ABSTRACT THE 5' TERMINI OF RIBOSOMAL RIBONUCLEIC ACID FROM HIGHER PLANTS BY Leroy Allan Watrud This thesis is based on a study of higher plant ribosomal ribonucleic acid (rRNA). The objectives were (a) to determine the identity of the 5' rRNA termini, that is, to ascertain whether these groups consist of specific nucleoside 3',5' diphosphates, and (b) to determine whether the terminal groups are the same in different higher plant species. A monocotyledon, wheat (Triticum aestivum, L.), and a dicotyledon, cauliflower (Brassica oleracea, L. var. botrytis), were the two species studied. RNA was isolated by one of three methods, depend— ing on the characteristics of the tissue to be extracted. Essentially, the techniques used were extraction and deproteinization with either (a) phenol-sodium dodecyl sulfate solution, (b) phenol-phosphate solution, or (c) phenol-oresol-naphthalene l,5 disulfonate. The l Leroy Allan Watrud procedures were developed to the extent that consistent quantitative yields were obtained from a given plant source. The purified rRNA was hydrolyzed in 0.3 N KOH at 37 C for 48 hours, and the solution was subsequently neu- tralized by the addition of a sulfonic acid resin (Dowex SO-H+ or Rexyn lOl). Components of the hydrolysate were separated by ion exchange on DEAE-cellulose or by gel fil- tration on Sephadex G-15. Nucleoside 3',5' diphosphates released from the 5' terminus by alkaline hydrolysis were identified by having identical properties as standards in paper chromatography, electrophoresis, as well as in anion exchange chromatography, and by comparison of absorption spectra. Adenosine 3',5' diphosphate was the only 5' ter- minal group obtained following alkaline hydrolysis of un- fractionated (258 + 165) cauliflower rRNA extracted by phenol-sodium dodecyl sulfate, and also from rRNA ex- tracted by phenol-phosphate from one source of wheat embryo. Similarly, this was the predominant group ob- tained from wheat embryo rRNA extracted from another source. In addition, traces of cytidine 3',5' diphosphate 2 Leroy Allan Watrud and guanosine 3',5' diphosphate were also found in the RNA hydrolysate from the latter source. These observations suggest that a common mechanism may control the initiation of rRNA synthesis in the species studied. Satisfactory methods for the synthesis of nucleo- side 3'(2')5' diphosphate standards, with which the 5' rRNA termini could be compared for identification were lacking at the beginning of this study. The synthesis of these standards, catalyzed by nucleotide phosphotransferase in a low energy phosphate transfer from p-nitrophenyl phosphate to 5' nucleotides, is also documented in this thesis. THE 5' TERMINI OF RIBOSOMAL RIBONUCLEIC ACID FROM HIGHER PLANTS BY Leroy Allan Watrud A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1973 a“? Q? This thesis is dedicated to my wife Lidia, our son Buck, and to my father, each for their unique inspiration. ii ACKNOWLEDGEMENTS I would like to express my appreciation to Dr. Clifford J. Pollard, my major professor, for his assistance and critical evaluation of this work. Thanks are due to members of my guidance committee, Dr. Everett S. Beneke, Dr. John A. Boezi, and Dr. Robert P. Scheffer for their suggestions and criticism of the manuscript. I wish to thank Dr. Aleksander Kivilaan for mean- ingful discussions and for liberally supplying materials and equipment during the course of this investigation as well as Dr. Albert H. Ellingboe and Dr. Norman E. Good for the use of equipment. iii TABLE OF CONTENTS LIST OF TABLES O O O O O O O O O O O O 0 LIST OF FIGURES I O O I O O O O O O O 0 INTRODUCTION. . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . Ribosomal Structure . . . . . . . . Ribosomal RNA . . . . . . . . . . . 5' Terminal Residues of Various RNA Nucleotide Separation Techniques. . MATERIALS AND METHODS . . . . . . . . . Experimental Materials. . . . . . . Extraction of RNA from Plant Tissue Species Extraction with Phenol-Phosphate. Extraction with Phenol and Sodium SUlfate O O O O O O O O O O I Extraction with Phenol and Naphthalene 1,5-Disulfonate . . . . . . . Tests for Purity and Integrity of the Isolated RNA. . . . . . . . . . . iv Dodecyl O Page viii 12 18 21 26 26 27 27 28 30 31 TABLE OF CONTENTS (cont.) Optical Purity. . . . . . . . . . . Sucrose Density Gradient Centrifugation Polyacrylamide Gel ElectrOphoresis. Dische Diphenylamine Test for DNA . Alkaline Hydrolysis . . . . . . . . . . Chromatography and Electrophoresis. . . Fractionation of RNA Hydrolysate by DEAE Cellulose (Formate) Ion Exchange Chromatography, with Increasing formate . . . . Chromatography of geners from RNA Cellulose (Cl') Linear Gradient Chromatography by Desalting . . . . Chromatography on Resin . . . . . Using Batch Elution Concentrations of Tris- Nucleosides and Con- Hydrolysate on DEAE Using a 0.0-0.3 M NaCl Paper Paper Isolation from E. RESULTS . . . Isolation Chromatography. . . . . . . . . . . ElectrOphoresis . . . . . . . . . . of Nucleotide Phosphotransferase c0110 0 O O O O O I O O O O O O O O of RNA from Plant Tissues . . . . . Page 31 31 32 33 34 34 34 36 37 37 38 39 40 40 43 43 TABLE OF CONTENTS (cont.) Page Isolation of Nucleotide Phosphotransferase. . . 47 Large Scale Preparation of Nucleoside 3',5' Diphosphate Standards Using Nucleotide Phosphotransferase. . . . . . . . . . . . . . 52 Resistance of Nucleoside 3',5' Diphosphate Standards to Sodium Meta-Periodate Oxidation. 55 Fractionation of Nucleotides and Congeners by DEAE Cellulose (Formate) Ion Exchange, Using Batch Elution with Dilute Salt. . . . . . . . 56 Fractionation of Nucleosides and Congeners by DEAE Cellulose (Cl‘) Ion Exchange Using a Linear Salt Gradient. . . . . . . . . . . . . 61 Separation of Nucleosides, Nucleoside Mono- phosphates, and Nucleoside 3',5‘ Diphos-r phates by Sephadex G-15 Gel Filtration. . . . 61 Ribosomal RNA 5' Termini. . . . . . . . . . . . 67 5' Termini of Wheat Embryo rRNA . . . . . . 67 5' Termini of Cauliflower rRNA. . . . . . . 72 DISCUSSION. 0 O O O O O O O O O O O O O O O O O O O 76 RNA Isolation Procedures. . . . . . . . . . . . 76 Alkaline Hydrolysis of rRNA . . . . . . . . . . 78 Nucleotide Phosphotransferase . . . . . . . . . 79 Separation and Identification of Ribosomal RNA Terminal Groups . . . . . . . . . . . . . 80 The Termini of Ribosomal RNA. . . . . . . . . . 83 Future Applications . . . . . . . . . . . . . . 85 vi TABLE OF CONTENTS (cont.) Page LITERATURE CITED 0 O O O O I O O O O O O O 0 O O I I 8 7 APPENDIX C O O O O I O O O O O O O O O O O O O I O O 9 9 A. Model 1 Analysis of Variance, Comparison of Means and Groups of Means of Nucleo— side Diphosphate % Recovery . . . . . . . 99 B. List of Abbreviations and Symbols . . . . . lOl vii 10. 11. LIST OF TABLES Partial 5' terminal sequences of viral RNA . Partial 5' terminal sequences of rRNA from micro-organisms. . . . . . . . . . . . . . Partial 5' terminal sequences of rRNA from eucaryotic organisms . . . . . . . . . . . Yields of rRNA from different plant tissues. Isolation and purification of nucleotide phosphotransferase from E. coli W. . . . . Time course of phosphotransferase activity . Chromatographic migration of standard nucleosides and nucleotides in three solvent systems. . . . . . . . . . . . . . Paper electrophoretic separation of nucleo- sides and nucleotides. . . . . . . . . . . Effect of periodate treatment on the electrophoretic and chromatographic properties of nucleoside phosphates. . . . Recovery of nucleoside 3',5' diphosphate standards following desalting and paper chromatography . . . . . . . . . . . . . . 0 Identification of nucleoside 3',5' diphos- phates resulting from alkaline hydrolysis ' of wheat embryo rRNA . . .‘. . . . . . . . viii Page 19 20 22 43 48 51 53 54 56 6O 71 LIST OF TABLES (cont.) Table Page 12. Identification of nucleoside 3',5'.diphos- phates resulting from alkaline hydrolysis of cauliflower floret rRNA by comparison of the properties with those of adenosine 3',5' diphosphate. . . . . . . . . . . . . 74 13. Comparison of the quantitative recovery of 3' nucleoside termini and 5' terminal 3',5' nucleoside diphosphates. . . . . . . 74 14. Summary of data on the 5' termini of ribosomal RNA. . . . . . . . . . . . . . . 75 ix Figure LIST OF FIGURES Separation of high molecular weight RNA species by sucrose density gradient centrifugation . . . . . . . . . . . . Electrophoresis of rRNA on 4.6% polyacryla- mide gels O O O O O I O O O I O O O O O Elution of nucleotide phosphotransferase and phosphatase of E. coli W from DEAE cellulose. . . . . . . . . . . . . . . Fractionation of nucleosides and nucleo- tides by DEAE cellulose (formate) ion exchange, using batch elution with formate salts. . . . . . . . . . . . . Elution pattern of standards from a DEAE cellulose (Cl’) test column. . . . . . Separation of nucleosides, nucleoside mono- phosphates and nucleoside diphosphates by Sephadex G-15 gel filtration. . . . Absorption spectra (220-300 nm) for stand- ard adenosine 3',5' diphosphate and for material eluted from wheat rRNA hydro- lysate O O O O I O O O O O O O O O O O Elution pattern of neutralized cauliflower RNA hydrolysate from DEAE cellulose (Cl') effected by a linear NaCl gradient . . Page 45 46 49 58 62 64 69 73 INTRODUCTION That ribosomes function in protein synthesis is well established. It is now apparent, however, that knowledge of ribosomal organization is necessary in order to further elucidate the synthetic mechanism (1). Base sequence analyses of ribosomal ribonucleic acids (rRNA) and studies of ribosomal structural proteins may be useful in determining the mechanism of RNA-protein interactions which result in the formation of biologically active ribo- somes. Little is known of the base sequences or functions of rRNAs; however, progress has been madeon the identifi- cation of ribosomal proteins (2-8). Investigations of high M. W. rRNA synthesis have demonstrated that formation of the ribosome begins in the nucleolus by the transcrip- tion of a high molecular weight precursor rRNA. Portions of this precursor molecule are methylated before it be- comes associated with ribosomal structural protein. Two ribosomal subunits are subsequently formed by cleavage at specific points in the molecule; concurrent with this process, part of the precursor RNA is lost (9). l The 5' end of rRNA was chosen as the subject of the present investigation for several reasons. Synthesis of RNA on a DNA template has been shown to occur from the 5' to the 3' end of the RNA molecule (10,11). Identifica— tion of the 5' terminus, the first nucleotide of the grow- ing chain, may therefore be important in studying the initiation of RNA synthesis. Knowing the identity of end groups may also be useful in determining RNA heterogeneity, the chain length of the molecules, and in elucidating the mechanism of ribosomal assembly. The objectives of this investigation were (a) to determine the identity of the 5' termini of ribosomal RNA, i.e., to learn whether these groups consist of specific nucleoside 3',5' diphosphates, and (b) to determine whether the same rRNA 5' termini occur on different plant species. Satisfactory methods for the synthesis of nu- cleoside 3',5' diphosphate standards, with which the 5' termini could be identified, were lacking at the beginning of this study. Nucleotide phosphotransferase was isolated from Escherichia coli cells, which had been commercially grown in high peptone medium and harvested in late log phase, and used for the synthesis of standards. The synthesis of these standards by enzymatic low energy transfer of phosphate from an organic substrate to the 3'(2') position of nucleoside 5' monophosphates is also documented in this thesis. LITERATURE REVIEW Ribosomal Structure There are several types of ribosomes; most bacter— ial ribosomes are of the 708 type, but in yeast, higher plants and animal cytoplasm, the ribosomes are of the 808 type, containing 40-50% RNA (20). Ribosomes are composed of two ribosomal subunits; the larger having a sedimentation constant of 50-605, the smaller an S value of 30-40. The molecular weights of subunits from plant or animal tissue are respectively about 1.5 and 2.5 x 106 daltons, while those from bacteria are 1 x 106 and 2 x 106 (12). The ribosomes contain 40-60% protein, depending on the organism from which they are extracted. The average molecular weight of the pro- tein, reported on the basis of N-terminal amino acids, was estimated to be 2.5 x 104 (21,22). Early models for protein synthesis postulated that ribosomes were passive assembly plants for translation. This concept fit into the models since specific protein 4 synthesis is programmed by a specific messenger RNA (mRNA). Messenger RNA (13) and transfer RNA (14) bind to the smaller subunit during protein synthesis, while the grow- ing polypeptide chain is bound to the larger subunit through the attached RNA (15,16). Some support for the concept of the ribosome as a passive structure or non- specific workbench was provided by findings that ribosomes could be used interchangeably between species of bacteria. Additionally, no new ribosomes are formed subsequent to T2 bacteriophage infection of bacteria, suggesting that pre-existing bacterial ribosomes become the site of attach- ment of the newly synthesized T2 mRNA (17). Ribosomes which are homogeneous in structure have been proposed in support of the concept that ribosomes are a non-specific worktable for protein synthesis. Reports citing the pres- ence of ribosomal proteins in the amount of one copy per ribosome (4) appeared to support the concept that homo- geneity exists among ribosomes. More recent evidence based on the results of acrylamide gel electrophoresis and reconstitution of ribosomal subunits, however, indi— cates a distinct heterogeneity of ribosomal proteins (5, 18,19). Analysis of purified ribosomes from E. 92;; yielded 21 ribosomal proteins from the 308 subunit and 34 proteins from the SOS subunit (18). The ribosomes from E. 33;; therefore have about 55 different proteins (6), whose molecular weights range from 10,000-65,000 (23). Thirty unique 408 subunit proteins and thirty-nine 60$ subunit proteins were found in mammalian ribosomes. Preparations of 808 ribosomes contained three proteins that were absent in each of the individual subunits. Eukaryotic ribosomes were estimated then to contain 68-72 different proteins (5). The proteins isolated were found to yield unique peptides when hydrolyzed with trypsin, indicating a hetero- geneous population (4). This was also supported by immuno- logical experiments in which no cross reactivity was indi- cated for antibodies to the proteins (7). The hetero- geneity of proteins in ribosomal subunits is therefore strongly supported. Preliminary results indicate that ribosomal proteins from eukaryotic cells have the same general characteristics as those from prokaryotic orga- nisms (5). Several lines of evidence now indicate that in addition to a major group of proteins occurring to the extent of one per ribosome, there are some "fractional" proteins present in much lower frequency (i.e., less than one per ribosome) (6,18). Two models representing dif- ferent interpretations of the heterogeneity of the 308 particles are present in the literature. Both models assume that a ribosomal "core" composed of "unit" proteins is formed, to which the fractional proteins are added. The "static" model assumes no exchange of proteins while the "steady-state" model predicts that the fractional pro- teins are exchanging from one ribosome to another during the protein synthetic cycle. These fractional proteins are suggested to determine the functional capabilities of the ribosome. They could influence the initiation of pro- tein synthesis, chain prOpagation or termination by the orderly exchange of fractional proteins (8,18). A high rate of protein exchange in ribosomal proteins was demon- strated in double labeling experiments using 14C leucine and 3H leucine; protein components rapidly exchanged with similar ribosomal proteins in the cytoplasm (24). The Ea XEEEE exchange of free ribosomal proteins with proteins of the intact ribosome has also been reported (8). The steady state model is further supported by activation of 308 ribosomal subunits. "Inactive" 30$ ribosomes were incubated with unfractionated 308 protein and SOS subunits, resulting in an 80% increase in poly-U—directed poly- phenylalanine synthesis. Ribosomal structure has been further studied through the use of proteolytic enzymes. Disk electro- phoresis on polyacrylamide gels was used to study the effects of enzymes (trypsin, pronase, chymotrypsin), on proteins of the ribosome. Protein banding patterns on the gels were altered as enzymatic cleavage of the proteins occurred. The effects on intact ribosomes were very se- lective, with the pattern of attack very similar for the different enzymes. Free ribosomal proteins, on the other hand, were rapidly broken down without the selectivity found for the same proteins in the intact ribosomes (25). This suggests that the proteins are not accessible to the enzymes while in the intact structure. The susceptibility may therefore reflect the structural arrangement of the proteins or the degree of binding to RNA. Shielding of mRNA, tRNA, and nascent polypeptides against enzymic breakdown has also been observed when these substances are ribosome bound. RNA itself may interfere with the specific functions of added proteolytic enzymes such as trypsin, which is highly dependent on positively charged side chains (25). A model of ribosomal structure based on dye stack- ing studies, has been proposed in which the rRNA exists in a non-helical single stranded configuration. This con- figuration in the ribosome would then be determined by interactions between nucleotide bases and ribosomal pro- teins, resulting in the exposure of nucleotide phosphates to the surrounding medium (26). The negatively charged phosphate groups may then interact with positively charged magnesium ions in forming linkages to other nucleotides. The association-dissociation of the ribosome appears to be governed by the Mg++ concentration, higher concentrations favoring association. This model suggests exchange by means of Mg++ salt bridges between RNA-phosphates of the different particles. Loosening of the ribosomal particles may then occur by dissociation of protein interactions upon the removal of Mg++, rather than by disruption of the secondary structure of RNA. The same principle may be applied to the binding of mRNA to ribosomes. Another ribosomal model presented in the litera- ture suggests that considerable double helical rRNA struc- ture exists in the ribosome, as well as in aqueous 10 solutions of rRNA. This conclusion is based on the close coincidence of the melting curves of rRNA and of the ribo- somes. This same evidence suggests that ribosomal protein binds to single stranded regions of the molecule. Pro- teins bound to nucleic acids in the double helical state result in molecular stabilization as suggested by an in- crease (15-20 C) in the melting temperature (100). Experiments in which the ribosome is broken down to its component parts and subsequently reassembled should yield information valuable to the structural knowledge. Reconstitution experiments in which the ribosomal subunit is re-formed from the completely dissociated respective proteins and rRNA have, in fact, been performed. Purified proteins derived from the 303 ribosomal subunit, sequen- tially added to purified rRNA resulted in the reconstitu- tion of functional 305 ribosomal subunits (98). This latter study and others suggested that specific sequences of rRNA nucleotides recognize their protein counterparts (28). It was reported further that 168 rRNA from yeast or 185 rRNA from rat liver cannot replace E. gal; l6S rRNA in the formation of functional 308 particles. Active hybrid 308 particles were formed, however, when 168 rRNAs from distantly related bacterial genera, such as 11 Azotobacter vinelandii or Bacillus stearothermgphilis, were used to replace E. 39;; 168 rRNA (19). These three genera of bacteria were reported to have portions of 168 rRNA in common, suggesting that a requirement for specific base sequences in portions of rRNA may exist. The comparison of rRNA sequences of several bac- terial species revealed a correlation between species and rRNA sequence. Definite differences were found in nucleo- tide distribution of rRNAs among Species unrelated taxo- nomically, while no differences were found between E. coli and its related species (39). Sugiura and Takanami (40) reported similar 5' terminal nucleotides from three species of Bacillus. Hydrolysis of rRNA from these species with T1 RNase and pancreatic RNase respectively produced frag- ments identical in length and in 5' termini. Alkaline hydrolysis also released the same 5' termini from each of the species. The results differed from those obtained when other genera were studied. 12 Ribosomal RNA The ribosomal RNAs have been postulated to have a single stranded linear chain arrangement, composed of four primary nucleosides, and occasionally modified nucleosides, connected by phosphodiester linkages. The 165 and 23S RNA molecules from E. 39;; contain respectively, about 1500 and 3000 nucleotides each, the larger molecule breaking down into two smaller chains under certain experimental conditions (31,32). The SS rRNA of E. coli has been found to contain 120 nucleotide residues (33). High molecular weight rRNAs vary in size among ribosomes of different organisms. The larger ribosomal (50-608) subunit contains a single molecule of 23-288 rRNA. Lower eukaryotic organisms and some plant cells, for example, contain 258 RNA with a molecular weight of about 1.3 x 106. The smaller (30-408) subunit, on the other hand, contains a single molecule of 16-188 rRNA with a molecular weight of 0.5 to 0.7 x 106 (1). Another rRNA with a sedimentation coefficient of SS is bound to the larger subunit (33,43—49). Several lines of evidence including nucleotide distribution, 5' terminal nucleotide sequences and 3' terminal nucleotide sequences, indicate 13 that 23-288 and 16-188 RNAs are independent high molecular weight species. This is further supported by genetic evi- dence for distinct cistrons for these classes of molecules (16), although both 23-288 and 16-188 RNAs are derived from the same precursor ribosomal RNA molecule (1). The SS rRNA, on the other hand, is transcribed separately from the high molecular weight rRNAs (93). The role of ribosomal RNA in the function of the ribosome has not yet been resolved. Ribosomal reconstitu- tion experiments indicate a direct involvement of the rRNA, however, chemical studies on the primary structure of rRNA are incomplete; hence it is impossible to draw meaningful conclusions. Methylation studies have been used to investigate the relationship of some aspects of rRNA synthesis to ribosome formation. Evidence that methylation is a highly specific process which results in the production of E. 92;; 168 and 23S RNAs, that are homogeneous with respect to methylated sequences, suggests that a special func- tional significance may be attached to this specific mod- ification (methylation) of the basic polymeric structure (34). When HeLa cells were grown under conditions of methionine "starvation," undermethylation of the 455 14 ribosomal precursor RNA resulted and functional ribosomes were not formed under these conditions. The methyl- deficient 4SS precursor RNA was found, however, to yield 328 RNA. The failure to form functional ribosomes was attributed to the lack of methylation. The degree of methylation in ribosomal RNA of plant, animal, and bacterial cells studied is similar. The 163 and 23S RNAs of E. 32;; contain, respectively, about 17 and 11 methyl groups per 1000 nucleotides (36), while the 163 and 288 RNAs of HeLa cells contain 19 and 13 methyl groups per 1000 nucleotides (37,38). A major dif- ference between these organisms is found in the pattern of methylation. In E. Egli' methylation occurs principally in the heterocyclic bases; in wheat germ and HeLa cells, the methylation occurs predominantly in the 2' position of the ribose moiety. The only positively identified function of the 16S rRNA is its role in the assembly of the ribosomal particle; however, it is conceivable that some parts of the 16S rRNA are involved in the binding of mRNA and or tRNA. Moore (41) studied the effects treatment with sev- eral chemical reagents (formaldehyde, nitrous acid, dinitrofluorobenzene and perphthalic acid), had on the 15 ability of ribosomes to bind to mRNA. Formaldehyde, which reacts with free primary amines in nucleic acids, did not affect amine groups involved in hydrogen bonding. Formal- dehyde treatment of ribosomes prevented binding of poly- nucleotide mRNA, inactivated the 308 subunit, prevented 7OS dissociation and subunit reassociation, and prevented incorporation of amino acids. Nitrous acid, used to test the hypothesis that amino groups might be involved in binding, affects compounds of the general type R-NHZ, by converting them to the corresponding alcohols. Treatment with this acid resulted in inhibition of polynucleotide (mRNA) binding and failure of 308 and SOS subunits to re- associate, suggesting that amino groups might be involved in the maintenance of the 708 structure. Dinitrofluoro- benzene was used to determine if protein groups are in- volved in binding, since it reacts with a wide range of protein groups, but is unreactive with RNA. Ribosomal binding of mRNA was unaffected and no effect was found on ribosomal structure. Conversely, a reagent which selec- tively oxidizes the N nitrogens of cytosine and adenine 1 but does not affect amino groups of protein was used. Thus, perphthalic acid treatment of ribosomes confirmed the idea that rRNA amino groups are necessary for binding, 16 as mRNA was inhibited by the reagent. Perphthalic acid also prevented 308-503 reassociation. The conclusion drawn from these experiments is that rRNA amino groups are directly involved in messenger binding, and that mRNA is bound by its backbone via hydrogen bonds. The results of these experiments could be strengthened by repeating the study in conjunction with reconstitution experiments to more specifically examine the functional groups of the respective ribosomal components. Specific streptomycin binding to E. gel; 168 rRNA has very recently been shown to prevent ribosomal reconstitution (42). This binding also occurs on 308 ribosomal subunits of E. 39;; strepto- mycin sensitive strains in the same amounts as on the iso- lated 16$ rRNA. The availability of the rRNA streptomycin attachment sites appear to be controlled by the binding of a protein, P10, which is present in streptomycin resistant strains (42). This further serves to strengthen the con- cept of a functional role of rRNA in "control" of protein binding. A suggestion of a possible function of rRNA which persists in the literature is as a template in the forma- tion of ribosomal proteins. During the recovery period from methionine starvation, or conditions where protein l7 synthesis has been inhibited, cells make ribosomal protein preferentially and much of the accumulated rRNA is con- verted to mature ribosomes (51), even under conditions of inhibition of RNA synthesis with actinomycin D (50). Ribo- somal RNA accumulated during inhibition of protein syn- thesis is thus suggested to have template capabilities in subsequent preferential synthesis of ribosomal protein. The literature also contains reports suggesting contamina- tion with non-ribosomal RNA as the reason for template activity for polypeptide synthesis during Eg.z£E£g exper- iments (52,53). Very recently, the influence of the secondary structure on the translation of rRNA was studied (101). Thermal denaturation of rRNA resulted in increased tem- plate activity, with temperature (40-65 C) when the anti- biotic neomycin was included in the reaction mixture. Neomycin (or streptomycin) interacts with protein P10, a core protein of the 30S ribosomal subunit. Once the primary block to template activity is overcome with neo- mycin the remaining influence of secondary structure on the template activity was demonstrated. 18 5' Terminal Residues of Various RNA Species The most completely analyzed RNA species, tRNA, in most cases yields the purine pGp at the 5' terminus. At least 15 tRNA species have been completely sequenced; others have been partially sequenced (102). A breakthrough in the endgroup analysis of high molecular weight RNA of bacterial origin came with the development of a method for specifically labeling S' ter- minal groups by alkaline phosphatase hydrolysis, followed by enzymatic phosphorylation with polynucleotide kinase and Y - 32F ATP (54). The label at the 5' terminus aids in identifying the terminal group following fragmentation of the molecule. The 5' terminal RNA residue has been found in sev- eral cases to contain a nucleotide triphosphate. RNA syn- thesized E3 yiggg with E. 32;; DNA-dependent RNA polymerase contained predominantly purine triphosphate on the 5' ter- minals (10,11). The nucleoside triphosphate pppG, has been found as the 5' terminus of RNA isolated from bacter- iophage R17 (55), R23 and QB (56), and F2 (57). RNAs from tobacco mosaic virus and turnip mosaic virus have been shown to contain predominantly adenosine bearing a free OH 19 as the 5' terminus (57,58,59). The 5' termini of these viruses are summarized in Table l. The viral RNAs in each case exhibited purines as the 5' terminal residues. TABLE l.--Partial 5' terminal sequences of viral RNA. Source of RNA 5' terminal sequence Reference Bacteriophage pppGpGpGpUp... 96 M82 pPupPupPy... 84 pppPupPupPy... Variant QB + strand PPP(Gp)4(Ap)2(Cp)2... 85 QB (68) pppGpGpGpApUp 99 QB Ea vitro pppGpGpGpGpAp(Cp)2... 86 QB pppGpflumamu mnu madcapcw mononucmumm :H mwwnfisz« mm om mm mod boa moa voa moa lows .maesom lose...mom Road...eoz Eumm among Ammusuasoc mHHmo a mfi>mma mschmx maamo mmsos mg mHHmo ”pun ooummcmxv msououom “omnsuasuv maamo mqom Ansocfioumuv maamo mm mocwwmmmm mocmsqmm Hmcflanmu .m .mowummm.¢zm OURfiOm r .mEchmmHo osuomnmosm Scum dzmu mo moosmsvmm amcflsump .m amauummll.n Manda 23 and nucleosides, for example, contain a variety of ion- izable groups. The presence of these groups permits the relative mobility of RNA hydrolysate components to be predicted at a given pH (62). This knowledge may be utilized in the separation and identification of the var- ious hydrolysate components by electrophoretic or chroma- tographic methods. Electrophoretic separation is based on the net charge of the molecules and the resistance to movement due to the size and shape of the molecule. Therefore the relative mobilities of molecules may be determined by the degree of ionization and the approxi- mate resistance to flow. Chromatographic separation in the simplest form is based on the degree of attraction to either a stationary or a mobile phase of the system. Three classes of ionizing groups are important in separating nucleotides and polynucleotides: i) the pri- mary and secondary phosphate groups, ii) the amino groups of adenine, guanine, cytosine or their derivatives, and iii) the enol groups of guanine, cytosine, or uracil. The effect of the primary and secondary phosphate groups and of the amino groups on migration are most important, since the enol groups have pK values of 9.5 or above. This is 24 significantly higher than the useful values for most nu- cleic acid separations (62). In aqueous solution each of the nucleic acid bases has a unique 220-300 nm spectrum which varies character- istically with pH (63). This absorption of light due to the presence of the conjugated double bond systems of purines and pyrimidines also aids in their identifica— tion (63,64). Cleavage of RNA molecules by any of the hydrolytic processes in which an intermediate cyclic phosphate link- age is formed (chemically or enzymatically), produces characteristic products. Alkaline hydrolysis, for ex- ample, produces a 3',5' nucleoside diphosphate at the 5' terminus, intermediate nucleoside monophosphates, and a nucleoside at the 3' terminus. Interruptions of hydrol- ysis occur when specific ribose moieties are methylated in the 2' position, and results in the production of di- or tri-nucleotides. The different extent to which the 5' and 3' terminals are phosphorylated, compared with the rest of the chain, allows their separation based on charge differences. The oligonucleotides produced by hydrolysis of RNA may be separated by ion-exchange chromatography on DEAE 25 cellulose or DEAE Sephadex. This process is affected not only by ionic interactions between the oligonucleotides and the exchanger, but also by secondary non-ionic inter- actions such as hydrophobic and hydrogen bonding (65). These secondary forces are minimized by the incorporation of 7M urea, although all secondary binding forces are not completely eliminated, especially with DEAE cellulose (66). The nucleoside monOphosphates, dinucleotides, trinucleotides and nucleoside 3',S' diphosphates have net charges of -2, -3, -4, and -4 respectively, at pH 7.8 (37). This charge difference constitutes the basis of separation by batch elution with dilute salt, or by a linear salt gradient. Fractionation of RNA hydrolysate eluted from columns of DEAE cellulose has a practical limit of oligo- nucleotides containing ten residues or less. MATERIALS AND METHODS Expgrimental Materials Wheat (Triticum aestivum, L.) embryo was a gift of Quaker Oats Co., Saint Louis, Mo. Cauliflower (Brassica oleracea, L. var. botrytis) and mung beans (Phaseolus aureus, Roxb.) were purchased from commercial sources. Broad beans (Vicia faba var. Windsor) were purchased from Vaughn's Nurseries. Most seeds were surface sterilized briefly with a 1% sodium hypochlorite solution before allowing them to imbibe water; broad beans required a 35-minute exposure in order to prevent fungal contamina- tion. Escherichia coli W cells were obtained from General Biochemicals, Chagrin Falls, Ohio. Frozen E. 92;; B cells were kindly supplied by Dr. John Boezi. DEAE cellulose (medium) was purchased from Sigma Chemical Co., St. Louis, Mo. Triisopropyl naphthalene sulphonic acid and m—cresol were purchased from Eastman Kodak Co., Rochester, N.Y. 26 27 Other chemicals used were reagent grade and were available locally. Extraction of RNA from Plant Tissue RNA was extracted by one of three methods, depend- ing on the characteristics of the tissue to be extracted. Considerations taken into account were: i) large quan- tities of RNA were needed for detection of terminal resi- dues, ii) noticeable degradation of the rRNA could not occur in the extraction process, and iii) the isolated RNA had to be free of interfering substances. Extraction with Phenol-Phosphate The method of Singh and Lane (37) was modified for the preparation of rRNA from wheat embryo. The plant material (60 gm) was added to 360 ml of 0.05 M phosphate buffer, pH 7.0, and an equal volume of water saturated phenol. This suspension was shaken at high speed on an Eberbach 60 cycle shaker for 20 minutes at room tempera- ture, cooled on ice for 30 minutes, and subsequently 28 centrifuged at 12,000 x g for 10 minutes in a Sorvall re- frigerated centrifuge. The aqueous phase was siphoned off and the RNA precipitated by the addition of two volumes of redistilled absolute ethanol by allowing the solution to stand 24 hours at 4 C. The precipitate was collected by centrifugation, washed two times successively with 80% ethanol, and suspended in l M NaCl by stirring with a magnetic stirrer for three hours. The high molecular weight RNA was collected by centrifugation after allowing the solution to stand for 24 hours. It was washed two times with 80% ethanol and again suspended as before. The resulting high molecular weight RNA was washed with 80% ethanol, dissolved in 0.05 M phosphate buffer, centrifuged, and examined spectrophotometrically before running other tests for integrity of the material. The material noted above that was soluble in l M NaCl was precipitated with two volumes of absolute ethanol and saved fer isolation of sRNA. Extraction with Phenol and Sodium Dodecyl Sulfate This method of preparing ribosomal RNA by direct extraction was used on cauliflower florets and other fresh 29 plant tissues. The procedures of Halloin (60) as adapted from McCarthy and Hoyer (67) were used. The tissue was homogenized in two volumes (w/v) of water saturated phenol and two vslumes (w/v) of solution A, which consisted of 5%ssodium dodecyl sulfate, 0.28 M lithium sulfate, 0.1 M sodium acetate, and 0.001 M MgCl pH 5.0 (60,67). After 2: siphoning off the upper aqueous layer which separated by centrifugation, the phenol deproteinization was repeated. Following the addition of two volumes of absolute ethanol, nucleic acids which precipitated were collected by cen- trifugation. The original method of separating low molecular weight RNA and DNA from high molecular weight RNA was modified in these experiments by using two washes with 1 M NaCl rather than using 3 M sodium acetate. The precipitated nucleic acids were suspended in 200 m1 of l M NaCl and stirred with a magnetic stirrer for three hours before allowing to stand overnight. High molecular weight RNA was again collected by centrifugation, and washed two times with 80% ethanol. 30 Extraction with Phenol and Naphthalene 1,5-Disulfonate The method of Hastings and Kirby (68) as modified by Loening (69) was used for extracting RNA from germi- nated tissue. Plant tissues were homogenized in a Waring blendor at 70% line voltage for one minute in five times their weight of grinding solution containing 30 mM Tris- HCl (pH 7.5), 0.15 M NaCl, 0.5% sodium naphthalene 1,5 disulfonate (NDS) plus an equal volume of phenol-cresol solution. The phenol-cresol contained water saturated phenol (650 ml), m-cresol (70 ml), and 8-hydroxyquinoline (0.5 gm). The homogenate was centrifuged at 3,000 x g for 15 minutes in a refrigerated centrifuge. The aqueous layer was removed and made 1% in sodium triisopropyl naphthalene sulfonate and 6% in sodium 4-aminosalicylate. The aqueous solution was homogenized with an equal volume of phenol-cresol and recentrifuged. The interphase and aqueous layer were removed and made 0.45 M in NaCl, homogenized with phenol-cresol and recentrifuged. RNA was precipitated from the resulting aqueous layer by add- ing two volumes of absolute ethanol and allowing the solution to stand overnight in the cold. Low molecular 31 weight RNA and DNA were removed by treating with l M NaCl, as in the previous RNA extraction methods. Tests for Purity and Integrity of the Isolated RNA Optical Purity Spectra were taken with a Beckman DB spectrophoto- meter in the range from 220-300 nm. The ratio of the readings at 260 nm/280 nm ranged from 1.95 to 2.15 for purified RNA. Estimations of RNA were based on the as- sumption that 1 mg of RNA yields 24 optical density units (O.D.) at 260 nm. Sucrose Density Gradient Centrifugation Samples of RNA (2 mg) were routinely sedimented on linear 4-20% sucrose gradients prepared in either saline acetate (0.05 M NaCl, 0.05 M sodium acetate, 0.001 M MgCl pH 5.3), or saline citrate (0.015 M NaCl, 2! 0.015 M sodium citrate, pH 7.0). Centrifugation was per- formed at 53,000 x g with a Spinco 25.1 rotor for 32 16-17 hours at 2 C. The bottoms of the centrifuge tubes were punctured, and seven drop fractions collected which were then diluted with 1.5 ml of the buffer used to pre- pare the gradient. The samples were read on a Beckman DB spectrOphotometer. Polyacgylamide Gel Electrophoresis Samples of RNA were examined for structural in- tegrity prior to alkaline hydrolysis. Purified acrylamide and bisacrylamide were used for preparation of the gels. Gels used in this study contained 4.6% acrylamide since both high and low molecular weight RNAs could be distin- guished at this concentration. Acrylamide and bisacryla- mide (respectively, 4.6 and 2.3 gm/lOO ml buffer solution), were mixed and degassed at room temperature under reduced pressure. NNN'N" tetramethylethylenediamine (0.033 ml) and 10% (w/v) ammonium persulfate (0.033 ml) were added per gram of acrylamide present. The solution was mixed, rapidly pipetted with a propipet into 5 cm vertical tubes to within 1.5 cm from the top and water layered over the solution to ensure a flat gel surface. The buffers used contained: 0.04 M Tris, 0.02 M sodium acetate, 0.002 M 33 sodium EDTA; acetic acid was used to adjust the pH to 7.8 at S C. The sodium acetate was added to maintain the secondary structure of the RNA (70). Current was applied for one hour to remove excess polymerization catalysts from the gels prior to application of the samples. The RNA sample, 0.5 O.D. units, was dissolved in 50 micro- 1iters of the buffer containing 5% sucrose and layered over the gel. Electrophoresis was carried out at about 5 C in a cold room, with up to 10 v/cm applied. The amounts of the respective nucleic acid species were deter- mined by reading the gels directly on a Gilford spectro- photometer with an attachment for reading gels. Dische Diphenylamine Test for DNA One ml of .l% nucleic acid solution was mixed with two m1 of diphenylamine reagent and heated for 10 minutes in a boiling water bath. The optical density was read at 600 nm and compared with a standard curve relating O.D. to micrograms of DNA (71). 34 Alkaline Hydrolysis RNA was dissolved in a small amount of water and hydrolyzed in 0.3 N KOH (one ml/mg RNA) for 48 hours at 37 C. The hydrolysate was cooled on ice before adjusting to pH 7.0 with a sulfonic acid, strong acid cation ex- change resin (Dowex 50 W-H+, or Rexyn 101). The neu- tralized hydrolysate was filtered with a sintered glass funnel and the resin thoroughly rinsed with distilled water before applying the diluted RNA hydrolysate to a DEAE cellulose column. Chromatography and Electrophoresis Fractionation of RNA Hydrolysate by DEAE cellulose (Formate) Ion Exchange Chromatography, Using Batch Elutign with Increasing Concentrations of Tris-formate DEAE cellulose was prepared by allowing the dry powder to settle in 1 N NaOH, thereby minimizing the occlusion of air. Fifteen ml of alkali were used for each gram of dry adsorbent (72). The cellulose suspen- sion was poured on a coarse sintered glass funnel and 35 washed with l N NaOH until the filtrate was colorless. It was then washed successively with 1 N HCl and 1 N NaOH and distilled water. Fines were removed by suspending the cellulose in large volumes of distilled water and allowing it to settle for one hour before decanting the supernatant solution. The pH of DEAE cellulose was ad- justed by adding the buffer with which the column was to be equilibrated. DEAE cellulose was diluted with buffer until a suspension was made which was thin enough so that the rising surface of the bed could be seen while pouring the column. After filling the column to the level desired, 0.025 M Tris-formate, pH 7.8, was passed through it until equilibrium was reached. This served also to compact the column by gravity, before the neutralized dilute RNA hydrolysate was applied. Nucleosides were collected during the loading of the column with the RNA hydrolysate and during subsequent washing with 0.025 M Tris-formate buffer, pH 7.8. Nucleo- side monophosphates and dinucleotides were eluted with 0.085 M and 0.17 M Tris-formate buffer, respectively. The buffers of the last two concentrations were made 7 M in 36 urea to minimize non-ionic interactions. Trinucleotides and nucleoside 3',5' diphosphates were eluted with l M ammonium formate, pH 9.2, or with l M pyridine formate, pH 5.4. Chromatographyyof Nucleosides and Congeners from RNA Hydrolysate on DEAE Cellulose (Cl‘) Using a 0.0-0.3 M NaCl Linear Gradient DEAE cellulose, prepared as above (materials and methods), was suspended in 1 M NaCl solution before pack— ing columns. The packed columns were then washed with 4 M NaCl until the eluate produced a reading of zero at 260 nm. It was then rinsed with distilled water until the excess salt was removed (as indicated by a negative test with AgNO3). The column was equilibrated with 0.02 M sodium acetate (pH 5.4) containing 7 M urea, beforeapply- ing the diluted RNA hydrolysate. The elution was effected with a linear gradient of 0.0-0.3 M NaCl in 0.02 M sodium acetate (pH 5.4) buffer at a rate of 60 ml per hour. 37 Chromatography by Sephadex Gel Filtration Sephadex was poured by allowing a measured amount of dry beads to swell in water (e.g., for G-lS Sephadex the swelling time was five hours), prior to pouring a column. A thin slurry was prepared and added in a single batch to a 2.5 x 45 cm column which had been filled to one-third of its volume with 0.025 M phosphate buffer, pH 7.0. Elution of nucleosides was effected with the same buffer at a flow rate of 20 ml per hour. Desalting Samples containing excess inorganic salt from being eluted from columns or chromatographic paper were desalted by one of two methods. The first was by adsorp- tion of the sample to acid-washed Norit-A charcoal in water solution. Each sample was washed 14-18 times by mixing the slurry with a Vortex mixer and centrifuging at 10,000 x g to pellet the charcoal. The samples were eluted from the Norit A charcoal twice with eight ml of 80% ethanol containing 1% ammonia, and once with eight m1 of 50% ethanol also containing 1% ammonia, the charcoal 38 being pelleted by centrifugation. The ethanol eluates were combined and the sample dried under reduced pressure at 40 C. The second method used was column desalting, using DEAE cellulose (carbonate). Columns (1.0 x 2.0 cm) were packed with DEAE cellulose suspended in 1 M ammonium car- bonate and subsequently washed with 2 M ammonium carbonate. Excess salt was removed by rinsing the column with dis- tilled water. The samples were layered onto the columns in dilute solutions containing less than 0.05 M salt, before washing with 50 m1 of 0.02 M ammonium bicarbonate. Elution was effected with 2.0 M ammonium carbonate. The _eluate was evaporated to dryness under reduced pressure at a temperature less than 30 C with a flash evaporator. Ammonium carbonate was volatilized by repeating the latter step, usually three times. Chromatography on Dowex-l Anion Exchangngesin Columns of Dowex-l (X 2, 400 mesh, Cl-) were packed with the suspended resin in l M NaCl and subse- quently washed with 4 M NaCl in 0.01 N HCl to remove 39 residual substances absorbing at 260 nm (54). The columns (0.6 x 15 cm) were washed with 0.01 N HCl to remove re- sidual NaCl. Nucleoside 3',5' diphosphate samples were applied to the resin in 0.01 N HCl solution and elution was effected by a continuous gradient of 0.0-0.3 M NaCl, also in 0.01 N HCl (pH 2.0). Nucleoside diphosphates were collected at an elution volume characteristic for each nucleotide. The columns were reused, but the washing procedure was repeated and the resin backwashed before each use to prevent compaction. Paper Chromatography All paper chromatography was of the descending type on Whatman no. 1 or 3MM filter paper unless other- wise indicated. Chromatograms were developed in the following solvent systems: System A) ethanol 75, water 25, paper impregnated with 10% ammonium sulfate solution (73); system B) l-propanol 11, ammonia 7, water 2; sys- tem C) saturated ammonium sulfate solution 79, 0.05 M phosphate buffer 19, isopropanol 2; system D) isopropanol OH 10, H BO , 20 (74). 70, concentrated NH 3 3 4 40 Systems B and C are listed in the 1967 catalog of Schwartz BioResearch Inc., Orangeburg, N.Y. Paper Electrophoresis Separation of nucleosides and congeners was achieved in a Beckman RD-2 electrophoresis unit. The solvent system of Chandra and Varner (75) as modified by Becker (76) was used. It contained 0.01 M EDTA (tetra- sodium salt) and 0.34 ml of pyridine per liter, and was adjusted to pH 3.5 with glacial acetic acid. A constant potential of 400 volts was applied across Whatman 3MM paper (3.0 x 30 cm) to achieve separation. Isolation of Nucleotide Phosphotransferase from E. coli The enzyme was isolated from frozen cells of E. 92;; B and E. EQli.W° The latter had been grown on a high peptone medium and harvested in late log phase. Methods patterned after those of Brunngraber and Chargaff (77) were used to purify the nucleotide phosphotransferase. All operations were carried out in the cold. Frozen cells 41 were ground with alumina and 0.1 M acetate buffer (pH 6.0) and the homogenate centrifuged one hour at 20,000 x g. The supernatant solution was made 30% of saturation in ammonium sulfate and the precipitate resulting from cen- trifuging at 20,000 x g for 30 minutes was discarded. The supernatant solution was then made 90% saturated in am- monium sulfate and allowed to stand one hour prior to cen- trifuging at 20,000 x g for 30 minutes. The resulting precipitate was dissolved in 0.001 M sodium acetate buffer (pH 6.0) and dialyzed against two changes (one liter each) of this buffer. The dialyzed partially purified enzyme from above was applied to a DEAE cellulose column (2.5 x 20 cm) pre- viously equilibrated with 0.001 M sodium acetate (pH 6.0). Elution was effected with a continuous gradient of 0.005 M to 0.3 M sodium acetate pH 6.0, at a flow rate of 20 ml per hour. The eluate was collected in ten ml fractions. The enzyme was assayed at each step of the purifi- cation. Aliquots (0.1 ml) were tested for total protein (Lowry-Folin), phosphatase (formation of p-nitrophenol from p-nitrOphenylphosphate), and for nucleotide phos- photransferase. Assay for the latter enzyme was based 42 on the transfer of phosphate from p-nitrophenylphosphate to uridine. Paper chromatography or paper electrOphoresis was used to determine uridine monophosphate formation. RESULTS Isolation of RNA from Plant Tissues The procedure for isolating RNA was developed to the extent that consistent quantitative yields were ob- tained from a given plant source (Table 4). In a typical preparation, 200 grams of cauliflower florets yielded approximately 180 mg of RNA. However, variation in RNA yields did occur between "batches" of cauliflower. As indicated in Table 1, RNA yields from two sources of wheat embryo were approximately 300 mg/60 gm of tissue. TABLE 4.--Yields of rRNA from different plant tissues. A260 nm RNA Pla t i s e Am t T f ' l t' n --—-- n t s u oun ype o 150 a 10 A280 nm Yield Cauliflower florets I 200 gm phenol-soln. A 2.10 181 mg Cauliflower florets II 200 gm phenol-soln. A 2.11 181mg Wheat embryo I 60 gm phenol-phosphate 2.05 310 mg II 60 gm phenol-phosphate 2.05 319 mg III 60 gm phenol-phosphate 2.12 306 mg 43 44 The molecular integrity of the extracted RNA was determined by means of sucrose density gradient centrifu- gation and acrylamide gel electrophoresis. Analysis of fractions collected from the sucrose density gradient tubes (A260 nm), following centrifugation, indicated a bimodal distribution of rRNA for both cauliflower and wheat (Figure 1). In each case, the area of the profile of the 258 peak was approximately twice that under the 168 peak. RNA used for the analysis of terminal groups contained neither DNA nor low molecular weight RNA as evi- denced by sedimentation profiles or by acrylamide gel electrophoresis (Figure 2). Negative diphenylamine tests also indicated that the RNA used was free of DNA. Degra- dation of the RNA species sedimenting in the 258 region was observed when the phenol—phosphate solution was used to extract tissue by prolonged shaking (50 minutes). No such problem occurred when the time of shaking was de- creased to 20-30 minutes. 45 .A .8~ .6‘ / g ,4‘1/ <3 ’/ KC 02" N a} 8 B 3 .4«» H O :3 3. fl . 02' .l - T I U I I F l 10 30 50 70 Fraction number Fig. l.--Separation of high molecular weight RNA species by sucrose density gradient contrifugation. Sedimentation profile of 2 mg of wheat embryo rRNA (A) ex- tracted with phenol-phosphate, and cauliflower rRNA (B) extracted with phenol-solution A. Purified RNA was centrifuged at 53,600 x g for 17 hours on 4-20% linear sucrose gradients, bottoms of the centrifuge tubes were punctured and seven drop fractions collected. Fractions ‘were diluted with 1.5 m1 of the appropriate gradient buffer (see Materials and Methods), and the absorbance at 260 nm determined. 46 .75" 258 ‘50— 168 u L. fie 4 A 260 nm I I I 1.5 3.0 4.5 Relative mobility (cm) Fig. 2.-—E1ectrophoresis of rRNA on 4.6% polyacrylamide gels. Twenty ug of RNA prepared from cauliflower florets by the ‘phenol-solution A.procedure was applied in 50 ul of buffer containing 5% sucrose. Electrophoresis was carried out at 5 C with 10V/cm applied for 3 hrs. Under these conditions 258 RNA just enters the gel. 47 Isolation of Nucleotide Phosphotransferase The isolation of nucleotide phosphotransferase was first attempted from E. 32;; B (Materials and Methods, p. 40). Only a trace of nucleotide phosphotransferase activity was detected. However, this was sufficient to warrant further investigation of the enzyme from another source. The enzyme isolation, with minor modifications, was repeated on E. 39;; W. The results of this isolation and purification are summarized in Table 5. An increase in total phOSphotransferase activity, concomitant with a decrease in hydrolase activity is observed as purification progresses. Thus it appears that the hydrolase activity, when present, counteracts the effectiveness of phospho- transferase activity. The elution pattern from DEAE cellulose (acetate) shows two peaks having hydrolase, but no transferase activity (Figure 3). A very sharp peak exhibiting trans- ferase activity follows closely the first phosphatase peak; some residual hydrolase activity is eluted in the peak nucleotide phosphotransferase fractions. The effect of this hydrolase is counteracted by the inclusion of a 48 .Hoon\cwmuoum m8\wmooponm m2: m0 mmHoE 1 ca wufl>fluom oamwommmt xvh v.mm mm.N NH no.0 om mmOHSHHmUIm4MQ .HHH XN m.mm mo.o mow vm.o om GOHMUMHM mucuasm Eowcossd .HH v.HN v0.0 0mm mm.o ov uomuwxm mvfiuo .H sue>euo< «mun>euu< cod mos Hus E A me .u .m. d Hence oemeommm less Hmuoe AH \.m V lass muw>fluo¢ mopm coHDMOHuflHom cflmuoum mEsHo> mmmumgmmocm wmmummmcmuuonmmogm opfluowaosz .3 Haoo um Eoum ommummmcmuuosmmonm mpfluooaooc mo GOMumonwnom can coflumaomHll.m mqmde 49 (uMoles of UMP/m1) m m m H r~ m E‘ .4 44 E vi 0 ‘3 m u 4.) .5 m 0 ° a _fi tn m w 4 E C) i V 'c 40-, 050-4 9 m 2 m H a: m m 1* 6 rs " +! -H — m +1 - a 8 u 8 I; .25— m c 1 o n: 32 Q + O I 4 + O y . If) I l r l I 10 20 30 '40 Fraction number .Fig. 3.——E1ution of nucleotide phosphotransferase and phos- phatase of E. coli W. from DEAE cellulose. Aliquots of 10 ml fractions (Step III) enzyme eluted from a DEAE cellulose (acetate) column by a linear acetate gradient (0.005- 0.3 M, pH 6), were tested for protein (- -) conc., phosphatase (+ +) activity and nucleotide phosphotransferase (o 0) activity as described in the Materials and Methods. Maximum nucleotide phos- photransferase activity occurred at fraction 32, with a bimodal dis- tribution of phosphatase activity, maximum activity being found at