Inhibition of initiation of protein synthesis by 7-methylguanosine- 5'-monophosphate

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1 Prc. Nat. Acad. Sci. USA Vl. 73, N. 1, pp , January 1976 Bichemistry Inhibitin f initiatin f prtein synthesis by 7-methylguansine- 5'-mnphsphate (prtein synthesis/methylated nucletides/mdified 5'-termini) EILEEN D. HICKEY, LEE A. WEBER, AND CORRADO BAGLIONI* Department f Bilgical Sciences, State University f New Yrk, Albany, N.Y Cmmunicated by Alexander Rich, September 17, 1975 ABSTRACT Translatin f rabbit glbin mrna in a wheat germ prtein-synthesizing system is inhibited b7y nucletide 7-methylguansine-5'-mnphsphate (m G5'p) the but nt by ther guansine nucletides withut the 7-methyl grup r with the phsphate in a different psitin. Translatin f RNA f tbacc msaic virus and ply(a) + HeLa RNA is als inhibited by m7g51p. We shw that m7g5'p prevents the assciatin f mrna with ribsmal subunits t frm an initiatin cmplex. We prpse that m7g5'p interacts with a site n initiatin factr(s) r ribsmes which is invlved in mrna recgnitin, presumably by binding t the 5'-terminal sequence m7g51ppp. m7g5'p des nt inhibit translatin f ply(u) and RNA f satellite tbacc necrsis virus, which d nt have the 5'-terminal sequence m7g5'ppp. In the case f RNA f satellite tbacc necrsis virus, sme stimulatin f its translatin is cnsistently bserved in the presence f m7g5p; pssible interpretatins f this finding are discussed. Rttman et al. (1) have described a dinucletide at the 5'- terminus f a variety f mrnas that cnsists f 7-methylguansine linked thrugh its 5'-hydrxyl grup via a triphsphate t the 5'-hydrxyl grup f a ribsemethylated nucletide. The 5'-terminal sequence m7g5'ppp... has been reprted fr revirus (2), vesicular stmatitis (3), vaccinia (4), and silkwrm plyhedrsis virus RNAs (5) and fr rabbit glbin (6), HeLa (7), myelma (8), and L cell mrnas (6). These reprts suggest that methylated blcked 5'-termini may be a rather general feature f eukarytic mrna. Bth et al. (9) have demnstrated a specific rle fr the 7-methyl grup f the 5'-terminal guansine f revirus RNA. Viral RNA can be synthesized in vitr by virin-assciated RNA plymerase. This RNA is methylated when S- adensylmethinine is added t the incubatin mixture, but is unmethylated when S-adensylhmcysteine, an inhibitr f methylatin, is added. The methylated RNA is translated effectively and with fidelity by a wheat germ prtein-synthesizing system; whereas the unmethylated RNA is translated nly after being methylated during the incubatin in the wheat germ extract (9). Additin f S-adensylhmcysteine t the wheat germ extract prevents methylatin and ablishes translatin f unmethylated revirus RNA. Similarly, the remval f the 5'-terminal m7g5'p frm rabbit glbin mrna causes a lss f the template activity f this mrna (6). The presence f the methylated 5'-terminal nucletide seems t be specifically required fr the interactin Abbreviatins: TMV, tbacc msaic virus; STNV, satellite tbacc necrsis virus; Hepes, N-2-hydrxyethylpiperazine-N'-ethanesulfnic acid; m7g5'p, 7-methylguansine-5'-mnphsphate; m7g, 7- methylguansine; m7g2 3)'p, 7-methylguansine-2(3)'-mnphsphate; G5'p, guansine-5'-mnphsphate. * T whm reprint requests shuld be addressed. f revirus RNA with ribsmal subunits in the frmatin f initiatin cmplexes (1). A functinal rle fr the m7g5'p... nucletide sequence in mrna translatin has nt yet been directly shwn. It seems pssible, hwever, that this grup acts as a recgnitin signal fr mrna. We reprt here that this is the mst likely functin f the 5'-end f mrna by shwing that the nucletide m7g5'p specifically inhibits translatin f mrnas with the sequence m7g5'ppp..., but nt f ther RNAs that d nt have this grup at the 5'-terminus. Other analgs f guansine d nt shw inhibitry activity. MATERIALS AND METHODS 7-Methylguansine and 7-methylguansine mnphsphates were purchased frm Terra-Marine Biresearch (La Jlla, Calif.). Glbin mrna was islated by repeated sucrse gradient centrifugatin as described (11). Tbacc msaic virus (TMV) was given t us by Dr. Bryan E. Rberts, Massachusetts Institute f Technlgy. TMV RNA was prepared by phenl extractin (12). Ply(A) + RNA was prepared frm HeLa cell plysmes as described (13). Revirus RNA synthesized by virins and labeled with S-adensyl[CHs- 3H]methinine (1) was a gift f Gerald W. Bth and Aarn J. Shatkin f the Rche Institute f Mlecular Bilgy, Nutley, N.J. Satellite tbacc necrsis virus RNA was given t us by Dr. Abraham Marcus, Institute fr Cancer Research, Fx Chase, Philadelphia. Sparsmycin was a gift f Upjhn C., Kalamaz, Mich. Radiactive amin acids were purchased frm New England Nuclear. Cell-Free Prtein Synthesis. Previusly published methds were used t prepare the wheat germ extract (14). The wheat germ prtein-synthesis assay cntains in 5,l: 15 Ml f wheat germ extract, 1.2 mm ATP,.25 mm GTP, 15 mm creatine phsphate,.67 unit f creatine phsphkinase (Sigma), 22 mm N-2-hydrxyethylpiperazine-N'-ethanesulfnic acid (Hepes)/KOH ph 7.1, 3. mm Mg-acetate,.1 M KCl, 2.6 mm dithithreitl, 1.8 mm meicaptethanl, 5,uM unlabeled amin acids minus lysine r phenylalanine, and 5 MC' f [3H]lysine (38 Ci/mml) r [3H]phenylalanine (59 Ci/mml). RNA, guansine nucletides, and inhibitrs were added at the cncentratins indicated in the figure legends. The incubatins were carried ut at 24, and 5-M1 samples were taken in duplicate befre starting the incubatin and at the specified time intervals. The samples were prcessed fr determinatin f radiactivity as described (15). Binding f Revirus [3H1RNA t Initiatin Cmplexes. The binding reactins were carried ut in 5 Ml f the wheat germ cell-free system cntaining 5 MiM unlabeled amin acids,.1 mm sparsmycin, 25 cpm f revirus 19

2 Bichemistry: Hickey et al. Prc. Nat. Acad. Sci. USA 73 (1976) c8 _- = 7~ G; _ '~~~~~~~~~~~~~2(' 6 2 4 12,125.25.5 1. [mm] FIG. 1. Inhibitin f translatin f rabbit glbin mrna by m7g5'p.

2 2 Bichemistry: Hickey et al. Prc. Nat. Acad. Sci. USA 73 (1976) c8 _- = 7~ G; _ '~~~~~~~~~~~~~2(' , [mm] FIG. 1. Inhibitin f translatin f rabbit glbin mrna by m7g5'p. The incubatins were carried ut in the wheat germ cellfree system as described in Materials and Methds. Aliquts (5 /1) were sampled in duplicate after 6 min at 24. Glbin mrna was added at 13 Ag/ml in all incubatins except thse measuring endgenus incrpratin (X---- X). The cncentratin f the guansine nucletides added is indicated n the absicissa. Translatin in the presence f the nucletides is expressed as % f the untreated cntrl; the endgenus incrpratin f the cntrl was 116 cpm. The cntrl incrpratin with glbin mrna added was 37, cpm. [3H]mRNA (specific activity 8 cpm/ltg), and the indicat- Pd cncentratin f m7g5'p. After 1 min at 24 the incubatins were stpped by additin f :45 ml f 1 mm cyclheximide in.1 M KCI, 2 mm TrisHC1 (ph 7.5), and 5 mm Mg-acetate. The samples were applied t 15-3% sucrse gradients in the same buffer and centrifuged at 22, rpm in a Beckman SW-27 rtr. The A26 f the gradients was mnitred in a Gilfrd recrding spectrphtmeter, and the radiactivity f the fractins cllected was determined after additin f 12 vlumes f 1 part Tritn X-1, 2 parts.8% butyl-pbd (C.I.B.A.) in tluene, and 1% water. RESULTS The presence f a 5'-terminal m7g5'p in rabbit glbin mrna affects its translatin by wheat germ extracts (6). When the 5'-terminal m7g5'p is remved by peridate xidatin and fl-eliminatin, the mrna lses mst f its ability t stimulate prtein synthesis (6). In experiments directed at establishing a specific rle fr m7g5'p in prtein synthesis, we have translated rabbit glbin mrna in the presence f mnnucletide analgs f the 5'-terminal end f this mrna. Only m7g5'p inhibits prtein synthesis, whereas the ther cmpunds tested (m7g, m7g2(3)'p, and G5'p) shw negligible inhibitin and nly at very high cncentratins (Fig. 1). This indicates that the chemical grups essential fr 12 c -L) ca-e 2 EL u c mm m'g5'p y(a) +RNA TMV RNA in mrna K.5 FIG. 2. Inhibitin f translatin f TMV RNA and ply(a) + HeLa cell RNA by m7g5'p. The incubatins are carried ut as described in Fig. 1. TMV RNA is added at 8 gg/ml; ply(a) + RNA is added at 36,g/ml. Translatin in the presence f m7g5'p is expressed as % f the untreated cntrl; the cntrl incrpratin fr TMV RNA is 41,143 cpm, and fr ply(a) + RNA 11,96 cpm. The curve fr glbin mrna translatin is the same shwn in Fig. 1. inhibitin f glbin mrna translatin by m7g5'p are the methyl grup in psitin 7 and the phsphate in 5'. Cmpunds with the phsphate grup in a different psitin r withut the methyl grup shw n inhibitin. Endgenus prtein synthesis f the wheat germ extract is als inhibited by m7g5'p (Fig. 1). We have bserved inhibitin f translatin f ther mrnas by m7g5'p in wheat germ extracts and als in a different cell free system. Translatin f TMV RNA and f HeLa cell ply(a) + RNA in the wheat germ extract is inhibited, thugh incmpletely r at slightly'higher cncentratins f the inhibitr (Fig. 2). Ply(A) + RNA f HeLa cells has been shwn t have 5'-terminal m7g5'p (7). Prtein synthesis prgrammed by endgenus mrna in a HeLa cell extract is als inhibited by m7g5'p (t be published elsewhere). Mechanism f inhibitin f prtein synthesis by m7g51p A direct demnstratin that m7g5'p acts at the level f initiatin f prtein synthesis has been btained by studying the frmatin f initiatin cmplexes between revirus RNA labeled in the methyl grup f the 5'-terminal guansine (2) and wheat germ ribsmes (Fig. 3). The binding reactin is carried ut in the presence f sparsmycin, an inhibitr f elngatin, as suggested by Darnbrugh et al. (16). In the cntrl experiment (Fig. 3A) 8% f the labeled RNA becmes assciated with ribsmes. Hwever, the assciatin f revirus RNA with ribsmes is partially inhibited by the presence f.125 mm m7g51p (Fig. 3B) and is inhibited by Fractins Binding f revirus [3H]RNA t the 8S initiatin cmplex. The binding assay is carried ut as indicated under Materials and FIG. 3. Methds. The incubatin mixtures are analyzed by sucrse gradient centrifugatin. (A) Cntrl; (B).125 mm; and (C).5 mm m7g5'p.

E 6 61 41 Bichemistry: Hickey et al. -A / Cntrl --I 754 14 1 5.2,. q 1 Prc. Nat. Acad. Sci. USA 73 (1976) 21 rly tu) * STNV RNA 21.25mM m7g5p 25 2 5 A E Q I 1 2 Mg/ml glbin mrna 3 4 a- Glbin mrna -.

3 E Bichemistry: Hickey et al. -A / Cntrl --I ,. q 1 Prc. Nat. Acad. Sci. USA 73 (1976) 21 rly tu) * STNV RNA 21.25mM m7g5p A E Q I 1 2 Mg/ml glbin mrna 3 4 a- Glbin mrna mm m7g5p FIG. 5. Translatin f STNV RNA and f ply(u) in the presence f m7g5'p. The incubatins are carried ut as described in the legend f Fig. 1. STNV RNA is added at 2 jig/ml and, in a separate experiment, at 5,gg/ml. The values reprted as % f cntrl (4186 and 6417 cpm, respectively) are the average f the tw experiments; the endgenus incrpratin in the presence f each cncentratin f m7g5'p has been subtracted. Ply(U) is added at 2 gg/ml; the cntrl incrpratin with ply(u) is 4529 cpm f [th]phenylalanine incrprated and the endgenus incrpratin is 366 cpm. The glbin mrna translatin curve is the same shwn in Fig. 1 and is presented here fr the sake f cmparisn. gg/mi TMV RNA FIG. 4. The effect f glbin mrna (A) and TMV RNA (B) cncentratin n the inhibitin f translatin by m7g5'p. The assays are carried ut in the wheat germ cell-free system as described in the legend f Fig. 1. ver 7% by.5 mm m7g5'p (Fig. 3C). N binding f revirus RNA t 8S initiatin cmplexes ccurs in an incubatin mixture kept at (nt shwn). The RNA that des nt bind t the initiatin cmplexes sediments at the tp f the gradients because f degradatin during the incubatin. Several ther labeled mrnas have been shwn t be degraded in a 1-min incubatin (ur unpublished bservatins). This degradatin des nt affect ur results since the label in the revirus RNA that we have used is in the 5'-terminal nucletide, which is prtected nce the RNA is bund t ribsmes (1). in7g5'p has n effect n the binding f initiatr MettRNAf t the 4S ribsmal subunit (t be published elsewhere). It seems pssible that m7g5'p interferes with the binding f mrna in the frmatin f the 8S initiatin cmplex by ccupying ne site with which the 5'-terminal nucletide f mrna interacts. This implies that the 5'-terminal nucletide acts as a specific recgnitin signal during initiatin. This suggestin is supprted by the experiments f Bth et al. (9), wh have shwn that unmethylated revirus RNA is nt translated by a wheat germ extract, whereas methylated revirus RNA is efficiently translated. Messenger RNA and m7g5'p may thus cmpete fr binding t the same site with different affinity. We have calculated that the cncentratin f m7g5'p at which we bserve 5% inhibitin f glbin mrna translatin (.63 mm; Fig. 1) is at least 1-fld higher than the mlar cncentratin f mrna in the assay. We have used 22, as the mlecular weight fr glbin mrna (17) and have made the assumptin that all the RNA added t the wheat germ extract is pure and active glbin mrna. In s ding, we have presumably verestimated the cncentratin f mrna. We pstulate that m7g5'p inhibits mrna translatin by binding t a site that interacts with the 5'-terminus f mrna. We have attempted t learn mre abut this interactin by translating increasing cncentratins f mrna in the presence f a cnstant inhibitry cncentratin f m7g5'p (Fig. 4). In the case f glbin mrna we have bserved that the inhibitin is cnstant ver a 1-fld range f mrna cncentratin (Fig. 4A). This can be explained by assuming that m7g5'p inhibits an initiatin factr in a dsedependent fashin. Even in the presence f relatively high cncentratins f the inhibitr there is sme active factr that can participate in the initiatin prcess. Translatin f glbin mrna is linearly related t the amunt f mrna added under these cnditins (Fig. 4A). In the case f TMV RNA we find that the inhibitin by m7g5'p is vercme by increasing the RNA cncentratin (Fig. 4B). We prpse a pssible explanatin fr the discrepancy between the result btained with glbin mrna and that with TMV RNA. We have seen previusly that m7g5'p des nt cmpletely inhibit translatin f TMV RNA even at the highest cncentratins tested (Fig. 2). This raises the pssibility that TMV RNA is hetergeneus with respect t inhibitin f translatin by m7g5'p. The result f the experiment shwn in Fig. 4B may be explained by the additin f increasing amunts f RNA mieties that are nt inhibited by m7g5'p. Since these mlecules may nt have this methylated nucletide at the 5'-terminus (see Discussin), it becmes extremely relevant t establish whether the translatin f this type f RNA is inhibited by m7g5'p. Satellite tbacc necrsis virus (STNV) RNA is unique amng a number f mrnas in that it has a 5'-terminal se-

22 Bichemistry: Hickey et al. quence f either ppapgpup... (18, 19) r pppapgpup... (2). Furthermre, accurate translatin f STNV RNA ccurs withut the attachment f a 5'-terminal m7g5'p miety (J. Brker, S.

4 22 Bichemistry: Hickey et al. quence f either ppapgpup... (18, 19) r pppapgpup... (2). Furthermre, accurate translatin f STNV RNA ccurs withut the attachment f a 5'-terminal m7g5'p miety (J. Brker, S. N. Seal, and A. Marcus, in preparatin). It is therefre f interest t ascertain whether r nt m7g5'p has any effect n the translatin f this mrna. As shwn in Fig. 5, in cntrast t the results btained with ther mrnas, STNV RNA translatin is nt inhibited, but is instead stimulated abut 5% by relatively lw cncentratins f m7g5'p. Bth et al. (9) have shwn that unmethylated revirus RNA can be methylated by an endgenus methylating enzyme f the wheat germ and is then translated as well as methylated revirus RNA. In sme incubatins we have added.32 mm S-adensylhmcysteine, a cmpetitive inhibitr f this enzyme (9), t the wheat germ extract. At this cncentratin S-adensylhmcysteine prevents translatin f unmethylated revirus RNA (9). We have bserved n effect n the translatin f STNV RNA, TMV RNA, glbin mrna, and sea urchin egg mrna in the presence f S-adensylhmcysteine (R. Ens, L. Weber, and C. Baglini, unpublished bservatins). The translatin f ply(u) is nt inhibited by m7g5'p (Fig. 5). We have translated ply(u) under the same inic cnditins used fr translatin f natural mrnas. At the relatively lw Mg2+ cncentratin used, stimulatin f prtein synthesis by ply(u) is relatively mdest, abut 12-fld ver the endgenus incrpratin at 2 gg/ml f ply(u). This restilt supprts ur hypthesis that m7g5'p inhibits translatin f nly thse RNAs having this nucletide at the 5'-terminus. DISCUSSION The experiments described abve shw that m7g5'p can blck the translatin f sme mrnas, whereas it des nt inhibit the translatin f RNAs that lack the m7g5'ppp... 5'- terminal sequence. Therefre, m7g5'p des nt inhibit prtein chain elngatin. We have shwn that m7g5'p inhibits the binding f labeled revirus mrna t 8S initiatin cmplexes. The inhibitry activity f guansine nucletides requires bth the presence f the methyl grup in the 7 psitin and the 5' phsphate. We have nt yet tested the activity f m7g5'pp r m7g5'ppp. These cmpunds may prve t be even mre effective inhibitrs, since they mre clsely resemble the 5'-terminus f mrna than m7g5 p. Translatin f rabbit glbin and HeLa ply(a) + RNA is inhibited by m7g5'p. These RNAs are knwn t have 5'-terminal m7g5'ppp (6, 7). Translatin f TMV RNA is als inhibited by m7g5'p. While this manuscript was being prepared fr publicatin, we learned that Keith and Frankel- Cnrat (21) and Zimmern (22) have examined the 5'-terminal sequence f TMV RNA by the methds intrduced fr the study f ther methylated viral and mammalian RNAs, and have determined that the majrity f the mlecules have m7g5'p at the 5'.terminus. Translatin f TMV RNA is inhibited by m7g5'p t a less degree than glbin mrna (Fig. 2). We believe that TMV RNA is a hmgeneus template f mlecular weight abut 2 X 16 (12), cntaining enugh nucletides t cde fr 1 prteins the size f rabbit glbin. We find that abut 1-15% f the translatinal activity f TMV RNA is resistant t m7g5'p inhibitin. The RNA translated in the presence f m7g5'p may crrespnd t mlecules that d nt have this nucletide at the 5'-terminus; hidden breaks in TMV RNA may result in the frmatin f these mlecules (22). Anther explanatin fr the translatin Prc. Nat. Acad. Sci. USA 73 (1976) f TMV RNA being resistant t m7g5'p is wrth mentining. TMV RNA is a plycistrnic mrna, and it is pssible that initiatin f plypeptide chains can take place at internal sites, thugh the preferred initiatin site invlves the m7g5'ppp... 5'-terminal sequence. The bservatin that the translatin f STNV RNA is nt inhibited by m7g5'p demnstrates the specificity f this inhibitr and als raises the pssibility f the existence f separate mechanisms fr the initiatin f prtein synthesis fr classes f mrnas that cntain different 5'-termini. This has intriguing pssibilities as a ptential translatinal cntrl mechanism. The stimulatin f STNV RNA translatin by m7g5'p is rather surprising. A pssible explanatin fr this bservatin is that m7g5'p inhibits translatin f endgenus mrna r mrna fragments present in the wheat germ extract, and thus eliminates cmpetitin between this RNA and STNV RNA. Translatin f HeLa ply(a) + RNA in the wheat germ extract des nt appear t be cmpletely inhibited at high m7g5'p cncentratins. This result is pen t different interpretatins. One pssibility is that, in the hetergeneus ppulatins f mrnas present in the ply(a) + RNA, there are mrnas that d nt pssess the m7g5'ppp... terminal sequence and are nt inhibited by m7g5'p. Whether the fractin f HeLa cell mrna resistant t m7g5'p inhibitin cdes fr prteins different frm thse cded fr by the HeLa mrna that is inhibited is a matter f speculatin. We believe that m7g5'p will be a very useful tl in elucidating the rle f methylated 5'-termini f mrna in prtein synthesis. Our preliminary results suggest that this cmpund might be used t determine whether a template fr prtein synthesis has the 5'-terminal sequence m7g5'ppp... It seems pssible, mrever, that labeled m7g5'p can be used t establish whether a specific initiatin factr r a ribsmal prtein interacts with the 5'-terminus f mrna. This wrk has been supprted by grants f the Natinal Institutes f Health and Natinal Science Fundatin t C.B. We are extremely grateful t Sandra E. Grant fr expert technical assistance and t Rbert Ens fr the preparatin f wheat germ extract. 1. Rttman, F., Shatkin, A. J. & Perry, R. P. (1974) Cell 3, Furuichi, Y., Muthukrishnan, S. & Shatkin, A. J. (1975) Prc. Nat. Acad. Sci. USA 72, Abraham, G., Rhdes, D. P. & Banerjee, A. K. (1975) Nature 255, Wei, C. M. & Mss, B. (1975) Prc. Nat. Acad. Sci. USA 72, Furuichi, Y. & Miura, K. I. (1975) Nature 253, Muthukrishnan, S., Bth, G. W., Furuichi, Y. & Shatkin, A. J. (1975) Nature 255, Furuichi, Y., Mrgan, M., Shatkin, A. J., Jelinek, W., Dalditt- Gergiev, M. & Darnell, J. E. (1975) Prc. Nat. Acad. Sci. USA 72, Adams, J. & Cry, S. (1975) Nature 255, Bth, G. W., Banerjee, A. K. & Shatkin, A. J. (1975) Prc. Nat. Acad. Sci. USA 72, Bth, G. W., Furuichi, Y., Muthukrishnan, S. & Shatkin, A. J. (1975) Cell 6, Jacbs-Lrena, M. & Baglini, C. (1972) Prc. Nat. Acad. Sci. USA 69, Rberts, B. E., Mathews, M. B. & Brutn, C. J. (1973) J. Ml. Bil. 8, Pembertn, R. E., Liberti, P. & Baglini, C. (1975) Anal. Bichem. 66,

Bichemistry: Hickey et al. 14. Rberts, B. E. & Patersn, B. M. (1973) Prc. Nat. Acad. Sci. USA 7,233-2334. 15. Jacbs-Lrena, M. & Baglini, C. (1973) Eur. J. Bichem. 35, 559-565. 16. Darnbrugh, C. H., Legn, S.

5 Bichemistry: Hickey et al. 14. Rberts, B. E. & Patersn, B. M. (1973) Prc. Nat. Acad. Sci. USA 7, Jacbs-Lrena, M. & Baglini, C. (1973) Eur. J. Bichem. 35, Darnbrugh, C. H., Legn, S., Hunt, T. & Jacksn, R. J. (1973) J. Ml. Bil. 76, Gaskill, P. & Kabat, D. (1971) Prc. Nat. Acad. Sci. USA 68, Prc. Nat. Acad. Sci. USA 73 (1976) Wimmer, E. & Reichmann, M. (1968) Science 16, Lesnaw, J. & Reichmann, M. (197) Prc. Nat. Acad. Sci. USA 66, Hrst, J., Frankel-Cnrat, H. & Mendeles, S. (1971) Bichemistry 1, Keith, L. & Frankel-Cnrat, H. (1975) FEBS Lett. 57, Zimmern, D. (1975) Nucleic Acid Res. 2,

The demonstration of lysosomes by the controlled temperature freezing-sectioning method By LUCILLE BITENSKY

The demonstration of lysosomes by the controlled temperature freezing-sectioning method By LUCILLE BITENSKY 205 The demnstratin f lyssmes by the cntrlled temperature freezing-sectining methd By LUCILLE BITESKY (Frm the Department f Pathlgy, Ryal Cllege f Surgens f England, Lincln's Inn Fields, Lndn, W.C. 2)