- Email: [email protected]
Intracellular distribution of ribosomes and polyribosomes in Bacillus megaterium
J. Mol. Biol. (1970) 52, 467-481
Intracellular
Distribution of Ribosomes and Polyribosomes in Bacillus megaterium ERIC CUNDLIFFEt
Department of Molecular Biology and Microbiology Tufts University Medical School Boston, Mass. 02111, U.S.A. (Received 30 April,
1970)
DNA-membrane complexes were prepared using the “Sarkosyl M-band” technique and were used to study the intracellular localization of ribosomes and polyribosomes in Btillua rnegaterium. Cell components are present in such Mbands by virtue of attachment, directly or indirectly, to the cell membrane. Under standard conditions M-bands formed from rapidly growing cells contain about 66% of the total polyribosomes; 26 to 30% being attached to DNA apparently ti nascent messenger RNA while at least 30 to 40 oh appear to be membranebound. When cells were incubated with actinomycin D, 60 s ribosomal subunits accumulated in the M-brand whereas 30 s subunits did not, suggesting that the cell membrane may selectively bind the larger ribosomal subunit. In other experiments the formation of polyribosomea on DNA and their subsequent release into the cytoplaem are demonstrated as is the attachment of 30 s riboeomea to nascent messenger RNA in viva.
1. Introduction Previous work in this laboratory has shown that crystals of the detergent “Sarkosyl” (sodium N-lauroyl sarcosinate) can be used to produce DNA-membrane complexes from lysates of bacteria (Tremblay, Daniels & Schaechter, 1969). The method utilizes the selective adsorption of membrane components to crystals of magnesium-sarkosyl and yields complexes which can be recovered as discrete bands (“M-bands”) in sucrose density-gradients. DNA is present in M-bands by virtue of being bound to the cell membrane and does not itself adhere to crystals of magnesiumSarkosy1. The M-band technique has been used as an assay for membrane-bound DNA in Bscheri&a co& infected by T4 phage (Earhart, Trembley, Daniels & Schaechter, 1968) and @I phage (Linial & M&my, 1970). Recently the method was used with other techniques to follow the distribution of messenger RNA in E. coli (Rouviere, Lederberg, Grrtnboulan & Gros, 1969). When the current work was started it was already known that M-bands contain ribosomes (Tremblay et al., 1969). I therefore set out to determine whether there were polyribosomes in M-bands, reasoning that, if so, at least some of them might be formed on nascent messenger RNA still bound to DNA (see Stent, 1966; Schaechter $ McQuillen, 1966). It seemed possible that the M-band technique might therefore be t Present address: Department of Ph armaeology, University Hills Road, Cambridge, CB2 3EF, England 467
of Cambridge Medical School,
468
E. CUNDLIFFE
useful in studying the attachment of ribosomes to messenger RNA in viva and for investigating the relationship between transcription and translation. In order not bo degrade polyribosomes nor to shear them from DNA during the prepara’tion of lysates, much of this work has ‘utilized protoplasts of Bacillus megat&urn, since they can be lysed simply by addition of detergent with a minimum of manipulation. As the work progressed it became necessary to modify the M-band technique somepolyribosomes bound to what; consequently it is now possible to isolate nascent DNA free from other (“mature”) polyribosomes.
2. Materials and Methods (a) Growth of bacteria
and preparation
of protoplasts
Bacillus megaterium KM was steady-state labeled with a given radioactive precursor (see text) by growth for 3 generations in protoplast medium supplemented with peptone and was converted to protoplasts using lysozyme as described elsewhere (Cundliffe, 1968a). Suspensions of protoplasts were incubated for 30 min before experiments were started, by which time the optical density (600 nm) was increasing rapidly. Protoplasts used in the current experiments were all in this state of active “growth”. In some experiments relating to Figs 3 and 4 whole cells were used and were subsequently lysed by the method of Schaechter (1963). Lysozyme (ZOO pg/ml.) was added for 30 set at 37”C, then the cells were poured onto an equal volume of frozen, crushed TMK buffer (see below). Then they were harvested by centrifugation, resuspended in a small volume of TMK buffer and lysed by addition of detergent (see text). (b) Sarn$ng
procedure
Samples of protoplast suspensions (usually O-6 ml.) were pipetted into thin-glass vials standing in an ice-bath. When many samples were to be taken, and immediate treatment of lysates was not possible, sodium azide was added to a final concentration of 10-G. The chilled protoplasts were transferred as soon as possible onto sucrose density-gradients and lysed to prepare M-bands as below. When total lysates, not involving M-bands, were required, protoplasts were sampled directly into vials containing deoxyribonuclease, Triton Xl00 and sometimes ribonuclease as previously described (Cundliffe, 1968a). Lysis was instantaneous and the use of azide was not necessary. Subsequent manipulations of all lysates were carried out in the cold. (c) Preparation
of M-bunds
Suspensions of chilled protoplasts (0.5 ml. with or without azide) were pipetted onto buffer-see below) and then sucrose density-gradients (15 to 40”/ w / v sucrose in TMK lysed by adding Sarkosyl to a final concentration of 0.2% W/V. Sarkosyl was mixed into the suspension by stirring gently but thoroughly. In some experiments (see text) RNase (10 pg/ml.) or Triton Xl00 (0.04% v / v ) was also added to the lysate at this time. The gradient plus lysate (11.6 ml. plus 0.5 ml.) was left in an ice-bath for 20 to 30 min and then centrifuged at 15,000 rev./min for 25 min at 0°C in the Spinco SW41 rotor. After centrifugation gradients were separated into 2 fractions: (i) the top fraction, namely all the material found above the M-band; and (ii) the M-band, which was present as a white viscous band about one-third of the distance from the bottom of the gradient. Usually a negligible amount of radioactivity w&s found below the M-band. In some experiments these 2 fractions were removed into trichloroacetic acidat afmal concentration of 5 to 10% w/v, bovine serum albumin (100 pg/tube) was added as carrier and precipitates were collected on glass-fiber filters, dried and counted by liquid-scintillation as previously described (Cundliffe, 1968a). (d) Re-fractionation and analysis of M-bands In experiments where the ribosome content of M-bands was assayed, the M-band removed into DNaae (10 pg total), sodium deoxycholate (1 y/o w/v final concentration)
was and
INTRACELLULAR
DISTRIBUTION
OF
POLYRIBOSOMES
469
sometimes RNase (10 pg total). This treatment degrades DNA and removes Sarkosyl crystals. Two vol. of cold TMK buffer were then added and a portion of the lysate was analyzed by sucrose gradient centrifugation (see below). (e) Sucrose
dewity-grc&Ws
All the sucrose gradients used here were linear gradients of 15 to 40% w/v sucrose in TMK buffer. TMK is 10 mM-Tris, 5 mM-magnesium acetate, 100 mM-potassium chloride, adjusted with HCl to pH 7.6 at 20 “C. The volume of the gradients was 11.6 ml. Total lysates or lysates prepared from M-bands were centrifuged in the Spinco SW41 rotor at 40,000 rev./mm for 210 min at 0°C. Gradients were fractionated by dripping into trichloroacetic acid and fractions were filtered and their radioactivity estimated as describotl elsewhere (Cundliffe, 196%). (f) Preparation
of ribosomal subunits
Cells were steady-state labeled with [32P]phosphate and incubated for 30 mm with actinomycin D (10 pg/ml.) Then they were harvested, resuspended in TMK buffer and lysed using lysozyme and Triton Xl00 (as above). Ribosomal subunits were then separated on sucrose gradients using the Spinco SW41 rotor at 40,000 rev./min as above. Gradient fractions containing ribosomal subunits were pooled and dialyzed overnight against TMK buffer. When portions were then re-run on sucrose gradients, 85% of the radioactivity of the 50 s preparation and 93% of that of the 30 s preparation sedimented as single peaks with the expected sedimentation properties. These preparations were used in the experiment described in Table 4. (g) Chemicals The following radiochemicals were obtained from New England Nuclear Corp., Boston, Mass. ; 32P-labeled phosphoric acid, [5-3H]uridine, [methyl-14C]thymidine and ]2-3H]glycerol. Bottromycin and actinomycin D were gifts from Merck & Co., Inc., Rahway, N.J.
3. Results (a) Preliminary experiments In order to obtain M-bands containing all the DNA it is necessary to lyse a given sample of protoplasts directly on a sucrose gradient and not, for example, in a separate vial. This creates obvious difficulties when experiments involve rapid sampling. Therefore controls were carried out in which protoplasts were chilled (but not lysed) in ice-cold glass vials containing sodium azide (see Materials and Methods) and later transferred to gradients and lysed. It was found that 10m2M-azide does not interfere with the M-band procedure nor does it alter the distribution of ribosomes and polyribosomes between the M-band and top fractions (see Materials and Methods) when compared with lysates produced directly on gradients in the absence of azide. The presence of azide is particularly useful in some of these experiments since, under these conditions, it promptly and completely stops protein synthesis and RNA synthesis and prevents the turn-over of pulse-labeled RNA (E. Cundliffe, unpublished data). (b) M-bands contain ribosomes and polyribosomes Figure l(a) shows a typical ribosome and polyribosome profile obtained after analyzing an M-band from 32P-labeled protoplasts. Both 50 s and 30 s ribosomal subunits were present together with polyribosomes which covered a broad size distribution. From such a Figure it is not possible to estimate accurately the number of polyribosomes present since many of them are pelletted. Therefore in some of these experiments M-bands were treated with RNase before analysis (see Materials and Methods) and polyribosomes were estimated as the 70 s material present in the subsequent sucrose gradient. This is possible since RNase converts polyribosomes
470
E. CUNDLIFFE I
I
70s .
(a)
I
I
I 40-
(b)
I
I
I
70s
6-
*E 2 3c a R
Fraction
no.
2c
IC
?
C
I
I
I
1
IO
20
30
40
Fraction no.
Fm. 1. (a) and (b) Sucrose density-gradient analysis of the ribosomes and polyribosomes of M-bands. Cells were steady-state labeled with [3aP]phosphate and converted to protoplasts which were incubated in non-radioactive medium for 30 min. Samples (0.5 ml.) were taken directly onto sucrose gradients and lysed immediately using 0.2% (W/V) Sarkosyl. These lysates were left in an ice-bath for 20 min, then centrifuged (see Materials and Methods). (a) M-bands were removed into DNase + sodium deoxycholate (b) M-bands were removed into DNase + sodium deoxycholate + RNase in a similar experiment. After 15 min at O”C, 2 vol. of TMK buffer were added and portions of each preparation were analyzed on sucrose gradients (see Materials and Methods). -a----•-, sap-radioactivity; steady-state label indicates ribosomes and polyribosomes in these and subsequent Figures. (c) Sucrose gradient analysis of a polyribosome preparation exposed to Sarkosyl. 32P-labeled protoplasts were sampled into thin-glass vials containing Sarkosyl (0.2% w/v). These were stood in ice for 10 to 20 min and then sodium deoxycholate (1% w/v) was added. When the lysates clarified (usually after less than 5 min) portions were analyzed on sucrose gradients (see Materials and Methods).
quantitatively to 70 s monosomes. When an M-band similar to that used for Figure 1 (a) was treated in this way, results shown in Figure 1 (b) were obtained. By comparison with a total protoplast lysate it was calculated that 65 to 70% of the total cell polyribosomes were present in the M-band used for Figure l(b). This M-band w&s prepared using 0.2% w/v Sarkosyl.
1
95 35 40
[14C-nzsthyZ]Thymidine (DNA) [VH]Uridine (RNA) [2-3H]Glycerol (membrane)
98 65 70
94 40 70
95 25 15
of total cell content M-band prepared with 0.2% Sarkosyl (c) Sarkosyl plus (b) Sarkosyl plus (a) Sarkosyl Triton Xl00 RNese alone
Percentage
Cells were steady-state labeled with [‘Wlthymidine, [sH]uridine or [3H]glycerol either singly or in double-isotope experiments (see text) to label DNA, RNA and membrane respectively. They were then converted to protoplasts and incubated in fresh medium for 30 min. Samples were taken directly onto sucrose gradients standing in an ice-bath and lysed immediately using Sarkosyl at various concentrations as in the Table. Some lysates were dso treated with RNase (10 pg/ml.) or Triton Xl00 (0.04% v/v) at this time. After 20 min at 0°C the gradients were centrifuged, Separated into M-band 8nd top fractions and these were precipitated with trichloroacetic acid, filtered and counted as under Materials and Methods.
M-band prepared with 0.1 y0 Sarkosyl
Steadystat0 label
Aruzlysis of M-bands prepared from protoplasts steady-state labeled with various radioactive precursors
TABLE
472
E. CUNDLIFFE
Since the M-band procedure involves the complexing of magnesium ions by Sarkosyl, it was necessary to show that polyribosomes were not degraded due to removal of magnesium under these conditions. Accordingly, lysates of protoplasts rich in polyriboaomes were prepared using either Sarkosyl (05% w/v) or Triton Xl00 (O*O5o/ov/v), and were left in ice for 10 to 20 minutes. Then Mg-Sarkosyl crystals were removed by adding sodium deoxycholate and both lysates were analyzed on sucrose gradients with and without RNase. It was seen that not more than 5% of the polyribosomes were degraded after exposure to Sarkosyl. Evidently the high magnesium content of the protoplast medium (20 mM) plus the fact that much of the Sarkosyl does not crystallize (but remains in a free form) allows sufficient magnesium to escape complex formation and to stabilize polyribosomes. Figure 1(c) shows a lysate after treatment with Sarkosyl(0.2°/o w/v) followed by sodium deoxycholate treatment. About 80% of the total ribosomes present were in polyribosomes. (c) Variations in the composition of M-bands When M-bands were prepared using different amounts of Sarkosyl they were found to differ in their content of certain cell components. Cells were steady-state labeled with [14C-methyE]thymidine (to label DNA), [LL3H]uridine (RNA) or [2-3H]glycerol (membrane) either singly or in double-isotope experiments and were then converted to protoplasts. These were incubated in fresh medium for 30 minutes, then M-bands were formed in various ways as in Table 1. It was found that M-bands which contained all the DNA could contain different amounts of RNA (a rough measure of ribosomes) and membrane material. The content of ribosomes and membranes varied together in response to changing the concentration of Sarkosyl. In other experiments the polyribosome content of M-bands was also found to vary with the amount of Sarkosyl used (Table 2). These experiments used 32P-labeled protoplasts, and polyribosomes were assayed as in Figure l(b). There was a correlation between the RNA content of M-bands (Table 1) and the polyribosome content (Table 2). TABLE 2
Polyribosome content of N-bands
Conditions
Sarkosyl Sarkosyl Sarkosyl Sarkosyl Sarkosyl
O/ototal cell polyribosomes in M-band
used to prepare M-band
alone (0.1%) alone (0.2%) (0.2%) + RNase (lo pg) (0.2%) + Triton Xl00 (0.04%) (0.2%) + RNaae (10 pg) + Triton
35 65 35t
2.5 Xl00
(0.04%)
1
t As 70 8 monosomes 3aP-labeled protoplasts were sampled onto sucrose gradients and lysed there under the various conditions used in Table 1. M-bands were then prepared, removed into DNase (10 pg total), RNase (10 pg total) and sodium deoxycholate (1% w/v) and stood at 0°C for 20 min. Then they were analyzed on sucrose gradients as in Fig. l(b). As indicated in the Table, when lysates were treated with RNase before preparing M-bands, the resultant M-bands contained 70 s monosomes rather than polyribosomes whether or not the M-bands were subsequently treated with RNase.
INTRACELLULAR
DISTRIBUTION
473
OF POLYRIBOSOMES
(d) Eflects of RNase on the composition of M-bands Tables 1 and 2 also show the compositions of M-bands prepared from lysates which had been treated with RNase (10 pg/ml. see Materials and Methods). This removed neither DNA nor membrane from M-bands, whereas RNA and polyribosomes were partially removed. It should be noted that RNase degraded all the polyribosomes to ‘70 s monosomes as usual but that 35 to 40% of these monosomes were subsequently found in the M-band (Table 2). (e) Minimal
M-bands
In other experiments, Triton Xl00 (0.04% v/v final concentration) was added to protoplasts together with Sarkosyl during the preparation of M-bands (see Materials and Methods). In this case M-bands containing relatively little membrane (10 to 16% of the total) were produced but their content of DNA was unaltered (Table 1). They also contained 25 to 30% of the stable RNA and about 25% of the total polyribosomes (Table 2). Such M-bands will be referred to as “minimal M-bands” and an analysis of their properties is the principal theme of this report. (f) &rn$ermentara’ty of the effects of RNase and Triton Xl00 on the composition of M-bands The RNA and polyribosomes which are not removed from M-bands by Triton Xl00 can be removed by RNase and vice versa. Treatment of lysates with both these (a)
I
I
I
(b) .
Y-
“b x .E s; “u a R
/
I
I
50s
6-
3-
O-
I IO
I 20
I IO
I 30 Fraction
I 20
I 30
no.
FIG. 2. Effects of RNase and Triton Xl00 on the ribosomal subunits of M-bands. 3aP-labeled protoplasts were sampled directly onto sucrose density-gradients and lysed by the addition of 0.2% (w/v) Sarkosyl. At the same time RNase (10 rg total) or Triton Xl00 (0.04% v/v) was added to some of these lysates which were stood at 0°C for 20 min before centrifugation. M-bands were then prepared, treated with DNase + sodium deoxyoholate + RNase and their ribosome content assayed by morose gradient analysis as in Fig. l(b). (a) -@-a--, Control M-band; -O--O-, lysate treated with RNase before formation of the M-band. (b) -e-a---, Control; -O--O-, lysate treated with Triton Xl00 before formation of the M-band.
474
E. CUNDLIFFE
reagents gave M-bands which contained all the DNA, about 15% of the membrane but no RNA or polyribosomes (Table 2 and unpublished data). This complementarity of action is also shown in Figure 2(a) and (b). In this experiment, which was similar to that described in Table 2, M-bands were prepared after treatment of lysates with either RNase or Triton Xl00 and their content of ribosomal subunits was assayed using sucrose gradients. As compared with control M-bands (similar to that in Fig. l(b) ) RNase specifically removes 30 s ribosomes (and some polyribosomes) whereas Triton Xl00 removes 50 s ribosomes (and some polyribosomes). In other experiments not reported here in detail, cells were steady-state labeled with [‘*C]uridine and converted to protoplasts which were incubated in fresh medium and then pulse-labeled for 12 seconds with [3H]uridine. From these protoplasts regular M-bands and minimal M-bands were prepared and their specific activities 3H/14C (trichloroacetic acid-precipitable radioactivity) were calculated. That for the minimal M-bands (containing 27% of the total ‘*C-labeled RNA) was 2.5 to 3 times higher than that for the regular M-bands (which contained 60% of the total l*C-labeled RNA). (g) M-b an ds f rm p rot opla sts incubated with actiwrnycin D contain only 50 s ribosornes Cells were steady-state labeled with [32P]phosphate and converted to protoplasts which were incubated in fresh medium for 30 minutes. Then actinomycin D (lOpg/ml.) was added for a further 30 minutes. When total lysates were prepared from such protoplasts under these particular ionic conditions (see Materials and Methods) all the ribosomes were found as 50 and 30 s subunits (Cundliffe, 19686; also Mangiarotti & Schlessinger, 1966). However, M-bands prepared from these protoplasts contained only 50 s particles (Fig. 3). In this case 55% of the total 50 s ribosomes of the cell were present in an M-band prepared using O-2o/oSarkosyl. When O.5o/oSarkosyl was used about 75% of the 50 s subunits were found in the M-band. In other experiments, similar to those in Table 2 but using protoplasts which had been incubated with
Fraction
no.
Fm. 3. Ribosome content of M-bands following incubation with actinomycin D. SaP-labeled protoplaats were incubated with actinomycin D (10 pg/ml.) for 30 min, then samples were lysed and M-bands produced using 0.2% (w/v) Sarkosyl. These M-bands were treated with DNase + sodium deoxyoholatte and their ribosome content analyzed by sucrose gradient sedimentation aa in Fig. l(a).
INTRACELLULAR
DISTRIBUTION
OF
POLYRIBOSOMES
476
actinomycin D, the effects of RNase (10 pg/ml.) and Triton Xl00 (0+4% v/v) upon the composition of these M-bands were tested. It was found that RNase had no effect upon the number of 50 s subunits in the M-band, whereas Triton Xl09 quantitatively removed them. Selective retention of 50 s ribosomal subunits in the M-band was also observed when whole cells were incubated with actinomycin D and then lysed using either Sarkosyl plus lysozyme or ultrasonic disintegration in the absence of lysozyme. In these experiments lysis was carried out in TMK buffer or in protoplast medium with similar results. The presence of 50 s ribosomes in M-bands in the absence of lysozyme suggests that artifactual attachment of ribosomes to membranes by lysozyme (Patterson, Weinstein, Nixon & Gillespie, 1970) is not primarily responsible for these observations. In any case the amounts of lysozyme (when used) in these studies (150 to 200 pg/ml., followed by dilution at least IO-fold) are within the “permissible” range of Patterson et al. In a further series of experiments (not reported here) protoplasts w&e incubated with rifampicin instead of actinomycin D. Again 50 s ribosomes were selectively retained in M-bands. Table 3 summarizes data from a typical experiment in which 0.2% w/v Sarkosyl was used to prepare M-bands from protoplasts before and after incubation with actinomycin D. TABLE 3
Ribosome and polyribosome content of M-bands and total lysates before and after treatment of protoplasts with actinomycin D Total lysate o/0 total ribosomal content (8) COXltI'Ol Polyribosomes 60 s subunits 30 s subunits
80-90 5 5
(b) After
of cell
actinomycin <5 65 35
D
M-Band o/o total cell content of 8 given component (a) Control (b) After actinomycin 60 90 50
D
0 60 <6
eaP-labeled protoplasts were sampled before and after incubation with aetinomycin D (10 pg/ml.) for 30 min. At each time M-bands were prepared using 0.2% (w/v) Sarkosyl and also total lysates were prepared using DNase (10 pg/ml.) and Triton Xl00 (0.05% v/v) as under Materials and Methods. Each of these w&s then 8n8lyzed on sucrose gradients with and without RNase. For total lysates the percentage of the total cell ribosomes present as subunits or as polyribosomes (inoluding ‘70 s meteri81) w8s calculated. Also the percentage of the total cell content of 8 given component present in the M-bands was computed.
(h) Entrainment of exogenous 50s ribosomal subunits in M-bands Ribosomal subunits (prepared as in Materials and Methods) were mixed separately with either Sarkosyl alone or a suspension of non-radioactive protoplasts to which Sarkosyl was subsequently added. The protoplasts used in this experiment had been incubated with actinomycin D as in Figure 3. M-bands were then prepared and were analyzed on sucrose gradients as usual. The results are given in Table 4. Evidently 50 s subunits (but not 30 s subunits) attached to some component of the lysate and
476
E. CUNDLIFFE TABLE
4
Entrainment of exogenous 50 S subunits in M-bands Conditions
Sarkosyl Sarkosyl Sarkosyl Sarkosyl Sarkosyl
used to prepare M-band
alone + lysate + lysate f RNase + lysate + DNase + lysate + Triton Xl00
o/0 entrained
50s 5 65 65 60 0
in M-band 30s 1 5 4 3 0
sap-labeled 50 s and 30 s ribosomal subunits were prepared as under Materials and Methods. Portions of these (corresponding in number to those derived from the amounts of protoplasts usually used to make M-bands) were then mixed with Sarkosyl (0.5% w/v) on top of sucrose gradients in th presence and absence of non-radioactive protoplasts in the usual amounts. At this time some of tIi e protoplast lysates were also treated with RNase (10 pg total), DNase (20 pg total) or Triton XI00 (0.04% v/v) in addition to Sarkosyl. After 20 min at 0°C the gradients were aentrifuged and M-band and top fractions were collected, precipitated with trichloroacetio acid, filtered and counted as under Materials and Methods.
were retained in the M-band; neither subunit attached to Sarkosyl crystals alone. As shown in Table 4, neither DNase nor RNase affected the retention of 50 s subunits in the M-band but Triton Xl00 completely removed them. (i) M-bands prepared from cells during recovery from actinomycin D The object of much of this work has been to examine the usefulness of the M-band technique as a method of studying the life cycle of polyribosomes and in particular for studying nascent polyribosomes. The following experiment shows that when polyribosomes are allowed to form in a cell previously depleted of polyribosomes, they appear first in the M-band fraction and are subsequently lost from this fraction. Cells were steady-state labeled with [32P]phosphate and then incubated in fresh medium with actinomycin D (10 pg/ml.) for 30 minutes. At this time analysis of a total cell lysate revealed that all the polyribosomes of the cell were degraded to subunits, and M-bands contained only 50 s subunits (as in Fig. 3). The cells were then collected on a Millipore filter, washed thoroughly with warmmedium, thenresuspended and incubated in the absence of antibiotic. Such cells usually show a long lag before growth commences and during this time they are not directly comparable to cells in the logarithmic phase of growth. However, they do re-form polyribosomes (Fig. 4(a)) which appear predominantly in the M-band at early times (Fig. 4(b)). Later in the recovery phase the proportion of the total polyribosomes found in theM-bandislower, although the absolute amount of polyribosomal material in the M-band is greatly increased. Figure 4(b) indicates that at late times the proportion of polyribosomes in the M-band became 20 to 25% of the total, similar to that in minimal M-bands prepared from actively growing protoplasts (Table 2). (j) The attachment of 30 S ribosomal subunits to nascent messengerRNA in vivo In the following experiment, polyribosomes were broken down without preventing RNA synthesis. Under these conditions it was hoped to study the attachment of ribosomes to new messenger RNA without allowing the re-formation of complete
INTRACELLULAR
4 (a)
/
I
I I j d odg$+*
I
I 3:s \ I ii,, 'y/y S'id\ I +y%d P I 40
5 30 Fraction
477
OF POLYRIBOSOMES
I
(b)
SOS -8
a :: l-
DISTRIBUTION
1
1
-; 40-
b
*'*I.
- $i 2
50
0
-
I IO
I 20
I 30
Minutes of recovery
no.
FICA 4. The ribosomes and polyribosomes of M-bands prepared during recovery of cells from actinomycin D. Cells were steady-state labeled with [saPIphosphate and then incubated with actinomycin D (10 @/ml.) for 30 min. Then they were collected on a Millipore filter, washed with fresh medium, and incubated in the absence of drug after resuspension in fresh medium. Samples were taken at various times, lysed as described under Materials and Methods, and M-bands produced using 0.2% (w/v) Sarkosyl. These M-bands were subsequently treated with DNase + sodium deoxyoholate + RNase as in Fig. l(b) and portions rerun on sucrose gradients. (a) Cells sampled 2 min (-a----•-) and 30 min (-O--O-) after removal of actinomycin. (b) A series of samples were taken at various times after removal of actinomycin. The amount of polyribosomal material in the M-band at any time point is compared to the total polyribosomal content of the cell at that time.
polyribosomes. The experiment uses the fact that the antibiotic bottromycin stops protein synthesis (but not RNA synthesis) and “freezes” polyribosomes without inhibiting the puromycin reaction (Cundliffe & McQuillen, 1967). Subsequent addition of puromycin is followed by rapid breakdown of most of the polyribosomes to subunits (E. Cundliffe, manuscript in preparation). RNA synthesis is not greatly inhibited in this system (E. Cundliffe, unpublished data). Thus, 32P-labeled protoplasts were treated with bottromycin (100 ,ug/ml.) for two minutes followed by puromycin (100 tLglm1.) for a further ten minutes. During the 6nal30 seconds of this incubation [3H]uridine (30 c/m-mole;83 PC/ml.) was added to label nascent RNA. Samples were then chilled in azide and a minimal M-band was prepared as in Figure 2(b). The results are shown in Figure 5. This minimal M-band contained mainly 30 s ribosomal subunits apparently associated with nascent RNA. When an M-band was prepared as above and treated with RNase before analysis, the 3aP profile was unaltered but now the 30 s subunits were devoid of 3H radioactivity. This indicates that the 3H found associated with 30 s subunits in Figure 5 was not mature ribosomal RNA integrated into the subunits but was, presumably, nascent messenger RNA.
4. Discussion The aim of this work was to examine the usefulness of the M-band technique as a means of studying the biogenesis of polyribosomes and their intracellular distribution. As the work progressed it became clear that although M-bands do indeed contain
478
E. CUNDLIFFE
Fraction
no.
FIG. 6. Ribosome content of M-bands prepared after incubation of protoplasts with bottromytin and puromycin. sap-labeled protoplasts were incubated for 2 min with bottromycin (100 pg/ml.) and then puromycin (100 pg/ml.) was added for a further 9.5 min. Then [3H]uridine (30 c/m-mole; 83 @/ml.) was added for a further 30 SW before the protoplasts were chilled in azide. Minimal M-bands were then prepared using 0.2% (w/v) Sarkosyl and 0.4% (v/v) T r1‘t on Xl00 as in Fig. 2(b). The M-bands were collected, treated with DNase + sodium deoxycholate and their content of ribosomes analyzed on sucrose gradients as in Fig. l(a). -e---a-, saP radioactivity, steady-state label indicates ribosomes and polyribosomes; -O--O-, aH radioactivity indicates pulse-labeled RNA.
DNA-bound polyribosomes, they also contain other polyribosomes and 50 s ribosomal subunits which do not appear to be bound to DNA. There are two likely reasons why polyribosomes might be found in M-bands: (i) they might be attached to membrane-bound DNA via nascent messenger RNA; or (ii) they might be bound to the membrane directly. The results presented here indicate that both these situations may exist in bacteria. One would expect that polyribosomes formed on nascent messenger RNA would readily be removed from the DNA template by small amounts of RNase and would subsequently not be found in M-bands. About 30% of the total cell polyribosomes normally found in regular M-bands are not present after treatment of lysates with RNase (Table 2). Conversely, minimal M-bands, prepared after treatment of lysates with Triton X100, contain DNA together with relatively little membrane and it is probable that polyribosomes present in these structures (about 25% of the total) are bound to DNA from which they are readily removed by RNase (Table 2). The agreement between these two estimates is suggestfive and I conclude that about 25 to 30% of the total cell polyribosomes were formed on nascent messenger RNA. Of the remaining polyribosomes 30 to 40% of the cell total were present in M-bands and could be removed by Triton Xl00 but not by RNase (the latter converts them to 70 s monosomes which remain in the M-band). These polyribosomes are clearly not bound to DNA and, since treatment with Triton removed polyribosomes and membrane from M-bands to approximately equal
INTRACELLULAR
DISTRIBUTION
OF
POLYRIBOSOMES
479
extents (Tables 1 and 2), it is suggested that these polyribosomes are membranebound. This figure (30 to 40% of the total polyribosomes) is clearly a lower limit for the number of membrane-bound polyribosomes since the M-bands used here do not contain all the cell membrane; it remains to be seen what the upper limit is. Comparable data have recently been presented by Rouviere et al. (1969). Using a different technique, involving co-sedimentation of DNA and membrane, they found about 45% of the RNA of E. COZY in centrifuged pellets. This value was reduced to about 30% following DNase treatment or to about 25% after treatment with sodium deoxycholate. Thus one can calculate that 20 to 30% of the total RNA may have been membrane-bound in their experiments, while 15 to 20% may have been associated with DNA. These results are not markedly different from those presented here. A further indication that M-bands contain ribosomes which are not attached to DNA (via nascent RNA) came when protoplasts were incubated with actinomycin D (Fig. 3) or with rifampicin. Under these conditions 50 s ribosomal subunits (at least 55 to 60% of the total) but not 30 s subunits accumulated in the M-band. Since the aim of this work was to study DNA-bound components, a technique was developed for removal of other cell components. Hence minimal M-bands were prepared and were found to be devoid of 50 s particles whether the M-bands were prepared from growing protoplasts (Fig. 2(b)) or from protoplasts incubated with antinomycin D. Again it seems likely that the 50 s subunits present in M-bands are membrane-bound since they are removed by Triton Xl00 (which removes membrane) but not by RNase. However, the physiological significance of this observation is not clear at present. As shown in Table 4, exogenously added 50 8 particles are also capable of entering M-bands. The presence of 30 s ribosomal subunits in M-bands is easier to understand. These particles are not removed by Triton Xl00 (i.e. they are still present in minimal M-bands) but they are removed by RNase (Fig. 2(a) and (b)). Apparently they are attached to nascent RNA and represent the first step in the formation of polyribodomes. Previously many authors have discussed the significance of membrane-bound ribosomes and polyribosomes in bacteria (for a review see Hendler, 1965). Several groups have suggested that membrane-bound polyribosomes are physiologically significant in B. megateriu?n (Godson, Hunter & Butler, 1961; Schlessinger, 1963; Schlessinger, Marchesi & Kwan, 1965; Yudkin & Davis, 1965). The ionic conditions influencing the binding of polyribosomes to membranes of Bacillus amylofaciens have been investigated by Coleman (1969) who reported that divalent cations (Mg2+) favor attachment (see also Schlessinger et al., 1965) whereas monovalent cations (K+) depress the binding. From Coleman’s data it seems that the conditions used here are not optimum for detection of membrane-bound polyribosomes, thus raising the possibility that the current work underestimates the extent of this binding. Aronson (1966) has related binding of ribosomes to membranes to the presence of a nascent peptide on the ribosome. This clearly does not account for the apparent binding of free 50 s particles to membranes reported here but may explain the binding of polyribosomes. If so, it would suggest that ribosomes bind to membranes by more than one mechanism. Finally, Moore & Umbreit (1965) suggested that 50 s ribosomes of Streptococcus faectzlis are membrane-bound. However, it is still not clear why some polyribosomes are bound to bacterial membranes. Thus special classes of proteins may be made exclusively by membrane-bound polyribosomes or alternatively each 31
480
E. CUNDLIFFE
polyribosome might pass through a stage of membrane-attachment (perhaps to help free it from the DNA). Another possibility is that the membrane may somehow regulate protein synthesis by sequestering free 50 s particles, quite apart from its function of binding polyribosomes. In addition to showing that polyribosomes are formed in association with DNA and subsequently released (as suggested by Stent, 1966; Schaechter & McQuillen, 1966), the experiment described in Figure 4(a) and (b) indicates that the M-band technique is selective in retaining only certain polyribosomes. This experiment rules out the possibility that either a constant number or a constant proportion of the total cell polyribosomes are artifactually entrained in M-bands. I have also demonstrated, under particular physiological conditions, the attachment of 30 a ribosomal subunits to nascent RNA (presumably messenger RNA) in viva (Fig. 5). This attachment represents the first step in the biogenesis of a polyribosome and in polypeptide chain initiation (Nomura & Lowry, 1967; Guthrie & Nomura, 1968; Ghosh & Khorana, 1967). Subsequent steps in polypeptide chain initiation involve binding of a 50 s particle to give a 70 s initiation complex, where upon the initiator aminoacyl-tRNA enters the ribosomal peptidyl donor site from which it can be removed by puromycin (Monro, Maden & Traut, 1967). Under the conditions of this experiment 70 s initiation complexes are broken down in the presence of puromycin and the over-all process is halted at the first step. To my knowledge, this is the first clear demonstration of the binding of 30 s ribosomal subunits to nascent mRNA in vivo. In conclusion, a technique has been developed for the preparation of minimal Mbands which contain DNA still bearing many of its physiological appendages. About 25% of the total cell polyribosomes are present in such structures, while other polyribosomes (30 to 40% of the total) and 50 s ribosomes appear to be bound to the membrane. This technique ought to yield further information concerning the genesis of polyribosomes on nascent messenger RNA and their subsequent “maturation” (release from DNA). This work was supported by United States Public Health Service grant Al 06103 from the National Institute of Allergy and Infectious Diseases to Moselio Schaechter, without whose insight and encouragement this work would not have been possible. I am also grateful to the Fulbright Commission for a travel grant. REFERENCES Aronson, Coleman, Cundliffe, Cundliffe, Cundliffe, Earhart,
A. I. (1986). J. Mol. Biol. 15, 606. G. (1969). BiocIxm. J. 112, 533. E. (196&z). J. Gen. Mi~robiol. 52, 425. E. (19683). Biochem. Biophya. Res. Comm. 38, 247. E. & McQuillen, K. (1967). J. Mol. Biol. 30, 137. C. F., Tremblay, G. Y., Daniels, M. J. & Schaeohter, M. (1968). Cold 8~.
Symp.
Quant.
Biol.
Harb.
38, 707.
Ghosh, H. P. & Khorana, H. G. (1967). Proc. Nat. Acad. Sci., Wash. 58,2466. Godson, G. N., Hunter, G. D. t Butler, J. A. V. (1961). Biochem. J. 81, 69. Guthrie, C. t Nomura, M. (1968). Nature, 219, 232. Hendler, R. W. (1966). Nature, 207, 1063. Linial, M. & Melamy, M. (1970). J. Viyol. 6, 72. Mangiarotti, G. & Schlessinger, D. (1966). J. Mol. BioZ. 20, 123. Monro, R. E., Maden, B. E. H. & Traut, R. R. (1967). Qetaetic Ekmente: Propdee Function. ed. by D. Shugar. New York: Academic Press Inc.
and
INTRACELLULAR
DISTRIBUTION
OF POLYRIBOSOMES
Moore, L. D. & Umbreit, W. W. (1965). Biochem. biophye. Acta, 101, 466. Nomum, M. & Lowry, C. V. (1967). Proc. Nat. Ad. Sci., Wash. 58, 946. Patterson, D., Weinstein, M., Nixon, R. & Gillespie, D. (1970). J. Bact. 101, 584. Rouvi&e, J., Lederberg, S., Grauboulan, P. & Gros. F. (1969). J. Mol. Biol. 46, 413. Schaaohter, M. (1963). J. Mol. Biol. 7, 561. Schawhter, M. & McQuillen, K. (1966). J. Mol. Biol. 22, 223. Schlessinger, D. (1963). J. Mol. BioZ. 7, 669. Schleesinger, D., Marchesi, V. T. & Kwan, B. C. K. (1965). J. Bad. 90, 456. Stat, G. S. (1966). Proc. Roy. Sot. B. 164, 81. Trembley, G. Y., Danids, M. J. BE Schaechter, M. (1969). J. MOE. BioZ. 40, 65. Yudkin, M. D. & Davis, B. D. (1965). J. Mol. Biol. 12, 193.
481
Intracellular
Distribution of Ribosomes and Polyribosomes in Bacillus megaterium ERIC CUNDLIFFEt
Department of Molecular Biology and Microbiology Tufts University Medical School Boston, Mass. 02111, U.S.A. (Received 30 April,
1970)
DNA-membrane complexes were prepared using the “Sarkosyl M-band” technique and were used to study the intracellular localization of ribosomes and polyribosomes in Btillua rnegaterium. Cell components are present in such Mbands by virtue of attachment, directly or indirectly, to the cell membrane. Under standard conditions M-bands formed from rapidly growing cells contain about 66% of the total polyribosomes; 26 to 30% being attached to DNA apparently ti nascent messenger RNA while at least 30 to 40 oh appear to be membranebound. When cells were incubated with actinomycin D, 60 s ribosomal subunits accumulated in the M-brand whereas 30 s subunits did not, suggesting that the cell membrane may selectively bind the larger ribosomal subunit. In other experiments the formation of polyribosomea on DNA and their subsequent release into the cytoplaem are demonstrated as is the attachment of 30 s riboeomea to nascent messenger RNA in viva.
1. Introduction Previous work in this laboratory has shown that crystals of the detergent “Sarkosyl” (sodium N-lauroyl sarcosinate) can be used to produce DNA-membrane complexes from lysates of bacteria (Tremblay, Daniels & Schaechter, 1969). The method utilizes the selective adsorption of membrane components to crystals of magnesium-sarkosyl and yields complexes which can be recovered as discrete bands (“M-bands”) in sucrose density-gradients. DNA is present in M-bands by virtue of being bound to the cell membrane and does not itself adhere to crystals of magnesiumSarkosy1. The M-band technique has been used as an assay for membrane-bound DNA in Bscheri&a co& infected by T4 phage (Earhart, Trembley, Daniels & Schaechter, 1968) and @I phage (Linial & M&my, 1970). Recently the method was used with other techniques to follow the distribution of messenger RNA in E. coli (Rouviere, Lederberg, Grrtnboulan & Gros, 1969). When the current work was started it was already known that M-bands contain ribosomes (Tremblay et al., 1969). I therefore set out to determine whether there were polyribosomes in M-bands, reasoning that, if so, at least some of them might be formed on nascent messenger RNA still bound to DNA (see Stent, 1966; Schaechter $ McQuillen, 1966). It seemed possible that the M-band technique might therefore be t Present address: Department of Ph armaeology, University Hills Road, Cambridge, CB2 3EF, England 467
of Cambridge Medical School,
468
E. CUNDLIFFE
useful in studying the attachment of ribosomes to messenger RNA in viva and for investigating the relationship between transcription and translation. In order not bo degrade polyribosomes nor to shear them from DNA during the prepara’tion of lysates, much of this work has ‘utilized protoplasts of Bacillus megat&urn, since they can be lysed simply by addition of detergent with a minimum of manipulation. As the work progressed it became necessary to modify the M-band technique somepolyribosomes bound to what; consequently it is now possible to isolate nascent DNA free from other (“mature”) polyribosomes.
2. Materials and Methods (a) Growth of bacteria
and preparation
of protoplasts
Bacillus megaterium KM was steady-state labeled with a given radioactive precursor (see text) by growth for 3 generations in protoplast medium supplemented with peptone and was converted to protoplasts using lysozyme as described elsewhere (Cundliffe, 1968a). Suspensions of protoplasts were incubated for 30 min before experiments were started, by which time the optical density (600 nm) was increasing rapidly. Protoplasts used in the current experiments were all in this state of active “growth”. In some experiments relating to Figs 3 and 4 whole cells were used and were subsequently lysed by the method of Schaechter (1963). Lysozyme (ZOO pg/ml.) was added for 30 set at 37”C, then the cells were poured onto an equal volume of frozen, crushed TMK buffer (see below). Then they were harvested by centrifugation, resuspended in a small volume of TMK buffer and lysed by addition of detergent (see text). (b) Sarn$ng
procedure
Samples of protoplast suspensions (usually O-6 ml.) were pipetted into thin-glass vials standing in an ice-bath. When many samples were to be taken, and immediate treatment of lysates was not possible, sodium azide was added to a final concentration of 10-G. The chilled protoplasts were transferred as soon as possible onto sucrose density-gradients and lysed to prepare M-bands as below. When total lysates, not involving M-bands, were required, protoplasts were sampled directly into vials containing deoxyribonuclease, Triton Xl00 and sometimes ribonuclease as previously described (Cundliffe, 1968a). Lysis was instantaneous and the use of azide was not necessary. Subsequent manipulations of all lysates were carried out in the cold. (c) Preparation
of M-bunds
Suspensions of chilled protoplasts (0.5 ml. with or without azide) were pipetted onto buffer-see below) and then sucrose density-gradients (15 to 40”/ w / v sucrose in TMK lysed by adding Sarkosyl to a final concentration of 0.2% W/V. Sarkosyl was mixed into the suspension by stirring gently but thoroughly. In some experiments (see text) RNase (10 pg/ml.) or Triton Xl00 (0.04% v / v ) was also added to the lysate at this time. The gradient plus lysate (11.6 ml. plus 0.5 ml.) was left in an ice-bath for 20 to 30 min and then centrifuged at 15,000 rev./min for 25 min at 0°C in the Spinco SW41 rotor. After centrifugation gradients were separated into 2 fractions: (i) the top fraction, namely all the material found above the M-band; and (ii) the M-band, which was present as a white viscous band about one-third of the distance from the bottom of the gradient. Usually a negligible amount of radioactivity w&s found below the M-band. In some experiments these 2 fractions were removed into trichloroacetic acidat afmal concentration of 5 to 10% w/v, bovine serum albumin (100 pg/tube) was added as carrier and precipitates were collected on glass-fiber filters, dried and counted by liquid-scintillation as previously described (Cundliffe, 1968a). (d) Re-fractionation and analysis of M-bands In experiments where the ribosome content of M-bands was assayed, the M-band removed into DNaae (10 pg total), sodium deoxycholate (1 y/o w/v final concentration)
was and
INTRACELLULAR
DISTRIBUTION
OF
POLYRIBOSOMES
469
sometimes RNase (10 pg total). This treatment degrades DNA and removes Sarkosyl crystals. Two vol. of cold TMK buffer were then added and a portion of the lysate was analyzed by sucrose gradient centrifugation (see below). (e) Sucrose
dewity-grc&Ws
All the sucrose gradients used here were linear gradients of 15 to 40% w/v sucrose in TMK buffer. TMK is 10 mM-Tris, 5 mM-magnesium acetate, 100 mM-potassium chloride, adjusted with HCl to pH 7.6 at 20 “C. The volume of the gradients was 11.6 ml. Total lysates or lysates prepared from M-bands were centrifuged in the Spinco SW41 rotor at 40,000 rev./mm for 210 min at 0°C. Gradients were fractionated by dripping into trichloroacetic acid and fractions were filtered and their radioactivity estimated as describotl elsewhere (Cundliffe, 196%). (f) Preparation
of ribosomal subunits
Cells were steady-state labeled with [32P]phosphate and incubated for 30 mm with actinomycin D (10 pg/ml.) Then they were harvested, resuspended in TMK buffer and lysed using lysozyme and Triton Xl00 (as above). Ribosomal subunits were then separated on sucrose gradients using the Spinco SW41 rotor at 40,000 rev./min as above. Gradient fractions containing ribosomal subunits were pooled and dialyzed overnight against TMK buffer. When portions were then re-run on sucrose gradients, 85% of the radioactivity of the 50 s preparation and 93% of that of the 30 s preparation sedimented as single peaks with the expected sedimentation properties. These preparations were used in the experiment described in Table 4. (g) Chemicals The following radiochemicals were obtained from New England Nuclear Corp., Boston, Mass. ; 32P-labeled phosphoric acid, [5-3H]uridine, [methyl-14C]thymidine and ]2-3H]glycerol. Bottromycin and actinomycin D were gifts from Merck & Co., Inc., Rahway, N.J.
3. Results (a) Preliminary experiments In order to obtain M-bands containing all the DNA it is necessary to lyse a given sample of protoplasts directly on a sucrose gradient and not, for example, in a separate vial. This creates obvious difficulties when experiments involve rapid sampling. Therefore controls were carried out in which protoplasts were chilled (but not lysed) in ice-cold glass vials containing sodium azide (see Materials and Methods) and later transferred to gradients and lysed. It was found that 10m2M-azide does not interfere with the M-band procedure nor does it alter the distribution of ribosomes and polyribosomes between the M-band and top fractions (see Materials and Methods) when compared with lysates produced directly on gradients in the absence of azide. The presence of azide is particularly useful in some of these experiments since, under these conditions, it promptly and completely stops protein synthesis and RNA synthesis and prevents the turn-over of pulse-labeled RNA (E. Cundliffe, unpublished data). (b) M-bands contain ribosomes and polyribosomes Figure l(a) shows a typical ribosome and polyribosome profile obtained after analyzing an M-band from 32P-labeled protoplasts. Both 50 s and 30 s ribosomal subunits were present together with polyribosomes which covered a broad size distribution. From such a Figure it is not possible to estimate accurately the number of polyribosomes present since many of them are pelletted. Therefore in some of these experiments M-bands were treated with RNase before analysis (see Materials and Methods) and polyribosomes were estimated as the 70 s material present in the subsequent sucrose gradient. This is possible since RNase converts polyribosomes
470
E. CUNDLIFFE I
I
70s .
(a)
I
I
I 40-
(b)
I
I
I
70s
6-
*E 2 3c a R
Fraction
no.
2c
IC
?
C
I
I
I
1
IO
20
30
40
Fraction no.
Fm. 1. (a) and (b) Sucrose density-gradient analysis of the ribosomes and polyribosomes of M-bands. Cells were steady-state labeled with [3aP]phosphate and converted to protoplasts which were incubated in non-radioactive medium for 30 min. Samples (0.5 ml.) were taken directly onto sucrose gradients and lysed immediately using 0.2% (W/V) Sarkosyl. These lysates were left in an ice-bath for 20 min, then centrifuged (see Materials and Methods). (a) M-bands were removed into DNase + sodium deoxycholate (b) M-bands were removed into DNase + sodium deoxycholate + RNase in a similar experiment. After 15 min at O”C, 2 vol. of TMK buffer were added and portions of each preparation were analyzed on sucrose gradients (see Materials and Methods). -a----•-, sap-radioactivity; steady-state label indicates ribosomes and polyribosomes in these and subsequent Figures. (c) Sucrose gradient analysis of a polyribosome preparation exposed to Sarkosyl. 32P-labeled protoplasts were sampled into thin-glass vials containing Sarkosyl (0.2% w/v). These were stood in ice for 10 to 20 min and then sodium deoxycholate (1% w/v) was added. When the lysates clarified (usually after less than 5 min) portions were analyzed on sucrose gradients (see Materials and Methods).
quantitatively to 70 s monosomes. When an M-band similar to that used for Figure 1 (a) was treated in this way, results shown in Figure 1 (b) were obtained. By comparison with a total protoplast lysate it was calculated that 65 to 70% of the total cell polyribosomes were present in the M-band used for Figure l(b). This M-band w&s prepared using 0.2% w/v Sarkosyl.
1
95 35 40
[14C-nzsthyZ]Thymidine (DNA) [VH]Uridine (RNA) [2-3H]Glycerol (membrane)
98 65 70
94 40 70
95 25 15
of total cell content M-band prepared with 0.2% Sarkosyl (c) Sarkosyl plus (b) Sarkosyl plus (a) Sarkosyl Triton Xl00 RNese alone
Percentage
Cells were steady-state labeled with [‘Wlthymidine, [sH]uridine or [3H]glycerol either singly or in double-isotope experiments (see text) to label DNA, RNA and membrane respectively. They were then converted to protoplasts and incubated in fresh medium for 30 min. Samples were taken directly onto sucrose gradients standing in an ice-bath and lysed immediately using Sarkosyl at various concentrations as in the Table. Some lysates were dso treated with RNase (10 pg/ml.) or Triton Xl00 (0.04% v/v) at this time. After 20 min at 0°C the gradients were centrifuged, Separated into M-band 8nd top fractions and these were precipitated with trichloroacetic acid, filtered and counted as under Materials and Methods.
M-band prepared with 0.1 y0 Sarkosyl
Steadystat0 label
Aruzlysis of M-bands prepared from protoplasts steady-state labeled with various radioactive precursors
TABLE
472
E. CUNDLIFFE
Since the M-band procedure involves the complexing of magnesium ions by Sarkosyl, it was necessary to show that polyribosomes were not degraded due to removal of magnesium under these conditions. Accordingly, lysates of protoplasts rich in polyriboaomes were prepared using either Sarkosyl (05% w/v) or Triton Xl00 (O*O5o/ov/v), and were left in ice for 10 to 20 minutes. Then Mg-Sarkosyl crystals were removed by adding sodium deoxycholate and both lysates were analyzed on sucrose gradients with and without RNase. It was seen that not more than 5% of the polyribosomes were degraded after exposure to Sarkosyl. Evidently the high magnesium content of the protoplast medium (20 mM) plus the fact that much of the Sarkosyl does not crystallize (but remains in a free form) allows sufficient magnesium to escape complex formation and to stabilize polyribosomes. Figure 1(c) shows a lysate after treatment with Sarkosyl(0.2°/o w/v) followed by sodium deoxycholate treatment. About 80% of the total ribosomes present were in polyribosomes. (c) Variations in the composition of M-bands When M-bands were prepared using different amounts of Sarkosyl they were found to differ in their content of certain cell components. Cells were steady-state labeled with [14C-methyE]thymidine (to label DNA), [LL3H]uridine (RNA) or [2-3H]glycerol (membrane) either singly or in double-isotope experiments and were then converted to protoplasts. These were incubated in fresh medium for 30 minutes, then M-bands were formed in various ways as in Table 1. It was found that M-bands which contained all the DNA could contain different amounts of RNA (a rough measure of ribosomes) and membrane material. The content of ribosomes and membranes varied together in response to changing the concentration of Sarkosyl. In other experiments the polyribosome content of M-bands was also found to vary with the amount of Sarkosyl used (Table 2). These experiments used 32P-labeled protoplasts, and polyribosomes were assayed as in Figure l(b). There was a correlation between the RNA content of M-bands (Table 1) and the polyribosome content (Table 2). TABLE 2
Polyribosome content of N-bands
Conditions
Sarkosyl Sarkosyl Sarkosyl Sarkosyl Sarkosyl
O/ototal cell polyribosomes in M-band
used to prepare M-band
alone (0.1%) alone (0.2%) (0.2%) + RNase (lo pg) (0.2%) + Triton Xl00 (0.04%) (0.2%) + RNaae (10 pg) + Triton
35 65 35t
2.5 Xl00
(0.04%)
1
t As 70 8 monosomes 3aP-labeled protoplasts were sampled onto sucrose gradients and lysed there under the various conditions used in Table 1. M-bands were then prepared, removed into DNase (10 pg total), RNase (10 pg total) and sodium deoxycholate (1% w/v) and stood at 0°C for 20 min. Then they were analyzed on sucrose gradients as in Fig. l(b). As indicated in the Table, when lysates were treated with RNase before preparing M-bands, the resultant M-bands contained 70 s monosomes rather than polyribosomes whether or not the M-bands were subsequently treated with RNase.
INTRACELLULAR
DISTRIBUTION
473
OF POLYRIBOSOMES
(d) Eflects of RNase on the composition of M-bands Tables 1 and 2 also show the compositions of M-bands prepared from lysates which had been treated with RNase (10 pg/ml. see Materials and Methods). This removed neither DNA nor membrane from M-bands, whereas RNA and polyribosomes were partially removed. It should be noted that RNase degraded all the polyribosomes to ‘70 s monosomes as usual but that 35 to 40% of these monosomes were subsequently found in the M-band (Table 2). (e) Minimal
M-bands
In other experiments, Triton Xl00 (0.04% v/v final concentration) was added to protoplasts together with Sarkosyl during the preparation of M-bands (see Materials and Methods). In this case M-bands containing relatively little membrane (10 to 16% of the total) were produced but their content of DNA was unaltered (Table 1). They also contained 25 to 30% of the stable RNA and about 25% of the total polyribosomes (Table 2). Such M-bands will be referred to as “minimal M-bands” and an analysis of their properties is the principal theme of this report. (f) &rn$ermentara’ty of the effects of RNase and Triton Xl00 on the composition of M-bands The RNA and polyribosomes which are not removed from M-bands by Triton Xl00 can be removed by RNase and vice versa. Treatment of lysates with both these (a)
I
I
I
(b) .
Y-
“b x .E s; “u a R
/
I
I
50s
6-
3-
O-
I IO
I 20
I IO
I 30 Fraction
I 20
I 30
no.
FIG. 2. Effects of RNase and Triton Xl00 on the ribosomal subunits of M-bands. 3aP-labeled protoplasts were sampled directly onto sucrose density-gradients and lysed by the addition of 0.2% (w/v) Sarkosyl. At the same time RNase (10 rg total) or Triton Xl00 (0.04% v/v) was added to some of these lysates which were stood at 0°C for 20 min before centrifugation. M-bands were then prepared, treated with DNase + sodium deoxyoholate + RNase and their ribosome content assayed by morose gradient analysis as in Fig. l(b). (a) -@-a--, Control M-band; -O--O-, lysate treated with RNase before formation of the M-band. (b) -e-a---, Control; -O--O-, lysate treated with Triton Xl00 before formation of the M-band.
474
E. CUNDLIFFE
reagents gave M-bands which contained all the DNA, about 15% of the membrane but no RNA or polyribosomes (Table 2 and unpublished data). This complementarity of action is also shown in Figure 2(a) and (b). In this experiment, which was similar to that described in Table 2, M-bands were prepared after treatment of lysates with either RNase or Triton Xl00 and their content of ribosomal subunits was assayed using sucrose gradients. As compared with control M-bands (similar to that in Fig. l(b) ) RNase specifically removes 30 s ribosomes (and some polyribosomes) whereas Triton Xl00 removes 50 s ribosomes (and some polyribosomes). In other experiments not reported here in detail, cells were steady-state labeled with [‘*C]uridine and converted to protoplasts which were incubated in fresh medium and then pulse-labeled for 12 seconds with [3H]uridine. From these protoplasts regular M-bands and minimal M-bands were prepared and their specific activities 3H/14C (trichloroacetic acid-precipitable radioactivity) were calculated. That for the minimal M-bands (containing 27% of the total ‘*C-labeled RNA) was 2.5 to 3 times higher than that for the regular M-bands (which contained 60% of the total l*C-labeled RNA). (g) M-b an ds f rm p rot opla sts incubated with actiwrnycin D contain only 50 s ribosornes Cells were steady-state labeled with [32P]phosphate and converted to protoplasts which were incubated in fresh medium for 30 minutes. Then actinomycin D (lOpg/ml.) was added for a further 30 minutes. When total lysates were prepared from such protoplasts under these particular ionic conditions (see Materials and Methods) all the ribosomes were found as 50 and 30 s subunits (Cundliffe, 19686; also Mangiarotti & Schlessinger, 1966). However, M-bands prepared from these protoplasts contained only 50 s particles (Fig. 3). In this case 55% of the total 50 s ribosomes of the cell were present in an M-band prepared using O-2o/oSarkosyl. When O.5o/oSarkosyl was used about 75% of the 50 s subunits were found in the M-band. In other experiments, similar to those in Table 2 but using protoplasts which had been incubated with
Fraction
no.
Fm. 3. Ribosome content of M-bands following incubation with actinomycin D. SaP-labeled protoplaats were incubated with actinomycin D (10 pg/ml.) for 30 min, then samples were lysed and M-bands produced using 0.2% (w/v) Sarkosyl. These M-bands were treated with DNase + sodium deoxyoholatte and their ribosome content analyzed by sucrose gradient sedimentation aa in Fig. l(a).
INTRACELLULAR
DISTRIBUTION
OF
POLYRIBOSOMES
476
actinomycin D, the effects of RNase (10 pg/ml.) and Triton Xl00 (0+4% v/v) upon the composition of these M-bands were tested. It was found that RNase had no effect upon the number of 50 s subunits in the M-band, whereas Triton Xl09 quantitatively removed them. Selective retention of 50 s ribosomal subunits in the M-band was also observed when whole cells were incubated with actinomycin D and then lysed using either Sarkosyl plus lysozyme or ultrasonic disintegration in the absence of lysozyme. In these experiments lysis was carried out in TMK buffer or in protoplast medium with similar results. The presence of 50 s ribosomes in M-bands in the absence of lysozyme suggests that artifactual attachment of ribosomes to membranes by lysozyme (Patterson, Weinstein, Nixon & Gillespie, 1970) is not primarily responsible for these observations. In any case the amounts of lysozyme (when used) in these studies (150 to 200 pg/ml., followed by dilution at least IO-fold) are within the “permissible” range of Patterson et al. In a further series of experiments (not reported here) protoplasts w&e incubated with rifampicin instead of actinomycin D. Again 50 s ribosomes were selectively retained in M-bands. Table 3 summarizes data from a typical experiment in which 0.2% w/v Sarkosyl was used to prepare M-bands from protoplasts before and after incubation with actinomycin D. TABLE 3
Ribosome and polyribosome content of M-bands and total lysates before and after treatment of protoplasts with actinomycin D Total lysate o/0 total ribosomal content (8) COXltI'Ol Polyribosomes 60 s subunits 30 s subunits
80-90 5 5
(b) After
of cell
actinomycin <5 65 35
D
M-Band o/o total cell content of 8 given component (a) Control (b) After actinomycin 60 90 50
D
0 60 <6
eaP-labeled protoplasts were sampled before and after incubation with aetinomycin D (10 pg/ml.) for 30 min. At each time M-bands were prepared using 0.2% (w/v) Sarkosyl and also total lysates were prepared using DNase (10 pg/ml.) and Triton Xl00 (0.05% v/v) as under Materials and Methods. Each of these w&s then 8n8lyzed on sucrose gradients with and without RNase. For total lysates the percentage of the total cell ribosomes present as subunits or as polyribosomes (inoluding ‘70 s meteri81) w8s calculated. Also the percentage of the total cell content of 8 given component present in the M-bands was computed.
(h) Entrainment of exogenous 50s ribosomal subunits in M-bands Ribosomal subunits (prepared as in Materials and Methods) were mixed separately with either Sarkosyl alone or a suspension of non-radioactive protoplasts to which Sarkosyl was subsequently added. The protoplasts used in this experiment had been incubated with actinomycin D as in Figure 3. M-bands were then prepared and were analyzed on sucrose gradients as usual. The results are given in Table 4. Evidently 50 s subunits (but not 30 s subunits) attached to some component of the lysate and
476
E. CUNDLIFFE TABLE
4
Entrainment of exogenous 50 S subunits in M-bands Conditions
Sarkosyl Sarkosyl Sarkosyl Sarkosyl Sarkosyl
used to prepare M-band
alone + lysate + lysate f RNase + lysate + DNase + lysate + Triton Xl00
o/0 entrained
50s 5 65 65 60 0
in M-band 30s 1 5 4 3 0
sap-labeled 50 s and 30 s ribosomal subunits were prepared as under Materials and Methods. Portions of these (corresponding in number to those derived from the amounts of protoplasts usually used to make M-bands) were then mixed with Sarkosyl (0.5% w/v) on top of sucrose gradients in th presence and absence of non-radioactive protoplasts in the usual amounts. At this time some of tIi e protoplast lysates were also treated with RNase (10 pg total), DNase (20 pg total) or Triton XI00 (0.04% v/v) in addition to Sarkosyl. After 20 min at 0°C the gradients were aentrifuged and M-band and top fractions were collected, precipitated with trichloroacetio acid, filtered and counted as under Materials and Methods.
were retained in the M-band; neither subunit attached to Sarkosyl crystals alone. As shown in Table 4, neither DNase nor RNase affected the retention of 50 s subunits in the M-band but Triton Xl00 completely removed them. (i) M-bands prepared from cells during recovery from actinomycin D The object of much of this work has been to examine the usefulness of the M-band technique as a method of studying the life cycle of polyribosomes and in particular for studying nascent polyribosomes. The following experiment shows that when polyribosomes are allowed to form in a cell previously depleted of polyribosomes, they appear first in the M-band fraction and are subsequently lost from this fraction. Cells were steady-state labeled with [32P]phosphate and then incubated in fresh medium with actinomycin D (10 pg/ml.) for 30 minutes. At this time analysis of a total cell lysate revealed that all the polyribosomes of the cell were degraded to subunits, and M-bands contained only 50 s subunits (as in Fig. 3). The cells were then collected on a Millipore filter, washed thoroughly with warmmedium, thenresuspended and incubated in the absence of antibiotic. Such cells usually show a long lag before growth commences and during this time they are not directly comparable to cells in the logarithmic phase of growth. However, they do re-form polyribosomes (Fig. 4(a)) which appear predominantly in the M-band at early times (Fig. 4(b)). Later in the recovery phase the proportion of the total polyribosomes found in theM-bandislower, although the absolute amount of polyribosomal material in the M-band is greatly increased. Figure 4(b) indicates that at late times the proportion of polyribosomes in the M-band became 20 to 25% of the total, similar to that in minimal M-bands prepared from actively growing protoplasts (Table 2). (j) The attachment of 30 S ribosomal subunits to nascent messengerRNA in vivo In the following experiment, polyribosomes were broken down without preventing RNA synthesis. Under these conditions it was hoped to study the attachment of ribosomes to new messenger RNA without allowing the re-formation of complete
INTRACELLULAR
4 (a)
/
I
I I j d odg$+*
I
I 3:s \ I ii,, 'y/y S'id\ I +y%d P I 40
5 30 Fraction
477
OF POLYRIBOSOMES
I
(b)
SOS -8
a :: l-
DISTRIBUTION
1
1
-; 40-
b
*'*I.
- $i 2
50
0
-
I IO
I 20
I 30
Minutes of recovery
no.
FICA 4. The ribosomes and polyribosomes of M-bands prepared during recovery of cells from actinomycin D. Cells were steady-state labeled with [saPIphosphate and then incubated with actinomycin D (10 @/ml.) for 30 min. Then they were collected on a Millipore filter, washed with fresh medium, and incubated in the absence of drug after resuspension in fresh medium. Samples were taken at various times, lysed as described under Materials and Methods, and M-bands produced using 0.2% (w/v) Sarkosyl. These M-bands were subsequently treated with DNase + sodium deoxyoholate + RNase as in Fig. l(b) and portions rerun on sucrose gradients. (a) Cells sampled 2 min (-a----•-) and 30 min (-O--O-) after removal of actinomycin. (b) A series of samples were taken at various times after removal of actinomycin. The amount of polyribosomal material in the M-band at any time point is compared to the total polyribosomal content of the cell at that time.
polyribosomes. The experiment uses the fact that the antibiotic bottromycin stops protein synthesis (but not RNA synthesis) and “freezes” polyribosomes without inhibiting the puromycin reaction (Cundliffe & McQuillen, 1967). Subsequent addition of puromycin is followed by rapid breakdown of most of the polyribosomes to subunits (E. Cundliffe, manuscript in preparation). RNA synthesis is not greatly inhibited in this system (E. Cundliffe, unpublished data). Thus, 32P-labeled protoplasts were treated with bottromycin (100 ,ug/ml.) for two minutes followed by puromycin (100 tLglm1.) for a further ten minutes. During the 6nal30 seconds of this incubation [3H]uridine (30 c/m-mole;83 PC/ml.) was added to label nascent RNA. Samples were then chilled in azide and a minimal M-band was prepared as in Figure 2(b). The results are shown in Figure 5. This minimal M-band contained mainly 30 s ribosomal subunits apparently associated with nascent RNA. When an M-band was prepared as above and treated with RNase before analysis, the 3aP profile was unaltered but now the 30 s subunits were devoid of 3H radioactivity. This indicates that the 3H found associated with 30 s subunits in Figure 5 was not mature ribosomal RNA integrated into the subunits but was, presumably, nascent messenger RNA.
4. Discussion The aim of this work was to examine the usefulness of the M-band technique as a means of studying the biogenesis of polyribosomes and their intracellular distribution. As the work progressed it became clear that although M-bands do indeed contain
478
E. CUNDLIFFE
Fraction
no.
FIG. 6. Ribosome content of M-bands prepared after incubation of protoplasts with bottromytin and puromycin. sap-labeled protoplasts were incubated for 2 min with bottromycin (100 pg/ml.) and then puromycin (100 pg/ml.) was added for a further 9.5 min. Then [3H]uridine (30 c/m-mole; 83 @/ml.) was added for a further 30 SW before the protoplasts were chilled in azide. Minimal M-bands were then prepared using 0.2% (w/v) Sarkosyl and 0.4% (v/v) T r1‘t on Xl00 as in Fig. 2(b). The M-bands were collected, treated with DNase + sodium deoxycholate and their content of ribosomes analyzed on sucrose gradients as in Fig. l(a). -e---a-, saP radioactivity, steady-state label indicates ribosomes and polyribosomes; -O--O-, aH radioactivity indicates pulse-labeled RNA.
DNA-bound polyribosomes, they also contain other polyribosomes and 50 s ribosomal subunits which do not appear to be bound to DNA. There are two likely reasons why polyribosomes might be found in M-bands: (i) they might be attached to membrane-bound DNA via nascent messenger RNA; or (ii) they might be bound to the membrane directly. The results presented here indicate that both these situations may exist in bacteria. One would expect that polyribosomes formed on nascent messenger RNA would readily be removed from the DNA template by small amounts of RNase and would subsequently not be found in M-bands. About 30% of the total cell polyribosomes normally found in regular M-bands are not present after treatment of lysates with RNase (Table 2). Conversely, minimal M-bands, prepared after treatment of lysates with Triton X100, contain DNA together with relatively little membrane and it is probable that polyribosomes present in these structures (about 25% of the total) are bound to DNA from which they are readily removed by RNase (Table 2). The agreement between these two estimates is suggestfive and I conclude that about 25 to 30% of the total cell polyribosomes were formed on nascent messenger RNA. Of the remaining polyribosomes 30 to 40% of the cell total were present in M-bands and could be removed by Triton Xl00 but not by RNase (the latter converts them to 70 s monosomes which remain in the M-band). These polyribosomes are clearly not bound to DNA and, since treatment with Triton removed polyribosomes and membrane from M-bands to approximately equal
INTRACELLULAR
DISTRIBUTION
OF
POLYRIBOSOMES
479
extents (Tables 1 and 2), it is suggested that these polyribosomes are membranebound. This figure (30 to 40% of the total polyribosomes) is clearly a lower limit for the number of membrane-bound polyribosomes since the M-bands used here do not contain all the cell membrane; it remains to be seen what the upper limit is. Comparable data have recently been presented by Rouviere et al. (1969). Using a different technique, involving co-sedimentation of DNA and membrane, they found about 45% of the RNA of E. COZY in centrifuged pellets. This value was reduced to about 30% following DNase treatment or to about 25% after treatment with sodium deoxycholate. Thus one can calculate that 20 to 30% of the total RNA may have been membrane-bound in their experiments, while 15 to 20% may have been associated with DNA. These results are not markedly different from those presented here. A further indication that M-bands contain ribosomes which are not attached to DNA (via nascent RNA) came when protoplasts were incubated with actinomycin D (Fig. 3) or with rifampicin. Under these conditions 50 s ribosomal subunits (at least 55 to 60% of the total) but not 30 s subunits accumulated in the M-band. Since the aim of this work was to study DNA-bound components, a technique was developed for removal of other cell components. Hence minimal M-bands were prepared and were found to be devoid of 50 s particles whether the M-bands were prepared from growing protoplasts (Fig. 2(b)) or from protoplasts incubated with antinomycin D. Again it seems likely that the 50 s subunits present in M-bands are membrane-bound since they are removed by Triton Xl00 (which removes membrane) but not by RNase. However, the physiological significance of this observation is not clear at present. As shown in Table 4, exogenously added 50 8 particles are also capable of entering M-bands. The presence of 30 s ribosomal subunits in M-bands is easier to understand. These particles are not removed by Triton Xl00 (i.e. they are still present in minimal M-bands) but they are removed by RNase (Fig. 2(a) and (b)). Apparently they are attached to nascent RNA and represent the first step in the formation of polyribodomes. Previously many authors have discussed the significance of membrane-bound ribosomes and polyribosomes in bacteria (for a review see Hendler, 1965). Several groups have suggested that membrane-bound polyribosomes are physiologically significant in B. megateriu?n (Godson, Hunter & Butler, 1961; Schlessinger, 1963; Schlessinger, Marchesi & Kwan, 1965; Yudkin & Davis, 1965). The ionic conditions influencing the binding of polyribosomes to membranes of Bacillus amylofaciens have been investigated by Coleman (1969) who reported that divalent cations (Mg2+) favor attachment (see also Schlessinger et al., 1965) whereas monovalent cations (K+) depress the binding. From Coleman’s data it seems that the conditions used here are not optimum for detection of membrane-bound polyribosomes, thus raising the possibility that the current work underestimates the extent of this binding. Aronson (1966) has related binding of ribosomes to membranes to the presence of a nascent peptide on the ribosome. This clearly does not account for the apparent binding of free 50 s particles to membranes reported here but may explain the binding of polyribosomes. If so, it would suggest that ribosomes bind to membranes by more than one mechanism. Finally, Moore & Umbreit (1965) suggested that 50 s ribosomes of Streptococcus faectzlis are membrane-bound. However, it is still not clear why some polyribosomes are bound to bacterial membranes. Thus special classes of proteins may be made exclusively by membrane-bound polyribosomes or alternatively each 31
480
E. CUNDLIFFE
polyribosome might pass through a stage of membrane-attachment (perhaps to help free it from the DNA). Another possibility is that the membrane may somehow regulate protein synthesis by sequestering free 50 s particles, quite apart from its function of binding polyribosomes. In addition to showing that polyribosomes are formed in association with DNA and subsequently released (as suggested by Stent, 1966; Schaechter & McQuillen, 1966), the experiment described in Figure 4(a) and (b) indicates that the M-band technique is selective in retaining only certain polyribosomes. This experiment rules out the possibility that either a constant number or a constant proportion of the total cell polyribosomes are artifactually entrained in M-bands. I have also demonstrated, under particular physiological conditions, the attachment of 30 a ribosomal subunits to nascent RNA (presumably messenger RNA) in viva (Fig. 5). This attachment represents the first step in the biogenesis of a polyribosome and in polypeptide chain initiation (Nomura & Lowry, 1967; Guthrie & Nomura, 1968; Ghosh & Khorana, 1967). Subsequent steps in polypeptide chain initiation involve binding of a 50 s particle to give a 70 s initiation complex, where upon the initiator aminoacyl-tRNA enters the ribosomal peptidyl donor site from which it can be removed by puromycin (Monro, Maden & Traut, 1967). Under the conditions of this experiment 70 s initiation complexes are broken down in the presence of puromycin and the over-all process is halted at the first step. To my knowledge, this is the first clear demonstration of the binding of 30 s ribosomal subunits to nascent mRNA in vivo. In conclusion, a technique has been developed for the preparation of minimal Mbands which contain DNA still bearing many of its physiological appendages. About 25% of the total cell polyribosomes are present in such structures, while other polyribosomes (30 to 40% of the total) and 50 s ribosomes appear to be bound to the membrane. This technique ought to yield further information concerning the genesis of polyribosomes on nascent messenger RNA and their subsequent “maturation” (release from DNA). This work was supported by United States Public Health Service grant Al 06103 from the National Institute of Allergy and Infectious Diseases to Moselio Schaechter, without whose insight and encouragement this work would not have been possible. I am also grateful to the Fulbright Commission for a travel grant. REFERENCES Aronson, Coleman, Cundliffe, Cundliffe, Cundliffe, Earhart,
A. I. (1986). J. Mol. Biol. 15, 606. G. (1969). BiocIxm. J. 112, 533. E. (196&z). J. Gen. Mi~robiol. 52, 425. E. (19683). Biochem. Biophya. Res. Comm. 38, 247. E. & McQuillen, K. (1967). J. Mol. Biol. 30, 137. C. F., Tremblay, G. Y., Daniels, M. J. & Schaeohter, M. (1968). Cold 8~.
Symp.
Quant.
Biol.
Harb.
38, 707.
Ghosh, H. P. & Khorana, H. G. (1967). Proc. Nat. Acad. Sci., Wash. 58,2466. Godson, G. N., Hunter, G. D. t Butler, J. A. V. (1961). Biochem. J. 81, 69. Guthrie, C. t Nomura, M. (1968). Nature, 219, 232. Hendler, R. W. (1966). Nature, 207, 1063. Linial, M. & Melamy, M. (1970). J. Viyol. 6, 72. Mangiarotti, G. & Schlessinger, D. (1966). J. Mol. BioZ. 20, 123. Monro, R. E., Maden, B. E. H. & Traut, R. R. (1967). Qetaetic Ekmente: Propdee Function. ed. by D. Shugar. New York: Academic Press Inc.
and
INTRACELLULAR
DISTRIBUTION
OF POLYRIBOSOMES
Moore, L. D. & Umbreit, W. W. (1965). Biochem. biophye. Acta, 101, 466. Nomum, M. & Lowry, C. V. (1967). Proc. Nat. Ad. Sci., Wash. 58, 946. Patterson, D., Weinstein, M., Nixon, R. & Gillespie, D. (1970). J. Bact. 101, 584. Rouvi&e, J., Lederberg, S., Grauboulan, P. & Gros. F. (1969). J. Mol. Biol. 46, 413. Schaaohter, M. (1963). J. Mol. Biol. 7, 561. Schawhter, M. & McQuillen, K. (1966). J. Mol. Biol. 22, 223. Schlessinger, D. (1963). J. Mol. BioZ. 7, 669. Schleesinger, D., Marchesi, V. T. & Kwan, B. C. K. (1965). J. Bad. 90, 456. Stat, G. S. (1966). Proc. Roy. Sot. B. 164, 81. Trembley, G. Y., Danids, M. J. BE Schaechter, M. (1969). J. MOE. BioZ. 40, 65. Yudkin, M. D. & Davis, B. D. (1965). J. Mol. Biol. 12, 193.
481