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The cyanelle: Chloroplast or endosymbiotic prokaryote?
II.MS l.ctters I (1977)7-12 ,~;)('opyright Federation of l-uropcan Microbiological Societies Published by Elsevier/North-llolland Biomedical Press
THE CYANELLE: CHLOROPLAST OR ENDOSYMBIOTIC PROKARYOTE? M. FIERDMAN Department of Biological Sciences. The University, Dundee DD1 41tN, Scotland
and R.Y. STANII{R btstitut Pasteur, 2.5 Rue du Docteur Roux. 7.5724 Paris. France
Received 8 November 1976
I. Introduction Cyam~phora paradoxa [ I ] is a small, photosynthetic bit'lagellate protist of uncertain taxonomic position. Each cell contains 2 - 4 structures termed cyanelles, which contain a pigmet~t system of the cyanobacterial and rhodophytan type [2]. Phycologists have interpreted the cyanellcs as unicellular cyanobacterial endosylnbionts, housed in a non-photosynthetic host cell 13,4]. Cyanelles have a fine structure closely resembling that of unicellular cyanobacteria, except that a typical cell wall cannot be resolved m electronmicrographs [41. They are, however, surrounded by a thin wall layer whose structural integrity is completely destroyed by lysozyme [5]. In view of the substrate specificity of lysozyme this layer is almost certainly composed of peptidoglycan, and is homologous with the innermost peptidoglycan layer of the cell wall of cyanobacteria and other Gram negative bacteria. This finding accordingly supports the hypothesis that cyanelles are endosymbiotic cyanobacteria which have lost the ability to synthesize the outer lipopolysaccharide cell wall layer characteristic of all freeliving members of this group. A rigorous discrimination between chloroplasts and endosymbiotic prokaryotes cannot be made on the
Address correspondence to: Dr. M. Ilerdman, Department of Biological Sciences, The University, Dundee DDI 4 I-IN, Scotland.
basis of ultrastructural data alone. One property which is in principle absolutely discriminatory is the degree of genetic complexity (genome size). The results of a study of the genetic material of C. paradoxa and its cyanelle are reported here.
2. Materials and methods 2.1. Organism Cyanophora paradoxa was obtained from l)r l.. Provasoli. The organism was grown in a medium (table 1)modified from that of Ramus [6], at 17°C in an atmosphere of air/CO2 (99 : 1, v/v) with a lightdark cycle (intensity 2000 lux) of 18 : 6 h. 2.2. k2v traction o f cyanelles
Cyanelles were extracted and purified by a method of F. Schaeffer (pets. commun.). The culture was centrifuged and resuspended in a small w)lume of 0.5 M sucrose in 0.03 M phosphate buffer (pH 7.6) for 15 rain at 4°C. Cell lysis was achieved by resuspending the culture, following further centrifugation, in 0.2 M buffered sucrose at 4°C. This osmotic shock generally lysed the majority (>95c7,) of the ceils, tile remainder lysing in two successive washings in 0.2 M buffered sucrose. The pellet produced by centrifugation of the lysate contained intact cyanelles and cellular debris.
"IA BLI'; 1
()'anol;hora culture medium ('ompound
Atnount (g/litrel
MgSO4 • 71120 (It3('OONll 4 ('asamino acids Sucrose Vitamin B 12 "l'ris a
0.1 0.2 2.5 2.5 10-6 0.3
Slock soln. ('yll b I'racc-metal mix PII c
50 lnl If) ml
pll of medium 7.6. a Iris (hydroxymethyllmelhylamine b Soln. Cyll contained, in g/litre: I.eCl 3 • 61120.0.04: ('aCl 2 ' 21120, 0.73; K('I, 0.60; K2gb'cerophosphate, 1.10 c Soln. PII contained, in g/litrc: II3BO 3, 1.14; I'I)'I'AINa21. 1.00; MnCI2 • 41120, 0.144" l'eCl 3 • 6tl20, 0.048; ZnSO 4 • 71120.0.022; ('o('12 " 6H20, 0.004
2.3. Pur(tTcati95',;~) of the remaining cellular DNA. This DNA was removed by i n c u b a t i o n with deoxyribonuclease ( D N A a s e ) ( 2 0 0 / a g / m l in 5 mM MgCI 2, 45 rain at .z;0 o ('} followed by two washings in 0.2 M sucrose, to yield a pure fraction of intact cyanelles from which cyanelle DNA was subsequently purified. Non-cyanelle I)NA was isolated by shaking the cyanelle suspension, without prior DNAase t r e a t m e n t , in an equal volume of ct'tloroform/isoamyl alcohol (24 : I } for 1 5 - 2 0 rain at r,~om temperature. Cyanelles remained intact during this treatment and were sedimented by centrifugation, non-cyanelle 1)NA being isolated from the supernatant. I)NA fractions were subsequently purified either by tile m e t h o d of Marmur 171 or on hydroxyapatite [8 I.
2.4. Characterisation oJ'DNA Thermal denaturation and renaturation of DNA was performed in a Gilford 240 recording spectrop h o t o m e t e r as previously described 19] except that DNA was denatured in the spectrophotometer cell and the renaturation reaction was initiated by the addition of sufficient volume of prewarmed 5 M NaCI to raise
the Na + c o n c e n t r a t i o n to 1 M. This pernlitted tile early stages of tile reaction to he followed. ('aesitnn chloride equilibriunl density gradient uhracentrifugalion t)t DNA was perforlncd in a MSF Centriscan 75 (45 000 rev./nlin at 20°C). I)NA contents were measured by lhe d i p h e n y l a m i n e nlethod [10].
3. Results ('yanelle I)NA inched conlpletely over a narrow range of temperat u re (fig. I a ) and the di fferen l ial melting curve (fig. lb)showect that the inajcnit.\. (t)8!:~) v,'as honlogeneous with a Tm (in 0.l Y SSC) nf 68.4 ( . corresponding to a b~,se conlposJtJon ¢7 .~>.6 nlol');. {(; + ('). A small fraction (2% of the total) exhibited a broad melting range and a inean base composition of 46.0 molS~ ((; + ('). In ci)llllast, iltlilcyanelle I)NA showed heterogeneil,,, ill melting: at least two major species, of base composition 50.3 and 69.8 molS~ (() + C). were present. Similar rest, Its were obtained by ultracentrifugation: crude extracts of whole cells of C paradoxa produced three major I)NA bands of densities 1.695. 1.709 and 1.729 g/cm 3 (corresponding to 3 5 . 7 . 5 0 . 2 and 70.2 mol plexity 0.12 X 108 dahons, was present in approximately two copies per single copy of tile main class. as calculated lronl the extrapolation to the origin of tile second-order plot of the reaction. Chemical analysis of the DNA c o n t e n t of isolated cyanelles yielded a vahie of 1.23 X 10-14 g I)NA (74.1 X 108 daltons) per cyanelle, t:'ach unique sequence of 1.17 X 10 ~ dahons is therefore present in approximately 60 copies per cyanelle. Renaturation of non-cyanelle I)NA also followed second-order kinetics" a rapidly renaturating fraction of 2.2 × 10 '9 daltons was observed ill addition to the main class of 2.4 × 10 I° daltons. Since the non-cyanelle I)NA c o n t e n t of C: parad¢ma was estimated by chemical analysis to be 12.7 X 10 I° daltons, it is evident ,hal
#
100
8,0
-
05
04
60
03
,.%
102
m
E
20/
40
6o
_.J
;4
do
9'0 Temperalure
6b
.2
rb
go
.
9'0
°C
l.'i,.z.I. Melting curves [AJ and differential melting curves [BI of cyanclle I)NA ( . . . .
) and non-cyanelle I)NA (o--
*).
a considerable number of repealed sequences are present in this fraction.
2.8 Ao-Aoo A -Am
4. Discussion
24
2.0
16
12
Time (ram)
Fig. 2. Second-order plot o f rcnaturation of cyanelle DNA.
DNA base composition and genome sizes of chloroplasts from a wide range of organisms are compared with those of tile cyanelle and of unicellular cyanobacteria in table 2. Chloroplasts vary, in mean DNA base composition from 26 -.41 tool% (G + ('}. Genome sizes also show variation which may result from experimental error in early work [ 11 ]. Covalently closed circular chloroplast DNA molecules have been observed in many organisms to be 9 X 107 daltons as measured from their contour length [ 1 2 - 1 4 ] , consistent with most recent estimates of kinetic complexity. The uncertain values for some genome sizes, and for total chloroplast DNA content, make estimation of the numbcr of copies of the genome per chloro-
1{} qA Bl.l'i 2 I)NA base compositions,
gellolllC
Organism
sizes and
gCII{}I'IICcolal,elll o f
DNA base cornposili{}n
Refs.
(;enome size (dallons X 1(} s)
No. of copies ofgenome
Refs.
[181 1191 12{~1 [21]
1.94 2.0 2.{} 2.3 {}.93 {}./'.;2 0.91
24 209 35
117231 115,161 [11,12]
(l]lOU: ((; + {'))
('hlorofflasts o12 (.'hlam.Pdomonas { "hh ~rclla I:)o,'lcna Itigher planls ('yanclle of C paradoxa Unicellular ('yanobac'~eria: Svnechocysti,~ spp. Svn(,ch(~(o{'(tis spp.
39.(} 33.{} 26.0 36.7 40.8
chloroplasls, ( paradoxa ~.'yanelles and unicellular C,Van{~bat.'teria
35.6 35.7 36.7 42.1 - 48.O 38.3 42.4 47.4 55.6 66.3 71.4
1.17 [221
18.9 17.6 29.0 16.423.2
52 4{} 72 100:'
1~41
60
23.9 23.O 39.2 30.6 25.3
I few I fev, 1 few
a Using lhc chloroplast contcnl of 0.5 -1.5 X 10 14 g in higher organisms 124] and gem)me size 0.9 × 108 dahons. ('yam> baclerial genome sizes and some base composition dala from our unpublished resulls.
plast difficult, l t o w e v e r it" the genome size o f 9 × 107 & l i o n s is correct chloroplasts probably contain
Acknowledgements
between 20 and 70 copies of lhe genome. The genome sizes o f 36 unicellular cyanobacteria so far e x a m i n e d are at least 1 0 20 times greater than those of chloroplasts (table 2) and a limited number o f measurements (M. l-lerdman, unpublished resuhs) indicate that the n u m b e r o f copies of the genome per cell is low. comparable to other prokaryotes. The genome o f the cyanelle of C. parado.va, present in a p p r o x i m a t e l y 60 copies o f 1.17 X 108 dallons, clearly resembles the chloroplast genome. In addition, ll~e rapidly renaturating cyanelle DNA c o m p o n e n t (kinetic c o m p l e x i t y 1.2 X 10 -7 daltons) has a counterpart in chloroplasts of Chlorella 115,161 and Cttlam.vd:mumas 117]. On genetic grounds, therefore, we c o n c l u d e thai the cyanelle is a p h o t o s y n t h e t i c o r g a n d i e rather than an e n d o s y m b i o t i c c y a n o b a c t e r i u m . However, it appears to differ from all other k n o w n chloroplasts by virtue of its enclosure within a peptidoglycan layer [51. J'his can be construed as powerful evidence for its evolutionary derivation from an e n d o s y m b i o t i c cyanobacterium.
This work was supported by a Royal Society European Programnae Fellowship. We thank Frances Burr and F. Schaeffer for providing details o f the modified culture m e d i u m and cyanelle extraction m e t h o d respectively.
References [I ] [2] [3] [4] [5] [6] [7} [8] 191 1101 [111 [121
Korschikov, A. (1924) Arch. Protistenk. 3, 57 74. ('hapman, D.J. (1966) Arch. Mikrobiol. 55, 17 25. (;eitler, L.(1959) Oster. Bt}t.Z. 106,464 471. ltall, W.f. andClaus, G.{1963) J. ('ell Biol. 19,551 563. Schenk, II.A.E. (1970) Z. Natt, rforsch. 25b. 656. Ramus, J.{1972}J.Phycol. 8,97 112. Marmttr, J. I1961)J.Molec Biol. 3,203 218. Brenner, l}.J., Fanning, G.R.. Rake, A.V. and ,IohnsotL K.E.(1969) Anal. Biochem. 28,447 459. Herdman, M. and ('arr, N.G. (1974~ Arch. Microbiol. 99,251 -254. Burton, K.(1956) Biochem. J.62.315 323. Slavik, N.S. and l lershberger, C.L. (1975) I'EBS l.,ctt. 52, 171 1 7 4 . Manning, J.E.,Wolslenhohne, I).R.,Ryan. R.S.,llunter,
11
[ 13 ] [14] 1151 [ 16 ] 117] [18]
J.A. arid Richards, O.('. (1971) Proc. Natl. Acad. Sci. USA 68, 1169 1173. Manning, J .1'i., Wolstenhohne, D.R. and Richards, O.C. (19721J. Cell Biol. 5 3 . 5 9 4 601. Kolodner, R. and Tewari, K.K. ~1975) F;iochem. Biophys. Acta 4(12. 3 7 2 - 3 9 0 . Baven, 'q. and R~dc, A. ~19731 l.lur. J. Biochem. 39, 413 420. I)almon, J. and Bayen, M. ( 19751 Arch. ~,iicrobiol. 103, 57-61. Wells. R. and Sager, R. (1971 ~ J. Molcc. Biol. 58, 6 1 1 622. Sager, R. and lshida, M.R. (19631 I'roc. Nail. Acad. Sci. USA 50,725 73().
[19] lwamura, T. and Kuwashima, S. (19691 Bi~Jchim. Biophys. Acta 174,330 339. [201 Brawerman, (;. and I:isensladl, J. (19641 Biochim. Biophys. A c t a 9 1 , 4 7 7 485. [21 ] Kirk, J.T.O. (1970) in: Autonomy and Biogenesis of Mitochondria and ('hloroplasts (Boardman, N.K., Linnane, A V,'. and Smillie, I,',.~,1.cds) pp. 2 6 7 - 2 7 6 , Syrup. Aust. Acad. Sci., North-ltolland, Amsterdam. [22] Stanier, R.Y., Kunisawa, R., Mandcl, M. and CohenBazire, G. ( 1971 ) Bacteriol. Rev. 35. 171 - 2(15. [23] Baslia, I).,Chiang, K-S.,Swift, ll. andSiersma. P. (19711 Proc. Natl. Acad. Sci. USA 68, 1157-.1161. [24] lewari, K . K . a n d W i l d m a n , S.(;.~1970) Svmp. Soc. Exp. Biol. 24, 147- 179.
THE CYANELLE: CHLOROPLAST OR ENDOSYMBIOTIC PROKARYOTE? M. FIERDMAN Department of Biological Sciences. The University, Dundee DD1 41tN, Scotland
and R.Y. STANII{R btstitut Pasteur, 2.5 Rue du Docteur Roux. 7.5724 Paris. France
Received 8 November 1976
I. Introduction Cyam~phora paradoxa [ I ] is a small, photosynthetic bit'lagellate protist of uncertain taxonomic position. Each cell contains 2 - 4 structures termed cyanelles, which contain a pigmet~t system of the cyanobacterial and rhodophytan type [2]. Phycologists have interpreted the cyanellcs as unicellular cyanobacterial endosylnbionts, housed in a non-photosynthetic host cell 13,4]. Cyanelles have a fine structure closely resembling that of unicellular cyanobacteria, except that a typical cell wall cannot be resolved m electronmicrographs [41. They are, however, surrounded by a thin wall layer whose structural integrity is completely destroyed by lysozyme [5]. In view of the substrate specificity of lysozyme this layer is almost certainly composed of peptidoglycan, and is homologous with the innermost peptidoglycan layer of the cell wall of cyanobacteria and other Gram negative bacteria. This finding accordingly supports the hypothesis that cyanelles are endosymbiotic cyanobacteria which have lost the ability to synthesize the outer lipopolysaccharide cell wall layer characteristic of all freeliving members of this group. A rigorous discrimination between chloroplasts and endosymbiotic prokaryotes cannot be made on the
Address correspondence to: Dr. M. Ilerdman, Department of Biological Sciences, The University, Dundee DDI 4 I-IN, Scotland.
basis of ultrastructural data alone. One property which is in principle absolutely discriminatory is the degree of genetic complexity (genome size). The results of a study of the genetic material of C. paradoxa and its cyanelle are reported here.
2. Materials and methods 2.1. Organism Cyanophora paradoxa was obtained from l)r l.. Provasoli. The organism was grown in a medium (table 1)modified from that of Ramus [6], at 17°C in an atmosphere of air/CO2 (99 : 1, v/v) with a lightdark cycle (intensity 2000 lux) of 18 : 6 h. 2.2. k2v traction o f cyanelles
Cyanelles were extracted and purified by a method of F. Schaeffer (pets. commun.). The culture was centrifuged and resuspended in a small w)lume of 0.5 M sucrose in 0.03 M phosphate buffer (pH 7.6) for 15 rain at 4°C. Cell lysis was achieved by resuspending the culture, following further centrifugation, in 0.2 M buffered sucrose at 4°C. This osmotic shock generally lysed the majority (>95c7,) of the ceils, tile remainder lysing in two successive washings in 0.2 M buffered sucrose. The pellet produced by centrifugation of the lysate contained intact cyanelles and cellular debris.
"IA BLI'; 1
()'anol;hora culture medium ('ompound
Atnount (g/litrel
MgSO4 • 71120 (It3('OONll 4 ('asamino acids Sucrose Vitamin B 12 "l'ris a
0.1 0.2 2.5 2.5 10-6 0.3
Slock soln. ('yll b I'racc-metal mix PII c
50 lnl If) ml
pll of medium 7.6. a Iris (hydroxymethyllmelhylamine b Soln. Cyll contained, in g/litre: I.eCl 3 • 61120.0.04: ('aCl 2 ' 21120, 0.73; K('I, 0.60; K2gb'cerophosphate, 1.10 c Soln. PII contained, in g/litrc: II3BO 3, 1.14; I'I)'I'AINa21. 1.00; MnCI2 • 41120, 0.144" l'eCl 3 • 6tl20, 0.048; ZnSO 4 • 71120.0.022; ('o('12 " 6H20, 0.004
2.3. Pur(tTcati
2.4. Characterisation oJ'DNA Thermal denaturation and renaturation of DNA was performed in a Gilford 240 recording spectrop h o t o m e t e r as previously described 19] except that DNA was denatured in the spectrophotometer cell and the renaturation reaction was initiated by the addition of sufficient volume of prewarmed 5 M NaCI to raise
the Na + c o n c e n t r a t i o n to 1 M. This pernlitted tile early stages of tile reaction to he followed. ('aesitnn chloride equilibriunl density gradient uhracentrifugalion t)t DNA was perforlncd in a MSF Centriscan 75 (45 000 rev./nlin at 20°C). I)NA contents were measured by lhe d i p h e n y l a m i n e nlethod [10].
3. Results ('yanelle I)NA inched conlpletely over a narrow range of temperat u re (fig. I a ) and the di fferen l ial melting curve (fig. lb)showect that the inajcnit.\. (t)8!:~) v,'as honlogeneous with a Tm (in 0.l Y SSC) nf 68.4 ( . corresponding to a b~,se conlposJtJon ¢7 .~>.6 nlol');. {(; + ('). A small fraction (2% of the total) exhibited a broad melting range and a inean base composition of 46.0 molS~ ((; + ('). In ci)llllast, iltlilcyanelle I)NA showed heterogeneil,,, ill melting: at least two major species, of base composition 50.3 and 69.8 molS~ (() + C). were present. Similar rest, Its were obtained by ultracentrifugation: crude extracts of whole cells of C paradoxa produced three major I)NA bands of densities 1.695. 1.709 and 1.729 g/cm 3 (corresponding to 3 5 . 7 . 5 0 . 2 and 70.2 mol
#
100
8,0
-
05
04
60
03
,.%
102
m
E
20/
40
6o
_.J
;4
do
9'0 Temperalure
6b
.2
rb
go
.
9'0
°C
l.'i,.z.I. Melting curves [AJ and differential melting curves [BI of cyanclle I)NA ( . . . .
) and non-cyanelle I)NA (o--
*).
a considerable number of repealed sequences are present in this fraction.
2.8 Ao-Aoo A -Am
4. Discussion
24
2.0
16
12
Time (ram)
Fig. 2. Second-order plot o f rcnaturation of cyanelle DNA.
DNA base composition and genome sizes of chloroplasts from a wide range of organisms are compared with those of tile cyanelle and of unicellular cyanobacteria in table 2. Chloroplasts vary, in mean DNA base composition from 26 -.41 tool% (G + ('}. Genome sizes also show variation which may result from experimental error in early work [ 11 ]. Covalently closed circular chloroplast DNA molecules have been observed in many organisms to be 9 X 107 daltons as measured from their contour length [ 1 2 - 1 4 ] , consistent with most recent estimates of kinetic complexity. The uncertain values for some genome sizes, and for total chloroplast DNA content, make estimation of the numbcr of copies of the genome per chloro-
1{} qA Bl.l'i 2 I)NA base compositions,
gellolllC
Organism
sizes and
gCII{}I'IICcolal,elll o f
DNA base cornposili{}n
Refs.
(;enome size (dallons X 1(} s)
No. of copies ofgenome
Refs.
[181 1191 12{~1 [21]
1.94 2.0 2.{} 2.3 {}.93 {}./'.;2 0.91
24 209 35
117231 115,161 [11,12]
(l]lOU: ((; + {'))
('hlorofflasts o12 (.'hlam.Pdomonas { "hh ~rclla I:)o,'lcna Itigher planls ('yanclle of C paradoxa Unicellular ('yanobac'~eria: Svnechocysti,~ spp. Svn(,ch(~(o{'(tis spp.
39.(} 33.{} 26.0 36.7 40.8
chloroplasls, ( paradoxa ~.'yanelles and unicellular C,Van{~bat.'teria
35.6 35.7 36.7 42.1 - 48.O 38.3 42.4 47.4 55.6 66.3 71.4
1.17 [221
18.9 17.6 29.0 16.423.2
52 4{} 72 100:'
1~41
60
23.9 23.O 39.2 30.6 25.3
I few I fev, 1 few
a Using lhc chloroplast contcnl of 0.5 -1.5 X 10 14 g in higher organisms 124] and gem)me size 0.9 × 108 dahons. ('yam> baclerial genome sizes and some base composition dala from our unpublished resulls.
plast difficult, l t o w e v e r it" the genome size o f 9 × 107 & l i o n s is correct chloroplasts probably contain
Acknowledgements
between 20 and 70 copies of lhe genome. The genome sizes o f 36 unicellular cyanobacteria so far e x a m i n e d are at least 1 0 20 times greater than those of chloroplasts (table 2) and a limited number o f measurements (M. l-lerdman, unpublished resuhs) indicate that the n u m b e r o f copies of the genome per cell is low. comparable to other prokaryotes. The genome o f the cyanelle of C. parado.va, present in a p p r o x i m a t e l y 60 copies o f 1.17 X 108 dallons, clearly resembles the chloroplast genome. In addition, ll~e rapidly renaturating cyanelle DNA c o m p o n e n t (kinetic c o m p l e x i t y 1.2 X 10 -7 daltons) has a counterpart in chloroplasts of Chlorella 115,161 and Cttlam.vd:mumas 117]. On genetic grounds, therefore, we c o n c l u d e thai the cyanelle is a p h o t o s y n t h e t i c o r g a n d i e rather than an e n d o s y m b i o t i c c y a n o b a c t e r i u m . However, it appears to differ from all other k n o w n chloroplasts by virtue of its enclosure within a peptidoglycan layer [51. J'his can be construed as powerful evidence for its evolutionary derivation from an e n d o s y m b i o t i c cyanobacterium.
This work was supported by a Royal Society European Programnae Fellowship. We thank Frances Burr and F. Schaeffer for providing details o f the modified culture m e d i u m and cyanelle extraction m e t h o d respectively.
References [I ] [2] [3] [4] [5] [6] [7} [8] 191 1101 [111 [121
Korschikov, A. (1924) Arch. Protistenk. 3, 57 74. ('hapman, D.J. (1966) Arch. Mikrobiol. 55, 17 25. (;eitler, L.(1959) Oster. Bt}t.Z. 106,464 471. ltall, W.f. andClaus, G.{1963) J. ('ell Biol. 19,551 563. Schenk, II.A.E. (1970) Z. Natt, rforsch. 25b. 656. Ramus, J.{1972}J.Phycol. 8,97 112. Marmttr, J. I1961)J.Molec Biol. 3,203 218. Brenner, l}.J., Fanning, G.R.. Rake, A.V. and ,IohnsotL K.E.(1969) Anal. Biochem. 28,447 459. Herdman, M. and ('arr, N.G. (1974~ Arch. Microbiol. 99,251 -254. Burton, K.(1956) Biochem. J.62.315 323. Slavik, N.S. and l lershberger, C.L. (1975) I'EBS l.,ctt. 52, 171 1 7 4 . Manning, J.E.,Wolslenhohne, I).R.,Ryan. R.S.,llunter,
11
[ 13 ] [14] 1151 [ 16 ] 117] [18]
J.A. arid Richards, O.('. (1971) Proc. Natl. Acad. Sci. USA 68, 1169 1173. Manning, J .1'i., Wolstenhohne, D.R. and Richards, O.C. (19721J. Cell Biol. 5 3 . 5 9 4 601. Kolodner, R. and Tewari, K.K. ~1975) F;iochem. Biophys. Acta 4(12. 3 7 2 - 3 9 0 . Baven, 'q. and R~dc, A. ~19731 l.lur. J. Biochem. 39, 413 420. I)almon, J. and Bayen, M. ( 19751 Arch. ~,iicrobiol. 103, 57-61. Wells. R. and Sager, R. (1971 ~ J. Molcc. Biol. 58, 6 1 1 622. Sager, R. and lshida, M.R. (19631 I'roc. Nail. Acad. Sci. USA 50,725 73().
[19] lwamura, T. and Kuwashima, S. (19691 Bi~Jchim. Biophys. Acta 174,330 339. [201 Brawerman, (;. and I:isensladl, J. (19641 Biochim. Biophys. A c t a 9 1 , 4 7 7 485. [21 ] Kirk, J.T.O. (1970) in: Autonomy and Biogenesis of Mitochondria and ('hloroplasts (Boardman, N.K., Linnane, A V,'. and Smillie, I,',.~,1.cds) pp. 2 6 7 - 2 7 6 , Syrup. Aust. Acad. Sci., North-ltolland, Amsterdam. [22] Stanier, R.Y., Kunisawa, R., Mandcl, M. and CohenBazire, G. ( 1971 ) Bacteriol. Rev. 35. 171 - 2(15. [23] Baslia, I).,Chiang, K-S.,Swift, ll. andSiersma. P. (19711 Proc. Natl. Acad. Sci. USA 68, 1157-.1161. [24] lewari, K . K . a n d W i l d m a n , S.(;.~1970) Svmp. Soc. Exp. Biol. 24, 147- 179.