Translational Regulation of Polysome Formation During

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1 JOURNAL OF BACTERIOLOGY, Nov. 1979, p /79/ /08$02.00/0 Vol. 140, No. 2 Translational Regulation of Polysome Formation During Dormany of Physarum polyephalum WILLIAM R. JEFFERY Department ofzoology, University of Texas, Austin, Texas Reeived for publiation 14 August 1979 The translational ativity of atively growing miroplasmodia and dormant miroslerotia of Physarum polyephalum was investigated by analyzing the distribution of ribosomes in polysomes. Miroplasmodial post-mitohondrial frations ontained substantial amounts of polysomes and ribosomal subunits but very few native monosomes. During the starvation period whih preeded miroslerotium formation, polysome levels remained onstant, whereas the subunit titer began to inrease. During enystment ribosomal subunits ontinued to aumulate as the level of polysomes gradually dereased. Dormant miroslerotia ontained a large surplus of stored ribosomal subunits but no detetable polysomes. However, inubation of miroslerotia with onentrations of yloheximide suffiient to slow polypeptide elongation without affeting initiation aused the gradual reappearane of polysomes at the expense of the subunits. Under these onditions the perentage of subunits driven into polysomes reahed values similar to those of atively growing miroplasmodia. Miroslerotia returned to nutrient medium ontained very low levels of polysomes during the lag period whih preeded germination. These were formed with preexisting, stored messenger ribonulei aid. During the germination period, polysome levels were markedly inreased. This elevation was dependent on new ribonulei aid transription. It is onluded that dormant miroslerotia ontain funtional messenger ribonulei aid and ribosomes whih are subjet to translational repression at the level of initiation. 490 As a response to adverse environmental onditions suh as starvation, dessiation, or suboptimal temperature, the plasmodial slime mold Physarum polyephalum an undergo a form of dormnany known as slerotization (15). During the slerotization proess the plasmodial ytoplasm is partitioned into small multinuleate ysts by intemal membrane formation (31). Aggregations of these ysts, alled slerotia, are haraterized by a low metaboli rate (10) and the ability to remain viable for up to several years (15). Return to favorable environmental onditions indues the dormant slerotia to revert to atively growing plasmodia. Dramati biohemial hanges aompany the indution of slerotization by nutrient depletion (25-27). During the starvation period preeding enystment, maromoleular synthesis ontinues with biosyntheti preursors derived from the partial atabolism of DNA, RNA, and protein (27) and the utilization of plasmodial glyogen reserves (11). Although the biohemial hanges whih our during the starvation period have been extensively studied, very little is known about the metaboli ativities of the dormant slerotium. For example, the fundamental question ofwhether ysts atively engage in protein synthesis during dormany or store mrna and ribosomes for use during plasmodial regeneration is unresolved. In the present study translational ativity during the transition from ative growth to dormany and during plasmodial regeneration from dormant slerotia has been investigated by analyzing the distribution of ribosomes in polysomes. It is demonstrated that plasmodial polysomes are gradually dissoiated during the enystment periods; that funtional, but relatively inative, mrna and ribosomes are sequestered in the mature slerotium; and that polysomes are reformed with stored slerotial mrna during plasmodial regeneration. It is onluded that protein synthesis is translationally repressed at the level of initiation during dormany. MATERIALS AND METHODS Culture methods. P. polyephalum (Carolina strain) was grown as miroplasmodia at 220C in agitated suspension ulture on a rotary shaker with the medium desribed previously (7). Dormant miroslerotia were fonned from mid-exponential-phase miroplasmodial ultures by starvation in a itrate-

2 VOL. 140, 1979 buffered (ph 4.6), nonnutrient salts medium (12). Germination of dormant miroslerotia was indued by their transfer bak to the routine ulture medium. Polysome preparation. Polysomes were prepared from miroplasmodia and miroslerotia by a modified version of the method of Brewer (4). Mid-exponentialgrowth-phase miroplasmodial ultures and dormant or germinating miroslerotial ultures were harvested by entrifugation at room temperature at 100 x g for 1 min. The supernatant was deanted, and the pellet was resuspended in 5 volumes of ie-old 50 mm Trishydrohloride (ph 7.2), 100 mm KCl, 100 mm MgCl2, 200 mm surose, and 75 mm EDTA and homogenized by 15 up and down strokes of a ground glass homogenizer. Inreasing the EDTA onentration from 25 mm (4) to 75 mm was onsistently found to yield larger polysomes. The post-mitohondrial fration was prepared from the homogenate by entrifugation at 20,000 x g for 10 min at 4 C. The supernatant frations were removed from the tubes and immediately layered on density gradients or stored in liquid N2. Zone sedimentation. Zone sedimentation of the post-mitohondrial supernatant fration was arried out at 40C through 12 ml, 10 to 50% linear surose density gradients. To preserve the integrity of the polysomes, it was neessary to dissolve the surose in 50 mm Tris-hydrohloride (ph 7.2), 100 mm KCl, and 100 mm MgCl2. The gradients were entrifuged in the Bekman SW41 rotor for 90 min at 35,000 rpm. The absorbane at 254 nm was reorded by pumping the gradients through an LKB Uviord I spetrophotometri ell. Determination of ribosome distribution. The absorbane profiles obtained were traed onto paper, and the area ontributed by the absorbane of surose (derived from blank gradients). was subtrated. The regions orresponding to ribosomal subunits (RSU), native monosomes, and RNase-produed monosomes and disomes (and oasionally trisomes) (Fig. 1A and B) were ut out and weighed. The level of polysomes was obtained by subtrating the weight of the native monosomal area from that of the total monosome and disome (and oasionally trisome) areas of RNasetreated samples. The level of RSU was estimated diretly from the weight of the area below the 60S and 40S peaks. The perentage of ribosomes in polysomes was obtained by dividing the weight of the polysomal area, obtained from RNase-treated profiles as desribed above, by the weight of the total ribosomal area. Measurement of poly(a) ontent. The polyadenyli aid [poly(a)] ontent of RNA, extrated from the post-mitohondrial fration of slerotia by proedures desribed previously (1), was determined by the method of Jeffery (14). This involved bringing portions of the RNA preparation to 10 mm Tris-hydrohloride (ph 7.6), 200 mm NaCl, and 5 mm MgCl2 and mixing them with an exess of [3H]polyuridyli aid (5.14 Ci/ mmol; New England Nulear Corp., Boston, Mass.). The mixture was inubated at 25 C for 15 min to promote annealing and then brought to 10,ug/ml with panreati RNase and inubated for 1 h at 37 C. Finally the digests were hilled, and the nulei aids were preipitated with 10% old trihloroaeti aid and olleted for liquid sintillation ounting on glass fiber filters. TRANSLATIONAL REGULATION OF DORMANT PHYSARUM 491 RESULTS Charateristis of miroplasmodial polysomes. The sedimentation proffies of post-mitohondrial frations prepared from exponentially growing, vegetative miroplasmodia are shown in Fig. 1. Ribosome distribution in polysomes and lighter partiles was somewhat different than that previously reported for Physarum miroplasmodia (4, 24). The major differenes observed were an enrihment of RSU and a defiieny in single ribosomes (Fig. 1A). RNase treatment of the post-mitohondrial fration before entrifugation onverted the polysomes to lighter partiles, mainly monosomes and disomes, but did not affet the RSU levels (Fig. 1B; no effet of RNase on RSU levels was observed in 25 separate experiments). An RNaseinsensitive, UV-absorbing material previously identified as glyogen partiles (28) remained in the high-moleular-weight region of the gradient after RNase treatment. Inomplete polysome digestion by RNase resulting in aumulation of disomes has also been reported in other euaryotes (19, 21). Sine it is unusual for atively growing ells to ontain proportionately larger RSU than monosome pools (2), further experiments were designed to determine whether this result was aused by the extration onditions. For example, it is possible that the high RSU levels were due to polysome runoff without reinitiation during the sample preparation. To test this possibility, yloheximide whih slows polypeptide elongation (29) was added to the extration media and the gradients. As shown in Fig. 1C, yloheximide addition did not redue RSU levels, suggesting that they were not aused by runoff. It is also unlikely that the high RSU levels were due to the exessive amounts of EDTA and Mg2e required to inhibit endogeneous RNase ativity in Physarum (4) sine they were also present when the onentrations of these omponents in the extration media were signifiantly redued (Fig. 1D). Similar levels of RSU were also observed when polysomes were extrated with various EDTA-Mg2+ ratios inluding the 25 mm/100 mm ratio originally employed by Brewer (4). The apparent exess of RSU ould also be due to seletive binding of polysomes and monosomes to membranes or other insoluble miroplasmodial strutures. This possibility was heked by the addition of Triton X-100 and sodium deoxyholate to the extration media. As shown in Fig. 1E, no alterations in the distribution of ribosomal material between polysomes, monosomes, and RSU were subsequently found. Thus, the results suggest that a high proportion of ribosomes exist in an RSU pool in Physarum miroplasmodia. Large

3 492 JEFFERY 0.5 E C: st I.) C O 0 5 -o 0. n D0.0~ 0.5 Distone Sedimented (m) A 60S 8 -I I I I I ~~~~I - I,I I II I ~~~~~~~~~~~~~~~~~~~~~~~~I I ~~~~~I FIG. 1. Zone sedimentation of the post-mitohondral firaton from P. polyephalum miroplasmodia. (A) Poatnitohondrial frations under normal extratin onditn. The arrows represent the position of40, 608, and 808 ribosomal markers derived from Ehrlih asites tumor eus entrifuged on parallel gradients. (B) Post-mitohondrial frations treated with 10 pg of panreati RNase A (RNase) per ml for 30 mmn on ie before entrifugatin. (C) Post-mitohondrial frations prepared under onditions in whih the extration media and gradient were suppemented with 20 pg of yloheximide per ml (D) Post-mitohondrial firations prepared with extration media omposed of50 mm Tris-hydrohloride (ph 7.6, 50 mm KCl4 I mm MgCI2, and 20 mm surose. Thegradients ontained the same onentration of salts. (E) Post-mitohondrial frations prepared in the presene of 0.5% Triton X-100) and 1% sodium deoxyhokate. The sedimentation diretion is from right to left. The vertial dashed lines represent the posion in whih the absorbane sale is halved. The hathed and shaded surfaes ui A and B represent the areas from whih the levels ofpolysomes and RSU, respetively, were akulated as desribed in the text. RSU pools are also found in yeast (20) and the fungus Muor (22). Polysome dissoiation and RSU aumulation during mirowlerotium formation. Dormant miroslerotia are formed from J. BACTERIOL. starved miroplasmodial ultures asynhronously between 24 and 36 h after the shift to nonnutrient medium. The sedimentation profiles of post-mitohondrial frations derived from ultures at seleted intervals in the starvation-enystment period and from mature miroslerotia are shown in Fig. 2A through D. Sine the high-moleular-weight regions of the gradients ontain glyogen partiles as well as polysomes, indiret methods are required for the measurement of polysome titers. This was failitated by measuring the levels of monosomes and disomes whih appeared in RNase-treated post-mitohondrial frations (Fig. 2E through H). Although lower proportions of ribosomes distributed in polysomes were observed in the present study (Table 1) ompared with previous measurements (24), it is believed that the urrent estimations are more aurate sine they exlude the glyogen omponents. The hanges deteted in polysome and RSU levels during slerotization are summarized in Fig. 3. It an be seen that polysome levels remained substantial during the starvation period, dereased markedly during and after enystment, and were undetetable in miroslerotia by 72 h after the medium shift. During a similar interval, the level of RSU was inreased threefold (Fig. 3), suggesting that enysting miroslerotia anuulate and store ribosomes. The initial deline in RSU and polysomes observed after the medium shift is due to "transfer shok" and not the starvation ondition itself, sine a similar effet ourred when miroplasmodia were swithed to fresh nutrient medium. The inability to detet polysomes in mature miroslerotia ould be due to depressed translatl ativity. This observation, however, ould also be explained by polysome ompartmentalization or the presene of an ativated yst-speifi, polysome-unoupling agent in the miroslerotia. The possibility of a polysomeunoupling agent ativated during the extration proedure was tested by mixing miroslerotia and miroplasmodia before homogenization. As shown in Fig. 4A the sedimentation profiles obtained from the post-mitohondrial frations of this mixture exhibited polysomes exluding the possibility of an unoupling agent. The absene of detetable polysomes in the miroslerotia was not due to ompartmentalization either, sine they were not released after detergent treatment (Fig. 4B) or by more extensive homogenizations. Thus, it appears that mature miroslerotia are translationally inative. Indution of polysomes in dormuant miroslerotia. The lak of an essential omponent, suh as mrna, or the ative repression of

4 VOL. 140, 1979 TRANSLATIONAL REGULATION OF DORMANT PHYSARUM 493 Is A lht /1H'4his ( ff, ht,8 l) /;', hf s 130 () W) 05 ('J,q C Li) IK{ 1 _/I IV. II, I/i, abi ~ Rf/oe 6 6hs Wi. H 7- hts HAi r acy SD I Distone Sedimented (m) FIG. 2. Zone sedimentation of the post-mitohondrial frations from P. polyephalum during the transition from ative growth to dormany. (A) 1 h, (B) 24 h, (C) 36 h, and (D) 72 h after the swith to nonnutrient medium. (E to H) RNase-treated ounterparts of A to D. Other details are similar to those in the legend to Fig. 1. an otherwise ompletely funtional protein syntheti mahinery ould ause the translational defiieny seen in dormant miroslerotia. It was therefore of interest to determine whether miroslerotial RSU ould be driven into polysomes. Low levels of yloheximide redue polypeptide elongation rates without affeting initiation (30) and an be used to saturate free ytoplasmi mrna with ribosomes (18). As shown in Fig. 5, inubation of mature miroslerotia with ltg 10 of yloheximide per ml, a onentration less than that neessary for maximal inhibition of protein synthesis in Physarum (6), resulted in the gradual appearane of polysomes at the expense of RSU. After 24 h of inubation, about 40% of the ribosomal material was observed in polysomes, a value whih approximated that found in exponentially growing miroplasmodia (Table 1). The indution of polysomes by yloheximide also shows that our proedures are apable of extration of these partiles from miroplasmodia, a finding onsistent with the idea that no polysome ompartmentalization ours during dormany. The indution of polysome formation in dormant miroslerotia by yloheximide annot be aounted for by new transription. Similar perentages of RSU were driven into polysomes in miroslerotia pretreated with ordyepin or atinomyin D before yloheximide addition to inhibit RNA synthesis (Table 1). Moreover, the relatively small inrease in poly(a) titer observed in yloheximide-treated miroslerotia (Table 2), whih was probably due to drug-speifi interferene with the normal turnover of this sequene (1), is inonsistent with the possibility that net poly(a)-rna synthesis is the ause of polysome formation. These results suggest that mrna, ribosomes, and the other omponents neessary for protein synthesis are stored in the slerotia and that translation is repressed at the level of initiation during dormany. Polysome reformation during miroslerotial germination. Dormant miroslerotia an be indued to form miroplasmodia by return to nutrient medium. Exystment ours after a lag phase of 24 to 36 h. As shown in Fig. 6 and quantitated in Fig. 7, low levels of polysomes an be deteted in miroslerotia as early as 1 h after the addition of nutrient medium. Polysome formation at this time is resistant to atinomyin D (Fig. 7). The low levels of polysomes present throughout the lag phase, whih inlude only about 10 to 20% of the available ribosomes, were not due to a defiieny in mrna sine, as was observed previously in dormant miroslerotia, 40 to 50% of the ribosomes ould be driven into the polysomes by yloheximide. However, in ontrast to the situation observed earlier in miroslerotia, 50% of the ribosomes were aumulated in polysomes after only 1 h of yloheximide treatment. Thus, the rate of polysome aumulation 20 10

5 494 JEFFERY in yloheximide-treated miroslerotia appears to be signifiantly stimulated after nutrient addition. During the exystment period, polysome levels dramatially inreased at the expense of RSU (Fig. 6 and 7). At this time the proportion of ribosomes in polysomes rose to the highest levels (53% at 48 h; 65% at 58 h) enountered in this investigation. Sine this rise was onsiderably depressed by atinomyin D administered during the lag phase, it is probably dependent on new transription in the exysted miroplasmodia. DISCUSSION The primary purpose of the present investigation was to determine whether translational ativity persists during slerotization, a form of dormany experiened by plasmodia of the aellular slime mold P. polyephalum. To avoid the unertainties related to variable preursor permeability and pool size during development, this question was approahed by examining the levels of ribosomes inorporated into polysomes. The results demonstrate that, although signifiant levels of polysomes are present throughout TABLE 1. Effet of drugs on polysome levels in exponentially growing miroplasmodia and dormant mirosierotia ofp. polyephalum % Polysonme Drug treatment Miroplasmodia Miroslerotiab Untreated 37.4 ± 4.2 (6) Undetetable Cyloheximided 44.2 ± 6.6 (6) 44.2 ± 2.5 (12) Cordyepine Undetetable Cordyepin and 36.4 ± 3.1 (3) yloheimidef Afinomyin I) Undetetable Atinomyin D and 46.2 ± 4.5 (3) yloheximide athe perentage of ribosomal material in polysomes was determined as desribed in the text. b Miroslerotia dormant for 12 weeks were utilized. The data are expressed as mean perentage ± standard error. The number of experiments appears in parenthese. d The yloheximide onentration utilized was 10 ig/nml. 'The ordyepin onentration utilized was 200 ug/ ml (9). f Miroslerotia were preinubated in ordyepin or atinomyin D for 2 h before the addition of 10 ug of yloheximide per ml Cordyepin and atinomyin D were shown to inhibit the inorporation of [3Hjuridine into the add-insoluble material of 96-h-starved miroslerotia by 92 and 83%, respetively (Jeffery, unpublished data). 'The atinomyin D onentration used was 300 plg/ml (26)..: FIG. 3. Relative levels of polysomes and RSU in the post-mitohondrial frations of P. polyephalum during the transition from ative growth to dormany. Polysome and RSU levels were determined as desribed in the text. The shaded area represents the approximate duration of the enystment period. E U-) qo 0 [ A 8 J. BACTERIOL. 0 v Distone Sedimented (m) FIG. 4. Zone sedimentation ofpost-mitohondrial frations derived from (A) a mixture of miroplasmodia and miroslerotia and (B) detergent-treated miroslerotia of P. polyephalum. In A, equal volumes of miroplasmodia and miroslerotia were mixed before homogenization. In B, the extration media ontained 0.5% Triton X-100 and 1% sodium deoxyholate. Other details are similar to those in Fig. 1. :3 3 the starvation period whih preede dormany and the lag phase before germination, translational ativity is severely repressed at the level of initiation in the dormant slerotium. The persistene of substantial levels of polysomes during the starvation period and the enystment proess is onsistent with earlier radioative preursor inorporation studies, whih suggested a ontinuation of protein sythesis at moderate rates during the slerotial indution period (27). The ontrast between Physarum plasmodia and starved mammalian ells is marked in this regard. Mammalian polysomes are rapidly dissoiated after the beginning of

6 VOL. 140, 1979 '.5 E ' 05 U-, C\'I _ D 0.5 A. /3 days, SIe,o/,um B Ihr,Cy/oherimide a 6 h s It Distane Sedimented (m) FIG. 5. Polysome formation in ylohexu TRANSLATIONAL REGULATION OF DORMANT PHYSARUM 495 exponentially growing ells. Sine this situation 30 ourred in the presene of an exess of RSU, it argues for the existene of similar proportions of potentially ative mrna in the ytoplasm of 2.0 both developmental stages. The possibility that low levels of protein synthesis our in dormant slerotia is not exluded v by the absene of detetable polysomes in grav dients prepared from their lysates. Indeed, that the gradual aumulation of polysomes after, polypeptide transloation is slowed by ylohexp imide suggests that initiation is ourring at low 3 rates in the slerotia. This is also supported by the detetion of low levels of radioative amino aid inorporation into the aid-insoluble fra- 2a0 tion during dormany (Jeffery, unpublished data). The existene of depressed, but not entirely eliminated, translational ativities in dor- mant Physarum is onsistent with what has been observed during dormany in other euaryoti organisms suh as fungal spores (3), plant seeds (23), and unfertilized sea urhin eggs (13, 19) Ṅutrient addition to dormant slerotia results mide- in the appearane of polysomes within 1 h. Powa lysome formation at this time appears to be treated miroslerotia of P. polyephalum. Dor miroslerotia, ultured for 13 days after the siwith based on the utilization of preformed mrna to nonnutrient medium, were supplemented wiith 10 onserved in the slerotium, rather than newly pg of yloheximide per ml. At various interva~ Is the synthesized transripts, sine it is ompletely post-mitohondrial frations were prepared ani sub- resistant to atinomyin D. This finding is on- sistent with the results of an earlier report jeted to zone sedimentation. (A) Post-mitohondrLal fration of whih the 13-day miroslerotiun. Posthondrial frations derived from mito- showed that ultures (B) I1 atinomyin D did not affet proh (C) 6 h, and (D) 24 h after yloheximide addition. I details are similar to those in Fig. 1. tein synthesis after slerotia were returned to nutrient medium (5). Although only low levels of polysomes were observed during the long lag period whih pre- eded slerotial exystment, the proportion of starvation, releasing the mrna as a free iytoplasmi partile (17, 32). Presumably, the mo- by starvation somehow prevents polysome dlis- TABLE 2. Poly(A) ontent of normal and bilization of plasnodial autoataboli proesses soiation in Physarum until the time of eniyst- yloheximide-treated mirosierotia of P. ment. polyephaluma Although protein synthesis appears to be ar- [3H]poly(U) rested in dormant slerotia, funtional mlina, hy- PoyA RNA bridized ribosomes, and the other fators and P)ly preursors MiCroslerotiumb (A2/ pm/ pg4tg % neessary to support tranalation must be preson of MYpm/ml Am1, of of ent. This is evidened by the aumulati4 RNA RNA polysomes in yloheximide-treated sle trotia d under onditions in whih new transriptiion Control ,840 4, is bloked. The appearane of polysomes in :ylo- Cylohexiniide ,110 6, treatede heximide-treated slerotia suggests that t rans- on in Poly(A) ontent was measured by [3H]polyuridyli a lation is repressed at the level of initiatii dormant Physarum. Potentially translaltable aid ([3H]poly(U)) assoiation as desribed previously mrna, stored in the small-partile fration of (14). b mammalian ells, an also be driven into Miroslerotia dormant for 12 weeks were used. poly- somes by yloheximide treatment (8, 16). It is A26, Absorbane at 260 nm. d The poly(a) ontent was alulated from the speifi ativity of [3H]poly(U). impressive that similar levels of ribosome are eventually aumulated in the polysomes ( Df y- in heximide per ml for 24 ' Miroslerotia were inubated in 10 jig of ylo- loheximide-treated slerotia as are observred h.

7 496 JEFFERY J. BACTERIOL. II. E 'ITI.5 0> fr o5raose E /hr, F24hrs, RNose G 34hrs, Rnose H 48hrs, RNose3r0t (0 to2) RNas-treate ounteparts fa-d. ther deails ar similr thse infgure10 05~~~I Distane Sedimented (m) FIG. 6. Zone sedimentation of the post-mitohondrial fration from Pplpau uigtetasto fr-om dormany to ative growth. (A) 1 h, (B) 24 h, (C) 34 h, and (D) 48 h after the return to nutrient medium. (E to H) RNa-se-treated ounterparts ofa-d. Other details are similar to those in Figure 1. &...: I 1..- :.t: J F FIG. 7. Relative levels of polysomes and RSU in the post-mitohondrial frations of P. polyephalum during the transition from dormany to ative growth. Polysome and RSU levels were determined as desribed in the text. In some experiments, 300X g of atinomyin D per ml was added to the ultures with the nutrient medium. The shaded area represents the approximate duration of the exystment period. 3j potentially translatable mrna in the ytoplasm, as measured by the ability of yloheximide to drive ribosomes into the polysomes, remained high. It is signifiant that the rate of polysome aumulation in yloheximidetreated slerotia was muh greater after nutrient addition. These observations suggest that the rate of translational initiation is markedly enhaned, although not to its potential maximum, after nutrient addition. The biohemial nature of the proesses responsible for translational repression in dornant slerotia and its ativation after nutrient addition are urrently unknown. Sine all the neessary requirements for protein synthesis are available in slerotia, it is oneivable that their translational ativities may be limited by relative quantitative defiienies of key substanes or by ative inhibitory mehanisms. The possibilities inlude a limited titer of one or more of the initiation fators or a protein-mediated repression of stored mrna, ribosomes, or other translational elements. These possibilities are presently being investigated in this laboratory. ACKNOWLEDGMENTS The expert tehnial assistane of Mary Robinson is gratefully aknowledged. Finanial support was provided by Publi Health Servie grant GM from the National Institute of General Medial Sienes and National Siene Foundation grant PCM LITERATURE CITED 1. Adams, D. S., and W. R. Jeffery Polyadenyli aid degradation by two distint proesses in the ytoplasmi RNA of Physarum polyephalum. Biohemistry 17: Baglioni, C., C. Veso, and M. Jaobs-Lorena The role of ribosomal subunits in mammalian ells. Cold Spring Harbor Symp. Quant. Biol. 34: Brambl, R., L. D. Dunkle, and J. L. Van Etten Nulei aid and protein synthesis during fungal spore germination, p In J. E. Smigh and D. R. Berry (ed.), The filamentous fungi: developmental myology.

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