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Detection of ribosomes and polysomes in Acetabularia mediterranea
Pol . 5, pp . 2113-2116, 1966 . Life Sciences Printed in Great Britain.
DETFCTI02i OF RIBOSOMES AND POLYSOMES IN ACET
rr .earA
Pergamoa Press Ltd.
.DTRTR
RexF
,
Janowaki, M . Laboratoire de blorphologie Animale . Université Libre de Bruxelles, Belgium .
(Received
3
June 1966 ; in final form 12 September 1966)
Anucleate fragments of the unicellular green alga Acetabularia mediterranea can synthesize proteins and active enzymes for several weeka l ' 2 and produce a species-specific "cap" 3 , These findings suggest that intermediates between nuclear DPIA and proteins, presumably messenger-RNA's, retain several weeks genetic information in the absence of nucleus . If this hypothesis is correct, we may hope to detect, in anucleate Prageients, polysomes which incorporate labeled amino-acids, but not uridine . On the other hand, nucleate fragments should incorporate both protein and RNA precursors into polysames . The first stage in this work has consisted in labeling the whole algae with both kinds of precursors, sad in isolating riboeames and polysomes by differential centrifugation . Materials and Methods . 20 adult whole algae were incubated for 120 min . at room temperature is natural seerwater containing either 100r.C of H~-uridine (The Radio-Chemical Center), or 50fr.C of reconstituted H 3 -protein hydrolysate (Schwarz Bioresearch) ; er they were cut into small pieces with scissors and gently homogenized by five strokes of a Teflon pestle in ten times their weight of the following medium
(at k°C) : 0 .01 t4 Tris-HC1 pH 7 .4, 0.01 M Y,C1, 0.5 ~ sodiu¢e deoxycholate, 0 .5 ~
lubrol (Imperial Chemical Industries) . Preliminary assays have shown that the . intracellular concentration of magne-
sium in the alga is 0 .05 ?~, For this reason, volumes giving a final Aig +f concentration of 0 .005 x4 suet be used .
The homogenate was divided into two equal parts . To one part ribonuclease (RNAeae Si~na) was added in order to obtain concentration of O .O~g/ml . The two samples were centrifuged for 10 min . at 15,000 RPb4 in a Beckman microfuge at 4° C in order to sediment the chloroplasta, and 25~ 1 of each of the two supernatants were layered an a 5 .0 ml gradient ca~mposed of 15 to 30 x sucrose in 0 .01 A4 Tris-HC1 pH 7 .4, 0 .01 M KC1, 0 .003 b1 Mg acetate .
2113
2114
RIB0801~8 AND POLYSOlIEB
Pol. 5, No . 22
The gradients were spun for 120 sin . at 37,500 RFC in the SW 39 rotor of a Spinco model L50 refrigerated centrifuge . In each experiment, the R'IAase exerted its action for 30 min . before the start of the centrifugation . F~Io appreciate RPFAase activity could be detected in the untreated sample under the conditions we used . The 5 ~ trichloroacetic insoluble radio-activity present in two-drops fractions was measured on millipore filters in a liquid scintillation counter (Nuclear Chicago) . S-values of the sedimenting material were determined by calibrating the sucrose gradients with non--radio-active Escherichia Coli 70, 50 and 30 S ribosomes . Results . a) Inçorporation of H3-uridine . It can be seen (fig .l) that a 120 min .exposure to H 3 -vridine results in the labeling of the ribosomal RT~TA in the monosomea as well as in the polysomes . The latter are sensitive to incubation in ~ .OR,K,g/ml R`FAase for 30 min . at 4 ° C and are degraded into monosomea having a S-value of 92 . RPFAase treatment shifts about three times more radio-activity to the ribosomen region than could be expected by the loss froaa the polysomes region ; it is thus highly probable that at least 2/3 of the polysomea have aedimented to the bottom of thè gradient in the untreated sample, and that they were not collected by puncturing the bottom of the tube . The proportion of single monosomes to polysomes is small ; this means that most of the ribosaaaes must be engaged in protein s;mtheais . Two other bands of FLYAese-sensitive material appear also in the gradient, sedimenting with S-values of 65 and 4ß . This materiel probably does not represent aubunita of the monosomea dissociated by a lack of ?Fg++ ions, since it is
very sensitive to IWAase (whïle Rscherichie Coli ribosa~mea were not affected under the same conditions) . Furthermore, increasing the Aig++ concentration in the homogenate up to O .OOß ti does not result in a lowering of the level of the 65 S and 4ß S radio-activity .
b) Incorporation of reconstituted Fi 3-protein hydrolysate . Fig .2 shows that a 120 min . exposure to tritiated amino-acids results in the labeling of ribosomal proteins in the polysomee and in the ribosomes . RNAase
treatment ahifte the radio-activity from the polysoaal region to the monosomes . Unfortunately, the 65 S and bß S peaks could not be seen in these experiments .
2115
RIBOSOI~S AND POLYSOMES
Vol. 5, No . 22 CPM S00~
400
300.
200-1
l00Î
0
FIG . 1 Sedimentation characteristics of 120 min. fi a-uridine labeled particles . Control ANAase treated sample
o 0 0
Conditions : see Materials and Methods . CPII
150 -
300
0
~
~
30
AWClIO"
FIG . 2 Sedimentation characteristics of 120 min. H3-amino-acids labeled particles . Control RNAase treated sample o 0 0 Conditions : see Materials and Hethods.
aiBOSO~s
21}s
~n
voi .
PoLYSO~s
Discussion . Our experiments show that labeling of polysomes and
82
5, No. 22
S ribosomes with tri-
tinted uridine and amino-acide can be detected in whole cells of Acetabularia mediterranes . Most of the ribosomes appear to be engaged in protein syntheâis. The rapid]y labeled
65
end
48
S components might represent "native ribosomes"
bearing messenger-RNA from the nucleus to the cytoplasm and acting as direct precursors of the polysomes . Such precursors have been found in rat liver and in HeLa cells 5 . Our techniques should enable us to find out whether stable messenger-RNA's are present in the cytoplasm of aaucleate fragments and if so, whether they are a part of active polysomes.
Sam.
Incorporation of tritiated uridine and amino-acids into whole Acetabularia cells allowed the detection of polysomes and 82 S ribosomes . Two kinds of rapid]~r labeled particles, sedimenting with S-values of
65
and
48,
were also
isolated . They seem to be different from the subunits of dissociated ribosomea . References .
~8,
1.
J . ARACHET, H. CHANTRENNE and F . VANDERHAEGHE, Biochim.Hiophys .Acte
2.
J. Hâh4~RLING, J. CLAUSS, K. KECK, G. ßICfiTER and G. WERZ, Exptl .Ce1]. Res.
544 (1955) "
~5~~ }6,
210
(1959) . (1934) .
3.
J . H1itR~RLING, Wilhelm Rqux's Arch .Entwicklungsm . ~~j., 1
4.
E .C .HE7SHAW, T.A .HOJAPSY.I and H .H .HIATT, J.t"!o1.Bio1. ~, 122
5.
W.K .JOhZIK sad Y, BECKFR, J.Mo1 .Dio1., ~,
496 (1963) .
(1963) .
DETFCTI02i OF RIBOSOMES AND POLYSOMES IN ACET
rr .earA
Pergamoa Press Ltd.
.DTRTR
RexF
,
Janowaki, M . Laboratoire de blorphologie Animale . Université Libre de Bruxelles, Belgium .
(Received
3
June 1966 ; in final form 12 September 1966)
Anucleate fragments of the unicellular green alga Acetabularia mediterranea can synthesize proteins and active enzymes for several weeka l ' 2 and produce a species-specific "cap" 3 , These findings suggest that intermediates between nuclear DPIA and proteins, presumably messenger-RNA's, retain several weeks genetic information in the absence of nucleus . If this hypothesis is correct, we may hope to detect, in anucleate Prageients, polysomes which incorporate labeled amino-acids, but not uridine . On the other hand, nucleate fragments should incorporate both protein and RNA precursors into polysames . The first stage in this work has consisted in labeling the whole algae with both kinds of precursors, sad in isolating riboeames and polysomes by differential centrifugation . Materials and Methods . 20 adult whole algae were incubated for 120 min . at room temperature is natural seerwater containing either 100r.C of H~-uridine (The Radio-Chemical Center), or 50fr.C of reconstituted H 3 -protein hydrolysate (Schwarz Bioresearch) ; er they were cut into small pieces with scissors and gently homogenized by five strokes of a Teflon pestle in ten times their weight of the following medium
(at k°C) : 0 .01 t4 Tris-HC1 pH 7 .4, 0.01 M Y,C1, 0.5 ~ sodiu¢e deoxycholate, 0 .5 ~
lubrol (Imperial Chemical Industries) . Preliminary assays have shown that the . intracellular concentration of magne-
sium in the alga is 0 .05 ?~, For this reason, volumes giving a final Aig +f concentration of 0 .005 x4 suet be used .
The homogenate was divided into two equal parts . To one part ribonuclease (RNAeae Si~na) was added in order to obtain concentration of O .O~g/ml . The two samples were centrifuged for 10 min . at 15,000 RPb4 in a Beckman microfuge at 4° C in order to sediment the chloroplasta, and 25~ 1 of each of the two supernatants were layered an a 5 .0 ml gradient ca~mposed of 15 to 30 x sucrose in 0 .01 A4 Tris-HC1 pH 7 .4, 0 .01 M KC1, 0 .003 b1 Mg acetate .
2113
2114
RIB0801~8 AND POLYSOlIEB
Pol. 5, No . 22
The gradients were spun for 120 sin . at 37,500 RFC in the SW 39 rotor of a Spinco model L50 refrigerated centrifuge . In each experiment, the R'IAase exerted its action for 30 min . before the start of the centrifugation . F~Io appreciate RPFAase activity could be detected in the untreated sample under the conditions we used . The 5 ~ trichloroacetic insoluble radio-activity present in two-drops fractions was measured on millipore filters in a liquid scintillation counter (Nuclear Chicago) . S-values of the sedimenting material were determined by calibrating the sucrose gradients with non--radio-active Escherichia Coli 70, 50 and 30 S ribosomes . Results . a) Inçorporation of H3-uridine . It can be seen (fig .l) that a 120 min .exposure to H 3 -vridine results in the labeling of the ribosomal RT~TA in the monosomea as well as in the polysomes . The latter are sensitive to incubation in ~ .OR,K,g/ml R`FAase for 30 min . at 4 ° C and are degraded into monosomea having a S-value of 92 . RPFAase treatment shifts about three times more radio-activity to the ribosomen region than could be expected by the loss froaa the polysomes region ; it is thus highly probable that at least 2/3 of the polysomea have aedimented to the bottom of thè gradient in the untreated sample, and that they were not collected by puncturing the bottom of the tube . The proportion of single monosomes to polysomes is small ; this means that most of the ribosaaaes must be engaged in protein s;mtheais . Two other bands of FLYAese-sensitive material appear also in the gradient, sedimenting with S-values of 65 and 4ß . This materiel probably does not represent aubunita of the monosomea dissociated by a lack of ?Fg++ ions, since it is
very sensitive to IWAase (whïle Rscherichie Coli ribosa~mea were not affected under the same conditions) . Furthermore, increasing the Aig++ concentration in the homogenate up to O .OOß ti does not result in a lowering of the level of the 65 S and 4ß S radio-activity .
b) Incorporation of reconstituted Fi 3-protein hydrolysate . Fig .2 shows that a 120 min . exposure to tritiated amino-acids results in the labeling of ribosomal proteins in the polysomee and in the ribosomes . RNAase
treatment ahifte the radio-activity from the polysoaal region to the monosomes . Unfortunately, the 65 S and bß S peaks could not be seen in these experiments .
2115
RIBOSOI~S AND POLYSOMES
Vol. 5, No . 22 CPM S00~
400
300.
200-1
l00Î
0
FIG . 1 Sedimentation characteristics of 120 min. fi a-uridine labeled particles . Control ANAase treated sample
o 0 0
Conditions : see Materials and Methods . CPII
150 -
300
0
~
~
30
AWClIO"
FIG . 2 Sedimentation characteristics of 120 min. H3-amino-acids labeled particles . Control RNAase treated sample o 0 0 Conditions : see Materials and Hethods.
aiBOSO~s
21}s
~n
voi .
PoLYSO~s
Discussion . Our experiments show that labeling of polysomes and
82
5, No. 22
S ribosomes with tri-
tinted uridine and amino-acide can be detected in whole cells of Acetabularia mediterranes . Most of the ribosomes appear to be engaged in protein syntheâis. The rapid]y labeled
65
end
48
S components might represent "native ribosomes"
bearing messenger-RNA from the nucleus to the cytoplasm and acting as direct precursors of the polysomes . Such precursors have been found in rat liver and in HeLa cells 5 . Our techniques should enable us to find out whether stable messenger-RNA's are present in the cytoplasm of aaucleate fragments and if so, whether they are a part of active polysomes.
Sam.
Incorporation of tritiated uridine and amino-acids into whole Acetabularia cells allowed the detection of polysomes and 82 S ribosomes . Two kinds of rapid]~r labeled particles, sedimenting with S-values of
65
and
48,
were also
isolated . They seem to be different from the subunits of dissociated ribosomea . References .
~8,
1.
J . ARACHET, H. CHANTRENNE and F . VANDERHAEGHE, Biochim.Hiophys .Acte
2.
J. Hâh4~RLING, J. CLAUSS, K. KECK, G. ßICfiTER and G. WERZ, Exptl .Ce1]. Res.
544 (1955) "
~5~~ }6,
210
(1959) . (1934) .
3.
J . H1itR~RLING, Wilhelm Rqux's Arch .Entwicklungsm . ~~j., 1
4.
E .C .HE7SHAW, T.A .HOJAPSY.I and H .H .HIATT, J.t"!o1.Bio1. ~, 122
5.
W.K .JOhZIK sad Y, BECKFR, J.Mo1 .Dio1., ~,
496 (1963) .
(1963) .