THE JOURNAL OF BrOLDFiCAL CHEMISTRY Vol. 255, No. 18, Issue of September 25. PP. 8529-8533.1900 Printed in 0 . S. A.

The Use of a-Amanitin to Inhibit in Vivo RNA Synthesis and Germination in Wheat Embryos*

(Received for publication, December 3, 1979, and in revised form, May 20, 1980)

Jerry Jendrisak From the Department of Botany, University of Minnesota, St. Paul, Minnesota 55108

Low concentrations (0.10 to 1.0 pg/ml) of a-amanitin inhibit wheat embryo germination. Labeled RNA, syn- thesized in vivo by embryos imbibed in the presence of [’H]uridine and various concentrations of a-amanitin, was analyzed by oligo(dT)-cellulose chromatography and by gel electrophoresis (acrylamide and agarose) coupled with fluorography. Low concentrations of a- amanitin (0.10 to 1.0 pg/ml) strongly and selectively inhibited in vivo poly(A)+ RNA synthesis in a manner which closely paralleled a-amanitin inhibition of puri- fied RNA polymerase Lt. These results suggest that de novo mF4NA transcription is required for germination. Higher concentrations of a-amanitin inhibited in vivo 5 S rRNA and tRNA synthesis in a manner which closely paralleled a-amanitin inhibition of purified RNA polymerase III. High concentrations of a-amanitin also inhibited accumulation of radioactivity into the rRNA precursor as weU as into mature 25 S and 18 S rRNAs in a manner which also closely paralleled a- amanitin inhibition of RNA polymerase In. The dis- crepancy of the in vivo inhibitory effect of high a-aman- itin concentrations on rRNA synthesis versus a lack of effect on purified RNA polymerase I, which presumably transcribes these genes, can be explained i f continued transcription of the large rRNA precursor by RNA polymerase I requires ongoing transcription by RNA polymerase III, or if there is degradation (wastage) of rRNA precursor and/or processed products in the ab- sence of transcription by RNA polymerase III.

It is generdy accepted that protein synthesis is required for resumption of embryo growth and radical emergence in the germinating seed (1-5). l t has not been resolved whet.her this protein synthesis for successful completion of germination is dependent on de novo mRNA synthesis (6, 7) or whether preformed mRNA, which was synthesized during embryogen- esis and stored in the dry seed (3, 8-12), plays an important role in germination after being mobilized during imbibition.

Studies with isolated wheat embryos indicate that RNA and protein synthesis commence very early during imbibition (2, 12-15). Since polysome formation in imbibing wheat em- bryos is not dependent upon transcription (10). stored mRNA is proposed to have an important role in early germination, perhaps to function primarily by allowing a rapid resumption of growth upon exposure of the embryo to water (8). Although preformed, stored mRNA is active during imbibition, it is

* This work was supported by a grant from the National Institutes of Health (GM 24294). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact,

important to determine whether it is indispensible for germi- nation, whether it is the sole source of translated mRNA in early gemination, just how far it continues to be a principal form of active mRNA, and if de nouo mRNA synthesis is obligatory for germination at all.

In eukaryotic organisms, three classes of DNA-dependent RNA polymerases (EC 2.7.7.6) are responsible for transcrip- tion of nuclear genes (reviewed in Refs. 16 and 17). The enzymes can be distinguished chromatographically, structur- ally, and by differential sensitivity to inhibition by the fungal toxin a-amanitin. Studies in mammalian systems indicate that RNA polymerase I is refractory to inhibition by a-amanitin and transcribes the large ribosomal RNA precursor in the nucleolus, that RNA polymerase I1 is inhibited by low con- centrations of a-amanitin and transcribes messenger RNA precursors (heterogeneous nuclear RNA) (181, and that RNA polymerase 111 is inhibited only by relatively high concentra- tions of a-amanitin and transcribes tRNA precursors and 5 S rRNA. Plant RNA polymerases have similar a-amanitin in- hibition properties, chromatographic properties, and subunit structures to the cognate mammalian enzymes (19). The role of RNA polymerase I in rRNA synthesis has been demon- strated in higher plants by analysis of nascent RNA elongated in vitro in isolated nuclei in the presence or absence of a- amanitin (20). It, has also been shown that a-amanitin selec- tively inhibits in vivo AMP-rich RNA synthesis in cultured plant cells (21). The transcription products of plant RNA polymerase 111 have not yet been demonstrated.

Roles of the multiple eukaryotic nuclear RNA polymerases were unambi~ously defined by hybridization andfor size analysis of radioactive RNA products synthesized in isolated nuclei in the presence of various a-amanitin concentrations (17). The use of a-amanitin as an inhibitor of in viuo RNA synthesis has also indicated that RNA polymerase I1 synthe- sizes heterogeneous nuclear RNA, but not rRNA (21-27). In some studies, however, ambiguous results have been obtained. Ribosomal (28 S and 18 S) RNA synthesis is also inhibited, for example in mammalian liver tissue, after in vivo adminis- tration of a-amanitin (28-33). It has been suggested that long term exposure of cells to a-amanitin may exert complicating side effects which result in inhibition of nucleolar RNA syn- thesis (17).

The following communication describes the use of a-aman- itin to study in vivo RNA synthesis in germinating wheat embryos, initially with the goal of defining the transcriptional role of RNA polymerase III in plants. Other results indicate that de ~ O U O mRNA transcription is required for embryo germination. In addition, results obtained from this study indicate that in this system, a-amanitin inhibition of in vivo rRNA synthesis is a result of impaired RNA polymerase I11 function.

8529

8530 RNA Synthesis and Germination

EXPERIMENTAL PROCEDURES’

car r ied Out a t T o o m temperature. Embryos wers prepared from wheat seed by the method of Johnston m d Stern 135) using the carbon tetrachlorlddcyclo- hexane f lo t a t ion s t ep t o SaPara t e smbryoe from endosperm.

Isalarron and GermmatLon Of Wheat Embrvoa--The followxng s teps were

i n t e r e s t yere Cut Out. Gel SllcBS were placed In to ec ln t i l l a t lon vials and were incvbated ovsrnxghr i n 1 m l of Pro toso l t i s sue so lubi l izer . Toluene base scintillation solution was added and radloaCtLvYlty determined by l lqu ld sc in t l l ln t ion cowl tmg.

Of Dynan al. 1441. S e n s i t w i t y Of trichloroacetic acld-preclpitable Zadioac t lv i tTto a lka l ine hydro lys is and t o RNwe A wae determinsd by

method (46) and Correctlone were made for color Contnbufion by DNA. DNA qeneral mthoda described ln re f . 45. RNA was deternrned by the OICL~DL

waa determined by the diphenylamine method 147).

Other Methods-RNA polymerase a c t l v i t m s were dstermxned by the method

RESULTS

The Effect of a-Amanitin on Wheat Embryo Germina- tion-a-Amanitin was found to be a potent inhibitor of wheat embryo germination as determined by fresh weight analysis (Fig. 1). Wheat embryo germination is characterized by a rapid, initial 2.3-fold increase in fresh weight during the fist 40 min due to imbibition of water, followed by a lag period of 5 h, and finally a phase of cell enlargement (growth) during which fresh weight increases a t a sustained rate. Low concen- trations of a-amanitin inhibit growth 100% such that the fresh weight of embryos imbibed for 36 h in the presence of these concentrations of a-amanitin weigh no more than embryos imbibed for 40 min to 5 h in the absence of a-amanitin. Relative growth was determined routinely after 36 h of ger- mination time using the following relationship: Relative growth ( W )

I Portions of this paper (including “Experimental Procedures” and Figs. 1 to 5) are presented in miniprint as prepared by the author. Miniprint is easily read with the aid of a standard magnifying glass.

Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Doc- Full sized photocopies are available from the Journal of Biological

ument 79M92417, cite author, and include a check or money order for $1.35 per set of photocopies. Full sized photocopies are also included in the microfilm edition of the journal that is available from Waverly Press.

GERMINATION TIME (HOURS1

OO L ’0101 I 1 0.1

INHIBITOR CONCENTRATION Iuglml) I .o IO 100

On a weight basis, a-amanitin is as effective at inhibiting wheat embryo growth as is the protein synthesis inhibitor cycloheximide (Fig. 2). These results contrast with the very high concentrations of actinomycin D required to inhibit embryo growth (Fig. 2). The Effect of a-amanitin on in Vivo RNA Synthesis-

Labeled RNA was synthesized in vivo by wheat embryos imbibed in the presence of [“Hluridine and various a-amanitin concentrations. RNA and DNA determinations indicated that identical recoveries of nucleic acids were achieved for all treatments with the extraction procedure used. Trichloroace- tic acid-precipitable ”H incorporation was almost totally (98%) sensitive to alkaline hydrolysis and largely (90%) sensitive to degradation by RNase in all cases. The results of “H incor- poration into various RNA classes and species is summarized in Fig. 3 where incorporation is expressed as a percentage of radioactivity incorporated in the absence of a-amanitin (per cent of control). These data are compared to the a-amanitin inhibition curves for purified wheat RNA polymerases I, 11, and I11 (19).

Poly(A)+ RNA was isolated from total extracted nucleic acids by oligo(dT)-cellulose chromatography and the effect of a-amanitin on r3H]uridine incorporation into poly(A)+ RNA and poly(A)- RNA (material not binding to oligo(dT)-cellu- lose) was examined. At low a-amanitin concentrations, incor- poration into poly(A)+ RNA was strongly inhibited whereas incorporation into poly(A)- RNA was largely unaffected (Fig. 3a). For example, when embryos were imbibed in the presence

RNA Synthesis and Germination 8531

of 0.10 pg/ml of a-amanitin, incorporation into poly(A)+ RNA was inhibited over 80% whereas incorporation into poly(A)- RNA was completely unaffected (0% inhibition). At higher a-amanitin concentrations, which resulted in total (100%) inhibition of incorporation into poly(A)+ RNA, incorporation into poly(A)- RNA was inhibited increasingly. At the highest a-amanitin concentrations tested (100 pg/ml), incorporation into poly(A)- RNA was inhibited 85%. a-Amanitin concen- trations required to inhibit incorporation into poly(A)+ RNA were virtually identical with and closely paralleled the con- centrations required to inhibit wheat RNA polymerase I1 activity. On the other hand, a-amanitin concentrations re- quired to inhibit incorporation into poly(A)- RNA closely paralleled the concentrations required to inhibit RNA polym- erase I11 activity.

Poly(A)- RNA was fractionated into high and low molec- ular weight classes by isopropyl alcohol precipitation. When the effect of a-amanitin on ["Hluridine incorporation into these two fractions was examined, it was found that inhibition in both cases occurred at the same a-amanitin concentrations. The a-amanitin concentrations required to inhibit in vivo "H incorporation into high and low molecular weight poly(A)- RNA were virtually identical with and closely paralleled con- centrations required to inhibit RNA polymerase I11 activity (Fig. 36) .

When "-labeled low molecular weight poly(A)- RNA synthesized in vivo in the presence of various a-amanitin concentrations was analyzed by 15% polyacrylamide gel elec- trophoresis and fluorography (Fig. 4), it was found that incor- poration into 5 S rRNA and tRNAs was relatively unaffected by low a-amanitin concentrations (0.10 to 1.0 pg/ml) but was strongly inhibited by higher concentrations (10 to 100 pg/ml). These low molecular weight RNAs were identified in fluoro- grams by comparison of their electrophoretic mobilities with those of various RNA markers, including RNA purified from wheat ribosomes (5 S rRNA) and ribosome-free crude extracts (tRNAs).

When incorporation into 5 S rRNA and tRNAs was quan- tified by counting radioactivity in gel slices, it was found that a-amanitin concentrations required to inhibit incorporation into 5 S rRNA were the same concentrations required to inhibit incorporation into tRNA. Moreover, the observed in-

STDS. STAINED GEL FLUOROGRAM

s 2 0 0 I I O IO 100 0 0.1 1.0 IO 100

E a-AMANITIN CONCENTRATION (ug/rnl)

g'&. 9. I n h i b i t i o n o f &! low m1ecuIa1 weight (5s ribosomal and trans- f e r ) RNA sync e s i s by --amanitin. Embryos were imhibed for 2 h in the presence of $HI uridine and the lndrcsted concentrationll Of -mani t in . Nucle ic sc lds were pur i f i ed and subjected to 15% polyacrylamide gal 01ectrO- phoresis as descr ibed in the text . The ethidium bromide s t a i n e d q e l and its f luoroqrm are presented. 55 rRNA and t R N A i d e n t i f i c a t i o n was m d e by sub-

a p e d supernatants) t o e lec trophores i s . j s c t inq RNA standards (pur i f i ed from ribosomes and from ribosonr-free high

STAINED GEL FLuOROGRAM

Q 0 01 1.0 IO I00 0 0.1 1.0 10 100 a - ~ ~ ~ ~ ~ ~ ~ ~ CONCENTRATION (ug/ml)

&.,I: Inh ib i t ion of lo VLVp h i g h l o l e c u l a r M i g h t rRNI aynthwi . by a- m a n l t m . Embryos n r e imbibed in the preeence of [)HI uridine and the Indicated concentrationll of c4-ame.nitin as descr ibed in the text . N U E l e i O acids n r e p u r i f i e d and subjec ted to 1.5% aqarose g e l e l e c t r o p h o r e s i s . The e t h l d h m brolnids s t a i n e d g a l and i t a f l u o m q r m are presented along with a track contamlnq rRNA markers.

hibition of incorporation into 5 S rRNA and tRNA closely paralleled a-amanitin inhibition of RNA polymerase I11 activ- ity (Fig. 3c).

When "H-labeled high molecular weight poly(A)- RNA synthesized in vivo in the presence of various a-amanitin concentrations was analyzed by 1.5% agarose gel electropho- resis and fluorography (Fig. 51, it was found that incorporation into the rRNA precursor, 25 S rRNA and 18 S rRNA was inhibited by high concentrations of a-amanitin (10 to 100 pg/ ml). These high molecular weight RNAs were identified in fluorograms by a comparison of their electrophoretic mobili- ties with RNA purified from wheat ribosomes.

When incorporation into these high molecular weight rRNA species was quantified by counting radioactivity in gel slices, it was found that incorporation into all three species was inhibited similarly by high concentrations of a-amanitin. Moreover, the observed inhibition closely paralleled a-aman- itin inhibition of RNA polymerase I11 activity (Fig. 3 4 .

DISCUSSION

De novo mRNA synthesis is required for wheat embryo germination. This conclusion is based on the results obtained from an analysis of in vivo RNA synthesis and embryo growth in the presence of various a-amanitin concentrations. Low a- amanitin concentrations, which inhibit only in vitro RNA polymerase I1 activity, selectively inhibit only in vivo

8532 RNA Synthesis and Germination

poly(A)+ RNA synthesis and germinative growth. The use of another RNA synthesis inhibitor, actinomycin

D, had little effect on embryo growth unless supplied at very high concentrations. The mode of action of actinomycin D is different from that of a-amanitin which interacts directly with the RNA polymerase molecule (48). Actinomycin D, a chrom- opeptide which forms nonionic complexes with DNA and in so doing, inhibits the action of RNA polymerases (49), is very effective at blocking RNA synthesis in animal cells (50). However, increasing evidence indicates that actinomycin D is only partially effective a t inhibiting RNA synthesis in germi- nating seeds (3), perhaps due to poor penetration of the drug into these tissues (51).

Cycloheximide, as expected, very effectively inhibits wheat embryo germination (100%) at low concentrations (10 pg/ml) by inhibiting protein synthesis (2). I t is important to rule out the possibility that a-amanitin inhibits protein synthesis in this system. It has been reported by others that a-amanitin neither inhibits in vitro translation of exogenous mRNAs in a wheat cell-free system (7), nor in vivo protein synthesis or polysome formation (7, 10) with preexisting mRNA. These results were verified2 and it is concluded that a-amanitin is acting with its expected specificity.

It appears that de novo mRNA synthesis is required for resumption of growth in germinating wheat embryos, a con- clusion which may very well be applicable to other plant species. These results suggest that the importance and signif- icance of stored mRNA in the dry seed and its role in germi- nation be re-evaluated, since stored mRNA is by itself insuf- ficient to allow for the resumption of germinative growth. I t cannot be ruled out, however, that preformed mRNA is insig- nificant in germination since the need for de novo transcrip- tion by itself may also be insufficient for resumption of embryo growth.

a-Amanitin is a very effective inhibitor of RNA synthesis in imbibing wheat embryos. Low a-amanitin concentrations re- sulted in selective inhibition of in vivo poly(A)+ RNA syn- thesis. The a-amanitin inhibition curve for in vivo poly(A)+ RNA synthesis is almost identical with the a-amanitin inhi- bition curve for RNA polymerase 11. These results suggest that a-amanitin readily enters the embryo tissues, probably at the same rate as ["]uridine, and results in immediate inhibition of endogenous RNA polymerase I1 activity. The degree to which the a-amanitin inhibition curve for in vivo 5 S rRNA and tRNA synthesis resembles the a-amanitin inhi- bition curve for RNA polymerase I11 also suggests relatively unimpeded uptake of a-amanitin. From these results it would appear that the intracellular a-amanitin concentrations, which are likely to reflect closely the concentrations of a-amanitin supplied in the germination medium, can be carefully con- trolled in this system. Since embryos were imbibed directly in the presence of a-amanitin, it is likely that a-amanitin is rapidly taken up along with water during imbibition (hydra- tion).

The fact that low a-amanitin concentrations inhibit in vivo poly(A)+ RNA synthesis in a manner closely paralleling the inhibition of RNA polymerase 11, and that higher concentra- tions inhibit in vivo 5 S rRNA and tRNA synthesis in a manner closely paralleling the inhibition of RNA polymerase 111, suggests that a-amanitin can be used as a highly selective inhibitor of in vivo RNA polymerase function in this system. RNA polymerase 111 transcription products have not yet been defined for higher plants. The data presented here suggest the involvement of RNA polymerase I11 in the transcription of small cellular RNAs in higher plants, which would agree with

' J . Jendrisak, unpublished observation.

the transcriptional roles determined for RNA polymerase 111 in animals (17) and yeast (52). Data obtained in this study would also suggest, however, that RNA polymerase 111 is responsible for transcription of the large rRNA precursor since the observed inhibition of in vivo rRNA ciynthesis by various a-amanitin concentrations closely paralleled a-aman- itin inhibition of RNA polymerase 111. In vitro transcription studies with nuclei of animals (17), plants (20), and yeast (52), on the other hand, established that RNA polymerase I tran- scribes these genes. This apparent discrepancy can be ex- plained if net in vivo synthesis of high molecular weight rRNA by RNA polymerase I requires ongoing transcription by RNA polymerase 111. That rRNA synthesis is inhibited in a manner closely paralleling the inhibition of RNA polymerase 111 sug- gests a close coupling of rRNA synthesis to RNA polymerase I11 function.

Ribosome biogenesis ultimately involves the participation of all three nuclear RNA polymerases. RNA polymerase I transcribes the rRNA precursor which is processed into ma- ture 25 S, 18 S, and 5.8 S rRNAs; RNA polymerase I1 presumably transcribes the mRNAs for ribosomal proteins; and RNA polymerase I11 transcribes 5 S rRNA. In addition, other RNA polymerase I, 11, and I11 transcripts may be required for ribosome assembly (small nuclear RNAs (?); mRNAs for methylases, nucleases, other assembly proteins). Some of these components could be important feedback fac- tors in nucleolar function. Limiting cellular concentrations of these products could result in the apparent inhibition of rRNA synthesis. For example, attention has been directed to the close correlation of the relationship of ribosomal protein syn- thesis and that of rRNA (53, 54). Presumably there exists in the wheat embryo a sufficient endogenous pool of protein components (at least for the duration that these labeling studies were carried out) since imbibition of embryos for 3 h in the presence of 10 pg/ml of cycloheximide, which inhibited embryo protein synthesis 98%, resulted in no inhibition of rRNA synthesis." Inhibition of endogenous RNA polymerase I11 by high a-amanitin concentrations could result in impaired RNA polymerase I function if the cellular concentrations of 5 S rRNA (or perhaps other RNA polymerase I11 transcripts involved in assembly) become limiting. On the other hand, if the rRNA precursor pool exceeds the available pool of 5 S rRNA, excess rRNA precursor and partially processed prod- ucts may be degraded. An excess of rRNA could be rectified by wastage. Either one of these mechanisms could explain the observed inhibition of rRNA synthesis in wheat embryos after in vivo administration of a-amanitin.

Acknowledgment-The excellent technical assistance of Ruby J. Larson is acknowledged.

REFERENCES

1. Marcus, A. (1969) Symp. SOC. Exp. Biol. 23, 143-160 2. Marcus, A,, Spiegel, S., and Brooker, J. D. (1975) in Control

Mechanisms in Development (Meints, R. H., and Davies, E., eds) pp. 1-19, Plenum Press, New York

3. Payne, P. I. (1976) Biol. Rev. 51,329-363 4. Fujisawa, H. (1966) Plant Cell Physiol. 7, 185-197 5. Walton, D. C., and Soofi, G. S. (1969) Plant Cell Physiol. 10,307-

6. Delseny, M., Aspart, L., and Guitton, Y. (1977) Planta 135, 125-

7. Cheung, C. P., Wu, J., and Suhadolnik, R. J. (1979) Nature 227,

8. Brooker, J. D., Tomaszewski, M., and Marcus, A. (1978) Plant

315

128

66-67

Physiol. 61, 145-149

J . Jendrisak, unpublished observation.

RNA Synthesis and Germination 8533

9. Dure, L., and Waters, L. (1965) Science 147,410-412 10. Spiegel, S., and Marcus, A. (1975) Nature 256, 228-230 11. Weeks, D. P., and Marcus, A. (1971) Biochim. Biophys. Acta 232,

12. Spiegel, S., Obendorf, R. L., and Marcus, A. (1975) Plant Physiol.

13. Marcus, A,, Feeley, J., and Volcani, T. (1966) Plant Physiol. 41,

14. Chen, D., Schultz, G., and Katchalski, E. (1971) Nature New Biol.

15. Sen, S., Payne, P. I., and Osborne, D. J . (1975) Biochem. J. 148,

16. Chambon, P. (1975) Annu. Reu. Biochem. 44, 613-638 17. Roeder, R. G. (1976) in RNA Polymerase (Losick, R., and Cham-

berlin, M, eds) pp. 285-329, Cold Spring Harbor Laboratory, New York

671-684

56,502-507

1167-1172

231,69-72

381-387

18. Lewin, B. (1975) Cell 4, 11-20 19. Jendrisak, J . (1980) in Genome Organization and Expression in

20. Gurley, W. B., Lin, C-Y., Guilfoyle, T. J., Nagao, R. T., and Key,

21. Seitz, U., and Seitz, U. (1971) Planta 97,224-229 22. Egyhazi, E., D'Monte, B., and Edstrom, J.-E. (1972) J. Cell Biol.

23. Hastie, N. D., and Mahy, B. W. J . (1973) FEBS Lett. 32, 95-99 24. Kedinger, C., and Simard, R. (1974) J. Cell Biol. 63, 831-842 25. Kuwano, M., and Ikehara, Y. (1973) Exp. Cell Res. 82,454-457 26. Tocchini-Valentini, G. P., and Crippa, M. (1970) Nature 228,993-

27. Tocchini-Valentini, G. P., and Crippa, M. (1970) Cold Spring

28. Hadjiolov, A. A,, Dabeva, M. D., and Mackedonski, V. V. (1974)

29. Jacob, S. T., Muecke, W., Sajdel, E. M., and Munro, H. N. (1970)

30. Jacob, S. T., Sajdel, E. M., Muecke, W., and Munro, H. N. (1970)

31. Schmid, W., and Sekeris, C. E. (1973) Biochim. Biophys. Acta

Plants (Leaver, C. J., ed) pp. 77-92, Plenum Press, New York

J. L. (1976) Biochim. Biophys. Acta 425, 168-174

53,523-531

995

Harbor Symp. Quant. Biol. 35, 737-742

Biochem. J. 138, 321-334

Biochem. Biophys. Res. Commun. 40, 334-342

Cold Spring Harbor Symp. Quant. Biol. 35,681-691

312, 549-554 32. Sekeris, C. E., and Schmid, W. (1972) FEBS Lett. 27, 41-45 33. Tata, J. R., Hamilton, M. J., and Shields, D. (1972) Nature New

34. Jendrisak, J. J., and Burgess, R. R. (1975) Biochemistry 14,4639-

35. Johnston, F. B., and Stern, H. (1957) Nature 179, 160-161 36. Wiegers, U., and Hilz, H. (1971) Biochem. Biophys. Res. Comrnun.

37. Hiatt, H. H. (1962) J. Mol. Biol. 5, 217-229 38. Perry, R. P., La Torre, J., Kelley, D. E., and Greenberg, J. It.

39. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A. 69,

40. Rhoads, R. E. (1975) J. Biol. Chem. 250,8088-8097 41. Zubay, G. (1962) J . Mol. Biol. 4, 347-356 42. Laemmli, U. K. (1970) Nature 227,680-685 43. Bonner, W. M., and Laskey, R. A. (1974) Eur. J. Biochem. 46,

44. Dynan, W. S., Jendrisak, J. J., and Burgess, R. R. (1977) Anal.

45. Davidson, J . N. (1972) The Biochemistry of the Nucleic Acids,

46. Schneider, W. C. (1957) Methods Enzymol. 3,680-684 47. Burton, K. (1956) Biochem. J. 62,315-323 48. Cochet-Meilhac, M., and Chambon, P. (1974) Biochim. Biophys.

49. Harbers, E., and Muller, W. (1962) Biochem. Biophys. Res. Com-

50. Goldberg, I. H., and Friedman, P. A. (1971) Annu. Reu. Biochem.

51. Neumann, J. (1964) Physiol. Plant. 17, 363-370 52. Schultz, L. D. (1978) Biochemistry 17, 750-758 53. Warner, J . R. (1974) in Ribosomes (Nomura, M., Tissieres, A,,

and Lengyel, P., eds) pp. 461-488, Cold Spring Harbor Labo- ratory, New York

5 4 . Wu, B. C., Rao, M. S., Gupta, K. K., Rothblum, L. I., Mamrack, P. C., and Busch, H. (1977) Cell. Biol. Int. Rep. 1, 31-44

Biol. 238, 161-164

4645

44, 513-519

(1972) Biochim. Biophys. Acta 262,220-226

1408-1412

83-88

Biochem. 79, 181-189

Academic Press, New York

Acta 353, 160-184

mun. 7, 107-110

40, 775-810