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Dissociation and reassembly of polyribosomes in relation to protein synthesis in the soybean root
J. Nol. Biol. (1967) 26, 237-247
Dissociation and Reassembly of Polyribosomes in Relation to Protein Synthesis in the Soybean Root C.Y.Lnv
AND JOE L. KEY
Department of Botany and P.&t Pathology and Department of Biology Purdue University, Lafayette, Indiana 47907, U.X.A. (Received 7 November 1966, and in revisedform 21 February 1967) Polyribosomes of the soybean root rapidly dissociate under energy-depleting conditions, namely anaerobiosis and dinitrophenol treatment. The loss of polyribosomes cannot be attributed to a depletion of messenger RNA, because polyribosomes re-form to about 75% of the original level following restoration of aerobic conditior24 in the presence of dactinomycin. The daotinomycin treatment resulted in greater than 90% inhibition of RNA synthesis during the aerobic recovery period. Nascent polypeptide is lost from the ribosomes coincident with or during the conversion of polyribosomes to monoribosomes during anaerobiosis. Cycloheximide blocks both the conversion of polyribosomes to monoribosomes and the loss of nascent polypeptide from ribosomes. The results suggest that polyribosome dissociation occurs as a result of completion of read-out and release of polypeptide and monoribosome, coupled with a failure of attachment of ribosomes to messenger RNA and subsequent initiation of new peptide chains.
1. Introduction During an investigation of the association of DNA-like RNA, which in a previous study was shown to have characteristics in common with messenger RNA (Ingle, Key & Helm, 1965), with ribosomes in the soybean root (Lin, Key & Bracker, 1966),
we observed that culture conditions which tended to be less aerobic than normal caused a decrease in the proportion of ribosomes in the polyribosome complex. Marks, Burka, Conconi, Per1 & Rifkind (1965) found that treatment of reticulocytes with fluoride caused a rapid shift of polyribosomes to monoribosomes ; and they concluded that there is a requirement for energy, possibly ATP or GTP, for maintenance of the polyribosome structure. Because of the rapid dissociation of polyribosomes and the pattern of dissociation, it was concluded that the process occurred as a single event. Williamson & Schweet (1964J965) presented evidence supporting the view of a dynamic equilibrium of attachment and detachment of ribosomes from polyribosomes during protein synthesis. They suggested that the rate of attachment is the rate-limiting step in protein synthesis. Villa-Trevino, Faber, Staehelin, Wettstein & No11 (1964) postulated that attachment requires a special mechanism which is separable from the read-out process, and emphasized a possible decisive role of the attachment
mechanism
in metabolic
control.
Based on the above observations, our experiments were designed to elucidate the mechanism of the loss of polyribosomes under energy-deficient conditions. The results 237
238
C. Y.
LIN
AND
J. L.
KEY
indicate that the dissociation of polyribosomes results from completion of read-out with release of polypeptide and ribosomes in association with failure of attachment and initiation of new protein synthesis. This interpretation emphasizes that the attachment mechanism may have an important role in metabolic control (VillaTrevino et al., 1964).
2. Materials and Methods Soybean seeds were germinated in rolls of moist paper (Ingle & Key, 1965). The various treatments were carried out at 30°C by placing seedlings after 60 to 66 hr germination in bundles of 50 to 60 and immersing the roots 1 to 2 cm in a solution, the composition of which varied with the particular treatment. Anaerobiosis was maintained by bubbling nitrogen gas through the incubation medium. The apical (tip) &mm sections of the soybean roots were cut onto dry ice and ground in dry ice by mortar and pestle. The powder was gently homogenized in a loose-fitting ground-glass conical hand homogenizer (usually two strokes with 8 to 10 quarter turns each) in a 0.25 M-sucrose solution containing 0.05 m-Tris buffer (pH 7*4), 0.015 M-KC& and 0.02 M-&$& (Lin et al., 1966). The homogenate was filtered through Mira cloth, and the filtrate was centrifuged at 13,500 g for 15 min. Ribosomes were prepared from the supernataut solution by layering the sample over successive layers of 0.5 and 1.6 Msucrose (the sucrose solutions contained 0.05 M-Tris buffer, O-015 ~-Kc1 and 0.005 MMgCl,) followed by centrifugation at 105,000 g for 4 hr (Wettstein, Staehelin 8r Noll, 1963). The ribosome pellets were suspended in 0.05 m-Tris buffer (pH 7.4) containing 0.015 ~-Kc1 and 0.005 M-Mgcl,. In one set of experiments, the 13,500 g supernatant fraction was layered directly onto a linear sucrose gradient and centrifuged (see below). All steps were carried out at 0 to 4°C. The ribosome preparations were layered onto linear 10 to 34% sucrose gradients and centrifuged at 23,000 rev./min in the SW25 rotor for 2 hr or used directly in amino acid-incorporating studies, following the methods of Mans & Novelli (1961,1964) and Williams & Novelli (1964). The distribution of ribosomes in sucrose gradients was determined by collection with continuous recording in an ISCO model D density-gradient fractionator (collection being from the top of the tubes). Collection was made at a syringe speed at 3+ ml./min with the O.D. profile being made at a wavelength of 254 ml*. When ribosomes were isolated from tissue which had been labeled with [3H]- or [14C]leucine lo-drop fractions were collected, and the O.D. was or [3H]adenosine and [3H]uridine, at 260 mp. The samples were precipitated with trichloroacetic acid, collected measured on nitrocellulose membrane fdters (type B-6 Schleicher & Scheull), and counted in a liquid-scintillation spectrometer. Total protein syntheis by the tissue was measured by following [lgC]leucine incorporation (Key, 1964). The mild homogenizing conditions necessary to allow isolation of polyribosomes from the soybean root did not result in quantitative rupture of the cells and thus in quantitative recovery of the ribosomes. Accordingly quantitative extraction of ribosomes was made, and the results showed that none of the treatments used in Fig. 1 resulted in a significant change in the ribosome population of the tissue over the treatment interval. There was a small preferential loss of monomer ribosomes under the conditions used routinely to isolate the ribosomes (see Table 4). Also, the amount of ribosomes placed on the sucrose gradients was selected, based on preliminary experiments, to give a monomer population which was within scale sensitivity of the recorder, thus accounting for the different total amounts of ribosomes on the different gradients. Radioactive amino acids and nucleosides were obtained from Schwarz BioResearch. Dactinomycin was a gift from Merck, Sharp & Dohme. Puromycin was obtained from Nutritional Biochemicals and cycloheximide from the Sigma Chemical Company.
3. Results The distribution grown
under
different
on sucrose gradients of ribosomes prepared from soybean roots culture
conditions
is shown
in Fig.
1. There
was
a lower
pro-
POLYRIBOSOMES
IN
THE
SOYBEAN
ROOT
239
1 I’ -I-
J -1 (1
23
Volume FIG.
1. ?hmwsc gradient
distributions
collected
of ribosomes
c
(ml.) isolated
from the soybean soot.
Ribosomos were isolated from the epicad 5-B cm of the root of 120 soybean seedlings treated in the Eolluwjng mauner for 1 hr at 30°C. (a) Normal culture method (Inglo & Key, 1965); (b) incubated in water with shaking; (e) incubated in w&or without shaking; (d) incubated in water with continuous nitrogen gas bubbling; (e) incubated in IO-“~~-dinitrophenol with shaking; and (f) incubated in 20 pg dactinomycin/ml. with shaking.
.Time (mid FTC. 2. Kinetics of [14C]leucine ticorporation by isolated ribosomes. Preparations (a) to (f) correspond to those shown ia Fig. 1. Tho incorporation medium consisted of 50 pnoles Tris (pH 7.4), 5 pmoles lUgCl,, 8 pmolcs KCl, 0.5 pmole ATP, 0.15 ,umole GTP, 6.4 poles phosphoenolpyruvate, 0.05 mg of pyruvic kin&se, corn supernataut (O-5 to 1-O mg protein), O-2 ml. of ribosomes (06 to 1-O mg protein) and O-5 PC [14C]leucine (240 rc/pmole) in a final volume of O-5 ml. at 37%. Incorporation is shown as cts/min/mg ribosomal RN-4.
240
C. Y.
LIN
AND
J. L.
KEY
portion of polyribosomes relative to monoribosomes in roots which were grown in solution culture with shaking (Fig. l(b)) and without shaking (Fig. l(c)) compared to the control conditions (Fig. l(a)). This loss ofpolyribosomes under the less aerobic conditions suggested that a decreased energy supply might be involved. Accordingly, seedlings were incubated for one hour in solution culture with continuous bubbling of nitrogen gas through the medium (anaerobic treatment). There was almost complete loss of polyribosomes under this condition (Fig. l(d)). Treatment of the roots for one hour with dinitrophenol likewise resulted in complete loss of polyribosomes (Fig. l(e)). The proportion of polyribosomes was only slightly lowered by a two-hour treatment of 20 pg dactinomycin/ml. (Fig. l(f)) which inhibited RNA synthesis by about 90% (see also Fig. 9). Polyribosomes were dissociated during anaerobiosis in the presence of dactinomycin. The in vitro amino acid-incorporating activity of the ribosome preparations shown in Fig. 1 is presented in Fig. 2. The level of incorporating activity corresponded closely to the relative amount of polyribosomes in the various preparations. The poly U-dependent incorporation of phenylalanine (Fig. 3) shows that the ribosomes remain “biologically” active after dissociation under anaerobic conditions.
Time (min)
FIG. 3. Kinetics
of [14C]phenylalanine
incorporation
with
polyuridylic
acid as t,emplate.
The conditions used for incorporation of [W]phenylalanine were as for [14C]leucine incorporation except that 10 pmoles of MgCI, and 80 pg of poly U were used. --e-e-, Ribosomes corresponding to Fig. l(b); -A-.--.--A---, ribosomes corresponding to Fig. l(f); -- 0 --0 --, ribosomes corresponding to Fig. l(d). Incorporation is shown as cts/min/mg ribosomal RNA.
Following transfer of the seedlings to the anaerobic condition, there was a rapid loss of polyribosomes (Fig. 4(a) to (d)) corresponding with a rapid loss in protein synthetic activity (Fig. 7). This dissociation of polyribosomes during anaerobiosis was almost completely blocked by cycloheximide (Fig. 4 (e) to (h) compared with (a) to (d)). Under normal conditions, cycloheximide inhibited protein synthesis by about 95% (Fig. 7) without altering the level of polyribosomes (Table 1). Lowering the temperature from 30 to 5°C during the anaerobic treatment greatly decreased the rate of dissociation of polyribosomes (Fig. 5). These observations suggested that protein synthesis possibly was required to accomplish polyribosome dissociation during anaerobiosis. If completion of read-out and release of finished protein were associated with the loss of polyribosomes, the monomer ribosomes following dissocia-
POLYRIBOSOMES
IN
THE
Volume
FIG. 4. Kinetics
ROOT
241
2
23 0
0
SOYBEAN
of polyribosome
collected
dissociation
(ml.)
during anaerobiosis.
Ribosomes were isolated from the root tips of 120 seedlings at the following times after initiation of the anaerobic treatment: (a) and (e) 15 min; (b) and (f) 30 mm; (c) and (g) 60 min; (d) and (h) 120 min. Samples (e), (f), (g) and (h) were isolakd from seedlings treated with 2pg/ml. cycloheximide 45 min prior to and during the anaerobic treatment.
TABLE
Inhibition
by cycloheximide
1
of puromycin-induced
Treatment
None Puromycin (1 X 10m4M) Cycloheximide (2 pg/ml.) Puromycin + cycloheximide
decrease in polyribosomes Polyribosomes (%I 75 58 75 73
Ribosomes were isolated from seedlings which had been incubated for 1 hr as indicated. Data show the percentage of total ribosomes present as polyribosomes on sucrose gradients. The percentage of polyribosomes was calculated from the area under the curves of sucrose gradient profiles.
242
C. Y.
LIN
AND
V&me
J. L.
KEY
collected(ml.) (bl
(a)
FIG. 5. Influence of temperature on dissociation Ribosomes were isolated from seedlings after
of polyribosomes a l-hr anaerobic
during the anaerobic treatment. treatment: (a) at 36’C; (b) at
5%.
tion would contain much less “nascent” polypeptide than polyribosomes actively engaged in protein synthesis. The nascent polypeptide of polyribosomes was removed from the ribosomes in vivo during or coincident with the dissociation of polyribosomes under anaerobiosis (Fig. 6(a) and (c)). In contrast, there was no loss of nascent polypeptide associated with the conversion of prelabeled polyribosomes to monoribosomes by an in vitro RNase treatment of the isolated polyribosomes (Fig. 6(a) and (b)). Table 2 shows that the loss of nascent protein from polyribosomes under anaerobiosis was blocked by cycloheximide just as was the dissociation of the polyribosomal structure. Cycloheximide also stabilized polyribosomes in the presence of puromycin (Table 1). The reassociation of ribosomes into the polyribosomal structure after prior dissolution under anaerobic conditions is shown in Fig. 8 and Table 4. There was rapid reassociation during the initial 15 to 30 minutes (Fig. S(a) and (b) and Table 4),
0 Tube no.
(a)
(b)
Cc)
6. Release of nascent polypeptide during polyribosome dissociation under anaerobiosis. (a) Sucrose gradient pro6le of ribosomes isolat,ed from roots following a 15-min labeling with 160 pc of [3H]leucine. (b) Sucrose gradient profile of ribosomes as in (a), except that the ribosomes isolated were treated for 1 min with 1 pg RNase/ml. at 37T prior to layering the ribosomes onto the sucrose gradient. (c) Sucrose gradient profile of ribosomes isolated from seedlings treated as in (a), followed by 1-hr anaerobic treatment prior to isolation of the ribosomes. Optical density; - - - -, radioactivity. -, FIG.
POLYRIBOSOMES
IN
THE
SOYBEAN
243
ROOT
Time (mid
FIG. 7. Kinetics
of in. viwo [W]leucine incorporation. 140 seedlings were incubated at 30% in 30 ml. of distilled water containing 5 PC of [Wlleucine control (incubation with shaking); (13 pc/pmole) under each of the following conditions. * -O--O, incubation with shaking following a I-hr anaerobic treatment ; - - n - - - A - -, --O---C---. incubation with shaking for 15 mm followed by incubationunder anaerobic conditions ;-A e-1 A-, incubation with shaking in the presence of 2 pg cycloheximide/ml. (the same curve shows [r4C] leucine incorporation in the presence of cycloheximide following a l-hr anaerobic treatment).
followed by a slower recovery up to completion by two hours (Fig. 8(c) and (d)). The recovery accomplished during the first 30 minutes was not dependent to a large extent on RNA synthesis, inasmuch as the initial rapid reassociation was only slightly impaired by dactinomycin (Fig. S(b) and (e), Fig. 9, and Table 4). The slower reassociation up to complete recovery was dependent upon RNA synthesis, since no additional recovery occurred in the presence of dactinomycin. Figure 9 shows that the dactinomycin treatment, which did not greatly impair the initial rapid recovery of polyribosomes, inhibited RNA synthesis by more than 90% during this same interval. Earlier work (Lin et al., 1966) showed that about 70% of the newly synthesized RNA associated with ribosome preparations following a 30-minute pulse TABLET
Preservation of polyribosome-associated nascent polypeptide during anaerobiosis by cycloheximide Specitlc aativity polyribosomes
Treatment
260 rnp)
(cts/min/o.n
Control Control Control Control
+ nitrogen gas (1 hr) + cycloheximide (45 min) + cycloheximide (45 mm) + nitrogen
gas (1 hr)
of
2260 47ot 2860 2840
Seedlings were labeled for 15 min with [WI1 eucine. After the 1%min labeling period, ribosomes were prepared immediately from one group of seedlings (control). Another group was incubated under anaerobic conditions for 1 hr, another with cycloheximide for 46 mm followed by I-hr anaerobic treatment prior to isolation of ribosomes. The ribosomes were centrifuged on sucrose gradients, collected and counted. Data are expressed as average cts/min/o.n. at 260 rnp for the polyribosome region of the gradient. $+Xnce no polyribosomes persisted after 1 hr in nitrogen gas, the specific activity is for the monoribosome region of the gradient (see Fig. 6).
244
C. Y.
LIN
AND
Volume
J. L.
collected
KEY
(ml.)
of ribosomes into the polyribosome structure following the anaerobic FIG. 8. Reassociation treatment. All seedlings were grown under anaerobic conditions prior to the following treatments and isolation of the ribosomes: (a) no aeration; (b) 30 min aeration; (c) 60 min aeration; (d) 120 min aeration; (e) 30 min aeration (plus a l-hr treatment in 20 pg dactinomycin/ml. prior to the anaerobic treatment and during the 30-min aeration period); and (f) 120 min aeration (the dactinomycin treatment was the same as in (e)).
with radioactive precursor was the DNA-like RNA which appears to be messenger RNA and about 30% was ribosomal RNA. Even though there appears to be some loss of RNA essential to polyribosome formation (presumably messenger RNA) during the anaerobic treatment (Fig. 8, Table 3), the rapid loss of polyribosomes cannot be attributed to a signikant loss of messenger RNA during the treatment. Even though the initial ribosome profIles following reassociation (Fig. 8) are similar to the normal control preparations, the data do not rule out the possibility of reassociation of ribosomes with fragments of messenger RNA. The latter seems unlikely, however, in view of the parallel recovery of protein synthetic activity. The relative rates of total protein synthesis during polyribosome recovery (Fig. 8) subsequent to the anaerobic treatment are shown in Fig. 7. The restoration of the normal rate of protein synthesis coincided with the appearance of a normal complement of polyribosomes.
4. Discussion Polyribosomes of the soybean root rapidly dissociate under energy-depleting conditions, specifically anaerobiosis and dinitrophenol treatment, consistent with
POLYRIBOSOMES
20
IN
30
THE
0 Tube no.
40
SOYBEAN
IO
(a)
245
ROOT
20
30
40
(b)
FIG. 9. Associationof newly synthesized RNAwithribosomesduring recovery from the anaerobic treatment. Seedlings were grown for 1 hr under anaerobiosis followed by 30.min aeration with shaking. During the 30-min aeration period, seedlings were labeled with 250 PC each of [3H]adenosine and [3H]uridine: (a) without dactinomycin and (b) with 20 pg dactinomycin/ml. (the daotinomycin was added 1 hr prior to the I-br anaerobic treatment and during the 30-min labeling period. -, Optical density; - - - -, radioactivity.
the observations of Marks et al. (1965) and more recently Felicetti, Colombo & Baghoni (1966), on the dissociation of reticulocyte ribosomes following fluoride treatment. Recent results (Ravel, Mosteller & Hardesty, 1966) indicate that the loss of polyribosomes associated with fluoride treatment in the retioulocyte system may relate to the inhibition of messenger attachment and binding of aminoacyl sRNA to ribosomes in addition to a possible effect on energy metabolism (Marks et al., 1965). The loss of polyribosomes under energy-depleting conditions cannot be attributed to the depletion of messenger RNA caused by degradation of messenger and/or lack of new messenger synthesis. Treatment with dactinomycin, which resulted in greater than 90% inhibition of RNA synthesis, caused only a slow loss of polyribosomes TABLET Influence of incubation time under anaerobic conditions on the initial reassociattion.of polyyribosomes Anaerobiosis time (hr) 0 1 2 4
rapid
Polyribosomes (%I 72 63 55 44
Seedlings were gram under anaerobic conditions for the indicated times. The recovery of polyribosomes was allowed to proceed for a 30-min period following transfer of seedlings to an aerated medium. The data are expressed as percentage of total ribosomes present as polyribosomes and calculated as in Table 1.
C. Y.
246
LIN
AND TABLE
Reassociation
J. L.
KEY
4
in vivo of rnonoribosomes into polyribosomes following
the
anaerobic treatment
Time (min)
15 30 60 120
The percentage polyribosomes length of the aerobic treatment
Controls 48 58 63 -
Percentage polyribosomes Control$ Dactinomycin$ -
-
62 69 73
54 62
was calculated as shown in Table 1. The times represent following the 1-hr anaerobic treatment.
the
7 The postmitochondrial supernatant fraction was layered directly onto the linear 10 to 34% sucrose gradients (i.e. the ribosomes were not centrifuged through 1.6 M-SUCIOSB prior to gradient fractionation). 3 Ribosomes prepared as described in Materials and Methods (i.e. ribosomes were centrifuged through 1.6 M-SUCrOSe prior to gradient fractionation). The dactinomycin treatment is described in Pig. 8.
relative to anaerobiosis. Following the anaerobic treatment, reassociation of ribosomes into the polyribosome structure, to about 75% of the original level, occurred in the absence of RNA synthesis, consistent with the results of Marks et al. (1965). The in viva conversion of polyribosomes to monoribosomes in the soybean root under anaerobiosis was dependent upon protein synthesis as judged by the inhibition by cycloheximide, and did not appear to occur as a single-event dissociation as proposed by Marks et al. (1965) for the dissociation of reticulocyte polyribosomes under energy-deficient conditions. The loss of polyribosomes during anaerobiosis was completely blocked by cycloheximide (under conditions in which cycloheximide alone inbibited protein synthesis by about 95%), in agreement with the recent results of Felicetti et al. (1966). Lowering the temperature from 30 to 5°C during anaerobiosis greatly reduced polyribosome dissociation. The pulse-labeled polypeptide associated with the polyribosome structure (nascent protein) was released from the ribosome during or coincident with the dissociation of polyribosomes under anaerobiosis. Cycloheximide prevented the release of nascent polypeptide during anaerobiosis. Thus the release of nascent polypeptide during the anaerobic treatment which was dependent upon protein synthesis strongly indicates that the dissociation of polyribosomes occurred as a result of the release of ribosome and protein from the messenger-ribosome-protein complex as protein chains were completed. With the dynamio equilibrium of attachment and detachment of the monoribosome to polyribosomes as new protein chains are initiated and released (Hardesty, Hutton, Arlinghause & Schweet, 1963; Williamson & Schweet, 1964,1965), the complete loss of polyribosomes under energy-depleting conditions (presumably dependent upon chain completion and release) implies that the attachment of ribosomes to messenger RNA and peptide chain initiation were severely impaired prior to cessation of peptide bond synthesis. An early aberration in the attachment and initiation mechanism prior to cessation of the energy-dependent peptide bond formation, or a structural modification within the cell placing a physical limitation on the attachment mechanism, would be compatible with the observed results. The attachment
POLYRIBOSOMES
INTHE
SOYBEAN
ROOT
247
of monoribosome to messenger RNA and polypeptide chain initiation are apparently a rate-limiting step in protein synthesis (Williamson & Schweet, 1964,1965), and the complexity of initiation is only now being understood (Adams & Capecchi, 1966; Webster, Engelhardt & Zinder, 1966; Clark & Marcker, 1966). Villa-Trevino et al. (1964) postulated that the attachment process may require a special mechanism which is separable from the read-out process, and emphasized the decisive role that the attachment mechanism might have in metabolic control, a view supported by other workers (Sidransky, Staehelin & Verney, 1964; Trankatellis, Montjar & Axelrod, 196&Q). We thank Mrs Linda Karpyak for excellent technical assistance and 1Dr Arthur Aronson for helpful discussions during the investigation and preparation of the manuscript. This work was supported by a contract from the U.S. Atomic Energy Commission, COO-1377-5. REFERENCES Adams, J. M. & Capecchi, M. R. (1966). Proc. Nut. Acad. Xci., Wash. 55, 147. Clark, B. F. 6. 8z Marcker, K. A. (1966). J. Mol. BioZ. 17, 394. Felicetti, L., Colombo, B. & Baglioni, C. (1966). Biochim. biophys. Acta, 119, 120. Hardesty, B., Hutton, J., Arlinghause, R. & Sohweet, R. (1963). Proc. Nat. Acad. Sci., Wash. 50, 924. Ingle, J. & Key, J. L. (1965). Plant Physiol. 40, 1212. Ingle, J., Key, J. L. & Holm, R. E. (1965). J. Mol. Biol. 11, 730. Key, J. L. (1964). Plant Physiol. 39, 365. Lin, C. Y., Key, J. L. & Bracker, C. E. (1966). Plant Physiol. 41, 976. Mans, R. J. & Novelli, G. D. (1961). Arch. Biochem. Biophys. 94, 48. Mans, R. J. & Novelli, G. D. (1964). Biochim. biophys. Acta, 80, 127. Marks, P. A., Burka, E. R., Conconi, F. M., Perl, W. & Rifkind, R. A. (1965). Proc. Kc& Acad. Sci., Wash. 53, 1437. Ravel, J. M., Mosteller, R. D. & Hardesty, B. (1966). Proc. Nat. Acad. Sci., Wash. 56, 701. Sidransky, H., Staehelin, T. & Verney, E. (1964). Science, 146, 766. Trankatellis, A. C., Montjar, M. & Axelrod, A. E. (1965a). Biochemistry, 4, 1678. Trankatellis, A. C., Montjar, M. & Axelrod, A. E. (19653). Biochemistry, 4, 2065. Villa-Trevino, L., Farber, E., Staehelin, T., Wettstein, F. 0. & Noll, H. (1964). J. BioZ. Chem. 239, 3826. Webster, W. D., Engelhardt, D. & Zinder, N. (1966). Proc. Nat. Acad. Sci., Wash. 55, 155. Wettstein, F. O., Staehelin, L. & Nell. H. (1963). Nature, 197, 430. Williams, 6. & Novelli, G. D. (1964). Biochem. Biophys. Res. Comm. 17, 23. Williamson, A. R. & Schweet, R. (1964). Nature, 202, 435. Williamson, A. R. & Schweet, R. (1965). J. Mol. BioZ. 11, 358.
Dissociation and Reassembly of Polyribosomes in Relation to Protein Synthesis in the Soybean Root C.Y.Lnv
AND JOE L. KEY
Department of Botany and P.&t Pathology and Department of Biology Purdue University, Lafayette, Indiana 47907, U.X.A. (Received 7 November 1966, and in revisedform 21 February 1967) Polyribosomes of the soybean root rapidly dissociate under energy-depleting conditions, namely anaerobiosis and dinitrophenol treatment. The loss of polyribosomes cannot be attributed to a depletion of messenger RNA, because polyribosomes re-form to about 75% of the original level following restoration of aerobic conditior24 in the presence of dactinomycin. The daotinomycin treatment resulted in greater than 90% inhibition of RNA synthesis during the aerobic recovery period. Nascent polypeptide is lost from the ribosomes coincident with or during the conversion of polyribosomes to monoribosomes during anaerobiosis. Cycloheximide blocks both the conversion of polyribosomes to monoribosomes and the loss of nascent polypeptide from ribosomes. The results suggest that polyribosome dissociation occurs as a result of completion of read-out and release of polypeptide and monoribosome, coupled with a failure of attachment of ribosomes to messenger RNA and subsequent initiation of new peptide chains.
1. Introduction During an investigation of the association of DNA-like RNA, which in a previous study was shown to have characteristics in common with messenger RNA (Ingle, Key & Helm, 1965), with ribosomes in the soybean root (Lin, Key & Bracker, 1966),
we observed that culture conditions which tended to be less aerobic than normal caused a decrease in the proportion of ribosomes in the polyribosome complex. Marks, Burka, Conconi, Per1 & Rifkind (1965) found that treatment of reticulocytes with fluoride caused a rapid shift of polyribosomes to monoribosomes ; and they concluded that there is a requirement for energy, possibly ATP or GTP, for maintenance of the polyribosome structure. Because of the rapid dissociation of polyribosomes and the pattern of dissociation, it was concluded that the process occurred as a single event. Williamson & Schweet (1964J965) presented evidence supporting the view of a dynamic equilibrium of attachment and detachment of ribosomes from polyribosomes during protein synthesis. They suggested that the rate of attachment is the rate-limiting step in protein synthesis. Villa-Trevino, Faber, Staehelin, Wettstein & No11 (1964) postulated that attachment requires a special mechanism which is separable from the read-out process, and emphasized a possible decisive role of the attachment
mechanism
in metabolic
control.
Based on the above observations, our experiments were designed to elucidate the mechanism of the loss of polyribosomes under energy-deficient conditions. The results 237
238
C. Y.
LIN
AND
J. L.
KEY
indicate that the dissociation of polyribosomes results from completion of read-out with release of polypeptide and ribosomes in association with failure of attachment and initiation of new protein synthesis. This interpretation emphasizes that the attachment mechanism may have an important role in metabolic control (VillaTrevino et al., 1964).
2. Materials and Methods Soybean seeds were germinated in rolls of moist paper (Ingle & Key, 1965). The various treatments were carried out at 30°C by placing seedlings after 60 to 66 hr germination in bundles of 50 to 60 and immersing the roots 1 to 2 cm in a solution, the composition of which varied with the particular treatment. Anaerobiosis was maintained by bubbling nitrogen gas through the incubation medium. The apical (tip) &mm sections of the soybean roots were cut onto dry ice and ground in dry ice by mortar and pestle. The powder was gently homogenized in a loose-fitting ground-glass conical hand homogenizer (usually two strokes with 8 to 10 quarter turns each) in a 0.25 M-sucrose solution containing 0.05 m-Tris buffer (pH 7*4), 0.015 M-KC& and 0.02 M-&$& (Lin et al., 1966). The homogenate was filtered through Mira cloth, and the filtrate was centrifuged at 13,500 g for 15 min. Ribosomes were prepared from the supernataut solution by layering the sample over successive layers of 0.5 and 1.6 Msucrose (the sucrose solutions contained 0.05 M-Tris buffer, O-015 ~-Kc1 and 0.005 MMgCl,) followed by centrifugation at 105,000 g for 4 hr (Wettstein, Staehelin 8r Noll, 1963). The ribosome pellets were suspended in 0.05 m-Tris buffer (pH 7.4) containing 0.015 ~-Kc1 and 0.005 M-Mgcl,. In one set of experiments, the 13,500 g supernatant fraction was layered directly onto a linear sucrose gradient and centrifuged (see below). All steps were carried out at 0 to 4°C. The ribosome preparations were layered onto linear 10 to 34% sucrose gradients and centrifuged at 23,000 rev./min in the SW25 rotor for 2 hr or used directly in amino acid-incorporating studies, following the methods of Mans & Novelli (1961,1964) and Williams & Novelli (1964). The distribution of ribosomes in sucrose gradients was determined by collection with continuous recording in an ISCO model D density-gradient fractionator (collection being from the top of the tubes). Collection was made at a syringe speed at 3+ ml./min with the O.D. profile being made at a wavelength of 254 ml*. When ribosomes were isolated from tissue which had been labeled with [3H]- or [14C]leucine lo-drop fractions were collected, and the O.D. was or [3H]adenosine and [3H]uridine, at 260 mp. The samples were precipitated with trichloroacetic acid, collected measured on nitrocellulose membrane fdters (type B-6 Schleicher & Scheull), and counted in a liquid-scintillation spectrometer. Total protein syntheis by the tissue was measured by following [lgC]leucine incorporation (Key, 1964). The mild homogenizing conditions necessary to allow isolation of polyribosomes from the soybean root did not result in quantitative rupture of the cells and thus in quantitative recovery of the ribosomes. Accordingly quantitative extraction of ribosomes was made, and the results showed that none of the treatments used in Fig. 1 resulted in a significant change in the ribosome population of the tissue over the treatment interval. There was a small preferential loss of monomer ribosomes under the conditions used routinely to isolate the ribosomes (see Table 4). Also, the amount of ribosomes placed on the sucrose gradients was selected, based on preliminary experiments, to give a monomer population which was within scale sensitivity of the recorder, thus accounting for the different total amounts of ribosomes on the different gradients. Radioactive amino acids and nucleosides were obtained from Schwarz BioResearch. Dactinomycin was a gift from Merck, Sharp & Dohme. Puromycin was obtained from Nutritional Biochemicals and cycloheximide from the Sigma Chemical Company.
3. Results The distribution grown
under
different
on sucrose gradients of ribosomes prepared from soybean roots culture
conditions
is shown
in Fig.
1. There
was
a lower
pro-
POLYRIBOSOMES
IN
THE
SOYBEAN
ROOT
239
1 I’ -I-
J -1 (1
23
Volume FIG.
1. ?hmwsc gradient
distributions
collected
of ribosomes
c
(ml.) isolated
from the soybean soot.
Ribosomos were isolated from the epicad 5-B cm of the root of 120 soybean seedlings treated in the Eolluwjng mauner for 1 hr at 30°C. (a) Normal culture method (Inglo & Key, 1965); (b) incubated in water with shaking; (e) incubated in w&or without shaking; (d) incubated in water with continuous nitrogen gas bubbling; (e) incubated in IO-“~~-dinitrophenol with shaking; and (f) incubated in 20 pg dactinomycin/ml. with shaking.
.Time (mid FTC. 2. Kinetics of [14C]leucine ticorporation by isolated ribosomes. Preparations (a) to (f) correspond to those shown ia Fig. 1. Tho incorporation medium consisted of 50 pnoles Tris (pH 7.4), 5 pmoles lUgCl,, 8 pmolcs KCl, 0.5 pmole ATP, 0.15 ,umole GTP, 6.4 poles phosphoenolpyruvate, 0.05 mg of pyruvic kin&se, corn supernataut (O-5 to 1-O mg protein), O-2 ml. of ribosomes (06 to 1-O mg protein) and O-5 PC [14C]leucine (240 rc/pmole) in a final volume of O-5 ml. at 37%. Incorporation is shown as cts/min/mg ribosomal RN-4.
240
C. Y.
LIN
AND
J. L.
KEY
portion of polyribosomes relative to monoribosomes in roots which were grown in solution culture with shaking (Fig. l(b)) and without shaking (Fig. l(c)) compared to the control conditions (Fig. l(a)). This loss ofpolyribosomes under the less aerobic conditions suggested that a decreased energy supply might be involved. Accordingly, seedlings were incubated for one hour in solution culture with continuous bubbling of nitrogen gas through the medium (anaerobic treatment). There was almost complete loss of polyribosomes under this condition (Fig. l(d)). Treatment of the roots for one hour with dinitrophenol likewise resulted in complete loss of polyribosomes (Fig. l(e)). The proportion of polyribosomes was only slightly lowered by a two-hour treatment of 20 pg dactinomycin/ml. (Fig. l(f)) which inhibited RNA synthesis by about 90% (see also Fig. 9). Polyribosomes were dissociated during anaerobiosis in the presence of dactinomycin. The in vitro amino acid-incorporating activity of the ribosome preparations shown in Fig. 1 is presented in Fig. 2. The level of incorporating activity corresponded closely to the relative amount of polyribosomes in the various preparations. The poly U-dependent incorporation of phenylalanine (Fig. 3) shows that the ribosomes remain “biologically” active after dissociation under anaerobic conditions.
Time (min)
FIG. 3. Kinetics
of [14C]phenylalanine
incorporation
with
polyuridylic
acid as t,emplate.
The conditions used for incorporation of [W]phenylalanine were as for [14C]leucine incorporation except that 10 pmoles of MgCI, and 80 pg of poly U were used. --e-e-, Ribosomes corresponding to Fig. l(b); -A-.--.--A---, ribosomes corresponding to Fig. l(f); -- 0 --0 --, ribosomes corresponding to Fig. l(d). Incorporation is shown as cts/min/mg ribosomal RNA.
Following transfer of the seedlings to the anaerobic condition, there was a rapid loss of polyribosomes (Fig. 4(a) to (d)) corresponding with a rapid loss in protein synthetic activity (Fig. 7). This dissociation of polyribosomes during anaerobiosis was almost completely blocked by cycloheximide (Fig. 4 (e) to (h) compared with (a) to (d)). Under normal conditions, cycloheximide inhibited protein synthesis by about 95% (Fig. 7) without altering the level of polyribosomes (Table 1). Lowering the temperature from 30 to 5°C during the anaerobic treatment greatly decreased the rate of dissociation of polyribosomes (Fig. 5). These observations suggested that protein synthesis possibly was required to accomplish polyribosome dissociation during anaerobiosis. If completion of read-out and release of finished protein were associated with the loss of polyribosomes, the monomer ribosomes following dissocia-
POLYRIBOSOMES
IN
THE
Volume
FIG. 4. Kinetics
ROOT
241
2
23 0
0
SOYBEAN
of polyribosome
collected
dissociation
(ml.)
during anaerobiosis.
Ribosomes were isolated from the root tips of 120 seedlings at the following times after initiation of the anaerobic treatment: (a) and (e) 15 min; (b) and (f) 30 mm; (c) and (g) 60 min; (d) and (h) 120 min. Samples (e), (f), (g) and (h) were isolakd from seedlings treated with 2pg/ml. cycloheximide 45 min prior to and during the anaerobic treatment.
TABLE
Inhibition
by cycloheximide
1
of puromycin-induced
Treatment
None Puromycin (1 X 10m4M) Cycloheximide (2 pg/ml.) Puromycin + cycloheximide
decrease in polyribosomes Polyribosomes (%I 75 58 75 73
Ribosomes were isolated from seedlings which had been incubated for 1 hr as indicated. Data show the percentage of total ribosomes present as polyribosomes on sucrose gradients. The percentage of polyribosomes was calculated from the area under the curves of sucrose gradient profiles.
242
C. Y.
LIN
AND
V&me
J. L.
KEY
collected(ml.) (bl
(a)
FIG. 5. Influence of temperature on dissociation Ribosomes were isolated from seedlings after
of polyribosomes a l-hr anaerobic
during the anaerobic treatment. treatment: (a) at 36’C; (b) at
5%.
tion would contain much less “nascent” polypeptide than polyribosomes actively engaged in protein synthesis. The nascent polypeptide of polyribosomes was removed from the ribosomes in vivo during or coincident with the dissociation of polyribosomes under anaerobiosis (Fig. 6(a) and (c)). In contrast, there was no loss of nascent polypeptide associated with the conversion of prelabeled polyribosomes to monoribosomes by an in vitro RNase treatment of the isolated polyribosomes (Fig. 6(a) and (b)). Table 2 shows that the loss of nascent protein from polyribosomes under anaerobiosis was blocked by cycloheximide just as was the dissociation of the polyribosomal structure. Cycloheximide also stabilized polyribosomes in the presence of puromycin (Table 1). The reassociation of ribosomes into the polyribosomal structure after prior dissolution under anaerobic conditions is shown in Fig. 8 and Table 4. There was rapid reassociation during the initial 15 to 30 minutes (Fig. S(a) and (b) and Table 4),
0 Tube no.
(a)
(b)
Cc)
6. Release of nascent polypeptide during polyribosome dissociation under anaerobiosis. (a) Sucrose gradient pro6le of ribosomes isolat,ed from roots following a 15-min labeling with 160 pc of [3H]leucine. (b) Sucrose gradient profile of ribosomes as in (a), except that the ribosomes isolated were treated for 1 min with 1 pg RNase/ml. at 37T prior to layering the ribosomes onto the sucrose gradient. (c) Sucrose gradient profile of ribosomes isolated from seedlings treated as in (a), followed by 1-hr anaerobic treatment prior to isolation of the ribosomes. Optical density; - - - -, radioactivity. -, FIG.
POLYRIBOSOMES
IN
THE
SOYBEAN
243
ROOT
Time (mid
FIG. 7. Kinetics
of in. viwo [W]leucine incorporation. 140 seedlings were incubated at 30% in 30 ml. of distilled water containing 5 PC of [Wlleucine control (incubation with shaking); (13 pc/pmole) under each of the following conditions. * -O--O, incubation with shaking following a I-hr anaerobic treatment ; - - n - - - A - -, --O---C---. incubation with shaking for 15 mm followed by incubationunder anaerobic conditions ;-A e-1 A-, incubation with shaking in the presence of 2 pg cycloheximide/ml. (the same curve shows [r4C] leucine incorporation in the presence of cycloheximide following a l-hr anaerobic treatment).
followed by a slower recovery up to completion by two hours (Fig. 8(c) and (d)). The recovery accomplished during the first 30 minutes was not dependent to a large extent on RNA synthesis, inasmuch as the initial rapid reassociation was only slightly impaired by dactinomycin (Fig. S(b) and (e), Fig. 9, and Table 4). The slower reassociation up to complete recovery was dependent upon RNA synthesis, since no additional recovery occurred in the presence of dactinomycin. Figure 9 shows that the dactinomycin treatment, which did not greatly impair the initial rapid recovery of polyribosomes, inhibited RNA synthesis by more than 90% during this same interval. Earlier work (Lin et al., 1966) showed that about 70% of the newly synthesized RNA associated with ribosome preparations following a 30-minute pulse TABLET
Preservation of polyribosome-associated nascent polypeptide during anaerobiosis by cycloheximide Specitlc aativity polyribosomes
Treatment
260 rnp)
(cts/min/o.n
Control Control Control Control
+ nitrogen gas (1 hr) + cycloheximide (45 min) + cycloheximide (45 mm) + nitrogen
gas (1 hr)
of
2260 47ot 2860 2840
Seedlings were labeled for 15 min with [WI1 eucine. After the 1%min labeling period, ribosomes were prepared immediately from one group of seedlings (control). Another group was incubated under anaerobic conditions for 1 hr, another with cycloheximide for 46 mm followed by I-hr anaerobic treatment prior to isolation of ribosomes. The ribosomes were centrifuged on sucrose gradients, collected and counted. Data are expressed as average cts/min/o.n. at 260 rnp for the polyribosome region of the gradient. $+Xnce no polyribosomes persisted after 1 hr in nitrogen gas, the specific activity is for the monoribosome region of the gradient (see Fig. 6).
244
C. Y.
LIN
AND
Volume
J. L.
collected
KEY
(ml.)
of ribosomes into the polyribosome structure following the anaerobic FIG. 8. Reassociation treatment. All seedlings were grown under anaerobic conditions prior to the following treatments and isolation of the ribosomes: (a) no aeration; (b) 30 min aeration; (c) 60 min aeration; (d) 120 min aeration; (e) 30 min aeration (plus a l-hr treatment in 20 pg dactinomycin/ml. prior to the anaerobic treatment and during the 30-min aeration period); and (f) 120 min aeration (the dactinomycin treatment was the same as in (e)).
with radioactive precursor was the DNA-like RNA which appears to be messenger RNA and about 30% was ribosomal RNA. Even though there appears to be some loss of RNA essential to polyribosome formation (presumably messenger RNA) during the anaerobic treatment (Fig. 8, Table 3), the rapid loss of polyribosomes cannot be attributed to a signikant loss of messenger RNA during the treatment. Even though the initial ribosome profIles following reassociation (Fig. 8) are similar to the normal control preparations, the data do not rule out the possibility of reassociation of ribosomes with fragments of messenger RNA. The latter seems unlikely, however, in view of the parallel recovery of protein synthetic activity. The relative rates of total protein synthesis during polyribosome recovery (Fig. 8) subsequent to the anaerobic treatment are shown in Fig. 7. The restoration of the normal rate of protein synthesis coincided with the appearance of a normal complement of polyribosomes.
4. Discussion Polyribosomes of the soybean root rapidly dissociate under energy-depleting conditions, specifically anaerobiosis and dinitrophenol treatment, consistent with
POLYRIBOSOMES
20
IN
30
THE
0 Tube no.
40
SOYBEAN
IO
(a)
245
ROOT
20
30
40
(b)
FIG. 9. Associationof newly synthesized RNAwithribosomesduring recovery from the anaerobic treatment. Seedlings were grown for 1 hr under anaerobiosis followed by 30.min aeration with shaking. During the 30-min aeration period, seedlings were labeled with 250 PC each of [3H]adenosine and [3H]uridine: (a) without dactinomycin and (b) with 20 pg dactinomycin/ml. (the daotinomycin was added 1 hr prior to the I-br anaerobic treatment and during the 30-min labeling period. -, Optical density; - - - -, radioactivity.
the observations of Marks et al. (1965) and more recently Felicetti, Colombo & Baghoni (1966), on the dissociation of reticulocyte ribosomes following fluoride treatment. Recent results (Ravel, Mosteller & Hardesty, 1966) indicate that the loss of polyribosomes associated with fluoride treatment in the retioulocyte system may relate to the inhibition of messenger attachment and binding of aminoacyl sRNA to ribosomes in addition to a possible effect on energy metabolism (Marks et al., 1965). The loss of polyribosomes under energy-depleting conditions cannot be attributed to the depletion of messenger RNA caused by degradation of messenger and/or lack of new messenger synthesis. Treatment with dactinomycin, which resulted in greater than 90% inhibition of RNA synthesis, caused only a slow loss of polyribosomes TABLET Influence of incubation time under anaerobic conditions on the initial reassociattion.of polyyribosomes Anaerobiosis time (hr) 0 1 2 4
rapid
Polyribosomes (%I 72 63 55 44
Seedlings were gram under anaerobic conditions for the indicated times. The recovery of polyribosomes was allowed to proceed for a 30-min period following transfer of seedlings to an aerated medium. The data are expressed as percentage of total ribosomes present as polyribosomes and calculated as in Table 1.
C. Y.
246
LIN
AND TABLE
Reassociation
J. L.
KEY
4
in vivo of rnonoribosomes into polyribosomes following
the
anaerobic treatment
Time (min)
15 30 60 120
The percentage polyribosomes length of the aerobic treatment
Controls 48 58 63 -
Percentage polyribosomes Control$ Dactinomycin$ -
-
62 69 73
54 62
was calculated as shown in Table 1. The times represent following the 1-hr anaerobic treatment.
the
7 The postmitochondrial supernatant fraction was layered directly onto the linear 10 to 34% sucrose gradients (i.e. the ribosomes were not centrifuged through 1.6 M-SUCIOSB prior to gradient fractionation). 3 Ribosomes prepared as described in Materials and Methods (i.e. ribosomes were centrifuged through 1.6 M-SUCrOSe prior to gradient fractionation). The dactinomycin treatment is described in Pig. 8.
relative to anaerobiosis. Following the anaerobic treatment, reassociation of ribosomes into the polyribosome structure, to about 75% of the original level, occurred in the absence of RNA synthesis, consistent with the results of Marks et al. (1965). The in viva conversion of polyribosomes to monoribosomes in the soybean root under anaerobiosis was dependent upon protein synthesis as judged by the inhibition by cycloheximide, and did not appear to occur as a single-event dissociation as proposed by Marks et al. (1965) for the dissociation of reticulocyte polyribosomes under energy-deficient conditions. The loss of polyribosomes during anaerobiosis was completely blocked by cycloheximide (under conditions in which cycloheximide alone inbibited protein synthesis by about 95%), in agreement with the recent results of Felicetti et al. (1966). Lowering the temperature from 30 to 5°C during anaerobiosis greatly reduced polyribosome dissociation. The pulse-labeled polypeptide associated with the polyribosome structure (nascent protein) was released from the ribosome during or coincident with the dissociation of polyribosomes under anaerobiosis. Cycloheximide prevented the release of nascent polypeptide during anaerobiosis. Thus the release of nascent polypeptide during the anaerobic treatment which was dependent upon protein synthesis strongly indicates that the dissociation of polyribosomes occurred as a result of the release of ribosome and protein from the messenger-ribosome-protein complex as protein chains were completed. With the dynamio equilibrium of attachment and detachment of the monoribosome to polyribosomes as new protein chains are initiated and released (Hardesty, Hutton, Arlinghause & Schweet, 1963; Williamson & Schweet, 1964,1965), the complete loss of polyribosomes under energy-depleting conditions (presumably dependent upon chain completion and release) implies that the attachment of ribosomes to messenger RNA and peptide chain initiation were severely impaired prior to cessation of peptide bond synthesis. An early aberration in the attachment and initiation mechanism prior to cessation of the energy-dependent peptide bond formation, or a structural modification within the cell placing a physical limitation on the attachment mechanism, would be compatible with the observed results. The attachment
POLYRIBOSOMES
INTHE
SOYBEAN
ROOT
247
of monoribosome to messenger RNA and polypeptide chain initiation are apparently a rate-limiting step in protein synthesis (Williamson & Schweet, 1964,1965), and the complexity of initiation is only now being understood (Adams & Capecchi, 1966; Webster, Engelhardt & Zinder, 1966; Clark & Marcker, 1966). Villa-Trevino et al. (1964) postulated that the attachment process may require a special mechanism which is separable from the read-out process, and emphasized the decisive role that the attachment mechanism might have in metabolic control, a view supported by other workers (Sidransky, Staehelin & Verney, 1964; Trankatellis, Montjar & Axelrod, 196&Q). We thank Mrs Linda Karpyak for excellent technical assistance and 1Dr Arthur Aronson for helpful discussions during the investigation and preparation of the manuscript. This work was supported by a contract from the U.S. Atomic Energy Commission, COO-1377-5. REFERENCES Adams, J. M. & Capecchi, M. R. (1966). Proc. Nut. Acad. Xci., Wash. 55, 147. Clark, B. F. 6. 8z Marcker, K. A. (1966). J. Mol. BioZ. 17, 394. Felicetti, L., Colombo, B. & Baglioni, C. (1966). Biochim. biophys. Acta, 119, 120. Hardesty, B., Hutton, J., Arlinghause, R. & Sohweet, R. (1963). Proc. Nat. Acad. Sci., Wash. 50, 924. Ingle, J. & Key, J. L. (1965). Plant Physiol. 40, 1212. Ingle, J., Key, J. L. & Holm, R. E. (1965). J. Mol. Biol. 11, 730. Key, J. L. (1964). Plant Physiol. 39, 365. Lin, C. Y., Key, J. L. & Bracker, C. E. (1966). Plant Physiol. 41, 976. Mans, R. J. & Novelli, G. D. (1961). Arch. Biochem. Biophys. 94, 48. Mans, R. J. & Novelli, G. D. (1964). Biochim. biophys. Acta, 80, 127. Marks, P. A., Burka, E. R., Conconi, F. M., Perl, W. & Rifkind, R. A. (1965). Proc. Kc& Acad. Sci., Wash. 53, 1437. Ravel, J. M., Mosteller, R. D. & Hardesty, B. (1966). Proc. Nat. Acad. Sci., Wash. 56, 701. Sidransky, H., Staehelin, T. & Verney, E. (1964). Science, 146, 766. Trankatellis, A. C., Montjar, M. & Axelrod, A. E. (1965a). Biochemistry, 4, 1678. Trankatellis, A. C., Montjar, M. & Axelrod, A. E. (19653). Biochemistry, 4, 2065. Villa-Trevino, L., Farber, E., Staehelin, T., Wettstein, F. 0. & Noll, H. (1964). J. BioZ. Chem. 239, 3826. Webster, W. D., Engelhardt, D. & Zinder, N. (1966). Proc. Nat. Acad. Sci., Wash. 55, 155. Wettstein, F. O., Staehelin, L. & Nell. H. (1963). Nature, 197, 430. Williams, 6. & Novelli, G. D. (1964). Biochem. Biophys. Res. Comm. 17, 23. Williamson, A. R. & Schweet, R. (1964). Nature, 202, 435. Williamson, A. R. & Schweet, R. (1965). J. Mol. BioZ. 11, 358.