- Email: [email protected]
Comparative studies on cytoplasmic ribosomes from algae
Vol . 7, pp . 327-336, Life Sciences Printed in Great Britain.
1968 .
Pergamon Press
COMPARATIVE STUDIES ON CYTOPLASMIC RIBOSOMES FROM ALGAE M . Rodriguez-Lopez and D . Vazquez Instituto MaraBÓn and Instituto de Biologia Celular,Velazquez 144 Madrid-6, Spain (Received 6 December 1967 ; in final form 26 ,January 1968) Ribosomes can be classified according to their sedimentation coefficient into two types . Ribosomes of the 70 S type are present in procaryotic cells and eucaryotic organelles whereas 80 S ribo-
somes are found in the cytoplasm of eucaryotic cells . Ribosomes of the 70 S type differ from those of the 80 S type not only in their sedimentation coefficient but also in composition, sedimentation coefficients of their subunits and RNA, and response to ionic environment and to certain antibiotics inhibitors of protein synthesis (1,2,3) .
However we do not know yet if all ribosomes of one given type (70 S or 80 S) have the same mechanism for protein synthesis, nor is there conclusive evidence that all ribosomes of a given type ha~-e the same behaviour towards antibiotics . It is conceivable that more than two types of ribosomes have evolved, and the existence of ribosomes having characteristics of both 70 S and
80 S types is possible . In this respect some interesting information is already available . The sedimentation coefficient of ribo sonies from the protocoan Crithidia oncopelti is 80 S and the requirements of these ribosomes for protein synthesis are similar
to other ribosomes of the 80 S type ; however, unlike other 80 S ribosomes those of Crithidia oncopr~lti are easily dissociated into subunits which can reaggregate under suitable conditions (4) . On the other hand, ribosomes from the slime mould Dictyostelium disc oideum have a sedimentation coefficient of 70 S but do not readily dissociate in 10 - 3 _~1 rig ++ and in this respect they behave like other 80 S ribosomes (S) . Ribosome from guinea pig liver have 80 S ribosomes which dissociate to give 60 S subunits with 60 ~ RNA (like 70 S ribosomes) and 40 S subunits with 43 ~ RNA (rather like 80 S ribosomes)(6) . Dtarcker and Sanger (7) found N-formyl-methionyl-s-RNA in yeast ; this compound is the initiator of protein syn327
328
RIBOSOMES FROM ALGAE
Vol . 7, No . G
thesis by ~0 S ribosomes but it has not been reported in eucaryotic cells other than yeast . The work oî Taylor and Storck (8) has shown that blue-green algae have ~0 S ribosomes whereas other workers found that green algae have 80 S ribosomes in the cytoplasm (q,22) . Chlamydo monas is phylogenetically interesting and contradictory reports have appeared concerning its cytoplasmic ribosomes ; it appears
that these ribosomes have some features in common with both ~0 S
and 80 S ribosomes (10,11,12) . The present work is a contribution to the knowledge of algal ribosomes and attempts to relate the properties of algal ribosomes with algal phylogeny . Material and Methods Organism and ,growth conditions . Anacystis montana (LB 1405 /3), Chloralla pyrenoidosa Chick (211/8 a) and Chlamydomonas rein hardü (11/326 wild type +) were obtained from the Culture Collect ion of Algae and Protozoa (The Botany School, University of Cam-
bridge, Cambridge, England) . A . montana was grown in medium D (13), C . reinhardü in medium I (14) and C . pyrenoidosa in the medium previously described by one of us (15) .
The algae were collected by centrifugation while in the exponential phase of growth, washed with cold 10 -2 _M Tris/HC1 buffer pH 7 .4 containing 10 -2 M Mgt and 5 x 10 -2 M NH4+, and all further steps for breakage and fractionation were carried out at
44 C . This standard buffer was used throughout this work ; the Mgt ion concentration being changed when required . Frozen Escher ichia coli B was obtained from. the Microbiological Research~Establishment (Porton, England) . A diploid yeast (Saccharomyces fragilis x Saccharomyces dobzanskü ) was grown and used for comparative studies of ribosomes (16) .
Cell breakage and fractionation . For breakage of the cells A . montana and C . reinhardü were resuspended in the standard buffer and 15 ~ (w/v) sucrose . C . pyrenoidosa was resuspended in the standard buffer containing 5 x 10 3 M Mgt and 15 ~ sucrose . Yeast was resuspended in the standard buffer containing 5 x 10 3 M Mgt. Cells were then broken in a Ribi cell fractionator at 30000 Psi ; under these conditions the cells were broken but not their chloroplasts . E . coli was broken by alumina grinding and the broken preparation extracted with the standard buffer .
Vol .
7, No .
6
329
RIBOSOMES FROM ALGAE
E . coli and yeast were fractionated following standard
procedures (16,17) . To obtain cytoplasmic ribosomes from algae
the broken cell preparations were first centrifuged at 4000 g for 15 min to separate the chloroplasts and ribosomes prepared from
the supernatant fluids by the methods used to obtain bacterial ribosomes (17) . Ribosomes were finally resuspended in the standard buffer containing either 5 x 10 -3 _M Mgt for yeast, C . pyrenoidosa and C . reinhardü ribosomea or 10 -2 M Mgt for E . coli , A . montana , and also in some cases for C . reinhardü ribosomes .
Ribosome analysis . The algal ribosomes obtained as above described were not pure enough for estimation of their RNA and protein content . For these determinations the ribosomes were
further purified by centrifugation for 90 min at 37000 r.p .m . in the SW 39 rotor of the Spinco model L-2 preparative ultracentrifuge through a sucrose gradient (5-20 ~ (w/v) sucrose) in the standard buffer containing 5 x 10 - 3 or 10 -2 M Mgt as above . 32 x 0 .15 ml fractions were taken, alternative fractions were diluted with 4 ml of water and O .D . estimated at 260 ~x . The remaining fractions
corresponding to the ribosomal peaks in the three tubes were pooled . Protein was estimated directly by the method of Lowry et al . (18) using as control a sucrose solution with the same concentration of sucrose as the ribosomal suspensions and using bovine serum albumin as a standard . For the estimation of RNA, the ribosomes-were treated twice with 0 .5 N perchloric acid for 2C min at 704 and the extracts were pooled . The O .D . of this extract at 260 mu was measured . The RNA content was determined by assuming that 1 mg of ribosomal RNA has an O .D . at 260 m~,t of 25 units . Fundamentally the same procedure was followed to estimate RNA and protein in the ribosomal subunits but here the ribosomes were centrifuged for 13 hr at 2¢000 r .p .m . in the SW 25-2 rotor of the Spinco model L-2, the sucrose gradient contained 10 - 4 _M Mgt and 1 ml fractions were taken . The fractions corresponding to the faster and slower subunits were pooled separately for analysis . Purity of the preparations of ribosomal subunits was always confirmed by centrifugation through sucrose gradients in
the SW 39 rotor and estimation of U .D . at 260 m~ of the fractions as indicated above . Bindins of antibiotics . Binding of 14 C-chloramphenicol and 1 4C-spiramycin I to ribosomes was studied by methods described else-
330
fol . ï, n'o . C>
RIBOSOMES ~ROM ALGAr
cNmnoo woNAS 0o uos z " c i.y ~ .v r 0
" os '
" os I
"
"
NlsosowAs .a
" ".
s~
a 10 ,
o
e-
+
"
_
wy .
" .. 10~
w
w y"
"
41
10
rq " CT :ON
CMLONfIy " 00
170 5
w3 i
F~ACTION
20
41~SOME}
WS
.OS
~
i
"
b
e . 1Ó M
~ .
"
" - 10 1 w
w~~
~
.-
tù" r
wr
+7
Flr, . 1 Seditnentation patterns of Anacystis montana , Chlamydomor2as reinhardü and Çhlorella pyrenoidosa ml 0 .05 of ribosome suspensions were centrifuged for 120 min at 3000 r .p .m . in the SW 3q rotor of the Spinco L-2 centrifuge through a 5-20 ~ (w/v) sucrose gradient containing the required concentration of Mg++ . 0 .1$ ml fractions were taken into q ml of water and O .D . at 260 m}r were estimated . The arrows indicate the position of the peaks obtained when Escherichia coli or yeast ribosomes, ribosome dimers or ribosomal subunits of known sedimentation coefficients were run .
331
RIBOSOMES PROM ALGAE
Vol . 7, No . 6
where (17,18) . 14C-chloramphenicol (specific activity 6 .81 ~C/umole) was obtained from The Radiochemical Centre (Amersham, Great Britain) and 14 C-spiramycin I (specific activity 6 y~C/2$ mg) was kindly supplied by Dr . Mc Fadzean (May and Baker Ltd ., Great Britain) . The sources of other antibiotics are given in an earlier paper (17) . Results The results obtained in studies on centrifugation of algal ribosomes through sucrose gradients with different concentrations
of Mgt are shown in Fig. 1 . These sedimentation patterns suggest that the sedimentation coefficient of A . montana ribosomes is 70S whereas that of C . reinhardü and C . pyrenoidosa ribosomes is 80 S ; this is in agreement tivith previous determinations carried out by other workers using more accurate methods (8,12,20) . Under the
experimental conditions used in this work it appears that there was some formation of dimers at 10 -2 Di Digs in the three types of ribosomes studied and there was some dissociation into ribosomal subunits at 10 - 3 ~I ~1g~ . This disr~ociation was more marked than in
TABLE 1 Chemical Composition of Algal Ribosomes and Ribosomal Subunits
Subunits Ribosomes Organism
~ protein i~ RNA
Faster subunit ~ protein
~ RNA
Slower subunit ~ protein ~ RNA
A . montana
37
63
31
69
37
63
Ç . reinhardü
35
65
31
69
30
70
C . pyrenoidosa
49
$1
47
53
50
50
Ribosomes or ribosomal subunits were purified by centrifugation through a sucrose gradient as indicated under Material and Methods . Protein was determined by the method of Lowry et al . (18) . RNA was extracted with 0 .5 _N perchloric acid at 70Q, the O .D . at 260 rr~ of the extract measured and RNA calculated on the basis that 1 mg of ribosomal RNA has an O .D . at 260 m}i of 25 units .
332
RIBOSOMES FROM AL~AF
Vol, î, No, i>
mammalian ribosomes but it was not complete as in bacterial ribo-
somes . The dissociation of algal ribosomal subunits was complete at 10 -4 M Mgt and has already been shown by other workers (12,20) . Chemical analysis has shown that in C . pyrenoidosa ribosomes the ratio RNA/protein is close to 1 whereas A . montana and C . reinhardü ribosomes are richer in RNA (Table 1), In this
respect A . montana and C . reinhardü ribosomes are like those from bacterial cells whereas C . pyrenoidosa ribosomed are like those from yeast and mammalian cells (1) . Similar results were obtained
when ribosomes or ribosomal subunits were analysed and no differ-
ence was found in the RNA/protein ratio between the ribosomal sub-
units of the same alga .
The sti` king selectivity of some antibiotics which are able to inhibit specifically protein synthesis by either the ;0 S or 80 S ribosomes is well known (2,3) . These antibiotics can be used to establish the type of system in algae . Cycloheximide is a a elective inhibitor of protein synthesis by ribosomes of the eucaryotic type (80 S) whereas chloramphenicol, erythromycin, spiramycin and lincomycin act selectively on ribosomes of th~z
procaryotic type (70 5)(3) . When the effects of antibiotics ti" Pr~: tested on intact algae it was found that on13- antibiotics acting
on ~0 S ribosomes strongly inhibited A . montana (Table 2) . Ur, the other hand only Cycloheximide completely blocked growth of C . py renoidosa at low concentrations . Cycloheximide also inhibited
growth of C . reinhardü but the higher concentrations required might possibly be due to a permeability barrier . In the behaviour towa~°ds antibiotics A, montana behaved like bacteria whereas C .
pyrenoidosa behaved like eucaryotic organisms . The uptake of 1~C-chloramphenicol by C . reinhardü and C . pyrenoidosa was studied in order to determine if the low sensitivity of these algae to the antibiotic could be due to a permeability barrier . Results not shuwr~ here showed that our strains of C . reinhardü and C . pyre noidosa were permeable to chloramphenicol and the extent of antibiotic uptake by these algae was comparable to that obtained pre-
viously with bacteria (2) . Chloramphenicol, erythromycin, spiramycin and lincomycin are known to be active on chloroplast ribosomes and this might explain the observed bleaching of the cells and the inhibitory effect of these antibiotics on growth of C . pyrenoidosa and C . reinhardü by high concentrations .
333
RIBOSOMES FROM ALGAE
Vol . 7, No . 6
TABLE 2 Effect of a Number of Antibiotica on Growth of Algae ;B inhibition of growth Antibiotic
ug/ml
A .montana
C .reinhardü
C .UVrenoidosa
1
10
12
l00
32
90
loo
Chloramphenicol 1 ++ l0 n 100
86
20
97 100
5
5o 80
So 80
10
1
100 100
3 85
100
100
90
50 87
100
1
100
5
25
Cycloheximide " "
Erythromycin +' Spiramycin ++ n Lincomycin "
10
21
l0 100
1 10
100
80
95 100
100 100
34 90
30 70
100
0
40
100
8
100
100
0
85
$ ml of the alga suspension (1 x 107 cells/ml) was taken in 25 ml conical flasks, and incubated in the presence of light at 2$Q with strong agitation for 72 hr in the presence or absence of antibiotic . The number of cells was estimated microscopically at the beginning and the end of the incubation period . Incubations were carried out in duplicate and the average figures obtained . Experimental evidence has shown that in cell-free systems
14 C-chloramphenicol and 14C-spiramycin I bind to 70 S but not to
80 S ribosomea (2,19,21,22) . A . montana ribosomea are found to bind these two antibiotics (Table 3) . On the other hand a much smaller binding to C .pyrenoidosa and C . reinhardü ribosomes was found and even this binding could be due to contamination with mitochondria] and chloroplastic ribosomes as this is very difficult to avoid in preparations from algae (22) . Lack of sensitïvity of cytopl .asmic~
ribosomes from green algae to chloramphenicol has already hc "+" n r~"ported by other workers (9,10, 22) . Erythranycin and ] i ncomyc"..i n +c l+ i c"1+
Vol . 7, No . 6
RIBOSOMES FROM ALGAE
334
TABLE 3 Binding of 14C-chloramphenicol and 14C-spiramycin I to Ribosomes uumoles radioactive antibiotic bound/mg ribosomes Ribosomes from:
Non-radioactive antibiotic added 14C-chloramphenicol
A .montana " " "
None Cycloheximide Erythromycin Lincomycin
C . reinhardü C . nyrenoidosa
14 C-spiramycin I
108 107 21 4
90 -
None
31
20
None
26
6
Suspensions of ribosomes (final concentration 1 mg/ml) in 10 -2 M Tris/HC1 buffer pH 7 .4 containing 10-2 M Mgt and 0 .1 M NHQ were used for all binding experiments . To study binding of 1 4C-spiramycin I the antibiotic was added to a final concentration of $ x 10- M and the ribosomes were harvested by centrifugation after the binding reaction ; the results were corrected by using a blank in which 1ZC-spiramycin III was added to a final concentration 1 .$ x 10-4 M and mixed with the 14C-spiramycin I prior to addition of ribosomes and centrifugation . A similar technique was used to study binding of 14C-chloramphenicol but this antibiotic was used at a concentration $ x 10 -6 _M and 1 2 C-chloramphenicol (1 .$ x 10-4 M) was also added in the blank . Cycloheximide, erytromycin and lincomycin were added to the tubes when required (final concentration 1 .$ x 10-4 M) and mixed with 14C-chloramphenicol prior to addition of ribosomes and centrifugation . are active in preventing binding of 14C-chloramphenicol to bacterial ribosomes also prevented binding of this antibiotic to A .mont ana ribosomes ; on the . other hand cycloheximide which is not active on 70 S ribosomes did not prevent binding of 14C-chloramphenicol to A .montana and C. reinhardü ribosomes . Discussion The results in this work appear to give some idea of how ribosomes have evolved in algae and their relationship with bacterial ribosomes . It is known that bacterial ribosomes are 70 S, contain ing RNA/protein in the ratio 63/37, are readily dissociated into their subunits in buffer containing 10 - 3 _M Mgt and $ x 10 -2 _M NH4+ and are able to bind chloramphenicol and spiramycin III . Ribo-
Vol . 7, No . 6
RIBOSOMES FROM ALGAE
335
somes from a blue-green algae ( A .montana ) are similar in these characteristics to bacterial ribosomes but-are not so readily dissociated . Ribosomes from C . reinhardü have a sedimentation coefficient of 80 S but their ratio RNA/protein is characteristic of 70 S ribosomes ; their ionic requirement for stability appears to
be intermediate between the bacterial and the eucaryotic~types of~ ribosomes . Within the range of properties studied, ribosomes from C . nyrenoidosa prove to be similar to those of higher organisms
but dissociate somewhat more readily into subunits . These studies thus support previous reports on the close connection between
bacteria and blue green algae (23) and on the position of Chlamydomonas in phylogeny (12) . Acknowled~ements We wish to express our gratitude to Dr .C .Heredia for advice and to Miss Sara González Obiol for valuable help in the preparation of yeast and yeast ribosomes and to D]rs . Concepción López
Polo for her technical assistance . Our thanks are due to Prof . E . F. Gale, F .R .S ., Dr .D . Kerridge and Dr . G .A .M . Cros s for advice during the preparation of this manuscript . References 1 . M .L . PETERDIANN, In "The Ph sical and Chemical Pro erties of Ribosomes" . Elsevier Publ .House, Amsterdam-London (1964 2 . D . VAZQUEZ, fature ?~, 257 (1964) " 3 . D . VAZQUEZ and R.E . D10NR0, Biochim .Bionhys .Acta ~, 155 (1967) " 4 . G .A .r1 . CROSS, Proc . Soc . Gen . Dücrobiol . ~, IX (1967) " 5 . J .~1 . ASHLiORTH, Biochim .Bioohys .Acta 122, 213 (1966) .
6 . R .P . PERKY and D .E . KELLEY, J .DIo1 .Bio1 . 16, 255 (1966) . 7 . K . DIr1RCKER and F . SAfGER, J . rIol .Bio1 . _8, 835 (1964) " $ . ~I .~] . TAYLOR and R . STORCK, Proc .Nat .Acad .Sci .Wash . ~2, 187$ (1964) " 9 . J . EISENSTADT and G . BRAWERDIAN, Biochim. Biophys . Acta 80, 463 (1964) " 10 . R . Sa GER, I .B . WEINSTEIN and I. ÁSHKENAZY, Science ~, 304 (1963) " 11 . E . STUTZ and H . A`OLL, Proc . Nat . Acad . Sci . Wash . ,~, 774 x967) " 12 . R . SAGER and D1 . G. HAMILTON, Science ~, 709 (1967) " 13 . W.A . KRATZ and J . MYERS, Amer .J .Botany ~_, 282 (1955) " 14 . R . SAGER and S . GRANICK, Ann .N .Y . Acad .Sci . i, 831 (1953) .
336
RIBOSOMES FROM ALGAE
Vol, 7, No . 6
15 . M . RODRIGUEZ-LOPEZ, J .Gen .Microbiol . ~, 139 (1966) . 16 . C.F . HEREDIA and H .O . HALVORSON, Biochemistry ~, 946 (1966) . 17 . D . VAZQUEZ, Biochim . Bioyhys . Acta ~, 277 (1966) .
18 . O .H . LOWRY, N .J . ROSEBROUGH, A .L . FARR and R .J . RANDALL, J . Biol . Chem . ,1~, 265 (1951) . 19 . D. VAZQUEZ, Life Sciences ~, 845 (1967) " 20 . I .W . CRAIG and N .G . CARR, Biochem .J . _1~, 64p (1967) " 21 . A .D . WOLFF and F .E . HAHN, Biochim . Biophys .Acta Q~, 146 (1965) " 22 . L .A . ANDERSON and R .M . SMILLIE, Biochem.Biophys .Res .Commun .2,3, 23 . R .Y . STANIER and C .B . VON NIEL, Arch . Mikrobiol . í~2,, 17 (1962) .
1968 .
Pergamon Press
COMPARATIVE STUDIES ON CYTOPLASMIC RIBOSOMES FROM ALGAE M . Rodriguez-Lopez and D . Vazquez Instituto MaraBÓn and Instituto de Biologia Celular,Velazquez 144 Madrid-6, Spain (Received 6 December 1967 ; in final form 26 ,January 1968) Ribosomes can be classified according to their sedimentation coefficient into two types . Ribosomes of the 70 S type are present in procaryotic cells and eucaryotic organelles whereas 80 S ribo-
somes are found in the cytoplasm of eucaryotic cells . Ribosomes of the 70 S type differ from those of the 80 S type not only in their sedimentation coefficient but also in composition, sedimentation coefficients of their subunits and RNA, and response to ionic environment and to certain antibiotics inhibitors of protein synthesis (1,2,3) .
However we do not know yet if all ribosomes of one given type (70 S or 80 S) have the same mechanism for protein synthesis, nor is there conclusive evidence that all ribosomes of a given type ha~-e the same behaviour towards antibiotics . It is conceivable that more than two types of ribosomes have evolved, and the existence of ribosomes having characteristics of both 70 S and
80 S types is possible . In this respect some interesting information is already available . The sedimentation coefficient of ribo sonies from the protocoan Crithidia oncopelti is 80 S and the requirements of these ribosomes for protein synthesis are similar
to other ribosomes of the 80 S type ; however, unlike other 80 S ribosomes those of Crithidia oncopr~lti are easily dissociated into subunits which can reaggregate under suitable conditions (4) . On the other hand, ribosomes from the slime mould Dictyostelium disc oideum have a sedimentation coefficient of 70 S but do not readily dissociate in 10 - 3 _~1 rig ++ and in this respect they behave like other 80 S ribosomes (S) . Ribosome from guinea pig liver have 80 S ribosomes which dissociate to give 60 S subunits with 60 ~ RNA (like 70 S ribosomes) and 40 S subunits with 43 ~ RNA (rather like 80 S ribosomes)(6) . Dtarcker and Sanger (7) found N-formyl-methionyl-s-RNA in yeast ; this compound is the initiator of protein syn327
328
RIBOSOMES FROM ALGAE
Vol . 7, No . G
thesis by ~0 S ribosomes but it has not been reported in eucaryotic cells other than yeast . The work oî Taylor and Storck (8) has shown that blue-green algae have ~0 S ribosomes whereas other workers found that green algae have 80 S ribosomes in the cytoplasm (q,22) . Chlamydo monas is phylogenetically interesting and contradictory reports have appeared concerning its cytoplasmic ribosomes ; it appears
that these ribosomes have some features in common with both ~0 S
and 80 S ribosomes (10,11,12) . The present work is a contribution to the knowledge of algal ribosomes and attempts to relate the properties of algal ribosomes with algal phylogeny . Material and Methods Organism and ,growth conditions . Anacystis montana (LB 1405 /3), Chloralla pyrenoidosa Chick (211/8 a) and Chlamydomonas rein hardü (11/326 wild type +) were obtained from the Culture Collect ion of Algae and Protozoa (The Botany School, University of Cam-
bridge, Cambridge, England) . A . montana was grown in medium D (13), C . reinhardü in medium I (14) and C . pyrenoidosa in the medium previously described by one of us (15) .
The algae were collected by centrifugation while in the exponential phase of growth, washed with cold 10 -2 _M Tris/HC1 buffer pH 7 .4 containing 10 -2 M Mgt and 5 x 10 -2 M NH4+, and all further steps for breakage and fractionation were carried out at
44 C . This standard buffer was used throughout this work ; the Mgt ion concentration being changed when required . Frozen Escher ichia coli B was obtained from. the Microbiological Research~Establishment (Porton, England) . A diploid yeast (Saccharomyces fragilis x Saccharomyces dobzanskü ) was grown and used for comparative studies of ribosomes (16) .
Cell breakage and fractionation . For breakage of the cells A . montana and C . reinhardü were resuspended in the standard buffer and 15 ~ (w/v) sucrose . C . pyrenoidosa was resuspended in the standard buffer containing 5 x 10 3 M Mgt and 15 ~ sucrose . Yeast was resuspended in the standard buffer containing 5 x 10 3 M Mgt. Cells were then broken in a Ribi cell fractionator at 30000 Psi ; under these conditions the cells were broken but not their chloroplasts . E . coli was broken by alumina grinding and the broken preparation extracted with the standard buffer .
Vol .
7, No .
6
329
RIBOSOMES FROM ALGAE
E . coli and yeast were fractionated following standard
procedures (16,17) . To obtain cytoplasmic ribosomes from algae
the broken cell preparations were first centrifuged at 4000 g for 15 min to separate the chloroplasts and ribosomes prepared from
the supernatant fluids by the methods used to obtain bacterial ribosomes (17) . Ribosomes were finally resuspended in the standard buffer containing either 5 x 10 -3 _M Mgt for yeast, C . pyrenoidosa and C . reinhardü ribosomea or 10 -2 M Mgt for E . coli , A . montana , and also in some cases for C . reinhardü ribosomes .
Ribosome analysis . The algal ribosomes obtained as above described were not pure enough for estimation of their RNA and protein content . For these determinations the ribosomes were
further purified by centrifugation for 90 min at 37000 r.p .m . in the SW 39 rotor of the Spinco model L-2 preparative ultracentrifuge through a sucrose gradient (5-20 ~ (w/v) sucrose) in the standard buffer containing 5 x 10 - 3 or 10 -2 M Mgt as above . 32 x 0 .15 ml fractions were taken, alternative fractions were diluted with 4 ml of water and O .D . estimated at 260 ~x . The remaining fractions
corresponding to the ribosomal peaks in the three tubes were pooled . Protein was estimated directly by the method of Lowry et al . (18) using as control a sucrose solution with the same concentration of sucrose as the ribosomal suspensions and using bovine serum albumin as a standard . For the estimation of RNA, the ribosomes-were treated twice with 0 .5 N perchloric acid for 2C min at 704 and the extracts were pooled . The O .D . of this extract at 260 mu was measured . The RNA content was determined by assuming that 1 mg of ribosomal RNA has an O .D . at 260 m~,t of 25 units . Fundamentally the same procedure was followed to estimate RNA and protein in the ribosomal subunits but here the ribosomes were centrifuged for 13 hr at 2¢000 r .p .m . in the SW 25-2 rotor of the Spinco model L-2, the sucrose gradient contained 10 - 4 _M Mgt and 1 ml fractions were taken . The fractions corresponding to the faster and slower subunits were pooled separately for analysis . Purity of the preparations of ribosomal subunits was always confirmed by centrifugation through sucrose gradients in
the SW 39 rotor and estimation of U .D . at 260 m~ of the fractions as indicated above . Bindins of antibiotics . Binding of 14 C-chloramphenicol and 1 4C-spiramycin I to ribosomes was studied by methods described else-
330
fol . ï, n'o . C>
RIBOSOMES ~ROM ALGAr
cNmnoo woNAS 0o uos z " c i.y ~ .v r 0
" os '
" os I
"
"
NlsosowAs .a
" ".
s~
a 10 ,
o
e-
+
"
_
wy .
" .. 10~
w
w y"
"
41
10
rq " CT :ON
CMLONfIy " 00
170 5
w3 i
F~ACTION
20
41~SOME}
WS
.OS
~
i
"
b
e . 1Ó M
~ .
"
" - 10 1 w
w~~
~
.-
tù" r
wr
+7
Flr, . 1 Seditnentation patterns of Anacystis montana , Chlamydomor2as reinhardü and Çhlorella pyrenoidosa ml 0 .05 of ribosome suspensions were centrifuged for 120 min at 3000 r .p .m . in the SW 3q rotor of the Spinco L-2 centrifuge through a 5-20 ~ (w/v) sucrose gradient containing the required concentration of Mg++ . 0 .1$ ml fractions were taken into q ml of water and O .D . at 260 m}r were estimated . The arrows indicate the position of the peaks obtained when Escherichia coli or yeast ribosomes, ribosome dimers or ribosomal subunits of known sedimentation coefficients were run .
331
RIBOSOMES PROM ALGAE
Vol . 7, No . 6
where (17,18) . 14C-chloramphenicol (specific activity 6 .81 ~C/umole) was obtained from The Radiochemical Centre (Amersham, Great Britain) and 14 C-spiramycin I (specific activity 6 y~C/2$ mg) was kindly supplied by Dr . Mc Fadzean (May and Baker Ltd ., Great Britain) . The sources of other antibiotics are given in an earlier paper (17) . Results The results obtained in studies on centrifugation of algal ribosomes through sucrose gradients with different concentrations
of Mgt are shown in Fig. 1 . These sedimentation patterns suggest that the sedimentation coefficient of A . montana ribosomes is 70S whereas that of C . reinhardü and C . pyrenoidosa ribosomes is 80 S ; this is in agreement tivith previous determinations carried out by other workers using more accurate methods (8,12,20) . Under the
experimental conditions used in this work it appears that there was some formation of dimers at 10 -2 Di Digs in the three types of ribosomes studied and there was some dissociation into ribosomal subunits at 10 - 3 ~I ~1g~ . This disr~ociation was more marked than in
TABLE 1 Chemical Composition of Algal Ribosomes and Ribosomal Subunits
Subunits Ribosomes Organism
~ protein i~ RNA
Faster subunit ~ protein
~ RNA
Slower subunit ~ protein ~ RNA
A . montana
37
63
31
69
37
63
Ç . reinhardü
35
65
31
69
30
70
C . pyrenoidosa
49
$1
47
53
50
50
Ribosomes or ribosomal subunits were purified by centrifugation through a sucrose gradient as indicated under Material and Methods . Protein was determined by the method of Lowry et al . (18) . RNA was extracted with 0 .5 _N perchloric acid at 70Q, the O .D . at 260 rr~ of the extract measured and RNA calculated on the basis that 1 mg of ribosomal RNA has an O .D . at 260 m}i of 25 units .
332
RIBOSOMES FROM AL~AF
Vol, î, No, i>
mammalian ribosomes but it was not complete as in bacterial ribo-
somes . The dissociation of algal ribosomal subunits was complete at 10 -4 M Mgt and has already been shown by other workers (12,20) . Chemical analysis has shown that in C . pyrenoidosa ribosomes the ratio RNA/protein is close to 1 whereas A . montana and C . reinhardü ribosomes are richer in RNA (Table 1), In this
respect A . montana and C . reinhardü ribosomes are like those from bacterial cells whereas C . pyrenoidosa ribosomed are like those from yeast and mammalian cells (1) . Similar results were obtained
when ribosomes or ribosomal subunits were analysed and no differ-
ence was found in the RNA/protein ratio between the ribosomal sub-
units of the same alga .
The sti` king selectivity of some antibiotics which are able to inhibit specifically protein synthesis by either the ;0 S or 80 S ribosomes is well known (2,3) . These antibiotics can be used to establish the type of system in algae . Cycloheximide is a a elective inhibitor of protein synthesis by ribosomes of the eucaryotic type (80 S) whereas chloramphenicol, erythromycin, spiramycin and lincomycin act selectively on ribosomes of th~z
procaryotic type (70 5)(3) . When the effects of antibiotics ti" Pr~: tested on intact algae it was found that on13- antibiotics acting
on ~0 S ribosomes strongly inhibited A . montana (Table 2) . Ur, the other hand only Cycloheximide completely blocked growth of C . py renoidosa at low concentrations . Cycloheximide also inhibited
growth of C . reinhardü but the higher concentrations required might possibly be due to a permeability barrier . In the behaviour towa~°ds antibiotics A, montana behaved like bacteria whereas C .
pyrenoidosa behaved like eucaryotic organisms . The uptake of 1~C-chloramphenicol by C . reinhardü and C . pyrenoidosa was studied in order to determine if the low sensitivity of these algae to the antibiotic could be due to a permeability barrier . Results not shuwr~ here showed that our strains of C . reinhardü and C . pyre noidosa were permeable to chloramphenicol and the extent of antibiotic uptake by these algae was comparable to that obtained pre-
viously with bacteria (2) . Chloramphenicol, erythromycin, spiramycin and lincomycin are known to be active on chloroplast ribosomes and this might explain the observed bleaching of the cells and the inhibitory effect of these antibiotics on growth of C . pyrenoidosa and C . reinhardü by high concentrations .
333
RIBOSOMES FROM ALGAE
Vol . 7, No . 6
TABLE 2 Effect of a Number of Antibiotica on Growth of Algae ;B inhibition of growth Antibiotic
ug/ml
A .montana
C .reinhardü
C .UVrenoidosa
1
10
12
l00
32
90
loo
Chloramphenicol 1 ++ l0 n 100
86
20
97 100
5
5o 80
So 80
10
1
100 100
3 85
100
100
90
50 87
100
1
100
5
25
Cycloheximide " "
Erythromycin +' Spiramycin ++ n Lincomycin "
10
21
l0 100
1 10
100
80
95 100
100 100
34 90
30 70
100
0
40
100
8
100
100
0
85
$ ml of the alga suspension (1 x 107 cells/ml) was taken in 25 ml conical flasks, and incubated in the presence of light at 2$Q with strong agitation for 72 hr in the presence or absence of antibiotic . The number of cells was estimated microscopically at the beginning and the end of the incubation period . Incubations were carried out in duplicate and the average figures obtained . Experimental evidence has shown that in cell-free systems
14 C-chloramphenicol and 14C-spiramycin I bind to 70 S but not to
80 S ribosomea (2,19,21,22) . A . montana ribosomea are found to bind these two antibiotics (Table 3) . On the other hand a much smaller binding to C .pyrenoidosa and C . reinhardü ribosomes was found and even this binding could be due to contamination with mitochondria] and chloroplastic ribosomes as this is very difficult to avoid in preparations from algae (22) . Lack of sensitïvity of cytopl .asmic~
ribosomes from green algae to chloramphenicol has already hc "+" n r~"ported by other workers (9,10, 22) . Erythranycin and ] i ncomyc"..i n +c l+ i c"1+
Vol . 7, No . 6
RIBOSOMES FROM ALGAE
334
TABLE 3 Binding of 14C-chloramphenicol and 14C-spiramycin I to Ribosomes uumoles radioactive antibiotic bound/mg ribosomes Ribosomes from:
Non-radioactive antibiotic added 14C-chloramphenicol
A .montana " " "
None Cycloheximide Erythromycin Lincomycin
C . reinhardü C . nyrenoidosa
14 C-spiramycin I
108 107 21 4
90 -
None
31
20
None
26
6
Suspensions of ribosomes (final concentration 1 mg/ml) in 10 -2 M Tris/HC1 buffer pH 7 .4 containing 10-2 M Mgt and 0 .1 M NHQ were used for all binding experiments . To study binding of 1 4C-spiramycin I the antibiotic was added to a final concentration of $ x 10- M and the ribosomes were harvested by centrifugation after the binding reaction ; the results were corrected by using a blank in which 1ZC-spiramycin III was added to a final concentration 1 .$ x 10-4 M and mixed with the 14C-spiramycin I prior to addition of ribosomes and centrifugation . A similar technique was used to study binding of 14C-chloramphenicol but this antibiotic was used at a concentration $ x 10 -6 _M and 1 2 C-chloramphenicol (1 .$ x 10-4 M) was also added in the blank . Cycloheximide, erytromycin and lincomycin were added to the tubes when required (final concentration 1 .$ x 10-4 M) and mixed with 14C-chloramphenicol prior to addition of ribosomes and centrifugation . are active in preventing binding of 14C-chloramphenicol to bacterial ribosomes also prevented binding of this antibiotic to A .mont ana ribosomes ; on the . other hand cycloheximide which is not active on 70 S ribosomes did not prevent binding of 14C-chloramphenicol to A .montana and C. reinhardü ribosomes . Discussion The results in this work appear to give some idea of how ribosomes have evolved in algae and their relationship with bacterial ribosomes . It is known that bacterial ribosomes are 70 S, contain ing RNA/protein in the ratio 63/37, are readily dissociated into their subunits in buffer containing 10 - 3 _M Mgt and $ x 10 -2 _M NH4+ and are able to bind chloramphenicol and spiramycin III . Ribo-
Vol . 7, No . 6
RIBOSOMES FROM ALGAE
335
somes from a blue-green algae ( A .montana ) are similar in these characteristics to bacterial ribosomes but-are not so readily dissociated . Ribosomes from C . reinhardü have a sedimentation coefficient of 80 S but their ratio RNA/protein is characteristic of 70 S ribosomes ; their ionic requirement for stability appears to
be intermediate between the bacterial and the eucaryotic~types of~ ribosomes . Within the range of properties studied, ribosomes from C . nyrenoidosa prove to be similar to those of higher organisms
but dissociate somewhat more readily into subunits . These studies thus support previous reports on the close connection between
bacteria and blue green algae (23) and on the position of Chlamydomonas in phylogeny (12) . Acknowled~ements We wish to express our gratitude to Dr .C .Heredia for advice and to Miss Sara González Obiol for valuable help in the preparation of yeast and yeast ribosomes and to D]rs . Concepción López
Polo for her technical assistance . Our thanks are due to Prof . E . F. Gale, F .R .S ., Dr .D . Kerridge and Dr . G .A .M . Cros s for advice during the preparation of this manuscript . References 1 . M .L . PETERDIANN, In "The Ph sical and Chemical Pro erties of Ribosomes" . Elsevier Publ .House, Amsterdam-London (1964 2 . D . VAZQUEZ, fature ?~, 257 (1964) " 3 . D . VAZQUEZ and R.E . D10NR0, Biochim .Bionhys .Acta ~, 155 (1967) " 4 . G .A .r1 . CROSS, Proc . Soc . Gen . Dücrobiol . ~, IX (1967) " 5 . J .~1 . ASHLiORTH, Biochim .Bioohys .Acta 122, 213 (1966) .
6 . R .P . PERKY and D .E . KELLEY, J .DIo1 .Bio1 . 16, 255 (1966) . 7 . K . DIr1RCKER and F . SAfGER, J . rIol .Bio1 . _8, 835 (1964) " $ . ~I .~] . TAYLOR and R . STORCK, Proc .Nat .Acad .Sci .Wash . ~2, 187$ (1964) " 9 . J . EISENSTADT and G . BRAWERDIAN, Biochim. Biophys . Acta 80, 463 (1964) " 10 . R . Sa GER, I .B . WEINSTEIN and I. ÁSHKENAZY, Science ~, 304 (1963) " 11 . E . STUTZ and H . A`OLL, Proc . Nat . Acad . Sci . Wash . ,~, 774 x967) " 12 . R . SAGER and D1 . G. HAMILTON, Science ~, 709 (1967) " 13 . W.A . KRATZ and J . MYERS, Amer .J .Botany ~_, 282 (1955) " 14 . R . SAGER and S . GRANICK, Ann .N .Y . Acad .Sci . i, 831 (1953) .
336
RIBOSOMES FROM ALGAE
Vol, 7, No . 6
15 . M . RODRIGUEZ-LOPEZ, J .Gen .Microbiol . ~, 139 (1966) . 16 . C.F . HEREDIA and H .O . HALVORSON, Biochemistry ~, 946 (1966) . 17 . D . VAZQUEZ, Biochim . Bioyhys . Acta ~, 277 (1966) .
18 . O .H . LOWRY, N .J . ROSEBROUGH, A .L . FARR and R .J . RANDALL, J . Biol . Chem . ,1~, 265 (1951) . 19 . D. VAZQUEZ, Life Sciences ~, 845 (1967) " 20 . I .W . CRAIG and N .G . CARR, Biochem .J . _1~, 64p (1967) " 21 . A .D . WOLFF and F .E . HAHN, Biochim . Biophys .Acta Q~, 146 (1965) " 22 . L .A . ANDERSON and R .M . SMILLIE, Biochem.Biophys .Res .Commun .2,3, 23 . R .Y . STANIER and C .B . VON NIEL, Arch . Mikrobiol . í~2,, 17 (1962) .