e x t

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s. KRAWlEC, J. M. EISENSTADT

Thermal denaturatwn analysis Thermally induced hyperchromicity spectra of E. coli rRNA (which consists predominantly of GC pairs aT) and bleached E. grac,l,s cytoplasmic and mito~hondrial RNA's were compared to one another and to spectra of denatured synthetic poly AU and poly GC (c[. ref. 23) in an effort to characterize the secondary structure of native E. graczlis RNA's. The patterns (Fig. 5) indicate that the organelle RNA is different from the cytoplasmic RNA's of both the eucaryote and procaryote. Both the temperature at which denaturation occurs and the spectra of the denatured RNA's indicate that base paired regions of mitochondrlal RNA contain largely AU pairs whereas E. grackles cytoplasmic RNA contains mostly GC pairs. The relatively unstable AU pairs in E. gracd,s mitochondrial RNA begin to melt when the ambient temperature increases from 25 to 42°, and melting continues as the temperature is increased to 59 °. Little additional hyperchromicity is seen when the temperature is raised to 720 . The spectrum at 720 contains one peak centered at 255-26o m#, a pattern similar to that of poly AU (ref 23). By contrast, E. grackles cytoplasmic RNA exhibits a denaturation spectrum similar to those of E. cola rRNA and synthetic poly GC. Few of the relatively stable GC pairs melt when the temperature is raised from 25 to 42°. Large hyperchromic shifts occur when the temperature is increased to 59 ° and then to 72o . The spectra of denatured E. grackles cytoplasmic RNA and E. coli rRNA exhibit two peaks; one centered at 245 m#, the other at 280 m/~, spectra similar to that of poly GC (ref. 23).

Chromatographw analysis The differences in secondary structure of E. grac,hs cytoplasmic and mltochondrial RNA's indicate that the compositions of these nucleic acids are different. Base compositions (Table II) were calculated from data obtained by descending paper chromatography of 2',3'-mononucleotides produced b y alkaline hydrolysis of I M NaCl-insoluble E. gracilis cytoplasmic and mitochondrial RNA's. (E. col, rRNA served as a control.) The agreement between the values determined for E. col, RNA

TABLE

II

BASE COMPOSITIONS OF HIGHLY POLYMERIC E grac*7*s R N A ' s n e q u a l s t h e n u m b e r of a n a l y s e s . The ± v a h i e s are t h e 95 % confidence i n t e r v a l d e t e r m i n e d w i t h S t u d e n t ' s t dmtrlbutloi1. F o r d e t a i l s of t h e h y d r o l v m s a n d c h r o m a t o g r a p h y , see MATERIALS AND METHODS

Source

A demne

Uractl

Guamne

Cytosine

A U/GC

Re[erence

21 9

21 O

29 6

27 5

O 75

39

2344__06

223-Lo4

31o~I2

234+1o

084

3° 6

25 o

27 o

17.o

i 26

395~2o 25 i 252~o6

332±19 21 i 2io~io

I49~I0 32 4 3I I = k O 6

I25~o9 21 4 226_-[06

2.65

Bleached

E.

grctCtl$S

Bleached

E. gracHts (n = 12) E grackles chloroplasts

3°

E grackles nl!tochondria (n=

E. colt E eolz (n ~ 23)

17)

Bzochzm Bzophys. Acga, 2i 7 (197 o) i 3 2 - i 4 I

38

EUGLENA MITOCHONDRIAL R N A . I I

139

and previously published figures 3s is excellent. The composition of cytoplasmic RNA's from the SB3 m u t a n t m a y differ slightly from those of other bleached Euglena 39. The large amounts of adenine and uracil and the small amounts of guanine and cytosine in mitochondrial RNA's are notable. The preponderance of the former can be expressed b y the ratio of adenine plus uracil to guanine plus cytosine. The value for mitochondrial RNA is 2.65 and suggests t h a t AU pairs as seen in the thermal denaturation analysis have a greater chance of forming than do GC base pairs. By contrast, the composition of E. gracilis cytoplasmic RNA which gives a ratio of 0.84 favors the formation of an excess of GC pairs.

DISCUSSION

The chemical and physical characteristics of mitochondrial RNA's from bleached

E. grac~lis are atypical 4. Contrasting the sedimentation profiles of native E. gracilis RNA's with the patterns ordinarily seen in eucaryotes and their organelles emphasizes the unusual nature of the E. gracilis macromolecules. i M NaCl-insoluble RNA's (highly polymeric rRNA's) from whole cells conslst of two species: a more rapidly sedimenting "o~" component and a less rapidly sedimenting "fl" component. Analysis of rRNA's from five species of bacteria and five species of fungi revealed two classes of rRNA: eucaryotic ("o¢" = 23.4 S, "fl" = 16. 4 S) and procaryotic ("o~" ---- 20. 4 S, "fl" = 15 3 S) 14These results were extended b y distmguishing bacterial rRNA's ("~." = 23.5 S, "fl" = 16. 5 S) from both animal (vertebrate) rRNA's ("~" = 31.7 S, "fl" = 17.6 S) and plant (angiosperm) rRNA's ("o~" = 24. 7 S, "fl" = 16.5 S) 15. Similar results were obtained with slightly different techniques and different organisms; specifically, procaryotic rRNA's ("~" = 23 S, "fl" = 17 S) were shown to be different from animal (mammalian) rRNA's ("o~" = 29 S, "fl" = 18 S), and plant (angiosperm, fungal and algal) rRNA's ("o~" = 25 S, "fl" = 16 S) 4,18. More recent electrophoretic analyses of rRNA's from a variety of animals including sea urchins, fruit flies, and m a m m a l s indicate that the "od' component of animal RNA's varies in size and is larger in the more recently evolved species 17. From the results with bean chloroplasts, Neurospora crassa mitochondria, and the bacterium E. coli, KiJNTZEL AND NOLL4 asserted that organelle and procaryotic RNA's have comparable sedimentation coefficients. The sedimentation coefficients of E. gracihs cytoplasmic and mitochondrial RNA's are not consistent with the generalizations of CLICK AND TINT15 or KUNTZEL AND NOLL4 (see above). Values for the "c¢" and "fl" components of E. gracihs cytoplasmic RNA have been reported as 24 and 20 S (ref. 28), 22 and 17 S (ref. 29), and 25 and 19 S. These data agree poorly and none is characteristic of either plant or animal rRNA. Our results are in good agreement with sizes based on the electrophoretlc mobilities of E. gracilis rRNA's 17. The data from all laboratories indicate that the "~" component of E. gracilis RNA's is unstable and is equivalent to oi" approaches the size of the "~" component of plants, whereas the stable "fl" component is larger than the "fl" component of bacteria, plants, and (possibly) animals. The low sedimentation coefficients of i M NaCl-lnsoluble RNA's from bleached E. gracilis mitochondria m a y indicate a relatively low molecular weight, low diffusion coefficient, and/or low density 4°. Thermal denaturation spectra indicate that, in the B*och*m. B*ophys. Acta, 217 (197 o) 132-141

140

S. K R A W I E C , J. M. E I S E N S T A D T

highly polymeric RNA's of bleached E g r a c , h s , AU pairs are characteristic of the mitochondrlal macromolecules and GC pairs are characteristic of cytoplasmic RNA's. GC pairs are more stable than AU pairs, a condition apparent trom the gleater energy required to disrupt the hydrogen bonds The weaker AU pairs may produce a more extended polynucleotide with a lower diffusion coefficient and, therefore, a lower sedimentation coefficient. With highly polymeric RNA, an increase in cationic strength causes a shift from a less rapidly sedimenting unfolded molecule to a more rapidly sedimentmg compact molecule. In the absence of divalent cations, the relatively slowly sedimenting E . g r a c i l i s mitochondrlal RNA's may assume a more extended or possibly rod-hke configuration 35. The Svedberg equation indicates that macromolecules with low densities sediment more slowly than denser macromolecules with an equivalent molecular weight. Such ribonucleic acids as E g r a c , l i s mltochondrial RNA which contain predominantly adenine and uracil are slightly less dense than Euglena, bacterial, plant, and animal cytoplasmic rRNA's which contain more guanine and cytosineal. The data in Table I I I relate the source, size, and composition of rRNA's. The pattern that emerges has two notable features: (I)organelle RNA's are smaller than RNA's of whole cells and (2) the AU/GC ratio of organelle RNA is often very high whereas the ratio of these nucleotldes in cytoplasmic RNA's is always less than i TABLE

IlI

RELATION OF SEDIMENTATION CHARACTERISTICS AND A U / G C RATIOS

Source o/ R N A

Sedzmentat~on coe[/zc~ents

"~. . . . . Animal

E graczhs Plant Bacteria

E grac*hs c h l o r o p l a s t s Saccharomyces cerewsme mltochondrla H e L a cell m l t o c h o n d r l a

A U/GC ratio

~. . . . 6

~-. . . . o 40

Re[emnce

3. . . .

31 v 25 24 7 23 5 19-21

17 19 16 16 14

22 21

15 12

3 oo I 4°

2.69 1.13

20 5 14

16 ~ II

1.93

1.75

~-"+"3"

o 7°

15,

4~

15, t5, 3 O,

43 43 44

o 84 5 5 i5

o 82 o 86

o 95 o 86 I 26

8 13

Neurospora crassa mltochondna

E. graczl~s m l t o c h o n d r l a

3,

4

2 65

The compositions and sizes of mitochondrial or chloroplast RNA's from lettuce, cauliflower, and mushroom differ slightly from cytoplasmic rRNA's 1. The authenticity of these compositions and sizes can be established by demonstrating in electron microscopy or reconstitution experiments 42that cytoplasmic ribosomes do not contaminate the organelles used as a source of mitochondrial RNA's. SUMMARY AND CONCLUSION

The extraction of RNA's from bleached E . g r a c t h s z cytoplasm and mitochondria under conditions that reduce nuclease activity yields atypical RNA's. The mitoB~oeh~m B~ophys. Acta, 217 (197o) 132 141

E U G L E N A MITOCHONDRIAL R N A . chondrial

RNA's

differ from both

similar to the former than

2I

141 procaryotic

and

eucaryotic

RNA's

but are more

the latter.

ACKNOWLEDGMENTS

This investigation was supported by grant AM-o7189 from the National Institutes of Health (to J.M.E.) and Public Health Service Predoctoral Fellowship 5 Toi GM 275-09 91oo-41-48929 . The experiments reported here were taken from a dissertation submitted by S. K. in partial satisfaction of the requirements for the degree of Doctor of Philosophy at Yale University. REFERENCES I 2 3 4 5 6 7 8 9 IO Ii 12 13 14 I5 t6 t7 18 19 20 21 22 23 24 25 26 27 28 29 3o 31 32 33 34 35 36 37 38 39 4° 41 42 43 44

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