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THE UNIVERSITY OF ALBERTA 


140 PHENYLALANINE INCORPORATION BY 
WHEAT-SEEDLING CYTOPLASMIC RIBOSOMES 


by 


(Cc) EDWARD. B. L. TUCKER 


A TESTS 
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES AND RESEARCH 
IN PARTIAL FULFILMENT QF THE REQUIREMENTS FOR THE DEGREE 
OF MASTER OF SCIENCE 


DEPARTMENT OF PLANT SCIENCE 


EDMONTON, ALBERTA 
PALEee ane 








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THE UNIVERSITY OF ALBERTA 


FACULTY OF GRADUATE STUDIES AND RESEARCH 


The undersigned certify that they have read, and 
recanmend to the Faculty of Graduate Studies and Research, for 


140 phenylalanine Incorporation 


acceptance, a thesis entitled, 
by Wheat-Seedling Cytoplasmic Ribosomes", submitted by 
Edward B. L. Tucker, in partial fulfilment of the requirements 


for the degree of Master of Science. 










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ABSTRACT 


V4 e_phenylalanine incorporation was induced by homogeneous 


preparations of wheat-seedling cytoplasmic and chloroplast ribosomes 
which had been isolated by zonal centrifugation. Wheat embryo 


synthetase enzymes in the presence of stripped yeast tRNA and a 


C-phenyl- 
alanine produced Mae -phenylalany] tRNA which was used in the incorpora- 
tion systen. The incorporation by cytoplasmic 79S ribosomes required 
high concentrations of ribosomes, high concentrations of magnesium, low 
concentrations of tris-HCl buffer pH 7.6, and was complete after 45 


minutes incubation at ave This transfer of 4 


14 


C-phenylalanine fron 
C-phenylalanyl tRNA into protein was not dependent upon added energy 
factors. Incorporation by both species of ribosomes was inhibited by 
puromycin, only the chloroplast ribosomes were inhibited by chlor- 
anphenicol, and neither species of wheat-seedling ribosomes was 
inhibited by cycloheximide. The cytoplasmic ribosomes dissociated into 
subunits which would reassociate to form the parent species. Puromycin 
pretreatment of ribosomes resulted in complete dissociation. The 
reformed parent species would not induce polyphenyialanine synthesis. 
The results led to the conclusion that the honogenization and separation 
precedures had cleaved the polysomes into monosomes which contained 


not only mRNA fragments, but nascent protein as well. 



























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_ a ftew an wtatong dasozan Jud ,edvampent 


ACKNOWLEDGMENT 


The research described in this thesis was performed under 
the supervision of Dr. Saul Zalik. His guidance, wisdom and assistance 
was given willingly throughout the study and in the preparation of 
this manuscript. 

I wish to acknowledge my colleagues for helpful criticism 
and direction during the research; and Dr. Sara Zalik (Department of 
Zoology) for the use of equipment. 

Appreciation is given to my wife, Lindis, for her 
encouragement and helpful discussion, 

The financial assistance was provided by the University 


of Alberta and the National Research Council of Canada. 











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7 


TABLE OF CONTENTS 


Page 
UU EC VON Meets csads Sieh irik tees @ ass xe eo od 8 sities hd sretin wt Or aee ] 
Oe Rem Vil © abet east st) aoel sos ee gin d-s-4.0 8184 pe oa efe wert aint > Gina oles 3 
eerie ChonT SH ROMEEFOLENTESVIICICS 1S v. cc cleo cloves een eliie-eicit ons 3 
Pree, DC COUU Ot meererrats etch a ay say's, sca pire ace sin aerate ole) 3 
BPEL 1d la colo ene user N cst na che eve lare's scaie sels ¢ e's cisfh ere mie a eca'e 5 
Cee OUG d UO tee rete ac cty vicietrue rs ee his a ec cis 7 
DLE LU Tica OUMENeiistrst. c 9% gots > cele per etree et els wie ee rrare ese 8 

II. Differences between Chloroplast and Cytoplasmic 
Prove eo Vines | Zt UM GOUD LCAae taigttee cenit wetn enc + << 6s 9 
Li eeaiinoeAGldsincoroonaciOn DY Plant SYSLEMS, 9% 7... «ess se 1 
A. Amino acid incorporation by wheat 80S ribosomes ., 12 
B. Amino acid incorporation in other higher plants .. 17 

C. Amino acid incorporation by chloroplasts and 
lu Olao live scam yal Ds CINE S mative rate amace ie rer 8 cle tet ses aia! 6 20 
1. Amino acid incorporation by chloroplasts ....... 20 

2. Amino acid incorporation by chloroplast 
ADU S ANG Semmerere taste as atete sche cia grate are coher eht el ete at nis i 22 
IVa BLISSUCTULlUNsULebUKaryOte RI DOSUNGSE tps... cc cnc nie siecs es 
A. Dissociation of ribosanes by high salt 

COrce urd GLO smamietrte ct abet tata cie tea wee seas © erect e 23 
B. Dissociation of ribosomes by dissociation factors . 23 
CePuronVGciieused LO SLinUlate™ GiSSOClatiON ls ncn as). © 28 
PATER LAU SS AN UaM bl MOD Siiiestts pista: se ets sietete’s sles yn, ciuticle cin nia le sl4 5! ess «8 4/0 30 
[ee NCUid Cr) G MEP ees ert ats alyielsin nts acacys nee Oates wists e pai ncwe 30 


LT EGU DCT Cesta es ores ra Ne eee Oe a aige cee ore sear arate 31 


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Haan acu isin ae 
ad 


Checking Chemicals used in the Incorporation Reaction. 
ee LACTIS, <P isn eee oer eee 


B. Creatine phosphokinase 


9 9 92.8.0 ©). 0 @ 0 6 0 Op C.e.9 9 © © 0 ¢ © 6 8 6 6 


C. tRNA check 


eee: (pie s).0 6 8. 5 8. Pe. 8. £58. 0.0 0 9 Rete  g © 9 2 2 6 6 68 8 € 8 9 © 6 6 


Chenical Preparation 


oor ereer rere ere eer eer ere eee reer eereeeevee 


A. Preparation of bentonite 


ower ere eee were ee ere ee em ee nese 


Bend LOSCULITGL IG mmraee prere ie ate gen Or er eR a 
CUR OLS Ciel aLONmO MeDiEllO Way Gir ai eile te we dcten teria 
D. Preparation of chromatographic matertal ........... 
ECC LICR EU eet tins Si ais ¢ Me UACE ERD Gees S eatece se 
CES COU OMSK EG OD Cir cic dee suis. ginid tats sheik ofa, sats saale 010 wes vis 


E. Treatment of sucrose with diethyl pyrocarbonate 
prem Li © | OMMreretet sista dtetrcerg cesta ste 5 ara,s% ele ee eval e catheters ere 4 
Preparation of and Procedures with Ribosomes ........ 
PE CCU LeM id \MMPM ae a ritate esis crs ey sen ke Cartes cine aes 
SL SU La CIO MEOIMRTOUSONES Mare grils ccinip piers cc aisle sete aiace lacs 
TERT DOSONeSEOr ZONaLesehardc1 ON sata ce sci csece’s «eas 
Cap RiDOSOMmeS WILMOUL-ZONd! S@ardalion ...2ecrsess« 


C. Separation of ribosomes by zonal centrifugation ... 


D. Dissociation of ribosomes and separation of 


SU DUNE Summer un tir aercitee ettre vis iysierae Reeetete ahersie seetaeainas ene ie.s 
Bemeova nc ano sDUCKALECeENL Yt TUdd CHOM a. leteeicias suis «i9ia'e-s 
POPLULONVCLUMLEE Aue DOL aGIOUSUIICS (.<icre sawaa ceess «ss 

1. Preparation of ribosomes for dissociation 
AOWN TOOL UUCKGLS) Gates sane aes cele acaeuipe a) ses ers 


2. Preparation of ribosomes for zonal dissociation. 


G. Estimation of RNase in prepared ribosomes ........ 
He @OCOIEN Lot OMOVAIUGS. “vccguesnt ys s Mey wees areata 
de SR@SC UG sce De eee ie di atime wets Co et ee 


2. Calculating sedimentation EU ie ree Serene 


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iio BP OLedUreceeLOVMCOePOY AL] Ol. vss o0s soe ss age selene wese 
A. Preparatton of synthetase enzymes ........cscecsees 
B, Preparation of ribosomes from wheat germ .,........ 
C. Preparation, of 4c phenylalany] CRNADBeGe et .* SR eas 
LPMELELCOCDOYe Witimncac L 1OMMMy 4 wit es gates ote gic actress ae 
ome CEDORG GhOLMU MME OGLOE 0s; gulp 'g sare pain sas cree steeren ee 
RESULTS AND: DISGUSSIUNSSray. and.e ceva santos . fort ener esc 03 : 
ee SOLACLONS OTR DOSONGSaar, as, Se TOON EE Sameera 
A. Characteristics of zonal separated ribosomes .,... 
B. Ribonucleases in zonal separated ribosomes .,,..... 
II. Phenylalanine Incorporation by Zonal Separated 
RIDOSONeSeUs INOmdal Gans Terao YS LOMem, yigutcmie far tea er 
Biwi DOS OMNES CONCEMCAL1 ONT ©. irre ely ste sce acaltee ine ales es 
Be weLNCUDALIONECIMNG TOF aNCOFDOratl ON wncinaiaes se < sees « 
(ee bUrreneer coc benim NCOVDOY ALTON er fy a ose ware sss write 
D. Dependency of incorporation upon added energy 
ROCUOVS oem ire ciel gheta seus nee © ie seats Peer Cor 
Eber Cem diecOl canind bLON ame c iis ce oe Pee Oe 
F. Rate of incorporation as a function of magnesium 
COME CMU tral) Ol mmepeee eter eens a sie ete a tee iiiaees fevers slats ty ecstatic 
G. Effect of inhibitors of protein synthesis on 
TIGL Om aia Ol emmmme neattteisrettc sr sincere sontetaeleig 00 ie Se verdaa es ely 
H. Species specificity of tRNA and synthetase enzyme . 
III. Dissociation Characteristics and Incornoravion with 


Zonal Separated Ribosomes ,......., Ae tr Pca RCC 


Dime 1 aS Game etter cs sie hive a ane pie os aE ee re 
Mee UTSSUCTALRON MMe crip e tee ts cua ns ak vere w me Kors ue 3 
2. Incorporation with recombined monomers een 
B, Puromycin otP. bw. £86. 8 eS ee PTA ted Ne) Re MS Ae O14 £2 Bee SGC e O° RS Pe 88 2) 
Fe eee DIS SOCTACA ON Maras sateen Care chat sas om etd 


2. Incorporation with recombined monomers ...... 


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B. Incubation time for NHC] washed ribosomes ....... 92 
C. Dissociation and incorporation of ribosones fron 
Dlantseopedi tT Tenindsages#ey, .s% APES Ae Rh Can ae Ce 94 
D. Conditions for dissociation of ribosomes from 
SPOECAVMO UMD CNUSM iene: « ~ air eit oir oid aims s oe aoe 99 
E. Reconbination and phenylalanine incorporation 
ee SUES GARR AM ia AN POR ane ne nicroes: 106 
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Table 


LISTaOF sl ABLES 


Effect of buffer on incorporation of 140 oheny1- 
alanine by cytoplasmic wheat leaf ribosones 


Requirement of energy factors for incorporation 
by cytoplasmic wheat leaf ribosomes ............ 


Effects of chloramphenicol, cycloheximide and 
puronycin on incorporation by 70S and 80S 
FIDOSOT es mi COleWied tas CAVES Ae. he as ae sos ce cele 


Incorporation by subunits of high salt dissociated 
SUS svied emled GelhbOSONCSEme rT. 2.0.11 cite cis ict ce. 


Incorporation by subunits of high salt dissociated 
80S wheat leaf ribosomes which had been pretread- 
SW Lila OLICONY Cal TMM ce Meas action tots cats ue fols sb cadiede io <oeeesete 


Factors required for incorporation by washed 
WEA Lealic dla DOSONC Sie an hiusueceaie eeiii als noe wieatiew:« 


Effect of addition of partially purified super- 
natant factor on the incorporation by NH C1 
WaShedsWhedlealPateriDOSOMNGS Boas ass ccs esse see sae 


Incorporation by wheat leaf ribosomes fran 
seedlings of varying ages .,.,. A ae ENS Sgr RRA lee 


Incorporation by subunits of high salt dissociated 
(300 m KC1), 80S ribosomes obtained from 3.5 
day-old wheat seedlings and pretreated with 

DU RON Y.CAL msatea tie sfe-pcs ceo al crp nate erevaree ots eyes He ora 


Page 


66 


67 


75 


80 


87 


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92 


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111 


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12 


13 


14 
15 


LIS TOR SF IGURES 


The mechanism of protein synthesis ..,,..... feiss 


The structural relationship of ea to an 
amino acylated tRNA ......... Penn a ak eer 


Analytical ultracentrifuge pattern of leaf 
ribosomes. from Manitou wheat ",..9,....02..6.<..- 


Zonal separation of wheat leaf ribosomes ....... 


Analytical ultracentrifugation pattern of zonal 
separated 70S and 80S ribosomes fron wheat leaves 


Assay for RNase on wheat leaf ribosomes ........ 


Zonal separation of wheat leaf ribosones through 
ceUEE Urea ceUsuu tL hC ramet rr Nis oor tees mts. went aye 


Activity versus concentration of 80S wheat leaf 
EIDOSONG SUM Bree scl r dt a ta al dk eee date te ede 


Wosphenylalanine incorporation by cytoplasmic 
wheat leaf ribosomes versus time .,......ee0- he 


Effect of tris concentration on the phenylalanine 
incorporation by cytoplasmic wheat leaf ribosomes 


Effect of magnesium concentration on M40 pheny1- 
alanine incorporation by the 80S wheat leaf 
PIBCSONESIC HON, VRINNS. OF i ant, MEPS, FI DSS eS... 


Comparison of the chloroplast ribosomal rate of 
incorporation as a function of magnesium 
concentration with that of the surrounding 

CY COP LasnriGri DOs ONES! OT MWNEAT S «cert sect tat alate! ake 


Analytical ultracentrifugation pattern of 80S 
wheat leaf ribosones suspended in Buffer IV 


High salt dissociation of 80S wheat leaf ribosane 


Puronycin stimulated dissociation of wheat leaf 
CYCLOP TAStC Mi DOSONGS= =... ar a Pigs relarieats eiaca ate aie 


Page 


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Figure Page 


16 Effect of various concentrations of KC] on the 

dissociation of puromycin treated 80S wheat 

LES tem sChGGmene Weer ti sk, te eee Pies rene 84 
17 Zonal dissociation and separation of 80S wheat 

leaf ribosomes pretreated with puromycin ....... 86 
18 Reassociation of subunits of wheat leaf cytoplasm 

cytoplasmic ribosomes ...,..... SBR nae iene 88 
19 Sedimentation values of ribosanes prepared by 

homogenizing wheat leaves in Buffer III. ........ a7 30 
20 40 phenylalanine incorporation by washed wheat 

leche DOsOlesmVercUS aU IMGscser ai ai, eet lt ba 93 
21 Analytical ultracentrifugation conparison of 

ribosones prepared from wheat leaves of different 

DUES eS MAM ee tore ue itean ae ee Ce eee er Pena, 96 
oe A comparison of the ability of ribosomes, fron 

different ages of wheat seedlings, to dissociate 

Teil dace lee see a eS Re Tee eee 98 
23 Puronycin stimulated dissociation of ribosomes 

prepared fron 3.5 day-old wheat seedlings ...... 101 
24 The dissociation effect of various concentrations 


of KCl on the ribosones fron 3.5 day-old 
wheat seedlings, treated or untreated with 


DUT ONy.ci meme te ee Sar ial ete a ner ae iter 
25 Sedimentation values of NH,Cl washed ribosomes 

and their subunits from 3, g day-old wheat 

SENG See ETI CACC Es eae 105 
26 Zonal dissociation and separation of ribosones 

froneashedaysoldyseadiings chlerenlest.and cee’ 107 
27 Reassociation of and cation effect on ribosome 


subunits from 3.5 day-old wheat seedlings ...... 110 


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INTRODUCTION 


Protein synthesis in eukaryotes is thought to occur by a 
system generally similar to that of prokaryotes. Dissimilarities in 
their ribosones and in the initiation of protein synthesis by then 
have been reported. Leaves of higher plants contain abundant amounts 
of the two classes of ribosomes in the same cell. ‘Therefore, despite 
the difficulties associated with sorting out homogeneous ribosome : 
classes, they afford a unique opportunity for comparative studies. 

The ribosomes of the plant cytoplasm are of the eukaryotic 
class, while the ribosomes found in the chloroplast and mitochondria 
have characteristics similar to those of prokaryotes. These three types 
of ribosones are apparently capable of synthesizing proteins. Lately, 

a great deal of research evidence has confirmed that the ribosomes found 
in the cytoplasm are different from those found in the chloroplasts 

or mitochondria. Various methods used to obtain relatively-hono- 
geneous preparations of ribosomes have included organelle isolation, 
plant etiolation and the use of seed germ. A different approach in 
studying protein synthesis of the individual types of ribosanes involves 
the use of specific inhibitors of protein synthesis. In the present 
study the approach was to separate the chloroplast and cytoplasmic 
ribosomes fron an heterogeneous mixture by means of zonal centrifugation. 
Using this method relatively-large quantities of chloroplast and 
cytoplasmic wheat leaf ribosomes were obtained and the intactness of 
these was demonstrated by their ability to incorporate 14 0_phenylalanine. 

This study was undertaken to characterize the general 


features of an incorporation systen containing these ribosomes. 


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Experiments on the factors required for incorporation, the effect of 
protein synthesis inhibitors, and ribosomal dissociation and reassocia- 


tion were performed. 


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LITERATURE REVIEW 


I. Mechanism of Protein Synthesis 


A. Introduction 


Although the procedure involved in the organization and 
formation of a protein is a dynamic process, it is often divided into 
three steps--initiation, elongation, and termination--for ease in 
comprehension. This section of the literature review is similarly 
divided; however, Figure 1 attenpts to show protein synthesis in a 
more operational state. 

Boulter (1970) states that "most of our knowledge of the 
mechanism of protein synthesis is derived fron experiments using 70S 


microbial ribosomes, particularly those of E. coli". This portion of 





the literature review, therefore describes the process with data obtained 
fron bacteria rather than from plants. As is discussed tater in this 
review, protein synthesis in eukaryotes is thought to occur by a mech- 
anism similar to that for prokaryotes. However, a statement made by 
Mahler and Cordes (1966) seans relevant here. "The very fact that a 
relatively simple, unified, and self-consistent model appears to have 
energed from these efforts might suggest that perhaps some of the gen- 
eralizations are overly facile and that once the problems are rexanined 
in search for quantitative rather than qualitative agreement, inconsis- 
tencies may emerge that may gnaw at the very foundation of the 


magnificient edifice”. 
























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B. Initiation 


The mechanism of protein synthesis has been comprehensively 
described in reviews by Boulter (1970), Kaji (1970), Lucas-Lenard and 
Lipman (1971) and Boulter, Ellis and Yarwood (1972). It is generally 
believed that methionyl tRNA is the "universal initiator" in that both 
eukaryotic and prokaryotic cells contain two species of met tRNA, one 
of which is used exclusively for initiation of protein synthesis while 
the other is used exclusively in the elongation step of protein 
synthesis. However, in the prokaryotic cells, formylated met tRNA is 
required for initiation while in the eukaryotic cells, the initiator 
met tRNA is not formylated. As Boulter et al. (1972) have summarized: 
" (a) micro-organisms contain a transformylase and a fommylatable 
initiating tRN Uh, (b) animals and yeast do not contain a trans- 
formylase, but the initiating trnamet is formylatable; and (c) although 


in plants the initiating trnanet 


is not formylatable, the supernatant 
enzyme fraction appears to contain a transformylase which might, however, 
originate in the cell organelles". The amino terminal methionine is 
usually removed by a methionine specific amino peptidase, after the 
removal of the formyl group by a deformylase enzyme from the amino acid. 


The methionine on the tRNATe 


is placed into the internal position of 
the growing peptide chain. 

Leis and Keller (1970) found two types of methionine tRNA's 
in chloroplasts of wheat leaf tissue and two types of methionine tRNA's 
in the surrounding cytoplasm. The met tRNA's of chloroplast acted in 


a similar manner to those of E. coli in that one could be formylated 


and was the initiator of protein synthesis, while the other could not 
























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be formylated and donated its methionine to an internal position of a 
peptide. Neither of the met tRNA's of the surrounding cytoplasm could 
be formylated, but one acted as the initiator met tRNA while the other 
placed its methionine at an internal position of a peptide. Likewise, 
Yarwood, Boulter, and Yarwood (1971) isolated two major met tRNA 

species fron cotyledons of Vicia faba (L), only one of which was the 
initiator. They also isolated a minor species which could be exchanged 
for the initiator met tRNA of E. coli and this was explained as possibly 
of cell organelle origin. Monasterio et al, (1971) have performed 
experiments to compare the ability of the two types of met tRNA's in 


wheat embryo, rat liver and E. coli, to bind ribosomes. In all cases 





the initiator met tRNA bound more efficiently that did the other 
met tRNA species. 

Thus, results suggest that during evolution of prokaryotes 
to eukaryotes, methionine was conserved for the initiation of protein 
synthesis. The sequence of formation of initiator f met tRNAeY, as 


given in a review by Lucas-Lenard and Lipmann (1971), is shown as 


follows: 





ATP + methionine enzyme oe alee 


eet 1 
tRNATeE_. met-tRNAN®* + AMP (internal) 


met 


nzyme + 
Sprint ae tRNA et ——*» met~tRNAy + AMP 


met-tRNAGe e n!0_¢ormyltetrahydrofolate 2. F-met-tRNAZ* 


1) methionyl tRNA synthetase 


2) methionyl tRNA transfornylase 


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Initiation of protein synthesis requires the presence of 
three factors: Fl, F2, and F3. All three factors are loosely attached 
to the 30S subunit and they can be removed by washing the ribosomes with 
a concentrated salt solution. Sabol et al. (1970) isolated the three 
initiation factors from the 1.0 M NHAC] wash of E, coli ribosomes. 

These factors have been extensively purified by Wahba et al. (1969) 

and some of their physical properties have been reviewed by Lucas-Lenard 
and Lipmann (1971). Figure 1 shows their sequence and depicts their 
function, as suggested by this review. 

Initiation probably takes place in two steps: binding of 
the 30S subunit to mRNA and binding of f-met tRNA to the 30S ribosomal 
subunit-mRNA conplex. The first step requires F2, and if natural mRNA 
is the template, F3 is required (Revel et al., 1969). The second step 
requires both Fl and F2 as well as GTP and magnesium, The Fé factor 
appears to possess GTPase activity and it is likely that the GIP binds 
to this factor. With the binding of the 50S ribosomal subunit to this 
30S ribosomal subunit-mRNA-f-met-tRNA complex, factor 1 is released 


(Hershey et al., 1969) and elongation commences, 





C. Elongation 


Peptide-chain elongation can be described in three steps: 
binding of charged tRNA to the ribosane, formation of a peptide bond 
between the inconing amino acid and its predecessor, and translocation 
of both the mRNA and newly-synthesized peptidyl tRNA with the release 
of the antecedent tRNA. Two factors Ce and Hed, have been described 


by Miller and Weissbach (1970) for the binding reaction and their part 


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a ai a 
aii zamacde ats gotdeanw gt hylary oa nen Qont one hnube 208 nate 
it hadelort (Viet) 8 J fodet .ontiviee ter bodievdnsag 
-tempogdis Figo 3 WO Gaby TM wT. 7 oid vert? 191304 sot mk 


1 Sarre 26) gh 























“ae 


oom} 7 
7 va? 
_ 


viewntea 10 en} avert 210324) 32 
eqy “Pet Yor ro: 


-_ 


0SEE) moet ote 


otis 


a 


itete tad 7 

— 
1 od strvduaee 
<a! geno Alte rt 
“3 , wlal geod ied: 


jt? $7 oT eaviconeen toe StF ae Pow ce 57 One 4 tod 8 
einta Fi oy Peny yl of x} tere yereliss szatT) 2228 20g wha 
270s 4 noguz (giGemiis doc seh to gafontd edt ati. .sodoeT S. 
+ 4? T wogas? , ale ta Pay ~Jera Po AVa Fines iemoadd ial 
¢ 7 j > ls 40 ie xeegb 
Phones muldegngls bas (CUf .. fa ety oie 
ee nutiepnof? 
™ ue 
reqate ooniy of Rot) peg ed fis not segnats: Alsita-sbttqg?. 


boned oh 


5 
jieae oy 


re a , 


‘Pod he: rab Preys. ‘svar - t itn A) i owt» Baten 


—_ so 


40q viet! bne Aut somet- een —_ | Brae | 


: = 


ol 


_ - 










> 


yn ty MO LPO, smngodty ofl od ANit bayserta co pert 
elena bie aaa ext baw bios ombin wort 
saeoton art tow Ale ebrtupy bostteanaie-<twam 


Tit: aie’ 


¥ 


in the reaction is summarized below. Unlike initiation factors, 


translocation factors are found in the soluble fraction of the cell. 





T,,-GDP a8 he > Maelly ee ie 


Tate + GTP + aa-tRNA Semi racos thonSTs Eth “s lip 


This aa-tRNA-T -GTP conplex donates the aminoacy] tRNA for its codon- 
directed binding to a ribosonal site (acceptor site) neighbouring the 
donor site which is occupied by the initiator or previously-formed 
peptidyl tRNA, as shown in Figure }. Peptidyltransferase described by 
Maden et al, (1968) as an integral part of the 50S subunit, now catalyzes 
the peptidyl transfer to the newly-bound aa-tRNA fron f-met-tRNA or 
fron the previously-formed peptidy] tRNA. The succeeding step of elon- 
gation requires a third elongation factor (factor G) plus GTP and 
magnesium, and is a translocation of both the mRNA and the newly- 
synthesized peptidyl tRNA fron the acceptor to the donor site. At the 
same time there is a release of the predecessor tRNA. A new aminoacyl 
tRNA may now enter the unoccupied acceptor site and the steps are 
recycled until the correct peptide is constructed and the signal for 
termination occurs, 

The two translational factors isolated by Legocki and 
Marcus (1970) are discussed in the section, "Amino acid incorporation 


by wheat 80S ribosomes". 
D. Termination 


Genetic experiments have shown that cleaving of the bond 
between the formed peptide and the last tRNA that had been added 


(termination), is effected when the nonsense codes UAA, UAG or UA are 


ae 
‘ 


fies 


=f 
Sit5 { 
by 


rt 4 


6 9 
be 
1 


V4 fA 
f 


ote 







,27otaw? fol Ie tial naka whats bas me 


+ 
VJs 


L763. wor 


Vy wei 


> ¢& é 
‘. 1 
: -< 
o b i 
i” TA 


ont Avis fyopentog or ee 


Hintar 
nil mL 


ah Beet 


| ees 


dudes 


ods bat AW 


tr ocd De she 


itt & 


¢ 


yee 


‘wotGs 


os 


Wie Pea ch 


wv 


vi ¢ 


re 


shove att To a2 


aaa ¢) 


e) 


* 


»? i. 
a ‘ Yh aao 


meri f) 


n pas ud bnselgat CVS 


ole te notiaey) atdutie silt: at slit Wi i 


righ welts 


agsie fenotod 


- a = 
Heya to WORSTRIAT aff yd boetquooa 2} foidlw otte vane 


Jf ewer al 


260 off Te. 246q ferteial as 
wiy PRat-ge Onyed~yiwoa oA 
pa aw fe aaT A! it ry 4 wo 
A ¥ 4 i 
i. 40S98)) TOFo87 riepree 


to mottiso 


4 a ¢ mone 
ore F | aTaqu DON 


mnt Faoottefgnesd owl oT 


| wre q Pe | Pe sear: 


A ets. _ Alia fi + we 


Lc eft fs route : 


ane 2 
Wit Tyo ttonq Seslzend, 


shan 6 at eved?. owt 


ehfigeq tosio sid fio betas 


















wa +18, TANI 
i" @ come Li 


Hn 
mene 2b AMI Fybtigeg 
s (9001) fe 39 asbal 


t~vh@ avor’ ‘ong § 


feansf eo at bes ,muteen 


> 


0 ect tetne wor yom AR 
a 
—_ 
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kes 


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nytiovoqueait Sis otal” Mortage sat at ferguaeth sr5. (axed woe 


ich 


tlie # 


| 


baod afd y eotyats, 63 mort: eet ei 


babe fated bait: 260 AY Rete hee pales "8 > L. hl 


a6 QUA" MAL Ant esboo sam orn 
an 


Bs iar 


m7 ale va i 








— vee 


read (Brenner et al., 1967). Two release factors R, (which causes 
peptide release in response to UAA and UAG) and Ro (which causes 
peptide release in response to UAA and UGA) have been isolated and 
purified by Scolnick et al. (1968). A third termination factor (factor 
S) has been isolated by Goldstein et al. (1970) which stimulated the 
recognition of the release factors with their respective nonsense 
codons. Termination commences when the proper R factor complexes with 
the ribosome. When the peptidyltransferase catalyzes the transfer of 
the peptide from its tRNA in the donor site to the acceptor site, which 
contains the nonsense codon, the peptide is released leaving its tRNA 
attacted to the ribosome. There are various theories on how the 
ribosone with bound tRNA dissociates into subunits which may be used in 
initiation. It is possible that a factor as described by Kaji (1970), 
the TR factor, could remove the tRNA, leaving the monosome intact or 

in its subunits. Work by Kaempfer (1970) described the subunits as 
coming directly from polysomes, but indicated that they could reassoc- 
jate to form 65S monomers. Miall et al. (1970) has described a 
dissociation factor (factor DF) which is probably initiation factor | 
and which causes the dissociation of monosones into subunits ready to 
begin initiation. Davis (1971) proposed that this DF is present in 


limiting amounts and therefore strictly governs the number of subunits, 


Il. Differences between Chloroplast and Cytoplasmic Protein 


Synthesizing Complex 


It is thought that protein synthesis in chloroplasts and 


jin the surrounding cytoplasm occurs by a mechanisin similar to that 




























2atuu? dteniy ha ets aban ot ; 
aaennn visti) of bite (dint tin paki a sevoens'd ~ r 

bre bodufoe? mood ayer) Cae: ban AAG os Ben! apa 4 ‘ afer ; at _ 
notae?) warns) nosso teed OW. (aver) ats Ie ‘alent xd Ds 
ont botslumita 4 sibel (O0es) fa ty ntas ebtod yd bateldiel re i 
sznsenon svi fgoqsey Vieete. ‘An ww0794t oanot et auld ws aa 

diiw oon Toman “ae G74 SI TMeriw e9sunsiimo wo bsawtinst at 
% saVenss? off zauyl eaten Seitgtannst! ybitqsa sid mum » sie Tat 
hottw ,ahte rotqeods. si}. oF otfe tanh sft wy AND €2i mort OF dqaq 5 


* 2 oad - 
He 247 gylyes! boreslay ef ebtIqeq Shit hobo .sénaenon mali oA PASTR. 
¥ 


12 wor no zekvoon! svOPav oatp seit onozadi one oF F 


. 


ni tee of egw cohlw ettaedie etal 2efataces > AW? wend a 220dF 
_~ 


(Ser) Teal yo bediwsesh 26 Wosse7 & det? Bidilezoo af 21 sat s5tat 


oY 
= 


= 
40 Josiah wepetnod_at! Marigel ,AWF ale ovoies bia wtony ATS 


- 


a6 at toudid sé Bediyaast, (UNEP) sotqnosy yd oud entail 
—" oe ie ech? has —— ae eon 7 nr fans 
-<jvezas) Diver ve iO, DPARPHOTNNT Zee , esto tog mort t ul foot 
sedMoees. gat LOVOT) . fa de Tiel vane ted mo? 0: 


ea, 


, P : = oa 
at yboor elivudye ote) gempamdot io nottatoozeth afd eeaues GSR 


f sotoot wohselahal Uldadas@’ at dotew (40 voto?) voted? note 


: 
&) tnazord 2-4 cht Dan? bazogoisg! (TREE) givsd orate: nF 


tiimedve to wetien ats aieevee Yitotise ayotetady bre Se neal 
; bh a 1 

alatonr ot atest apayo bins Jestgorolt) msewlod | Bane 

; | mite Pee 


my 





I 
; 9 


bis! 23 eel ratty at Resins aero wit i pol 
- %) i 


and a sehen a _ ag Ginatys 
SS A neil aah 





10 


described for E. coli. It has been established that the protein- 
synthesizing conplex of the chloroplasts is equivalent to that in 
bacteria (Lyttleton, 1962; Ellis, 1969; Loening and Ingle, 1967; 
Sissakian et al., 1965), but that it is different from the complex of 
the surrounding cytoplasm which resembles that in animals (Marcus and 
Feeley, 1965; Allende and Bravo, 1966). Boardman et al. (1966) 
working with tobacco leaves, stated that ultracentrifuge analysis 
showed that the ribosomes of the chloroplast had a sedimentation coef- 
ficient of 70S; whereas the ribosomes from the surrounding cytoplasm 
had a sedimentation coefficient of 80S. This phenomenon has been 
observed many times now and is accepted as conmon knowledge. Hadziyev 
et al, (1968) using ion exchange chromatography showed chloroplast 
ribosonal RNA (rRNA) to have a higher cytidylic and a lower adenylic 
acid content that the leaf cytoplasmic rRNA, whereas the guanylic and 
uridylic acid content of all preparations was the same. Mehta et al. 
(1968) found wheat chloroplast ribosomes contained 16S and 235 RNA 
while the cytoplasmic ribosones contained 17S and 25S RNA. The 
proteins of chloroplast and cytoplasmic ribosomes have also been found 
to be different. Odintsova and Yurina (1969) using disc gel electro- 
phoresis found 25 bands for cytoplasmic particles, but only 22 bands for 
chloroplasts. Gualerzii and Cammarano (1969) calculated approximately 
90 proteins in cytoplasmic ribosomes, and 60 proteins in chloroplast 
ribosomes. Jones, Nagabhushan, Gulyas, and Zalik (1972) reported 

75 proteins in the 70S ribosomes and 85 proteins in the 80S ribosomes 
from wheat leaf. It was concluded that the proteins in cytoplasmic 


ribosomes are not at all like those found in the chloroplast. Payne 


i 
eye 
- . _ rag 7 wi 

— i a . 
-atetovq dt fad bata eaten read sui thee 
nt 3rid of doaTevicpp sid erentqnaten oat to ki pod 


r¥aer ,slont ina oatend "i Wes ai 13 ehaer sentir 


th xefqnms oy me) ToeeT ef TF Meat Jud Oh ig to 


of 
























whee M 
a 


= oo 

ine evovem) efsatns Wt SeN) aeitmmeey Horm mantgoige geibe rte 
(S000) fa ds neabGanl TBR) .overd une annetW :2aeF + XeF 
ateylanm ogutovtimostitee teats baisty ,zorne! WIGS 4H MEIC 


-i96n toitsionumited 6 fet Jecfamwwlds aly jo coeeds ony 3 ash ie _ 


efuetys gothmerrwis att Govt eatogodis afd epee (e0T Ye : a 





nosd zat sonsumnety <idT chs to Inots Youre HOMetnoMpe 
voyishel ptalveuts nies da Maiqepas of Pes won Somes yoann ’ ins 


Yuatqorosta Brewer Vit wm testis HMaa Aa her BAiew (80h) fe 36 
in, 7 Ve _ 


alivests vowo! & bos DifebT geo catyis & overt of (Ae) ANG bzod . 


se 
7 


Lie ‘Ti qnewy et Geetotw | AMy omen > Goto taal old ged int n> bi Sf 
; ' vt snr 
72 Foren wme2 82 W BIOTIC Tae 6 TD THINGS DTD PUVE 
. sil 
4 = = 7 a : . A eal 
) benletems eniersodiy teal govelia Jan bal Se 
: ‘ 
if AVA eS. fit Of beet oynow eaenz0dh atmesioot ; 
; 3 i ’ t <6 qo W ong = 
; ‘ . 7 . 2 oa ae 
htt | f yd} ‘ Raf 4) eh a ty el ¥ 5 ms Laqtiv’ ) ‘b Jealquriotha bia) rae 


riloels (ai Get patau (CAPT) ariyul bag gverinibs .dosvattpiagn 
et 

+ 35 git tit ,eo0gtdwag: vies \qoeys sot ebesd of orwok efe 
“iS honey hase levis Leaet } ons ws bak bay faut «2326 oor 
‘ paar, 

a{ametta at antatong Ge ben ,2amrodin aimatqogys 4 af 2ntede 







- 


hateqay (SVQr) aves Dobe < emeie enastawdatggatt #90 -tam 2 

conmreniin 2OG-arld wt enter 28 bre. sandy 20% si ie 
a mest QotyS nt antbeng wld. yodt batt 29-8 yet) A 

aye asta eal ni meer 


=? 7 


and Dyer (1971) have isolated a 5S rRNA conponent fron the larger 
subunit of both the cytoplasmic and chloroplast ribosomes of broad bean 
leaves. Electrophoretic mobilities indicated 118 and 122 nucleotide 
residues respectively for the cytoplasmic and chloroplast 5S rRNA. 
More recently Payne and Dyer (1972) have reported yet another 5.8S RNA 
component, which was hydrogen-bonded to the 25S rRNA of the 80S broad 
bean ribosome, This RNA could not be found on the rRNA of the broad 
bean chloroplast ribosomes and gave further evidence that the chloro- 
plast and cytoplasmic ribosones were different. Comparative electron 
microscopic studies of chloroplast amd cytoplasmic ribosomes showed 
their general shape to be the same; however, some of the chloroplast 
ribosomes showed a cleft which was not seen on the cytoplasmic 
ribosomes (Bruskov and Odintsova, 1968). Dimensions given for cyto- 


plasmic and chloroplast ribosomes in pea and bean are as follows: 


Cytoplasmic Chloroplast 
260-10 A long (pea and bean) 220-10 A long ( pea and bean) 
190-10 A wide (pea) 170-10 A wide (pea and bean) 


220-10 A wide (bean) 


Probably the most important difference between cytoplasmic 
and chloroplast ribosomes is that the latter have been reported to 
carry on protein synthesis ata faster rate. Boardnan et al. (1966) 
found the 70S tobacco ribosomes were 10 to 20 fold more active in 
protein synthesis than the 80S ribosomes. Working with wheat, 

Hadziyev and Zalik (1970) reached similar conclusions. Boardnan et al. 


(1966) found that optimum activity of 70S ribosomes was obtained at 


7 es Le ie ° as 
o | . \ - a We 
if oni : t 


J i _ ‘ 
































f T . 
vegist edt aos? dasnegeg Ali a iniaeea wr te q 


nied heer to eomonodi+ tes! erm tds tins stmclgyin pt ton! 10 
shitesfoun SST ke BFF ho deathet iin an ers 
AYMs 22 Saslqatotid brig atima qo wit 9 oy (Pavetosqaen & ubt 
AU 78.2 sattone doy hedvogeNeund (StET) a ban erent erinnoery . 
beord 208 att to AWD 22S G2 - bsbHodnapovngs aw dahaw 20 “— 


brow! wit to AM eth no Paws? 62 donq fluoo AMR 2tdF esnot0itby fans» 


erin off lett ponektyy vrei) even bne eon _* v2 sshaorohts i 


a. Bs) 
al % re ; i] fz ots) ra.) , dee ee Ph o7 3W es Ti 20 i] __ meat gad bag. 5} 


bawute epupraded otmcalqetys bm Szelqoreldo to eptbuta atgd 2003 FI 


desiqoiotrto, adit Ya ame ,resewed cowne edd od of agate ievonag Perk 
i. — 
ots ged: 53 fe nage Jan 260 delehe Stolp o bewode 29 


a a 


Th 


eT") a1 nevi anosenomd, .{8cbi ,svesinvid hag vita de 


et y= 


(oO 26. Pk shed Dae esq 1) zomcodhs deel eovefs hae >¥aeel 


vi 
F2bT R00) ¥ : 5 Piers pal goty) _ 
( 13 (je bag 4 whol AD S38 . iiteed b&b ie saat enok, A. porta 
/ , nk Tetee F< 
Ota Ee Se? WOle By Or=c . (seq) odin A of =o 


¥. a 

(ngad). obhw at Te 
| pa 
footy > pecnehad sptstaT th Vite? tat Sion st sham 


rt 


of botvoqey muh eet qe? ade 687 ZF eomncodty teatget otia't 
14a 58 onwerehoS stirs ‘eH woe 2! eatttings niatougn a yt 
NT “vices aon STOP OS ed tt ove 29008 seit. ante oles 
tein dhiw giidaow semmazodt 206 an ans 

als 2 vibra woes reuoean “oat nt conihe 


ioe ee a 
+* batacele, 7 savocidya m7 sen 


iv, cn pe 


_S 





i ren 


12 


whereas optimum activity of 80S was at 5 mM Mg*?, and 
2 


11 to 15 mM Mg*@ 


> 
furthermore at low Mg” concentrations the 70S ribosone would dissociate 
while the 80S would only dissociate after dialysis. The chloroplast 
70S ribosone can be dissociated to give subunits of approximately 
30S and 50S, whereas the 80S cytoplasmic ribosomes can be dissociated 
to give subunits of around 40S and 60S. 

Protein synthesis by 80S plant ribosomes is inhibited by 
antibiotics which affect animal ribosomes (eg. cycloheximide), while 
antibiotics which affect bacterial ribosomes (chloramphenicol, lincomycin) 


inhibit protein. synthesis by plant 70S ribosomes and not the 80S 
ribosomes (Boulter, 1970; Ellis, 1969, 1970; Ellis and Hartley, 1971). 


III. Amino Acid Incorporation by Plant Systems 
A. Amino acid incorporation by wheat 80S ribosones 


Most of the work on cytoplasmic amino acid incorporation 
in wheat has been done on wheat germ, because of the problen of obtaining 
80S cytoplasmic ribosanes free of 70S chloroplast ribosomes. The 
work of Leis and Keller (1971), however, shows that the initiator 
f met-tRNA for the chloroplast system is present in the wheat germ, 
which does indicate the presence of the chloroplast system in wheat 
germ. In addition to this, there is the ever-present problem of a 
mitochondrial system being present. 

The fact that wheat germ ribosones are free of the active 
messenger RNA, but protein synthetic activities could be stimulated 
by poly U addition or by allowing the seeds to imbibe water, has been 


reported a number of times (Marcus and Feeley, 1965; Allende and Bravo 


ST 






























» - 7 

bes , "yh Mae ty aa BORD wielie wut 300, » 
sishaweets by now seeeedt+ LOT a? pao Fagan St, ‘DM Wi my i 
ten! qovolds aff _atevieth aef¥a stshineeth ylno blag 
Uliana weve Sr etinudieg dere oy batetso2r 
tiJalssertd ed aig esnGaddy Sinentqorys’ 205 ail enervedw’ 208 bin | 
,208 bis 204 taupe Fo adtnodue | avi 

vd Gestdfint 2) comcedin Shale é0o ut sheattnys neaorF 


ofidw .(obisbestefaye os) eemisydls Feminn footie! dbiniw eotords 


fisymmont!  footietgtewetyis) compte (whedon too) ts-aeaw 2079 > 


wo oi eel, OAR ec BiEsvowrrs eat nett VO ¢€| ative? aieaihin 


ATT ela has etfs Over peeer , afft2. ente! wieTuouy a 


— . 


pm yzy? the! 4 vl aeMotoqreonl bik conta 
- 


sodt+ 200 heer ud ne tsave ask? bsg OATH 


rma ttavewosnl bie t (ee huaraicetw> a0 1Ow agi bie] Jeg lt 
ntatarde io molded off Fo gajeosd .wwe SwodW no Saal teed epi SHorM 
any). Momadlie Peer cvelta OO. to sont eo@saedly ohmestapiye 
-_ Patt 
oy 
/ oa 


TOA HFT ee Dott cute ,wevewed .(TTOl) settee One efal 4a 
a 


‘op tohw ad? at ated ef medeye tenlamvolito, sd 40} was. 
Joovte nt mugye Jeetgoret@a ay ¥o otianhh, otd steothnr aeot ya 


5 Yo onl org Te tid 23 sent ~ztat of aotsibbs at 
‘ny 6 


: . Jne2e%q pnted stage fol at no Ri 
ovigoa aa? Yo sort ov zamAEEy mean Ipusiw sadtt ad | ti « 
ae 


betel watts sd biugg entstytion atasttnye niade AMR 18 : 
need eat .tadew ot dnt of pean weak ¥ ots aotatybs t 


aoe ye Bie wa @ 
U rs 


’ : 
i 1 


| eat : 


gv bins slat fA eter a sai 6 But! nar) 


a ~ m 





J 


1966). Incorporating systems used to examine protein synthesis of 
wheat germ and other plants usually contain: Tris-HCl buffer, pH 7.5 to 


7.8; MgCl, 4 to 10 mM; KCI, 20 to 50 mM; GTP; ATP; an energy regener- 


2° 
ating system (creatine phosphate and creatine phosphokinase or phos- 

phoenolpyruvic acid and pyruvate kinase); ribosomes 2 to 10 E560 units ; 
2-mercaptoethanol and supernatant protein. If a poly U system is used, 


+c-phenylalanine. However, if another 


then poly U is added plus 
radioactive amino acid is used, then a natural messenger or an artific- 
ial messenger such as TMV-RNA is needed and the 19 other amino acids 
are added. 

A conplete system refers to a system where the radio- 
active amino acid is added unattached to the tRNA, and in this system, 
besides the radioactiye amino acid added, tRNA plus the synthetase 
enzymes must be added. If the tRNA is charged with the radioactive 
amino acid, then the synthetase enzymes need not be included and the 
system is referred to as the transfer system. 

Using a complete system, Marcus and Feeley (1965) reported 
protein synthetic activity of imbibed or poly U treated wheat embryos. 
Allende and Bravo (1966) found similar results using both a complete 
systen and a transfer system. For both systems they found activity 
dependendent upon poly U, supernatant fraction, magnesium, potassium, 
GTP, and an energy regenerating system, Maximum activity was found at 
60 minutes with a magnesium concentration of 7.5 mM and 25 mM KCl. 

The samples were counted in a gas flow counter with efficiency of 39%. 
The highest activity that they reported for the transfer system was 


294 cpm and the counts decreased, which they felt demonstrated 





























mS 
te eiaaddnne atntovg eatenaa adit mane 

no? 2.0 hy wettud feet catntend ei Tewew tang 4g 

-\anone. ygreda na : 91) pW gade oF Of 2738 1H or 


“20 VH Seer dornpony pbseete Ane | od wOrs ikl 2A 
S @ pe ie 
PASI nnod OY Gd S somone geageniy aleviryyy rin htop aha To 


+ 


shoev ai -wataye U hed 6 +f crtedeen tnetere que ite fovautboos sort 


ia a 
yudPOnh VI - 16 veWelt Sobnntal yay ¥" rity We Ciiyie et Wy on Wai 


z 7 ¢ ay Ld 
“ait )ow fis “a voneseren Ceyatenee feult \bety 2) bhos untms avidosolb 


: > Pte f’ Le i. aHHse of @ 47 } (Wh balhy 2 Ne ey ° ¥ 


«a dgus vsenbee sn Te 
j ae 


] oa 
+WSP f 


~f 
i; 


“a ( gy ah). aretw ooteye got ante: 4 Cave diotqam A 


, ey ve ning al th AY CA O29 botocl lane pads at Dias onlay 
| a. im 
arsvatiay? aly culo AMY oben Lins ontan witiaelbert ot? 
ovitoanthen gfe. dttw beeied> ot At ene. 41 Ubebhe ad Seam 


sh. lad G tan | 3a) oc b wie Si} Aid ‘no OR art: ye a4 rar? bios ont 
’ 


. 4 ; a q ee Oh ve 4 « a @ ‘ v > 
TAIZ KS VOTRGE9T O15 cH OF Pots ION af rot2y 


Ne 
hod ine eT) ¥ 1 Nee toate , wePaye stotanms a paball wt 


; ; ; : 4 d P : ~~ ™ 
eogions Siw Besterd  yleq so boda Yo vit vidos af dadtave ntade 
a 


P j ¥ « po : * ab i 
5 6 \fod pilav 22Tgaey valfore baunt (aeel) overs bas sbnslt 


Hi . 
Ph. Le oe, 


ov gat ameseyr tied 1 \éeteve 19'tenert io “bas x 


vibeehtog mu lesagem fella) Jieianide UW yfog pine ‘thobe a 
pce 


‘i. 


me trot yew ot vidos mumtaeh “werd aye entveransge, wrens ie bn 


p alin 


0A We 2% ban Mi 2,9 Fo noiteninaznyy mit zangain yh “it w 20! shies f 
oe To vongispite Aity vat mip wor’ 200 a nt hepa a meant sf 1 
pew Aiptay? vatenwts ing sot batiagar ait a | 0 Sand ot 


tase trom sis ont Lach . 


ma) 


ere a 


14 


dependence of factors. Mehta et al. (1969) reported incorporation of 
14 0-phenylalanine in a system containing cytoplasmic polysomes, which 
were obtained fron the supernatant after pelleting the wheat-leaf 
chloroplast fragments. Chloroplast polysomes have been reported 

mainly on membranes (Filippovich et al., 1970), and thus the method of 
pelleting the chloroplast fragments to obtain a supernatant containing 
almost entirely cytoplasmic ribosomes is effective. Mehta et al. used 
10 mM magnesium and stated that at lower concentrations they isolated 
ribosome pellets containing less polysomes. In addition they found 
more cytoplasmic polysones at 4 days than at 7 days, and this phenomenon 
was re-emphasized by the fact that poly U stimulated 7-day seedlings, 
but not 4-day seedlings. 

In 1970 Legocki and Marcus described the isolation of two 
translational factors which are needed for in vitro poly U phenyl- 
alanine synthesis. They used a transfer system containing 6.5 ™ MgAc, 
and 71 m KCl, which was incubated for 10 minutes at 30°C. The two 
translocational factors were isolated fron the wheat germ supernatant 
and were purified by DEAE-cellulose chromatography , (NH) 504 fraction- 
ation, and hydroxylapatite chronatography, The ribosomes they used had 
been washed twice with 20 mM KCl - 1 ™ MgAcy - 3 mM 2-mercaptoethanol 
buffer containing 1% desoxycholate. In the system with added trans- 
locational factors, 11.8 pmoles of the added 20 .pmoles of the charged 
phenylalanyl tRNA were incorporated. If these factors were onitted 
there was negligible incorporation of the phenylalany! tRNA. With 
binding studies, Legocki and Marcus demonstrated that factor I 


stimulated the binding of anino-acy1-tRNA to ribosomes in a reaction 


by 
























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dependent upon poly U and GTP. Factor II acted as a translocase rather 
than a binding factor. Either factor was active in catalyzing ribosome 
dependent hydrolysis of GTP. 

Marcus (1970) described a tobacco mosaic virus RNA-wheat 
germ ribosone system which incorporated amino acids into peptides. A 
5 to 8-minute lag which appeared at the beginning of the protein synthesis 
reaction was shown to be a rate-limiting ribosane messenger attachment 
reaction. This reaction was found to require ATP and two soluble 
factors (initiation factors) and did not occur in systems where the 
ribosones were already bound to messenger RNA. Again supernatant 
factors were isolated by DEAE chromatography. Marcus makes the state- 
ment that the possibility does exist that one or more of the factors 
found to be soluble in extracts of dry embryo may subsequently become 
attached to the ribosome early in germination. It should be realized 
that the method used for the isolation of these factors would not 
exclude RNase; however, as is mentioned later in this thesis, RNase 
has been reported absent in wheat germ supernatant. To demonstrate 
that the rate-limiting step which he observed was actually the formation 
of this initiation conplex, Marcus showed that when radioactive TMV-RNA 
was incubated with the wheat germ ribosome system, depleted of tRNA, 
there was formed a ribosame-TMV-RNA "initiation" complex which sed- 
jmented on a sucrose gradient slightly faster than the ribosome. 
Puronycin or cycloheximide, which inhibit later steps of protein 
synthesis, had no effect on this system, When tRNA was added and the 
reaction extended for 5 minutes, a peak which represents polysone 


formation appeared in front of the ‘initiation’ complex peak. This 







a | ag 
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step was inhibited by the addition of cycloheximide or puromycin, 
When the incubation time was further increased, the radioactivity was 
found in a more dense region. Marcus, Bewley and Weeks (1970) used 
aurintricarboxylic acid (ATA), a compound which specifically inhibits 
the attachment of messenger RNA to the ribosome, to inhibit the formation 
of wheat germ initiation complex. If ATA was added after the initiation 
complex had formed (to stop further formation of this canplex) then 
the peptides could be synthesized following the addition of the two 
translational factors to the system. However, the initiation conplex 
formed only if both wheat germ initiation factors were added to the 
system prior to the addition of ATA. The formation of this conplex 
required ATP instead of GIP, but did not require the participation 
of f-met tRNA nor any other tRNA. This system, described by Marcus, 
differs fron the bacterial system where f-met tRNA is required and 
where GTP is required instead of ATP, 

However, Weeks ets alan 1972), fron the same laboratory, 
have since reported that the initiation complex was a 40S ribosomal 
subunit-mRNA-met-tRNA complex. In the presence of TMV-RNA, the 
initiator type of methionine tRNA bound only to the 40S subunit. 
Further, with the addition of ATA (to stop initiation conplex formation), 
the 60S subunit was required before amino acids were incorporated into 
peptides. 

Leis and Keller (1970) have separated, by BD-cellulose 
chromatography, two chain initiating methionine tRNA's from wheat 


met 


germ. The major initiating tRNA was not formylated by wheat germ 


extracts, and functioned in chain initiation without formylation, 



























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dew J? vitaseitey oft (heenaeyt sally mm zaw watt oh 
been (Otol) zaes¥ how osfwoll .auocnh “neh gen per 
, 
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notigunoet an) Ftdlnat of Saree PY il) a) AML “ay Naeony v0 508 
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foo wore int sie ,erewot feteye att of peey tenot sate Ucar 
at) OF Doobe eiaw Fiolve? AAT iat mee Inetw Wied TH yine bs mo 
Tams et Yo tian) oAT OATA% Netti hon OT 8 kr 5 8 
otiagtoleiad sft eh unr Aan UN Tue vate to Habre Ora A td 
minh v4 badbiseet males 2M Mad Azide ure ton Anse Sex | 
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SUK ty Dewt ee ney tupaiiet aT ; erie 

4 ni 


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a Tv 
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The other initiating trnavet 


could be formylated by a wheat germ 
cytoplasmic transformylase. They demonstrated that the unfomylated 
initiator met-tRNA was bound to the initiator site of wheat gem 

80S ribosomes in the presence of AUG by showing formation of methionyl- 
puromycin after the addition of puromycin to the initiation system, 

Marcus, Weeks, Leis and Keller (1970) studied protein 
chain initiation in the wheat germ system which contained TMV-RNA as 
messenger, Upon amino terminal analysis of these in vitro products, 
they showed that methionine was found in the N-terminal of the peptides 
and thus suggest that methionine was the initiating amino acid. At 
low magnesium concentrations (1.3 mM) unformylated met-tRNA binds to 
ribosomes in a reaction requiring viral RNA, ATP, GIP, and supernatant 
factors. 

Schultz et al. (1972) found endogenous messenger RNA on 
ribosomes from ungerminated wheat germ and suggested that the messenger 
RNA was preserved in a complex form with the ribosones. They observed 
seven fractions of RNA when total RNA was electrophoresed. Fraction 
I and II were 24S and 17S RNA respectively, while fraction VII was 
5S RNA containing 50% tRNA. Fraction V stimulated amino acid incorpor- 
ation and was believed to be mRNA. Endogenous activity was not found 
on fractions of the 74S monosome peak, but was found on fractions 
sedimenting at 90S and 45S. This suggested that messenger RNA was 


preserved in a complex form with the monomer. 
B, Amino acid incorporation in other higher plants 


App and Gerosa (1966) reported a rice embryo transfer 


Wl 


























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poly U system which demonstrated an absolute requirement for poly U, 


+ 
te K or NH’, GTP, and supernatant factors. They found that the 


Mg 
incorporation decreased when the ribosomes were washed with desoxy- 
cholate and that incorporation was almost completely eran when 
supernatant factors were added. They noted that the transfer reaction 
of unwashed ribosanes was enhanced by washing and by the addition of 
supernatant factor. Later, App (1969) purified two factors obtained from 
the rice embryo supernatant, which were also found on the ribosomes. 
Factor I on the ribosome was easily washed off with desoxycholate; 
however, factor II was released only after further washing with 0.5 M 
KCi. He isolated factor I from the supernatant while factor II was 
isolated from the ribosome, and both were purified with Ca3(P0,), 
followed by gel electrophoresis. It was noted during these experiments 
that the magnesium concentration for optimum polyphenylalanine synthesis 
varied with the character of the ribosome and supernatant used. 
Analytical ultracentrifugation snowed KCl-washed ribosomes to be 
dissociated in |] mM Mg*?, whereas the desoxycholate-washed ribosomes 
were not. Increasing the magnesium concentration resulted in the 
disappearance of subunits and appearance of material on the leading 

edge of the monosome peak, an occurrence which, according to App, 
represents polysome formation. When these two "transferases" as 

App named them, were added back to washed ribosomes, incorporation 

was stimulated. App noted that activity was found on a large number of 
fractions in polyacrylamide gel and not just in one band. App et al. 
(1971) reported that rice enbryo ribosones could more easily be 


dissociated in 0.5 M K€l if the seeds had been imbibed. This ease of 



























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in a“ 
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itl 


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om il WJor) si hwy snprermegye otf oor? fT am oat bataton?. oh 1 
ol O) no he Cottieag stew Avod bus .wpeodh aa wry bageloe) 
esiant sate uver? ati nul baton epw 3] stan ar opailal fay és b pant | i y 

trodiey2 on nal biyreiaylag quate 107 sors DieQHOy, ma! RSM ; ey 
yecy Jesus bos gtweOd + of Fo Yosseveda add attw nas 
ad 09 sommdedty Sadzow-T3% fawode ae tata 
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3 


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neotiaveqiaons ,eamZodts. bategw of sped bsths ao wit be 

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af6 29 qh .bued en At Paul gon bas fap btenyers 
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a 
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+! oe 


dissociation, they stated, parallels activation of protein synthesis 
in vitro. The ribosanal subunits fron either imbibed or non-imbibed 
embryos could be separated and reassociated to re-form active ribosomes. 
Their transfer polymerization system contained 10.5 ™ MgAcy, 60 mM KCl, 
and crude supernatant, All the density gradient centrifugation was 
done in a resuspension medium containing 20 mM tris, 15 mM KCl and 
5 mM 2-mercaptoethanol, in spite of the fact that reassociation 
incorporation studies were done with ribosanes dissociated with 500 mM 
KCl. The density gradient profile of dissociated ribosomes fran imbibed 
rice embryos contained a shoulder between the 40S and 60S peak. Dissoc- 
jation profiles based on analytical ultracentrifugation were obtained 
by addition of 500 mM KC1 and the 40S peak was then the same size as 
the 60S peak, They found both subunits to be required for active 
polyphenylalanine synthesis, and that dry embryo subunits could be 
replaced by imbibed subunits. 

Parthier (1971) described a poly U transfer system used 
with pea seedlings. When 0.4 ing (6 E560) units ribosanes, 1.0 mg 
of rat liver supernatant protein, 100 ug poly U, 0.75 mg of tRNA and 
13 mM MgCl. were incubated at ae polyphenylalanine synthesis was 
conplete after 5 minutes. Gulyas and Parthier (1971) found that the 
pea seedling ribosomes could be further activated for poly U directed 
polyphenylalanine synthesis by washing with high concentrations of 


NH,C1, KCl, detergent or by Sephadex gel filtration. They stated that 


4 
pea seedling supernatant would not stimulate incorporation because of 
its high content of nucleases, A single washwith 0.5 M NH,CT or 

1M KC1 enhanced poly U incorporation two to three times, and successive 


washing with 0.5 NH,C1, up to 4 washings, increased incorporation four 






























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Ded) dnt~ean sa hedi dat ands, wih ashes . — 
coments ayvisae ame veo eg beta frorseon infra us Steger ae 7 
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bow notrepy Pings Jepthawp yittaash aie 141A Seung 49 


7 
+ 
ial 


ing tot My a , 20 72, Mn O Dy OPW aiags ihe rohemadouesn nt ae 
wal tvoteney TENE Pant GAS a wthqe ab , ‘apnea Qaon ie 
t 


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ov ena ORE: a a ta myer 0 yo sae 
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: aa init ' ” a 


20 


times. It was also demonstrated that washing the ribosomes with the 
detergents desoxycholate, dodecylsulphate or triton X-100 increased 
ribosome activity. It was noted that by the addition of pea super- 
natant protein, formation of polyphenylalanine decreased and tests 
showed the pea supernatant to affect only poly U. The "nuclease" was 
heat resistant, and dialysable. They stated that this substance is only 
present in etiolated seedlings, particularly those of leguminous plants. 
Marei et al. (1972) have isolated ribosomes fron fig fruit 
which in a complete system, were dependent upon GTP, ATP, and magnesium 
for 140 phenylalanine incorporation. This incorporation was only 
Slightly dependent upon poly U and an examination of the ribosome 
preparation on a sucrose density gradient revealed many polysones 


which proved to be responsible for the observed incorporation. 


¢. Amino acid incorporation by chloroplasts and chloroplast 


ribosomes. 
1. Amino acid incorporation by chloroplasts 


Chloroplasts have all the components necessary for protein 
synthesis. They contain 70S ribosones, tRNA's, aminoacy1l-tRNA 
synthetases and soluble enzymes for the transfer of amino acids into 
peptidyl material, and they can synthesize at least some amino acids 
(Boulter, 1970). Amino acid incorporation in chloroplasts of the 
following plants have been reported; wheat (Banji and Jagendorf, 1966), 
Ranalletti et al., 1969); tomato (Davies and Cocking, 1967), (Hall and 
Cocking, 1966); spinach (He ber , 1962), (Spencer, 1965); tobacco 


(Spencer and Wildnan, 1964), Hampton et al., 1966), (Chen and Wildman, 















| y ‘ ai 
ott dilw eomegedis ote petteletiy fret) bade ek arvamealy il sn ~ 
boeserion) M-% moped sidgilahust quae qSt6 * oii erat 


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5 on 
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: 7 
eineiq evontoubel te scolie gered t end , apalthaee SAITO Seay ae 
ae - 
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my brangem-Orh , "Vs ie iPiteherct 0) a i an 
vid cu maint 2101 ,hotsawirho in Fie DB iayis 
sipenat Tear ) Mm Se + Ue ore Toe re 
< eu} i y baP 7 oy fl oan? 6 wh Ve ] 
7 A 
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csrueod ry 
a 
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nipioiw “Gy) ¥rase Sil ; Dalal ite ti ts avyan : lent Ge oi 


{ipertttio. .2 Aled » eamzodey 25), gtasnos WET, « 
oint ebice Ofte To Gotan mI A 10% panier of dues — 
zhtoe autumn swe Jzae!/ 7H peleotinye Meo Yad) beg, .]e7 

ol? Wy. atdet qowiTilaoot nphasyaquion? bios anim _@ sf 

. (4a@? , Probnaosl hey Fyetl) teed rhelesge- mood bia rr 
brn fan} q{Sae? .qeldndd bas envi) ound ” 
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eerie AM ane mad foie desi 
Vil el nn ome 


at mh 





21 


1970); bean (Margulies and Parenti, 1968), (Ranallett? et al., 1969); 


pea (Filippovich et al., 1970), (Lozano and Griffiths, 1970); and 





sunflower (Ranalletti et al., 1969). 

When doing amino acid incorporation with chloroplasts, 
the research worker must obtain chloroplasts free of bacteria and free 
of other subcellular organelles. Boulter (1970) suggests a limit of 
10" bacteria per ml incorporating systen can be tolerated. By using 
freshly-distilled water Bamji and Jagendorf (1966), could decrease 
the bacterial counts of agar plates incubated 24 hours, to 10° per 
0.5 ml reaction mixture. They stated that 10° bacteria per 0.5 ml 
would cause serious problens with their results. Parenti and Margulies 
(1967) observed 10° to 5x10" bacteria per ml chloroplast suspension 
when they used sterile water, chilled the tissue rapidly and washed the 
tissue with a solution of calcium hypochlorite. Gnanon et al. (1969) 
stated that "the incorporation of amino acids by chloroplasts isolated 
fron higher plants by ordinary biochemical techniques (using hypotonic 
buffers without protective colloids) could be ascribed largely if not 
completly to contaminating bacteria". They studied protein synthesis 
in chloroplasts without interference fron contamination by isolating 
chloroplasts with the Honda medium, and by adding respiratory inhibitors 
to the reaction. Lozano and Griffiths (1970) using aseptic tech- 
niques obtained chloroplasts containing only 10° to 10" bacteria per 
ml chloroplast suspension. Nuclei, mitochondria, bacteria and entire 
cells may contaminate the chloroplast fraction; however, relatively- 
intact, clean chloroplasts were obtained by isolating in a medium 
containing the protective colloids, Dextran and Ficoll, and by succes- 


sive washing of the chloroplasts. With the addition of a detergent 


2 {08eT 4 Se 29 rrettanal) taaet upeelil 


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22 


(triton X-100) to the systen, chloroplast membranes were selectively 
ruptured leaving the contaminants unaffected and the incorporation was 
taken to indicate the percent contamination. Parenti and Margulies 
(1967) estimated 2 to 10% of their incorporation was due to contamination, 
while Lozano and Griffiths (1970) reported 5%. 

As pointed out by Lozano and Griffiths (1970) different 
requirements for chloroplast protein synthesis have been reported; 
this is probably due to differences in intactness and purity of the 
isolated chloroplasts. For chloroplasts isolated fron young pea leaves, 
they obtained aptimum conditions of 12 mM MgCl,, 100 mM KCI, 16 mM 
2-mercaptoethanol, 2.75 mM ATP, 8.3 mM PEP, 10 ug/0.3 ml pyruvate 
kinase, to which they needed to add a mixture of GIP, UTP and CTP. 

Age appears to have an effect upon activity of chloroplasts 
as noted by Bamji and Jagendorf (1966) who found chloroplast preparations 
fron 4 or 5 day-old wheat seedlings to be more active than chloroplast 


preparations from 7 and 8 day-old plants. 
2. Amino acid incorporation by chloroplast ribosomes 


Sissakian et al. (1965) obtained amino acid incorporation 


by ribosomes isolated from pea seedling chloroplasts. Neither ATP nor 
anino acids stimulated incorporation, and dialysis of the ribosomes 


lowered activity which could not be regained. Hadziyey and Zalik (1970) 
reported chloroplasts of 4-day-old wheat seedlings contained a greater 
amount of polysomes than chloroplasts of 7-day-old plants. The 
incorporation was decreased if the polysones were broken into single 
ribosanes by RNase treatment, and could not be regained by the addition 


of poly U. The presence of two types of polysomes in chloroplasts-- 


oy 
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23 


free and membrane bound--was noted by Payne and Boulter (1969). They 
suggested that each type may synthesize a different group of proteins. 
After Chen and Wildman (1970) incubated intact tobacco chloroplasts with 
140 valine, they observed half of the incorporated amino acids to 

be in nascent protein on ribosomes in the soluble phase, while the rest 
was in nascent protein on ribosomes attached to membranes. When they 
isolated the membrane portion of chloroplast and added M40 amino acids 
and supernatant, the ribosomes bound to these membranes synthesized 
protejn. Work by Filippovich et al. (1970) indicated that protein 
synthesis in chloroplasts of pea seedlings takes place mainly on 


lamellar membranes. The free and membrane-bound ribosanes had been 


incubated in a medium containing all the factors for a complete system, 
IV. Dissociation of Eukaryote Ribosanes 
A, Dissociation of ribosomes by high salt concentrations. 


Martin et al. (1971) have reported a method for the dissocia- 
tion and reassociation of cytoplasmic ribosones from animals, plants, 
fungi and protozoa. Large quantities of subunits could be isolated 
by zonal centrifugation through a 10 to 30% sucrose gradient containing 
0.88 M KCl in the buffer. They reported inactivation of plant ribosomes 
if they were separated at a higher temperature (28°C) and if they were 
stored frozen at -20°C. However, isolation of the subunits at cooler 
temperatures (4°C) caused aggregation of subunits and thus contamination 
of the 60S fraction with the dimerized or conpacted 40S subunit. 
Aggregates of 60S subunits had an S value of 90S and 105S. Pretreat- 


ment of ribosomes with puronycin was required for conplete dissociation; 




























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24 


however, this treatment may have caused a modification of the subunits 
which exhibited an increased tendency to aggregate and a different 
magnesium dependency of polyphenylalanine synthesis, 

Subunits mixed in a ratio of 2.5: ] (Eo6g units) would 
reassociate to form active, poly U dependent 75S monosomes. Martin et al. 
reported pea and wheat to reassociate only fifty per cent. No activity 
was reported in the 40S subunit and the 20% activity of the 60S subunit 
was attributed to 40S contamination. Undissociated 75S particles were 
active in incorporation and were unresponsive to poly U. 

Peterman (1971) reported dissociation of rat liver ribosomes 
by the addition of urea to ribosanes pretreated with 0.6 M KCl and 
dialysed. Treatment with 2.7 M urea gave 60S subunits free of 40S 
subunits, while treatment with 2 M urea gave 40S subunits free of 60S 
ones. When these two subunits were isolated and recanbined, poly U 
directed incorporation was only 25% of control undissociated ribosomes. 

Staehelin and Falvey (1971) have isolated fron mouse and 
rat liver, ribosonal subunits which reassociate 100% to form active, 
poly U dependent 80S ribosomes. When purified polysones were incubated 
with all the components for protein synthesis, Manmerise. monosomes were 
formed which, when treated with 0.5 M KCl and centrifuged through a 
buffer containing 0.3 MKC1 and 3 mM MgACo» dissociated 80 to 90% into 
60S and 40S subunits. These workers precipitated the subunits by adding 
0.7 volume of 95% cold ethanol. They also noted that when run-off 
ribosomes were spun through a sucrose gradient containing the incorpora- 
tion buffer, the majority of ribosomes sedimented in a sharp 70S peak 


clearly separated fron the 80S complexes still carrying peptidy1-tRNA. 



























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25 


Analysis of the subunit RNA demonstrated the 40S to be 100% pure, but 
the 60S was contaminated with 40S to about 10%, 

Battaner and Vazquez (1971) found subunits were produced 
when yeast ribosanes, which had been washed with 0.5 M NHACl, were 
dialyzed for two hours against 1000-fold excess of buffer containing 
0.2 1™ MgAc. Separation of these subunits was performed by placing 
them on top of a 7 to 35% sucrose gradient in a buffer containing 
50 mM NH C1 and 5 mM MgAc,. At the end of the centrifugation, the top 
90% of the supernatant was discarded and the remainder of the supernat- 
ant (containing. only the 40S subunit) was collected, while the pellet 
(enriched in 60S subunit) was resuspended, recentrifuged and then 
collected. Reassociating the subunits restored 80 to 100% activity, 
the 40S did not incorporate at all, and the incorporation by the 60S was 
explained on the basis of 4 to 8% contamination by 40S. 

Wolfe and Kay (1967) dissociated wheat germ ribosomes by 
dialysis against a buffer containing 0.05 M KC1, 0.025 M tris-HCl 
(pH 7,5), 0.0003 M MgCl, and 0.006 M 2-mercaptoethanol. They obtained 
60S, 40S and 26S subunits, which could not be reassociated by increasing 
magnesium concentration to 0.005 M. Jones, Nagabhushan, and Zalik (1972) 
reported dissociation of wheat cytoplasmic ribosomes into 42S, 49S, 
and 61S particles after centrifugation through a 7 to 35% convex 
sucrose gradient in a buffer containing 20 mM tricine (pH 7.5), 10 mM 
MgCl. , 400 mM KC1, and 5 mM 2-mercaptoethanol. The 42S and 615 
reconbined to form the 80S monomers upon returning the magnesium 
concentration to 5 mM. Wheat chloroplast ribosones could easily be 


dissociated by the same method; however, the subunits did not readily 


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26 


recombine to form the parent 70S species. 

Nolan and Arnstein (1969) could reassociate subunits of 
rabbit reticulocyte free ribosomes to form the parent 80S species, but 
these were not active in a poly U incorporating system. By dissociating 
rat liver ribosones in 1M KCl, Terao and Ogata (1970) obtained subunits 
which would easily recombine to give parent 80S species active in poly U 
directed incorporation. Gravela (1971) reported dissociation and 
reassociation into active particles of Yashida hepatoma ribosomes. 

These ribosomes dissociated with a comparatively-mild treatment of 


KCIBOTEE DIAG 
B. Dissociation of ribosomes by dissociation factors, 


Subramanian et al. (1968) discovered in E. coli a protein 
factor found only on native 30S subunits which pronoted the almost 


complete dissociation of 70S ribosomes prepared from starved E. coli 





cells. The DF-I was extracted fron native subunits, which had been 
separated by 10 to 30% sucrose gradients, by storing them on ice in 1M 


NH,C1. The supernatant fron a high-speed centrifugation of this material 


4 
was dialyzed, and the fraction precipating between 30 - 70% saturated 
(NHy) S04 after being dialyzed was collected as the DF-I. They found 
that all the ribosomes would not dissociate and stated that their 
preparation might contain a small mmber of nondissociable ribosomes 
(presubably fragmented polysones). These authors observed that increas- 
ing the magnesium concentration, reduced the rate of dissociation, 


Their results support the cycle which hypothesizes that after termination, 


free 70S ribosomes are produced which must be dissociated for initiation, 



























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Garcia-Patrone et al. (1971) established that antibiotics which affect 
the 30S subunit modify the DF stimulated dissociation, whereas anti- 
biotics which affect the 50S subunit do not affect dissociation, 
This agrees with Subramanian et al. (1968) who state that the goal of 
the DF is the smaller subunit, 
| To show an analogy between DF and initiation factor, 
Subramanian and Dayis (1970) tested each initiation factor for its 
ability to dissociate. They concluded that only initiation factor F3 
would cause dissociation. Albrecht et al. (1970) fractionated the three 
initiation factors by DEAE column chronatography and agreed with 
Subramanian and Davis that F3 and DF were in the same protein fraction. 
They observed GIP to have no effect on the 100% dissociation they 
obtained. 

By DEAE-cellulose, phosphocellulose and Sephadex G-25 or 
G-100 chromatography, Miall et al. (1970) purified the DF to yield a 
single band on gel electrophoresis which had an apparent M.W. of 
8,350 to 9,200. GTP had no effect on the ability of this protein to 


dissociate 80 to 90% of the 70S E. coli ribosones. Because of similar- 





ities in purification (the protein was not adsorbed on DEAE cellulose, 
but was adsorbed on phosphocellulose) and molecular weight, Miall et al. 
concluded that DF was equivalent to Fl and not F3 as reported by 
Subramanian et al. 

Petre (1970) isolated a specific protein factor which would 
dissociate yeast ribosomes. Yeast "native" subunits were separated 
by differential centrifugation and a protein fraction was removed 


by washing these subunits with ammonium sulphate. The precipitate fran 

























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blvow rive dome? ctagig Sttioage » betaine! (OVer) Saiet 
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bi wuecers enw he haope wrens . belt empha ¥ spin ad j fan 


rei atet hylan ope state taro ome woh 


‘ty ,: hm 





28 


35 to 75% saturation was collected, dialyzed and heated at 40°C for 
30 minutes. After being cleared by slow centrifugation this crude 
protein fraction fron "native" subunits would cause a temperature- 
dependent dissociation of yeast ribosomes. Because this dissociation 
factor would pronote only 40 to 50% dissociation, Petre suggested the 
presence of two types of ribosomes. Experiments done to observe the 
hydrolysis rate of isolated yeast ribosonal RNA indicated that the 
amount of RNase present in the dissociation factor protein was much 
lower than would be required for the observed dissociation. 

Lawford et al, (1971) extracted from ribosomes enriched 
with native subunits of rat liver, a protein mixture which they partially 
purified by (NH) 550, fractionation. This partially-purified protein 
stimulated only a small amount of dissociation when ribosomes reformed 
fron subunits were used as substrate. The DF was active only if the 


ribosomes had been freed of both their messenger RNA and nascent protein. 
C. Puronycin used to stimulate dissociation. 


As stated above, runoff ribosomes will accumulate if protein 
synthesis is blocked by puromycin. Figure 2 indicates the obvious 
structural relationship of puronycin to an amino acylated tRNA. As 
described by Mahler and Cordes (1966), puromycin terminates protein 
synthesis by acting as a substitute for aminoacyl tRNA, thus causing 
the formation of a peptidly puranycin which is incapable of further 
chain growth and which is thus released. As stated by Boulter (1970), 
puranycin inhibits amino acid incorporation by cytoplasmic ribosones, 


chloroplast ribosomes, mitochondria and nuclei, and furthermore it is 




























yo? 3°02 36 hagend ‘tow barge .baseat too any 
shove 2/97 coliedothyéiss Wole vt borne! ontod eat iL) 
swims? 6 sruse Shaye ePhiodvr “avisan" ogee | se ) nba: 
naifafouaeth 2149 etuaage pempdin J2nay. Vo notaeragzeth Sad 
of) belewoue 298" (notaetaeeelt ot of O) ytao arenas Het ge 
tit riaede oF anaes on daewend paverzedh) Fo egit ond pe we: 
ti S009 bas i Qi Tetweend?y trasy beteloat * sie iid 
vt tw 7 edQ4Nn WEOET MOTT sere’ h al ai rq eraig oveeR uaa NS 
‘Tetaatald Soviapte odt 70? havtwpes ad bivaw inetd Soll 


butspran senimagiy mat tegaayexe (799) le Se Pata ta 
be « 2 J J 
Pietivsg yotd daliiw ene sia eopeey a ,veyel te ETE avé wi Ty 


nfss 7 , t i a a Lf Bad ? . r 4 eno’ es ae J f rt) xd We 
» ) 3< _ 


bouieveri rehaiedt? varw ietgafoogzit ta grew I leue a yiip be Tut 
: ¢ 


tl yin Wile ey “is Sy cevsidue zh beg ™ 3 #7 Prd ec 


regan Inara 14 FI Ro vigtececsu Vw fied “eo bsovtd ased Ue symoeod 
A : 
wit lwsgeth efals water a? baw arovirw’ -.2 
an 
‘ : 
ales notice (iiw seeped oud ,ovice botase 2A 


hyo ai? awash “ i2it wend yd hauls aly aha ‘eat 
, A Dey shy oe OF im no wt nivevug To gtirnotyetey rend 
hiastovg esteylergd wagebwg ,(000T) eaten) bis miner a i it 
patewes eult , AU. yee. yet otus edve 6 ** we i 1 ah 
vartiqut 9 sidaggamt 7) AS1Ne Ao gmetig Untinel 9 J = 

, (OTe) witiet ut pabete 24° ,beapelex varie oti 
6 copes mantras wit eeenioncie-) 1 
21 ay iadaaesiatatind ne habe 


i ; bane 
- a ii 


29 


specific to only protein synthesis, 


Figure 2 
Bee x pep 
N 3N SN 
4 mes ¢ 
niin e La O Kaiie x wy, 
oi SAHIN OH oem 
| 
NH, NH) 
I t 


Terminal adenosine of an amino 
acyl- (or peptidyl-)/-RNA 


Puromycin 

Puromycin has been used by a number of workers to produce 
free ribosomes which could be dissociated more easily to give more 
active subunits. Blobel and Sabatini (1971) held rat liver polysomes 
on ice in a buffer containing a high concentration of KCl and treated 
then with puronycin. After 15 minutes most of the nascent protein was 
released, but the ribosanes remained as 80S particles; however, when 
the mixture was thenincubated at 37°C for 10 minutes, the runoff 
ribosomes completely dissociated into subunits. These subunits did not 
contain tRNA, and would readily reassociate to form monomers which were 
active in poly U-directed incorporation. This method has also been used 
by Lawford et al. (1971) to obtain subunits which combine to form 80S 
rat liver ribosones active in poly U-dependent ‘year aN et aie 
Stahl et al. (1968), Lawford (1969), and van der Decken et al. (1970) 
used a method which involves the incubation of polyribosomes under con- 
ditions for protein synthesis by using untreated cell sap in the absence 


14 


of ‘C-labelled amino acids, but in the presence of puromycin. Both 


rat liver and skeletal muscle active subunits were prepared. 


Soe 
bob Venrt 224 fed (78@L) | WIS be “200, fadath ae 


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mm Pal q 
vemtgmnel™ 
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i he Go sree i eve? j im Ww ( 
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‘e uf . * mS a i — 1 _ 


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| oF- arhcites Ap hiw ef tancug NED (eet). te te ae? 


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* 


MATERIALS AND METHODS 
I. Chemicals 


Disodium’ 4H,0 AT Pemet ial cri Ula Gi bes disodium -4h.0 creatine 
phosphate, creatine phosphokinase (specific activity 81 I.U./mg), 
a salt-free lyophilized powder obtained fron rabbit skeletal muscle, and 
the potassium salt of polyuridylic acid were Calbiochem A grade (Los 
Angeles, California, USA). The stripped tRNA obtained from General 
Biochemicals (Chagrin Falls, Ohio, USA) was soluble RNA (sodium salt) 


V4 c-phenylalanine 


isolated from fresh yeast. The uniformly labelled L- 
(specific activity 405 mCi/mmole) which was in a sterilized aqueous 
solution containing 2% ethanol, POPOP, and PPO were purchased from the 
Radiochemical Centre (Amersham, Searle). The other chemicals were 
analytical grade: KCl, MgCl... THAME tris( hydroxymethyl) aminomethane], 
bentonite, sucrose, (NHq) SOQ» Trichloracetic acid and scintanalyzed 
toluene were from Fisher Chemicals (Edmonton, Alberta, Canada); 
2-mercaptoethanol from Eastman Organic Chemicals, (Rochester N.Y., USA) ; 
tricine[N-tris(hydroxymethy] )methyl-glycine] from Calbiochem; 

triton X-100, MgAC» phenol and NH Cl fron Baker Chemical Co, 
(Phillipsburg N.J., USA); sodium desoxycholate fron K&K Laboratories, 
INC. (Plainview N.Y,, California, USA); and diethyl pyrocarbonate from 
Research Chemical Corp. (Sun Valley, California, USA). The puromycin 
hydrochloride was obtained fron Nutritional Biochemicais (Cleveland, 
Ohio, USA); chloramphenicol U.S.P. fron Parke, Davis and Co. Ltd. 
(Brockville, Ontario, Canada), cycloheximide from Sigma Chemicals 


(St. Louis, Missouri, USA); and the nutrient agar was obtained fran 


Difco Laboratories (Detroit, Michigan, USA). 

























abisai Oyiie. “im rapeth qT autty tts - 0, alte” aii a 7 
7 ie) a hy 
om 0.8 84 yt ividos 3 Hinee) ont des Nevis Mi rhe idee 


hos ~atotum hersiaedy hams a teaherdo yshwot bast ue . 


aot) shety A wed ineahOT ed yf dew) iin 34/ chewy iag. 0 tier Ww: 


ey 


fevanod sv} Senhetep AY aa rc ‘tap 7) satinovitnd ips 
(ofn2 quthoe) AM eldufine com (ARO obit 0 eta oagela éteohan 


é i 


aE 7 : 
ontnelafynentqes <i) Retleast yhateti, stT . tener, dete mot ’ ators 
suosupe bast fing a] 6 1? yew ah Leto Bok ettvitse: on rtseqe) 


eA? WEY? bozemewip wit, +O beth Ta ve bina rs shaun: oi WTO. 


wow 2featmads terto ant ~fat Gee maavedt } ead = wns ve 
n Sal ce 
ten ‘) ae Al Piya iv v ov vii } ter) pee : ie! ite) P Tat 4 sebstty’ reah bibl 


o 
, =F 


‘ ‘ See & ‘ ® Pa ‘ : | r j ce a ’ 
sy liwerntoe bap Dias atoeserelsot 1! ,. ORL 2S) RBONRNE, - ae 
: ; i 
si , eeredtA shea nontit), shen Pret nee 7%: “ett nue 
PCA ..¥. a vevesegon) 42 wot mee, ayer en seas rere. Fotimttiid gs 


lao - 


| Wea? afd ad mor. ti Yip y res py! Tete srg ti 


O23 165 Pom) tothe went FO, 00 bee ia ray ashe O0t« aa 


‘ bee im dg 
« Bt “UCcey i 


cowry! Wa mye? goafodyeoesh nities g taeu tig 

er) -ataned tani Net ‘giveth tag (AGU wineries) Ye wo 
nto ol thu toro thfad pyohiay aud ) + QV02- tanta 
beafove'S) elsotweigot® Tenote busi poy hantegde: enw 6 at ae 
ee ee een 9 3A 

sina tm) saptz Met sbhaPtet oy «(ebang 


‘spl 


won? bantatad mahenh dtm we oh 
: ate | 7 ere 
my | 





3] 
II. Equipment 


Analytical ultracentrifugation analysis was done on a 
Spinco ultracentrifuge model E with schlieren optics. High speed pre- 
parative centrifugation was done in a Beckman L2-65B ultracentrifuge 
equipped for zonal centrifugation. The rotors used were Ti 60, SW als 
and Ti 14, Low-speed preparative centrifugation was done in an IEC 
(International Refrigerated Vacuum centrifuge), model BD-2, using 
rotor model 970. Fractionation of column effluents was performed by an 
ISCO ultraviolet analyzer model UA-2 in a refrigerated cabinet, while 
all other spectrophotonetry was performed with a Beckman Recording 
Spectrophotometer model DK. Radioactivity was measured in a Nuclear 


Chicago Mark I liquid scintillation counter. 
III. Checking the Chemicals 
A. ATP and GTP 


These two nucleotides were checked as suggested in 
circular OR-10 1969 Pabst Laboratories. The absorption spectra of ATP 
at pH 2 and 7 matched those shown for ATP in the publication and the 
GTP spectra at pH 1.7 and 11 matched those for GIP. Paper chromato- 
graphy in an isobutyric acid: concentrated NH ,0H :H0 (66:12:33) 
showed only minute amounts of ADP and AMP in the ATP, while no evidence 
of contaminants was shown in the GTP. The ratios of 250/260, 280/260 


were as stated by the publication. 
B. Creatine phosphokinase 


Creatine phosphokinase was assayed by the method described 


if 























or a me 
anes a 


rol 
& fo Sob wow SrevPens mubeega tinea fe ssi - 


iq Ba fF a act gt 


qe Apt) 2ohtqo eoetidee ciety D0 febon ens 
eau’ Pefrersn nwo) widade @ nk anata banaytirs 
tS #2 OFT deew foey ere. sre! feu ia pa Gy ae 
. J7i ns ot ongh old HPPMOTT Ine ov) Powter bein) t 
fits | ta mirigy, wii i ere Cm ryt tt ns 


m% Vd Dw iy ebw Caghliagetes Yo mol te 7 ate 1 


a 9 
Tey wir 3 hori + ote inal Q02r 


o als) iA 
PATIMOUSA riieaad & MPiW Geng isn aAw vt? whats raeqe otto te 
ave eo} as 7 Py me 
“ 5 om "i rpepun see us ivitsiathes (20 Tobe ‘vatenod ons a 


Ses fet ett j vhupit I vu ah 


Ssaewl) ane eat ee 


i) ae 
179 bao STR AS 


Gd tel cliiee oe Oeebdls wrW anit sontgun int wat GN) 
Teun f¢ 24) .aoprieded Seded CaP Gt my stud 
ail? | wileeoe iy ted AR ATA 40° coals Seat bedpten ¥ bas SH 
-olnnoeds voeel WTO eee he ew | ong ‘sf hq te eeciven 
ra) Og heater desonoy hte atrgsinaet a 
sonsbive of oT hiw , TTA aap OP “PA bow WA 45 aiid onunita 
CANONS , 0a \eeg oy antiaw onF =. STS and it mee ont 
a 
| ss ; 


2a ntaorlgat of 
badisoyeh bartsony old ve baanes tne evan 
. 


a a ae 
Cony a~) ee. |e 


8y4 


in the Worthington Manual [Worthington Biochemical, Corporation 
(Freehold, New Jersey, USA)]. The formation of phosphocreatine was 
followed by measuring the inorganic phosphorus liberated in a perchloric 
acid-molybdate digestion. Using K HPO, as a standard, an average rate 
of 0.029 ymoles phosphorus produced per minute was calculated, and 
evaluated to give an average rate of 34 E.U. per mg protein. Calbiochem 
stated the specific activity as being between 10 and 18 E.U. per mg with 
creatine as substrate, so it was concluded that the higher amount of 
activity could be due to a different lot than was specified in the 


catalogue. The lot was specified as having 81 I.U, activity. 
C. tRNA check 


Yeast-stripped tRNA (0.002 gm) was dissolved in 1 ml of 
0.01 M KAc buffer pH 5.0 and 0.8 ml was centrifuged on the analytical 
ultracentrifuge. Only one peak was observed. When 0.001 gm of bovine 
pancreatic RNase was added to 1 ml] of the above solution and the sol- 


ution incubated for 1 hour, the previously-noted peak had di sajpeared. 
IV. Chemical Preparation 
A. Preparation of bentonite 


One hundred grams of bentonite was stirred overnight with 
50 volumes of water. This suspension was then centrifuged at 1500 xg 
for 15 minutes and the upper gelatinous part of the pellet was scooped 
off and collected, This pellet was suspended in 2 1 of 0.05 M EDTA 
pH 7.2 solution and stirred overnight. After centrifugation the pellet 


was resuspended in distilled, deionized water and centrifuged. This 







not jure: qv? .f wide qetenritioltt fa 


bea La 7 " 


ae 


SINa fated 6 ‘at badotodh avindqanrig align? on? grt “a coil) 


2ow aetigaiwodigeate, jo sokheine? oat Page a wae i Ps ‘or 
of a: 


pidisd .riutorg he alla ME Wo otey ogee, meaty 8 ot bas 



















ote apeave ie , boGhend? ote pO MA arti ay nor sagt pie 


bag <botelwates — bonibor eng 


ones 


a 


dttw on veg U3 Bf Dan Of ‘naapied unied as tives athiosge 7 bet 
to wndom rApit ott Jedd bebwfanoe caw Zt 02 scoeteaslhi 
at bel lisane acu nedd sot tre wtih # od ord. “ad biwoa, a tvisa6 
setivitdos .U.1 (A gnlget es bol thosee eew bol edt GUS itsteg 
Me rena anes 5" 
i tal pia 
i a - 
Tei-rt navioeath cow! (mp 300.6) AVA? boouyadestreeT - : 
(oolieFenn of) ne Bedotiatnes tau fn .8,0 bes 0,2 Wy attod SAW JA 
atived to op (O0s0-wtW .bovisadd 2ew deed are’ ythg cspet hinngetd 
-fwe ot bee wortetGe ovuode att Yo taf oF tebba aow cee phieovon 
5 antl hel aie tafon-ytevatverq odd , ation f +04 batadeoalce sot 
; ye 
not eysqey? teat ome "j 
av tire aee To ihariesyctirt . 
Atiw lithe ry winner? aow Si topiasd to mwrp cmb ae 
ca (027 te bog hhuined edt 260 nelenag ans id], net fos 


jal tou ont res SOARES: a, ath 
emit ib ooutindga mint 






ened 2am £0) jaq aut to nay room dale yea onthe + aaduat Ww 2 
ards, # ii, Oe 10 f . wv bobaeqave 26 es abt bets $< if 







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33 


washing procedure was repeated three times and the pellet from the last 
wash was weighed, dried in a vacuum oven at 85°C overnight, and 
reweighed. A factor of 0.078 was calculated which when multiplied by 
the total wet weight of the pellet gave the actual dry weight of ben- 
tonite, and from this a 4% W/V suspension of bentonite was calculated 
and made, 

wet weight of pellet x 0.078 = dry weight 


dry weight x 25 = final volume of solution, 
B,. Plate counting 


To dissolve the nutrient agar, 23 gm was suspended in 1000 ml 
of distilled, deionized water and heated in a boiling water bath. One 
hundred ml aliquotes were sterilized for 15 minutes at once 16 psi 
and stored at roon temperature under sterile conditions until used. 
Dilutions of the ribosone solution were made in a ¥% strength ringer 
solution pH 7.0 containing in gm/4 1: NaCl, 2.25; KC], 0.105; CaCl.; Ole 
and NaHC0,, 0.05. Sterile pipettes were used to place 1 ml of sample 
into a disposable petri dish. A layer of agar at 45°C was poured onto 


and mixed with this sample. Incubation was for 48 hours at 27508 
C. Redistillation of phenol 


Phenol was placed into a round bottom flask and heated by a 
heating mantle. The first 3/4 of the condensed vapor was collected and 
the colorless crystals were stored in a dark-colored, tightly-closed 
storage bottle, in the dark. When distilled phenol was required, the 


crystals were melted by placing the bottle in a hot water bath. 


bE 






















ji a - . a 
des! wit nee veto ‘aa ory sents eave “pete 
bro. . 2iphavery 4° ww 46 here iMiuohy 6 we ers 
vi totfeitfum monw dott —— zom 809.0 ms 


tafuniea ssw ot inogned. te pibeneyrve vw a & oan 


: jos 
a Ne Bie 


Sigtae yob = 50,0 & tal fom Yo n shat 
nsatoe  anwtov fant? ¢ as 7 tte 


Paes 


¢ 


7 


ape 7 
pectaniiaeh hte ° c. 7} 


™ : ei 


hay 


1 —_ 

ie COG alo bebepceue 2dw ob ES aH Jonsson ons avinzeth 71 

; a ? g Pa al ” r ei 7 7 = 

died sore pawl hod & ob Sates bon +edaw bestaofed bel heh ae 
2 otttiae 

‘ rm oF , ‘ -~< ‘i — ion! P nit 

wr 7 Jb ARal 6 en nim ef 407 Poh.+ (82S Sw esiouph ta. fmt athe 
head tity euriIjbseoelieste seb oveyevaniad pon a6 peor 


“apals Atoneywe gs nt otte a3ew polivios oemmaadm eft to er ota 
a 


A 
% 


ef fH * Pot SW c2S 5, fdas? Oye wb patatetnos 8) Ng 9 


[aes 


yo (ot aaele od teen arte rvsagin ofivat2 , 20,0 “,<hath bag 
aFio bag 2p Da te vagy to says W yatb Fndeg oldezoqeth’ e aah 
'4 


je ermd OD. 909 BAW Abi taduant .afqmee zidd thw boxtae 


ae 
Taperiq 6 rio Feat Lo 2 hb al ‘a 


7 
fe bossa! hae deat) ieayd s0e haley a ath tessa 25H 7 cr at - 


bfk beige! for ew “qe beanabion elt YW A\e Ta iit 7 ~~ 
teag Fog fesigat tio a dyeh 8 at bavwode ovow sty i Hee 
ca [ees 


wit? , dasha fhw Fee Kayt hhetts wut ay ff sel elt 
ra ¥ yd 1 re ry . 
tod i: une r 


’ f rie 


- adlees a 


34 


D. Preparation of chranatographic materials, 
1. Cellex-D 


Cellex-D (DEAE cellulose) obtained from Bio: Rad Lab- 
oratories (Richmond, California, USA) was regenerated by consecutive 
washing with the following solutions, and filtering through Whatman No. 1 
filter paper in a Buchner funnel: 1.5 1 of 0.1 N NaOH; 1.5 1 of 
double distilled water (to neutral); 1.5 1 of 0.1 N HCl; 1.5 1 of 
double distilled water (to neutral). The regenerated cellulose was 


placed in the eluting buffer and packed into the column, 
2. Sephadex G-25 


Sephadex G-25 (fine) obtained fron Pharmacia (Uppsala, 
Sweden) was sterilized in its respective eluting buffer by autoclaving 


it for 40 minutes at 120°C and 16 pSi% 
E, Treatment of sucrose with diethyl pyrocarbonate (DEP) 


Six ml of DEP was added to 1.0 1 of 40% sucrose solution in 
the buffer II. The solution was heated in a boiling water bath for 20 
minutes and allowed to return to room temperature before being stored 
overnight at note Aliquotes of this solution were appropriately 


diluted with buffer and were used to make the zonal gradient. 
Powe butters 


The canposition of the buffers used routinely in this study 


are listed below according to the numbers by which they are designated. 


bE 




















> ore raatstt sfijereranne Fo note 
; us a 
7 71 

WGes bah 009 neve bon tee aides 0 a 


oviMwsenns ul Preieeeea pen fay fs: terviy? > «oes 
7 aa 
eae Lhd tas ony f i 


: on nord: (lw neug wits ai yer? Dis. .ena' ; - « ' we 
; a : 
vo | 6 @.E Se 0. tye 7 2.! sfenrur 4 nando we ay 
a i. 
ve f @.f pte FO %e PS ol lertaem of) vod bal heib old 
= 
= 7 rs 
; weal vite? badaqwonpe af acJuon Gt) a nottieet 75 an 
al 


f . ; ‘TARE Wenioo: ya Viud. re ot 1 
PS mig oles que a 


7 
seat’ atornc et) ran? bewteids (=n?) €2-d apborge °F 


te 

AS VG POA OID ty 6d? Tones, 277° OI yet frgde coy 

o S a, 

oa af iq] ( ’ st 7 cour ein. oii 
i ba : . . , >» 

D) wertyoatyy, (yuitnth «PW eeore 50. TAREE ae 

’ 3 a 
i] mae ti As aueTy ii ; [ i) ] a ft bin. ena % 4 y iy aie 7 
{ TO7 AJHG Hlae i > ys oar og ry huver { tu mi] rh, gifT Mt 


& a 
bauer: oajed aeptad SNdags Sigpint rma oF inter oO) haw TR LN 


j «a 
\) Oa! qo" ar ath aoe wl ae ert Ly) a toup! a) a3 Ss 


Peethoh AdoY att Oyom Os bate. siay Son votue at 


wiuTe ebit: VF qtont ior cots Ret} Wy. to 
7 a 7 _ a - 

totais Sn yertrodotaw yt evodaen and at? geitr 
on ie ; 


6) 


Buffer I 0.7 M sucrose, 100 mM tricine pH 7.5, 5 mM MgCl., 50 mM KCl 


5 nM 2-mercaptoethanol 
Buffer II 10 mM tricine pH 7.5, 10 mM MgCl., 4 mM 2-mercaptoethanol 


Buffer ILI 10 mM tricine pH 7.5, 1 mM MgClo, 100 mM KC1, 5 mM 2-mercap- 


toethanol, 10% triton X-100, 0.4 M sucrose 


Buffer IV 20 mM tricine pH 7.5, 400 m™ KC1, 10 mM MgCl., 4 mM 2-mercap- 


toethanol 
V. Preparation and Procedures with Ribosomes 
A. Plant material 


Seeds of Triticum aestivum variety Manitou were placed 
touching each other on 2 meneetor sterilized 3:2:1 soil mix (soil: 
peat:sand) and covered with 1 inch of vermiculite. After being soaked 
fron the bottan, the flats were placed in a Fleming-Pedlar Coldstream 
growth cabinet and grown for 4.5 days at a temperature Of e20eTC 72h 
24 hours illumination of 1200 to 1400 ft. candles, relative humidity 
of 45 to 50%, and watered daily with tap water. Prior to harvest for 


ribosome extraction, the plants were chilled in a cold room at ne 
B. Isolation of ribosomes 
1. Ribosones for zonal separation 


In a cold room (4°c) 100 gm of wheat leaves were cut with 
scissors into 1/8 to 1/4 inch pieces and were placed into a glass con- 


tainer kept at -9°C by a CaCl. salt-ice bath, Two hundred ml of 





















ae 


(3A Mn OF .9f Jem a B £:£ dalgstotss a ll a - 


= - formleyet apna 


. i ee 
- ~ * 
* “ bs _ 


-tovicdtond main Ny # este in bY Sa. Kong aia or ¥ 
Ve, Sie Aw ee =" aA a3. Se pth 
“Ayre Mo 104, Me got pg Bet 2.8 Mas gata tag hs Mh Y 
: eae REOaUE ft +0 AONE) 70 ray oe ’ 


¥ 
7 


~ 


S mM ‘i i : te sigue Sap “ 

-yarcon-$ Med’, of IgM MOT, TO Man OCF .2.% Ao SRSA wie 
: % i yet paneer + 

eBay ne 
: a4 ; puree te Ta 
ta] a 
¢ eoege ¢ te PW e Ree: Lh Re, 
© ’ i re 4 . ” es 
~ * meh Sy —. a hee vas AW , 
i ev “ist SNER sha 
H&K ed na 
ey 
basihy @igw Hola wOtyEv aovidens prota inT Ya goeee 


i ~ : * 4 4 : en ¥, fh e" : a 7 
rltew) xin figs (obo faxthtrete % 2siont $ no aetito Woes Gene 


ey 


Dedso2 ooiad qT ethianty e Hoot 1 dotw, ba vO RD hos (basen 

maotSzbio2 wel Dotegnrmal i 6. at Bente o15y oant 7 any... mmoszod § a 

.2 3° 09 Gl WO All eteqesd ¢ ih. eyed 2.% OT NYOP bane aatde a 

GD 42 QUAL 9g? OOS) 6. ge itantmul tte we 

ai ieowiet of ters .vetew qed diy yl heb, berstgw tae ,R0Lees 
a ; = ' 


jn of nm uty rp A’ DOTT fia pv aw einety ens ,motianeie / oF 


sepcedis? IW te brake “a 


a 
nOTIHILes2 Tono~ wot wk 


dtiw 25° oveW court ‘toot * me OOF (37%) mayo? GHB 
~f093 bit y' % oaat sasute’ tied bas apie — a 
fot fn perio sp slaaial snag 


>. an 


4) an Ae i; 


36 


sterile Buffer I containing 10% triton X-100, 5 ml of 4% bentonite and 
0.3 mg sodium desoxycholate was added. The mix was honogenized three 
times intermittently with a Polytron honogenizer type PT 200D. The homo- 
genate was clarified by passing through an Acme Supreme Juicerator 

lined with Whatman No, 1 filter paper. In later experiments to obtain 
higher yields of ribosomes, an additional 100 gm of chopped wheat leaves 
was added to the clarified supernatant and the honogenization and clar- 
ification steps were repeated. The heavy material was then sedimented by 
spinning 20 minutes at 30,000 xg and the supernatant obtained was layered 
on top of 10 ml of sterile buffer II containing 1 M sucrose, and was spun 
for 2 hours at 340,000 xg. The ribosomal pellets were resuspended in 


8 ml of Buffer II. 
2, Ribosomes without zonal separation 


The method of isolation was identical except that the leaves 
were ground in Buffer III. When the ribosomes were washed with NH,Cl, 
the 10 ml Buffer II underlay contained 0.5 M NH)C1. The pellet fron 
this high-speed centrifugation was resuspended in Buffer II and an 
equal volume of 1M NH,CI was added. The mixture was held on ice for 
15 minutes before being recentrifuged through the Buffer II containing 


1 M sucrose, 
C, Separation of ribosomes by zonal centrifugation 


The above-mentioned ribosomes were brought to a final 


volume of 10 m1 containing 3% sucrose and were layered on top of a 


Zr 
7 7 : 


at 


































si 


ons S9thosned te tore (sot woster OF enters ; 
seid béxtasgonen esw xtw oft =, betbe aeW ome ‘ —_ ae 
-ohot sfT =, ROOS TY equ? testenggmed petylot « 9. viens tema eo 
riaotvh srorqud sHON Ae dguenit? poleanq 46 bat oa We 

ntemdo 0) etrantrsdnd satel WE trong vost it T Ae nd a3} 

esvacl Ssorw boqqeds W om O0f fewolsrhen me ‘nal non et 
“als bee aoteactosgomod ed bae Sastao19ce pal vingts one. as , bbe 
(0 bathamitige ood? 2ow stasdem ened pr betesea ed { 29088 
Petts Jog2eragwe of? bas ox YOO, 08 Fe apart 08 
mage cw has ,seovsve © C patiinyaos 11 sotbod afixede Yo Te of 4 seis t 
nt pabreypures evew eseffag fempzodty o@T ox OOOORE 16-gwol $ 


= TT ant % 


whfetegee Inaos Juecliiw gomeodis .§. 


i 


rein q if Taeoee fTe5)peeh) sew nol gefor! te boltse eAT 
,\ 2,0 (fiw badesw arew asmraipd’y $q9 wad ae verte at “bnuom 


wer! feffeq aT 21300 4 2.0 bentedago arn bi I voit 
ns tine lL] sett al onners coe téw wor? ago rtsanes teagan 


“oi «*f so blef gehw sravetn eat J Sebbe baw (hi “wT % ti 


ee 


nitmietioco IT «ettae eft a tale bsmvTh Vinee preted snoted 


a 


ies 


wolseguttednag fenos yd reimzadin to not 184 


how? 6 ot artnet opal apenrodi4 tone Nt nsMeg —_ aft 
a tw ner oo beret wing ha avovang tee 
ery s. err 


Sf 


convex gradient ranging from 7 to 38% sucrose in the sterile Buffer II. 
The centrifugation in a Beckman Ti 14 zonal rotor spinning at 47,000 RPM 
was carried out for 3 hours at 25°C or for 7 hours at AP CR after which 
the gradient was analyzed via a flow cell in a Beckman model DK 
Recording Spectrophotoneter at 290 nm. The large quantities of 
ribosonal material used in the zonal separation made absorption at 

290 nm a requisite even though maximum absorption was at 260 nm. The 
peaks corresponding to the 70S and 80S ribosomes were collected and 
pelleted by centrifugation at 340,000 xg for 3 hours. These pellets 
were resuspended in Buffer II and were stored less than 24 hours at 4° 
or for longer periods under liquid nitrogen (-196°C) before being used 
in the incorporation experiments. Ribosomal concentration was determined 


by measuring the absorption at 260 nm. 
D. Dissociation of ribosomes and separation of subunits 


Ribosomes were suspended in Buffer II and centrifuged 
through Buffer IV which contained the sucrose gradient as described 
above. The peak corresponding to the subunits was collected and 
pelleted by centrifuging at 340,000 xg for 3.5 hours. Subunits were 


used immediately for recombination or incorporation studies. 
E. Swinging bucket centrifugation 


Three to five E560 units of ribosomal material was applied 
in a 0.1 to 0.2 ml sample on top of a 5 to 20% linear sucrose gradient 
in the appropriate buffer. The two sucrose solutions were mixed in a 


Universal Density Gradient Mixer, obtained from Buchler Instruments 


at ra ”) 






aa 
nine —— 


dsinw verte 2°) Je exuod T.4eP 4e J a te cued t. 105. be 


14 gett, af teste ott mt oeonsus a ap ner 


Mw O00,Th to ewanhas yotd: tapes Hf 4 reaintan at 






























1) (abou ateloel a at Map wal? & viv neagtens om vot 


14 cst iitnayn yal enb..a9 OOS te Teen OtReONME. OF 
«oa wT SQeerdés shew BOERS form: self At Beew’ Totvetom. im pdt 
‘= wy att tie. nal rikk¢ i? r4 9aeds ies a " i ee rp ay agta supe — 


ad 


has deofaation maw ehependiy G68 bas 2% alt op enna Be 
alloy get] ewan © ae? ge. O.9 if eget etnes «ud batst fee 


>) 3h ayeW Fo nad? bael henee2 eiey bon ia? Palh ae bebnaqeuest. gM 
j i _ 





boa on Gil. mie iy ii) : Ot. ra | pel Hb thotdaq Masta 
bawtrvatah zyw. “nv Fempeodih .-z eal ‘ja? not reroqroant oF 


im CVS ts mp tteproadin elt pata 


wa 
limwidue * fc an att Zoberagd)* ie nnitntogaelt 
# 
iy aay oH | r At S@iiew 2 wigw zaumrodhd 
j 244 ge Sra eavs et fe? Dentale daztiw Vi antud 4 eon 
: a 
S}10S fe ehigidye 4 oF gytbupgee ws. dang ad 


mpive .2ybot 8.0 teF on GO ORE ge mrewtiathes ¢d, rasore. 


. P J a 
sthuty naldawbasaue> so aottentdnaeo: Ww? \foteibenm 


mitapuliygnss geaaud er beget a: | 
. Ml 7 
ballqqs tnw (nivqtad (asmendie Yo 2dieu cay? wvt? oF ay 


— 


Tiethiny aeoppue Veni OS Gs ww Be “" % ‘slgma. tm $a 
sat hestm exew dngheaein naonoue eve att. & e vd. as rT 
Fjasmast onl veldeed & wert bembKddo .wwnrh nat bak ) vate , 


A) ae 


38 


(Fort Lee, New Jersey, USA) and the gradients were formed in nitro- 
cellulose tubes obtained from Beckman Instruments (Palo Alto, Califor- 
nia, USA). After spinning for 4.25 hours at 85,000 xg in a Beckman 

SW 27 swinging bucket rotor, the nitrocellulose tube was punctured at 
the bottom and the gradient was pumped by a peristaltic pump (LKB- 
produkter AB, Stockholm-Bronma 1, Sweden) through a flow cell at a rate 


of 1 ml per minute and the absorbance at 260 nm was recorded, 
F., Puronycin treatment of ribosomes 


1. Preparation of ribosomes for dissociation in swinging 


buckets 


The method used was similar to that described by Blobel 
and Sabatini (1971). A ribosome suspension which contained 100 E4¢4 
units/ml and which had been obtained either by zonal separation or by 
the method using Buffer III was chilled on ice; 0.2 ml of this solution 
was added to a solution containing 0.25 ml ice-cold, double-concentration 
Buffer IV and 0.05 ml of 0.01 M puromycin pH 7.0. This solution was 
held on ice for 15 minutes, transferred to a s7t Watae bath for 10 
minutes, and then 0.2 ml aliquotes of it were layered on top of sucrose 


gradients in the appropriate buffer. 
2. Preparation of ribosomes for zonal dissociation 


Four ml of a ribosane solution which contained 750 E569 
units/ml was added to a solution containing double~concentration 
Buffer IV plus 0.5 m1 0.01 M puromycin and was incubated 15 minutes 


at egos followed by 10 minutes at a7 Ge before being placed on top of 






















HZ 


: 
“ong he nt bane) araw esheets 7% sive (ABU ae 
wiptited ,offA afse) edromoienl npeniesll wiyt bon 
fjcatiss o nt gt COO, 88 a3 swe GS.2 yy entantge 4 ‘ 

c hewwPonty few atl sdolutt agora) ty ait J Oto ret mtn #2 
Tare hat 6 GE Berg =u STON we ‘bie ataod 
oe’ ¢ tt , » epoares Unehwwe .| oxi 2-fttood2 ae Tb al! 


Py Pen 1 Pot me iat 3% gonad we iq a bene adunte oq Tut ‘ 
igs aan 


eome2osiy to tromnsst ghoul 2) 


yy 7 
Qual pitt we ay nwigeF potTh: YOY Seaeodi Ve ao Figveqeyt cf - 
aioioud 


7 





fevafh ad | | eg? of Swldwl + caw tere bedded? Mp — 
4 Ope yt? Li ‘ais 2) pa A PSN! ‘ at a i A (ft for) terved 
m (olievedse 1eacs iste tapinite moe) SON its Peleg bis fo - 


fetiul  *e Tw 9.0 eest no bet) ida gow 11] aeThe oni inthe : 


Tole nity faueh cbitseant [a eo0 prtrnidadmn ao foltulog 56.09 bat ba 26 
wi eed?) =. 0.9% Oe whoever BF 10.40 Yo fm.d00 ba Vie n } ty) 
‘ 
Os « 
iow \t 6 Seve d? ,catuaha é( sot oat, ob 
g2orjue Sp gat fe Seveyeh etow a) to eadeupite fa. $.0 nodd bas. 29300 


Nia atwlrqorqqs od at aig 
1 5%: 


gag? oa berinines ¢oeiW Mort iloe gimepodts 5% Tim wo? 


abt? a Ttmsonad-atdunt Pie oe ee ng 
aa 








roitatnorsth Panes yet seegandis io noltersqay 


acJintm. SF baabosvaeh saw sngengce 


to got no hovaly givteed 4 
v 


>=, 4 N : 
a - & , - 







‘ 





39 


the zonal dissociation gradient. 
G. Estimation of RNase in prepared ribosomes 


The method described by Sodek and Wright (1969) was fol- 
lowed, except that wheat germ soluble RNA and ribosomes were used as 
substrate instead of purified RNA and enzyme. The assay mixture 
consisted of 1 m1 of the substrate solution, enzyme (up to 0.4 ml), and 
0.1 M NaAc buffer, pH 6.0, to give a final volume of 2m]. The assay 
mixture was incubated at e7eGe and 0.5 ml samples withdrawn 10, 40, and 
70 minutes after the addition of the enzyme and pipetted into a 0.5 ml 
5%perchloric acid containing 0.25% uranyl] acetate. The tubes were 
chilled and the precipitates spun down. A 0.5 ml sample of the super- 
natant was made up to 5.0 ml with water, and the absorption at 260 nm 
measured. One unit is equivalent to the production of 0.1 umole of 
acid-soluble nucleotides per hour, assuming an average Ea 6gof 10 for a 


umole/ml mixture of nucleotides. 
H. Sedimentation values 


Sedimentation velocity runs were made at 20°C of the 
Spinco Model E ultracentrifuge equipped with schlieren Optiicsae:.Lccures 
were taken at 0, 4, 8, 12, and 20 minutes after a speed of 39,460 


rev/min was reached. 
1. Reading the plates 


The plates were read backwards fron 5.0000 starting at the 


inside reference hole (see diagram below) on a Gaertner micro-comparator. 




















ee, pie 


amend terre 4 af aeons ton 


-to? aow (gael) sped tng eke? vd bail ramen aa 
a8 bseu view comaodia big BR eidvtue ray vaveutw apes 
ewivia yarzs of! gmat ban AAR hottteug tw Abupten? ¢ 

bie .(?o 4.002 qu) amesae ,wotdalos ata~tediie aie 1 Tmt tat “on 
yiers ofT- , fm S$ to cuufov fanfts evip oF ,0.0 Mg ‘eThud et WW Ty 
kas .0) 0! oeayblttw aelomms 1 , oi is | Ts eededuont — a 
in 2.0 6 ofnt batiedty has saree ‘ens to votethbe Oy aadte essuntn 


vis Fee | .etedJene low 245.0 grieled “0 bisa ahvofedl 


a"AQueP Sih Ww : ii: : fi 2.0) fh . ee nw [2-45 e714 Jog | Sis been be val tt 
wy O88 te Anttqnoed bow Vere tie te O.2 of qu -otem ssw SON 

a 

> ofoms (2) To melooaton sai oo Seofavives ct-2iwi end \bawa 

eat Oy W ; a+ y * qufiaveng ,“uon % ) 24? hfoslT aun ofdytt Le 


eobitooTsun To eruyEta ime om 


esu’cv nohisinemtbeZd | 














1 6 PO se wi Gal atgu ean vilcefsy nobistnenibe? C 
vie BT! ine mvistiipe dite bagatvna goutitossertty rn 
ia bosge a Voote zetunim OF bre Sf 8% 10 om 

bony ‘ 


; a 

asautg ode patbeat 
sa 

ett Ju quidunde Mone, s wor. abr senita ge! boon sow aot ig ont 


Ot rieqiian arate soniye) pire tap —0 wn v tof 29 a: 


er ae 






40 


The center of each peak was read for every time interval and these values 
were substracted from 5.0000 to obtain the distance the peak had moved. 
This value was divided by the Magnification Factor (1.9435 obtained by 
Dr. Jones) to obtain the distance the ribosome species had moved in 

the cell, and this distance was added to 5.71 (the distance of the cell 
fron the center of rotation). This final addition gave the distance of 
the species fron the center of rotation at the particular time. 


a— inside reference hole 


memiscus 


80 





I 


et 
4.4546 5.0000 
Sample calculation: 


5.0000 - 4.4546 = 0.5454 
0.5454 








oat tt 5.71 = 5.9906 = the distance the particle is from the 
; center of rotation. 
2. Calculating sedimentation values 
Sedimentation values were calculated using the equation 

d 

dt 2.303 (d log x) 
SM which upon integration gives S = ——y— = 

WX w dt 


w = radians/sec. Since | revolution = 2 radians, the speed used 
(39,460 rev/min) gives 4134 radians/second. If log x (the log distance 


the particle has moved from the center of rotation) is plotted against 


_ a ” ie: 




















giitnv ened bie farianel ind wave 10? wana 
bavi boa men ort? ewueteth wt ido Jo oo ono. a a t 
ud bons m Gfh0. 1) wine notbeat +0 pat welt ‘ed tabi zi 
a! hovont bat 2oPsage smecelly oa) «6 nude ty ante nia sco 0 
{tsp add Yo somutierb erit) Ga ot habhe cow aovetelb ats | 
Yo wneret af? oveg ante tebe tet? era? . retetor Tooneimse gil 
raieolpyeq at? 26 Wallatos % wnsees wnt mrt 


Nf 
nial 
5M n vo 


as, 





7 
ottetwatas af 
b2h2,0 = BPR, 8 ~ ONO 


tw + Wee «= a + See 


| #7 ele iiaee aly aware! > ni 
gina as 


mahiatoy, tea 
esuTny qotierseuifane pwttafin tas = : 
e 


nolteups oft? gulew bedetuates axaw eattev rt esrneg 


«ff fx nor b) 7) a) ‘ 


7 ae = 2 vevip nena wae i 
_ o, f 





_ wy booq? o6o cautery S « aston pare 


sompselh Gel oft) x poh Y scene te > eavio | (at 
Jentope wine P Lerpivees sto ast ie {¥ bovom & 


a in 
7 le . 7 
\ ) ae . 


4) 


d log x 


t (the time (sec) for the particle to move that distance), then Ae 


equals the slope of the line. Because a number of peak distances 
were measured, these points are plotted on a graph fron which a best 


fit line can be determined and the slope can be calculated, Svedberg 


units are in ieee sec, 
Pee SAO: 

$= Yemec Ole 
ee aa 


The calculations for the distance the particle had moved fron the center 
of rotation (x) and calculation of S value fran the log values of x 
and time in minutes was programmed on an Olivetti-Underwood desk 


conputer for efficient calculation. 
VI. Procedure for Incorporation 
A, Preparation of synthetase enzymes 


Twenty-five g of commercial wheat germ was ground with a 
mortar and pestle with 100 ml of the sterile hanogenizing buffer (0.45 M 
sucrose, 5 mM MgC1,, 0.05 M tris pH 7.5, and 5 mM 2 mercaptoethanol) 
used by App (1969) for rice embryos. The supernatant derived after the 
homogenate had been spun for 15 minutes at 11,000 xg--to remove large 
debris--was spun at 340,000 xg for 2 hours. Twenty ml of the resulting 
supernatant was chronatographed on a 30.0 x 3.0 cm column of fine 
Sephadex G-25 equilibrated with the sterile eluting buffer (0.01 M 
tris, pH 7.6; 6 m™ 2-mercaptoethanol). The portion corresponding to 
the first fraction absorbing at 280 nm was collected and 14.3 m1 of it 


was immediately used for the aminoacy] coupling reaction. All 








ve 
i) 


tt | ry y ; 





Me Dy ae 42 , (oonetath arte wba = algae 


zesonateth Jeng Ya veda “ ets20) part 10 i 
. . - a 
Sead ¢ dotiw mrt doeeb o An bintto he oth ethoxy em ob ; 


1 
etedkoais 


wiadiav2 .vtaleniés of ned agate aft tee b stead i a 
- ante Mor at rte a 
were 


_ -F c.3 


deep 





Ole K 8G 























yatiso oF vont. boven bed statidgn-een? sonkscth ant vot ena of 
Oo e@nlav ae! off cwrvt ant sv 2 to norteiwoles Dew ix} not tes 1 to 
f2aob bomen nis Teevild Gé a baciar9wwe ew estbelp nit a 

| , fe Meluetes inet 297% 107 vi3uq 


. - 


te AP 
Holsbiwreanl Ww? enibede 


a 
> 
comer condorignys to noldaveqer? JA 


a 
A Ai lw Orie 2G firtep Jao ww letotonms.? 


> @ ovit-yiaowt 7 
MW eh_.O) ot tod gbstaemomd ef tiere arie to Te OOT Ha hy oftanq bn 4aF 
(lonarlivesiharan. Me 2-bae.ceS Race 20,0 y voaetdagoi ti 

aft vet's bated. Tindieringad 607 . 20yrihie gohy 407 (88th) aA 
comet of--<pn QOD, ga warunhe BR <6t avqs aoe mile 
gittiveey af? Yo fe ype thin § » VOT Bx OOD, ODE Js wae 
ont) Yo amitoo wo-04 » a. at 60 besiqan ge zemeris: caw SO 
4H 10.0) +t potavie a biede any date: beset fbupar we 
o3 miidqoqseiTe narerog adT Petre) 
tt Yo tn E.0f Bap betast tom tow wt OOS tee Cary 


} : 
rita wh Sapir net quan Fypnontns-s Mee , 


a 










cimraa| Je 


42 


glassware used was soaked for 24 hours in chromic acid before being 
washed, to help deplete the amount of nuclease present, Plastic 
gloves were worn and other precautions against these nucleases were 


taken. 
B. Preparation of ribosomes fron wheat germ 


Multiples of the homogenization mixture described above for 
the preparation of synthetase enzymes gave the yields of wheat gem 
ribosomes required. The supernatant fron the 11,000 xg centrifugation 
was recentrifuged at 30,000 xg for 20 minutes, and this supernatant 
was underlayered with 10 ml of Buffer II containing 1 M sucrose. 
Further steps were the same as described for the purification and 


dissociation of ribosomes prepared in Buffer III, 
14 
C. Preparation of ‘C-phenylalany1-tRNA 


The incubation mixture, as described by App (1969), con- 
tained in a final volume of 20.0 ml the following: 100 mg creatine 
phosphate, 20 mg ATP, 800 ug creatine phosphokinase, 8.0 mM MgAcy 5 
Fe SUED) 140 _phenylalanine, 0.19 M tris pH 7.6, 27.6 mg stripped yeast 
tRNA and 14.3 ml wheat germ aminoacy] tRNA synthetase as prepared 
above. The reaction took place in 15 minutes at 36°C; after which 
0.1 volume of 20% (W/V) KAc buffer pH 5.0 was added and the mixture was 
extracted with an equal volume of 88% (V/V) redistilled phenol. After 
centrifugation at 11,000 xg for 15 minutes, the aqueous top layer was 
carefully removed and the RNA was precipitated by adding 1 ml of 202 


NaCl and 2.5 volumes of ice-cold ethanol, After standing overnight at 


oh 























eriod stoted hice aihewmts 5 ab prve % vt 
stizet? eq Sete a o 19 31 wont ui! 
wi somgetoon opel Heelege Ssehius ONG ship bins 


* Re 7 
mio tee tr? eororodin “fh hob se A 
497 avodo badrtog ntuFaele nese neporen Bd Fe aenon 
met foady Yo alety ef? aebp trate sz88 deaerye notye ‘ - say 


! 7 - 
A ritmea gr OU To ai? et The teeyogee SAT hartoper ee omg ay" 


5 


insterdyicy eid? Den , eptakie GS «otf ba OOGLE 1s bowut Ite wr 2 ; 


ny néctaes TT sethd Yo Ta Of hw Sevnestaaiee 
: 7: Vy = 
Mans rigo) bivey aff) ac? bod ocob sg emer. et? ayew enade Sane 
Ye ” ac 
JU “Yad a) townqere comteod is toma eae 
a 


7 


AAT ie r ' _— aT * r ® 7 : 
halt =~ (gy bet one 4 1) notfsyaqerll - 


now», Of) og Gl bodrenaeh ts ,otwi4ta ooldaduanh eft 
: 
io pe OR sonéwollo? sf? (er B,08 50 oavfow Tpyht eat Same 


re iy ui Pe ib) MENTS: 3G penn tsoi> Ru O08 «SIA pa ms atone 


Sanex bogulars pu 3. T5.. 8. 'Yily eted WOL0- orton ioe 
Derraqetg 2 o8R nitove Pwd ry peoatha. sreap Toad tw £08 be 


Asiviw aerts . "a8 


2nw oaprxte ett bon bebbeoaew O.2 Aa sattpd oft AWN} ye: 
etth = . Forme bet lacie ww f5 to sawloy te we 
eam VANE God Samana vat cratuiatn 2 To aat: a cont et riot 
POs to Tah paibbe. Wiveiateytoiny aw te A add. is tein 


i 
me 
+6 FARTING pribiiute wa Menene' p90 } z 
| i ' : fi | 
— 


i | 
av he - : ¥ ‘ 
_ —_ i 


fee wptaithe 1 mt sdatq tos mpronticns f 


43 


Sey the RNA was pelleted by centrifugation, and then redissolved in 

20 ml of 0.01 M KAc buffer pH 5.0. The precipitation and centrifugation 
was redone, and this time the pellet was suspended in 4.0 ml of the 
buffer. Any traces of phenol were removed by chromatographing the 

4 m1 of suspended RNA through a sterile 28 cm by 2,5 cm column of 
Sephadex G-25 equilibrated with 0.01 M KAc buffer pH 5.0, The first 
fraction absorbing at 280 nm was collected and the RNA was reprecipitated 
as above. The pellet was resuspended in 4 to 10 ml of the 0.01 M KAc 
buffer and stored at -2°c. Again precautions as mentioned above were 


taken against nucleases, 
D. Incorporation reaction 


Incorporation reaction mixtures containing 8-9000 cpn of 
M4 phenylalany1-tRNA, 25 mM KCl, 16 m™ MgC1,, 12 mM 2-mercaptoethanol, 
10 mM tris-HCl buffer pH 7.5, 1 mM ATP, 0.5 mM GTP, 5 mM creatine 
phosphate, 10 ug creatine phosphokinase, and 80 ug of poly U were 
prepared. To this were added 45 E560 units of ribosanes. After 60 
minutes incorporation at 37°C, 0.5 m1 of 0.1 M cold phenylalanine and 
0.5 ml of 20% trichloroacetic acid (TCA) were added. The samples were 
kept at 80°C for 30 minutes and filtered through a 0.45 » millipore 
filter. The filters were washed 10 times with 1.5 ml aliquots of 20% 
(W/V) TCA and finally with 25 ml of water. The filter discs were dried 
under a stream of hot air before being piaced in 15 ml of scintillation 
fluid (3 g PPO and 100 mg POPOP/1 toluene) in polyethylene vials. The 
channels ratio method was used to calculate disintigrations per 


minute (dpm) from counts per minute (cpm). As a precaution against 





























Ly 
i 


a 
ie 


vs : 
nt hovfucetien moa? ban noltagant tir, os aon oy | oh 


notingutiutnss bow, qobtedt dlaere ahi a. iit d rE 
aft to Im 06 af bobmegeme gee talres 4 ids enka é 

ody ontice reedantrnty, ye Wegener oy oM on Ya 

Yo patos 1 0,5 na Betinaie « gue’ wae: stinegt 


ce 


J2a+ FF any .0.2- 4e GS) OR MH TG.0 Wiis bets vag 2 


a 


betes ‘ulhsovers. eov At ot tee Satoallao enw on OBS ~ 


SAY B 10.0 off to fof oF Bok bebtugeveet ten _ vit. 
giow svode Lonar?nam, es. 2ae (ores a4) a abe t& sou e 
.eseatoun dank i 


vet 
_— 


~ ry 
ae 





i 

initisey nohfebarnont ” af 

wv » Mf, a 

i > 

b fps ha] 2 NCOP a pi FnioInes 22s RAG ott “1 ig! no? tarormperl 19m 
,lonetieotgsoreies Mer tf ..Foet Mw ot foe ie eS oP etee 


“ “~- ue anit . 7 te ; } 
anisteors fe Mie ir .Y gsr Po) .2.\ Aig Ve ae {IK-2 1 
? a 7 
visw U ylog. 40 On O8.brh sapurtiddgeods snttesra, gull alin 
1, 
i 
02 yorTtA 7aepeaats th 2sten 3 26 bebee =rau eter et: 


_ bere ontnatalyioda bing # 1,0 Io lm 80.0 Ch te neriotogagnl 
iqus2 ofT bebe ayew (291) bine ahMoanotetiotst BOS Ae 
ee | 


swogiittw au 4.0/6 Apanryes baris: iy haa eetunten 06 wt JOBS 


. 


“a 2 
rd 


05 ‘Yo ctqupi Ns fe. 2. day 2yanis Of bodenw orca eiagt 73 


boiyh osu earth qail?? ahr , vada Ta tees sit Fw gl iant? ‘ona 
nctiaifiantge Vo tn 2) eh baselg: anted sated a6, a” 16 sendy de 
wit .2fahy anor Aaa: int (gnict oz hy wo, v8 

89, 201 SVG E TIED, atetuates a2 byew § — ty 
ent hon, Og ttuanang 4 i, stra) aomtn ong 


rn 





ot 


ar | 


ou 









- 


44 


bacterial contamination, sterilized glassware and water were used 
throughout. Aliquots of reagents for single experiments were frozen in 
acid-washed, sterilized tubes until used and were discarded after once 
being opened, Reaction mixtures were prepared inside a fiberglass 


tissue culture hood under a germicidal tube, 
E. Preparation of T factor 


The method used for the isolation of T factor was described 
in detail by Legocki and Marcus (1970). Wheat germ, 30g _ was blended 
at low speed for 50 seconds (five blendings each of 10 seconds 
duration) in 240 ml of 1 ™ MgAc,~2 mM CaCl,-50 mM KCl. After this was 
centrifuged for 10 minutes at 24,000 xg, 0.01 volume of 0.1 M MgAco 
and 0.025 volume of 1M tris, pH 7.6, were added. Before centrifuging 
at 340,000 xg for 2 hours, the heavy material in the mixture was 
removed by a centrifugation for 20 minutes at 30,000 xg. Fifty ml 
of germ supernatant fraction were dialyzed overnight against two 500 ml 
changes of medium | (2 mM tris, pH 7.6; 5 mM 2-mercaptoethanol) 
containing 2 m™ MgAc, and 50 mM KCl. This protein solution was chroma- 
tographed on a DEAE-cellulose column, 1.8 x 19 an, equilibrated with 
medium I plus 50 mM KCl. Because high purity and separation of T 
factors was not required, the method of Legocki and Marcus (1970) 
was modified at this point. Medium I plus 0.3 M KCI was applied to the 
column and a void volume of 24 ml was discarded before 100 m1 of 
effluent was collected. The protein precipitate resulting fran the 
addition of (NHa) SOQ, to 65% saturation was pelleted by centrifugation 


peg 
at 30,000 xg for 10 minutes and was dissolved in 5 ml of medium [. 


bd 




























ul nNesav? otaw 2inatlhteges Sipats <e ied te. - 
eh vetts bebisorty orpw Gite Begy Tine esdut pestthe 
ciel preci? « Ghian botagntg wien SWAT ootesielt 
e008 Tab? =tevey 6 vane boad witne 


a ad 


+ut0* 7 4p pty, 


bediaszab EW. tod T Yo nOffeloxt ot? oc) bego pideSdT q, 


at 


in 


ted f ‘y a ? _ Hiei sh it) eT) ruven bas ow) va (tadab wt 
—s" ; > See 
banowe OF Fo foep 2yntlnwi< av/}) abnesoe TE aed base yor = 


t~wadth 14 fm Oes 20a Me Sok te fe Pet nb (it 

4 ; ; 
gk 7 aw ame'oyv 10.0 te OG) fai ea pelm df. ot bape? 
yt ‘fes9 ‘10%9 babes ayew .a.' ‘iq .cind fF 8 oppose ao 
a ® «ft ob Tobvetaln weed oat corned oe ae 090, DAE Sn 

ti VIVA » Uy. 6 ratentm OD oF catia mrrsgnes § Yas a 
bin 103 colags. SAciara we ylors seen nvttcery toatansque ino 6 
ae horamF Ms 2 33,1 Ny ete Mak) Tmthem to Beeeeee 
r holtvloe atgdortys elit . 13% fw OF bas cahem Ma § entated 
it CF RET yoomlog sporurlso-3Aad » no bsgrigs 
mane hee were Notd eauegel ©, i31-Min sa auth ‘bai 
(OT) owiehe. lpege ( to bohio Al , bay tupss son ad aa 
aye Os ‘ane 0 CO) 8 0,0 eta | outiell date: ets ist 


to (# OOF sve ted beberath ew tor be Fe. omitov htow 
arts naytt pnt tus 7 ated grag ntetoyg, oT -batsst 8 268 
Wo 7 hga? Peinas go vorel fogs 2a my I Peaudae tee a esti vt fi) ov Ae 


opened - 
Y ulbon to Te 2 at ‘Bavfoabre b do tee ee sunt. 
: - : 


. 
> 


This crude T factor suspension was dialyzed overnight in two 500 ml 
changes of medium I before the protein concentration was estimated 


by using the Folin reagent. 


45 











lw SE wee st eh nm ; 
ee ; @ew ne Pye desman At: $o°@ er: 
: aT 


one 
a 
— 


aie 
i 


RESULTS AND DISCUSSION 
I. Isolation of Ribosomes 
A. Characteristics of zonal separated ribosomes 


Ribosomes, prepared for zonal separation from the leaves of 
4.5 day old wheat seedlings, contained five peaks in the analytical 
ultracentrifuge (Figure 3A). The sedimentation values measured at 20°C 
were 47, 68, 79, 120, and 151S. It was assumed that the 79S peak was 
ribosones of cytoplasmic origin, while the 68S peak was ribosomes of 
chloroplast aire Other evidence bearing on this will be given 
later. The 47S peak was believed to contain ribosonal degradation 
products, while the 120S and 151S were believed to be respectively 
dimers and trimers of the 79S material. Ribosomes prepared for zonal 
separation fron leaves of 5.5 day-old wheat plants contained the same 
five peaks in the analytical ultracentrifuge (Figure 3B); however, the 
percentage of 70S peak had decreased in area while the 47S degradation 
peak had increased, 

During tissue preparation it was observed that the older 
plants were tougher and more difficult to grind, and it was concluded 
that the increase of degradation product in the 5.5 day-old plant 
preparations was caused by the more rigorous grinding needed. Therefore, 
4.5 day-old instead of 5.5 day-old wheat plants were used in further 
studies, 

The monosomes of the 70S and 80S were separated from each 
other by zonal centrifugation for either 7 hours at 4°C or 4 hours at 


25°C, Both methods gave essentially the same separation. Besides 

























Wlemaiere cin ey 


soadin Petaiees 


. 7 
rovest al? ote) en hi ive TERS vo? banger ge 
iedliieioenn art Wf | 7 GUL? Ben l wings... linc 


iso bovezesn Tauléy NOPCRIneDey 4 
aj i 1A! ati i, 4 ; “wa 79 a3 i‘ ; } en | hie ost a io 
9 od 4 sit \ roth? a rr rg t) times dates Ww 29m 20 


i 


ave j C aa . Pah * , 1, vi 1 A wh i is ee! | io) tiers 4 ny " 
s } 


mot Pnbe nemles whetawa of bovailed gaw tase. . \ aT ‘ 
i - 
—s ee ‘ 

‘ J Ju = ; 1 gw i bet a asus | ait of iW, 3 


iene 107 oo'vS ki eos 20F tara 4 ce end 70 ershiag 


any: and yout }ugo odie nh Ty ib Ae Se zavasl rey?) ae 4a 
ond ,1ovewod 7(60 wiuyTy, hrinsoastiy Teotiyl enw amp at 
aor? ef Ke) e+ Wi \ ‘A ¥ oO mn; Teesersad bal : PT: eeu! | a oF ¥ otean 


tesebuieel: bse 


Dan SIG ey 1} ‘ el 2 javGg svzels on bye] : ’ 
: By 4. 

it tna ,baitb oF Jiustthih ovom bas vodgued sven 238 
* _ 7 7 7 


* 


uilg@ him-ysh @.¢ ghd wl. Jouhorw ncliebevoeb Ye ——«< 
| ,Behoor prébcbwe, 2aeeepty ovom mff vd be asta tw eng fable 98 
vt At boaw wtow 2gata Jdcdw ble-veb 2.2 b.. voosant bf il ; 


- 


2 ' 7 


: 
fee vy) beiaveqee cian O08 bas 201 off to esmazongm aT 


jn orvad b 46 3° h 36 zitad f THT TS said olga uy i ti 


Be ebor 


aod baeG en amped ate 
os 





* io a 





Figure 3, 





47 





Analytical ultracentrifuge pattern of leaf ribosomes fron 


Manitou wheat. A. 4.5 day-old seedlings; B. 5.5 day-old 
seedlings. Wheat leaves were honogenized in Buffer I (plus 
10% triton X-100, 5 ml of 4% bentonite, 0.3 mg Na desocy- 
cholate). The ribosonal pellet obtained was resuspended in 
20 mi Buffer II, ‘clarified by centrifuging at 30,000 xg for 
15 min., and was layered on top of 1 M sucrose in Buffer II 
before being centrifuged for 2 hours at 340,000 xg. The pel- 
let was suspended in Buffer II and was analyzed. These 
pictures were taken 8 min. after a speed of 39,460 rev/min 
was reached and sedimentation values were calculated as 7s 


described in the "Methods". 


i 













7 t 
.? 
? 
i iteg aie etty teorteenk | 
of :zo0 ieee toe © 2 Uregilw cabin 
Pury rayient ater covget ised’ .2zpaplbsag 
1303 ri iiwetied 26 to [Ne Ol -% pried OF i 


*” iw honinddy Soffey Temeadh oT .(etetede 
« p 
yo UOU,OR Te ot Gutev neg we Do! titefs. tf) sania Ta OF 


sf veThd 6) 6oormue WP AogeT mo teva esiebés nti t 


-lon net) poe 080, O84 ge telad § fa? gettin ented eyeh 4 
2 r b % r ; J [ : 4 a - 
eeocT ba, ons vew Ure U2 vay af i al wa 

oh Wy wert ORK, OF Yo Vote? 2 957% oa 8 agtad | “sy aaah 


—— 

iol ; 7 

ef ch Cslalintéo saw aalow no ttneniigeig 6 
. 7 : i. a a 


_ = 
q 


7 i 
tw PGE ar r 





48 


requiring a longer running time, the centrifugation at 4°c was formidable 
because of condensation in the centrifuge tub and temperature fluctua- 
tions of buffer solutions during the loading operation. However, 
centrifugation was done at A°C to help retain biological activity. 
Figure 4A illustrates the separation attained when 1500 E560 units 

were placed on the zonal gradient. Using the triangulation method for 
calculating the areas of the two peaks, one may infer that the prepara- 
tion contained 20 to 25% 70S ribosomal material, and 75 to 80% 80S 
ribosomal material. Figure 4B illustrates that as much as 3000 E560 
units of ribosomes could be placed on the gradient and still attain 
sufficient separation. However, higher concentrations overloaded the 
gradient and one large fraction resulted, It should be noted that 
ribosones of 4B have sedimented further down the gradient than the 
ribosomes of 4A. This phenomenon of non-reproducibility occurred often 
and was thought to be caused by slight differences in the temperatures 
of the buffer. The density of the sucrose buffer will change with 
temperature, thus causing ribosomes on cooler gradients to sediment 
slower. Because this method was used only for routine separation of 
the ribosomes, this problem of non-reproducibility was not considered 
serious, 

The 80S ribosome fraction and the 70S ribosome fraction 
were isolated by centrifuging the collected fraction indicated on 
Figure 4B. The ribosomes obtained were assessed for purity on the 
analytical ultracentrifuge, and as is seen in Figure 5A and 5B, the 80S 
ribosonal fraction was almost devoid of 70S contaminantion and the 70S 


fraction was almost devoid of 80S contamination. 


Sp 
a! j if a’) 
“Ht 
Pe 
ea Ti 
ty 
'} 
i 
fii 
ot 
> Te) 


COS eas (Oh a@Piqbafmerqg Bah V6 beam Tee 


r4 ig if 
y 
o 
41 
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‘ a) 
iy 
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‘ 
tt? 
Ds 
Pe > le 


a _ ) 
- 7 ma) 


, 


Pres. 


7 ie « fy 
TY srvdewmamy Sop guy) 2 Aa i) adi it wt aa an 
4 


nel 


“4 
Fok 





















th1390 eau! wii onvbrdh andi Tee 
ntatd Ate at ak, 7H 1%, sic Ma soon 
nett vowtayis —winee emu ccna 
iy ; Pale _sealherp fanas of no tor tq 

: 


it CA OAR Seahas owl 499 36 eLYNB. ae pfs 


ont  Ferintem fpioccdr 20° 2e¢ ar OS DenNneAb: 


7 
" 4 a ' — 
jel? eirywirc!!) Ub ewe) fete Paeagery 


Hy) ibarg BSL weuélg od hleea @enogeety 
7 
117% ate n> rey 1 , even (ig (so eGeae I pl a 
b ' —€ 7 
yi brie rte tal 1? oman? of Wom: er ree 
ae 
bays od apoh wets) > bodisaehese SUN? Wa 
tit /Weit j* So “rif } Te Tr T AY bly 
owh ) 846. aria? | Locons ad od Spot e 
Wi 64 an Live SAP *V) ¥ Pena at} intt 1 > 7 : 
» be iG 40 ee | mi @ #6.) aunty > : da 
) mio bose Sew boiien 2) alee 
y ee ry , Goete ee 
shia 
. 
i 


mgr Pot Meme 2) ay be cathe 


1 aw bis oolong? enozaghy 26 gh 
‘ — 









: va 7 
in| | ea tay x44 Bi, etd Di pe Teva wl hateto d 


horegpe wiew Peiiaide a iw! 


| 


node ime, 208° 
; tains 




















Tee) fevdw Yo noMeraged. [sa53 


hh Hl bobavocne eem2cd?+ Yeal 


Yo Col no beoala ofow eeorte 


Bi ms? iodo nt atossue BeE'ok 4 





beBaléme anw inslbewp of? > | 75 
uf ogee" pdatiw 1 bee t eontyoas? bes 
2 0008 @ restau 4) 2, 4 .desostioo siou evomnom 


eztv 


5 sogrt 


Figure 4. 


Zonal separation of wheat leaf ribosomes. Ten ml of wheat 
leaf ribosones suspended in Buffer II containing 32 

sucrose were placed on top of a convex gradient ranging fron 

7 to 35% sucrose in Buffer II. After centrifuging for 7 hours 
at ete the gradient was analyzed via a flow cell at 290 nm 
and fractions I and II which were respectively 70S and 80S 


monomers were collected. A. 1500 £ Uni tS seb =e oUUURE 


260 260 


units, 


50 





1.6 





WUQSZ so.ueqiOsqe 





sedimentation 










fo) intnweib o2 





Sy 





2.0 





wWUQOSZT sdueqiosqe 


sedimentation 





Figure 5, 





52 





Analytical ultracentrifugation pattern of zonal separated 


80S and 70S ribosomes from wheat leaves. A. 80S; B, 70S. 
Ribosomes were zonal separated as is described in the "Methods" 
suspended in Buffer II and analyzed. These pictures were 

taken 8 minutes after a speed of 39,460 rev/min was reached. 


Sedimentation is from left to right. 


Se 


f 7 
ssiusoig seedT .bemgtone bad IL, 





fenon “: awviiteq maltepubirtaaoetztin foknyfant 
A .ewvie! tuertw vo? caldeodiy ZT ae! 


"7 
®. 
e 


t nl ned ged 2! 2f DaJe1EqRe 
, - 





apw ném\yer GOA, CE Ty beoge & 


.20B97 of Tiel at are 


7 a 
7 










bi 


53 
B. Ribonucleases in zonal separated ribosomes 


Ribonucleases have been reported in wheat. Matsushita (1959) 
reported RNase activity in the endosperm of seeds, the cytoplasmic 
solution, and the cytoplasmic particles (microsomes) of leaves and roots 
of wheat. He found the RNase on the microsomes to have a pH optimum 
of 6.0, to be activated by divalent cation, to be situated on the 
surface of microsomes because it was solubilized by adding taurocholate 
or BuOH, and to be activated by organic bases such as tris (hydroxy- 
methyl anino methane) and basic amino acids. Hadziyev et al. (1969) 
reported higher RNase activity in wheat chloroplasts than on the ribo- 
somes. They reported that the RNase degraded mRNA's as well as the 
ribosonal types of RNA, and that this degradation could be inhibited 
50% by the addition of bentonite to the grinding medium. Igarashi (1969) 
demonstrated the presence of nucleases in E. coli B ribosones which 
degraded poly U before in vitro protein synthesis could begin. 

The incorporation experiments required ribosomes which were 
active in protein synthesis and which would not contaminate the 
incorporation reaction mixture with nucleases that would degrade the 
poly U and charged tRNA. It was therefore pertinent to examine the 
ribosomes for RNase activity. From Figure 6 it was calculated that 


J0G5E units of ribosones contained 1 unit of enzyme, taken that one 


260 
unit is equivalent to the production of 0.1 umole of acid-soluble 
nucleotide per hour, assuming an average Aen of 10 for 1 umole per ml 
mixture of nucleotides, 


vyen and Farkas (1971) studied in detail the ribosome- 


bound nuclease in Avena leaf. They concluded that the ribosomes had 


te 


7 
. 
ie of) eT ?tdavederh poate nt % ' 
7 _ 






















in sf note » aA ot ae | ;inhne ante ad ' 
. _ 
atooy bra eaveel Fo fy valli hd veq phate 
bt of 


i iy w ave ef esnnepesti al? AG, 9eeK al 

‘no balaite staal in aia frolewtb is hea 4 ye ae 

sali ue? Gloag 2 oslt titles seu Tt gaugond is 1719 oe > 
i ha? ce ue aa, 1a 40 badawtis co at xt on o 

| os de veyrsoet - ab tas oniew otaed tne (aon vag 

Pig wbeht f emia tan a? esiviioe st ate heed 

vir ce Tiew a Oe bsbarveat neewn aff sary benvoqey ‘ah 
fons coteavadeen! rr ‘eS ben Ae wo vag bm 

1 aioe, AMOR, 42 eS Inodned to nord tbh te 


- 
2 gf 


oll tt flete '2 SF peecetcun Po. songesg orto sh naa 
itil DFuGd. &f 2oqdiye Pen toty ree iveR? gepted & ton 
Ly Fits heady Ody i ipey Sirranieg's, Aol faneqmant aft 
‘o) wei hietide Ut aioe rive hay ateodtines nlotevg : 
“mt wale it? anal orn Abt -qexka saat te ot 
24 Noe eotartart ae7 Kile ts lana 
fh OON Ti ater ya fan . agiytiae ret a 
‘i ) edi, aaeegetig: fin 1 ha sahaawee enauandi we 
fovtneSiok 14 3Pomeit@ te nor aetiary sie an | _ , 
iim sq Shoe T WOT bi i Bitoveen “ mn pn 


~Surnary wAt io ie 


2 r om : 7 r 547". 
bat eaheud)a att bs ua I 
oon ,* 
7 , = 


, 













i 6 
a 7 f 
£ 
H 7 7 , 
: é 
A 
J _ 
‘a 
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sf 
J 
ef 
e 
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g 
7 sf 
o ‘Yau! Je00% .esengodts Wel fseiw sell vol yseod = .3 eel 
* , 


L434 f 
gO “Kk masias sof euf aq) Cattul a) byfaqavy 199 eamzodh 
ss. | 


a. 


a w Jetiog stAT .esuod & 


: a . 7 
Si tsq srew Soe (oislodoynceab sv pm 6.0 ,sdinogned fm ¢ 


Sv ot 009,000 pntoutininss yd 
f 
‘ 
‘ 


L vyevtut Tm OF ai bbbaagqeuees 


WW) notiesttinsis whic bee 


. 7 >. : _— 7 
yorvaye! 25 PF 000,08 Js estuntm ef pal ouitvines 


Ox 000,00. Ya foA-© auge bye 17 +a¥tud at servsue AT 
 \mapdulde alavigtes tm { sbenlalaoe swusaie qpaie 220th ont 


7 ' | hobiyhha eepiod!« Tut.0 , (be\AVer andy Soorw 09 8.4) 
an -~ ~— — «® 


= _ —s .-- —~{ & ’ @ - — = o@ =. . 
7 _ 6 tes OF Th we AO tes ine gas* ORF) 
7 nf al ommaentis 2it tottiess..ta S$ to Seley [sath 
es mm 70 


é . 


7 : ic mi nut rressenpeodts 208 


Figure 6. Assay for RNase on wheat leaf ribosomes, Wheat leaf 
ribosomes were prepared in Buffer I (plus 10% triton X-100, 
5 ml bentonite, 0.3 mg Na desoxycholate) and were pelleted 
by centrifuging 340,000 xg for 2 hours. This pellet was 
resuspended in 20 ml Buffer II and after clarification by 
centrifuging 15 minutes at 30,000 xg was layered on top of 
1M sucrose in Buffer II and spun 2 hours at 340,000 xg. 
The RNase assay mixture contained: 1 ml substrate solution 
(4.8 mg wheat germ tRNA/m1), 0.4m1 ribosome solution 
(490 ED 69 units/ml), 0.1 M NaAc buffer pH 6.0 to give a 
final volume of 2 ml. seedling 80S ribosome sme. 5; germ 


80S riboOSOMeG == «m = , 


oo 


6 
wu ggg a2 


“ 
uequosge 





time (minutes) 


Ay 


i <A * he rn ge wang 












= omer 
~~ 7 su, Ly 
~ imei re aie te on Xe] BS 
‘— Am, i wt? vite) cal " 
an i ( tiers,» ee vt was 

Pat tt - Aig at fter ae 


Pat) 
‘ wv 


pun 2 a Cr. 
interes eee ttnedi a NY 2 
tl, 0. the —- 
“aT ot ae TNT. 


: beri neutee iy 
Qa ae vans = | 
rerun ita) omit 


1 





® ) fea 1 


a 
yy 
a) 





56 


bound substantial amounts of the cytoplasmic RNase during isolation 
and that only part of this RNase could be removed by washing--the 
rest being tenaciously bound to the ribosomes. Igarashi (1969) 
treated the ribosomes with a modified high salt washing procedure to 


remove the nucleases fran E. coli ribosomes, but the salt washing 





concomitantly removed a poly-U directed binding factor which had to be 
isolated and added back before the ribosanes would regain their activity. 
Personal conmunications with Igarashi indicated the isolation of 

this factor would be impracticable. Cohen (1970) reported that when 
polyribosomes of rabbit reticulocytes were washed with 0.6 M KCI, they 
remained intact, but they lost their ability to incorporate amino acids 
into peptides. Huvos et al. (1970) treated E. coli polysomes with 0.13 
ug pancreatic RNase, and found that even though the 28S rRNA had 
degraded 75%, incorporation had decreased only 50%. Although they 
stated that the enzyme had been purchased from Reanal, Budapest, they 
did not state the specific activity of the enzyme. However, 0.13 ug of 
a conparable pancreatic RNase purchased from Worthington enzymes would 
be equivalent to 3 enzyme units. Fron the RNase determination of 

wheat leaf ribosomes, 0.5 enzyme units were added per 45 E560 units 

of ribosomes (per incubation) and it was therefore relevant to note 

that sone decrease in incorporation could be expected due to the presence 
of RNase. It was decided that the RNase activity in the ribosomes 
themselves would be ignored for these first experiments to assure 
incorporating activity, but precautions against intruding nucleases 

(fron the apparatus, hands, etc.) were taken. 


Lanzani and Lanzani (1968) demonstrated RNase activity 
























- 
en i 


s ws iy 


are 
ay 


in 


Aa | "% 
7 


notislor? pittiuh H2an, ‘patitigno 2 on 
i3--gntaeaw Ye hevonte! ‘sdittuo veh 


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envbave ty pirideaw Jfme sel bal ithom © tot ase 
pootay | Mind siipedets ioe" her 
iif (haba batoow lh Uey veg & be 
| | i a mf 
bs to) chopon blew Saaeg)s en2-o vata aed ‘bab wre 5 
‘ts ta 
Nii bateohiwtt (pew ol 2 tw eat 5: tno me ai ioe 
oe UT) wee 
i 4 } faite afd ogee mt on 

z Ave | bi Fags ae 
ry (raw tee Coty ot watgen Stdder 25 mz2odtyy 
wr f ass 
Low i ) no; of g@ LORME Nias 128 i vod tud 4; of 7 
ah ines nt wh) fs an sev pai a 


Cin ht 1s eS wild hnwot va 008 tam : a 
ff j we : iti Daze rot by a4 nohtsroraant Wa 


ie - 7s 
a 


1 Laverrlon uy seed Sud vn ne ‘yeaa Pe 

tno aft th varyiioe 2 oan 8 998 it 20 eat 
rol wos". hemuapslag auit otinaieg, ate 6 
ee + ttn ssi £ of 3 ; rf > 
‘bie yaw eer teu somes e. is eeu 
ear byoald, aye Xt 2% (rotsesvont va) om 
ae Set Biijoo wie 


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56 

7 
ea 
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Te C6 00 8 bobt: 

SNE Ghee Tae | ah ° aan : 

estnalaun pnthun dat See sto Lut ni a, | 
es swrowdel vt 


civ iow vane bes extent {WaOf SA 


57 


on wheat germ microsomes only after the microsomes had been degraded 
with urea. There was no nuclease activity present in undegraded 
microsomes nor in the high speed supernatant. The wheat germ ribosomes 
prepared as described in "Methods" were assayed for RNase activity, 

and the results as shown in Figure 6 indicated no enzyme activity. 
Studies in this laboratory on the isolation and characterization of RNA 
by Dr.s Jones and Nagabhushan demonstrated that the ribosomal RNA 

of wheat germ was not degraded, while that of the wheat leaves was. 
These results confirmed the opinion that nucleases are not present in 
the supernatant of wheat embryo preparations, and thus, in the reaction 


Mae -phenylalanine, the supernatant 


whereby tRNA was charged with 
enzymes were always obtained from wheat germ. 

The sucrose used in the zonal separation of ribosomes of 
cytoplasmic and chloroplast origin was probably contaminated with 
nucleases which might damage the ribosomes directly or be retained on 
them. Solymosy et al. (1968) described a method for removing these 
enzymes fron sucrose. Diethylpyrocarbonate (DEP) was used as described 
in "Methods" in an attempt to produce nuclease-free sucrose, and 
Figure 7 indicates that results obtained from the zonal centrifugation 
were not typical and suggested that the ribosomes had agglonerated. 
Because the characteristic 'apple' odor of DEP was noticed in the 
prepared sucrose, and because the same separation results were obtained 
by Dr. Nagabhushan when she attempted to increase ribosonal yield 
by adding DEP to the isolation medium, 7t was concluded that the DEP had 


not been conpletely eliminated during the heating process. Wolf et al. 


(1970) described the action of DEP to be non-specific for proteins and 


7 7 a fe 
. 































by} POUNDED 7) basi Selec eight soae dail sae 5 
Lie oh Sieg ‘ysl yisae ease fou ots " 

* ee ' - 

diy imep Se@iw eat ,2aeeyone ton | out idl 29 MG 
JAM Set here a ve suena @ et Tih peck p a bet is 
v Ot alate Q.wught gt i 
' > ite Anant er? aid ie eld 
1 oMjendcruired neteutdsye 
iia. 
ta: Dog ef lw  babarypab YOM zeW 
it Tens av iiya qe wi? stn 8 rue 
: ! i@n wrravan } ‘ (ie sory % siti 


t 


j 
j 


no Talynse a et bw ries. 2aw A 
wong. yeadw my), fathead oysnte one 
wa feuds att at Oped deveault oft 
| baru tink) igiadd avah T) pensar 
, 08 yed ant lind amu sable 

Satie siaiieiiale (uae) lp ds a vamctod 

era aareiytya ann ‘oan Y 7 

24) nt Soko ae tio i it ob = ; 

fas’ ‘ont ment Wrpaddd sities bon estiotbat E 
nS aT of) dais ‘Wstenoae bre Tier en 


haoiod, enw Wa8 iy “oug Saf 


ny TOR pyitie’ wag 

Hidty Tortiddh eekaaat 4a Be Fiat on 
ait Jvc bebe emmeaaag ae toupee kahit vet any O 
Pia ePonaRg ‘phi wit ati bas ni ; ti tate 
ago rig set SIF saqé-inda actian'4 se ii | 
. vi - mi 


ert ht 


1.0-4 


290m: 


absorbance 


58 





sedimentation 


wo ne ee ee 


Figure 7, 


Zonal separation of wheat leaf ribosomes through a DEP 

treated buffer. Six ml diethyl pyrocarbonate was added to 

1.0 1 of a 40% sucrose solution in Buffer II, The solution 
was heated in a boiling water bath for 20 minutes and allowed 
to return to room temperature before being stored overnight 

at 4°c, 2500 E560 units of ribosones were centrifuged (47,000 
rpm for 7 hours at 4°c) through a 7 to 38% gradient prepared 
fron approprialely diluted aliquots of this DEP treated 


sucrose solution. The gradient was analyzed via a flow cell 


at 290 nm. 






~ — - = —ap : - - 
oi eingmibas 2 4s 









aadhy aot agai - ies ne vo 

ook I NAtah Tim 2, situs beseaty : 

wl al habiotien senaie OP 8 to €2 

oth one eetyntm OS vol Medeaien et at bo 

ovfed avoted-avud ste " ne amuse a 

0,0) Beau Piws ‘onpzndh, Fo 27 Vol sai ¥ hy "4 ba 

eae hee Ge ohh iii ad ” sual S 9 
beteay? Cy st4e Fe atenlp hi bomutt ss iste 


a a _- 
fico wt « ety betelane Zewetnerbaye On al 







59 


"that the general feature of this reaction is a formation of inter- 
molecular peptide-like bonds involving amino acids and carboxylic 
groups which yield bizzare polymetric proteins", "The intramolecular 
formation in these bonds results in inactive conformations". Anderson 
and Key (1970) reported the use of DEP to isolate intact polyribosanes 
in the presence of pancreatic RNase; however, the DEP concentration 
needed concanitantly caused the polyribosomes to completely lose their 
incorporating ability. Weeks and Marcus (1969) reported DEP to protect 
polyribosomes isolated from imbibed wheat embryos. These polysones 
had lost much of their ability for in vitro amino acid incorporation, 
It was concluded that although DEP has been used success- 


et al. 


fully for the isolation of undegraded nucleic acids (Solymosy 
1968), even small traces of it were enough to alter the protein 
structure of the ribosanes. However, further experiments aimed at 
obtaining complete hydrolysis of DEP in the sucrose, might be tried as 
an inexpensive source of nuclease-free sucrose would be an asset to 
this work. Nuclease-free sucrose may be purchased fron General 
Biochenical (Cnagrin Falls, Ohio, USA) but because large quantities 
were needed for zonal separation, the price made its use prohibitive. 
Therefore, the nucleases, if they were present in the sucrose, were 


tolerated, 


II. Phenylalanine Incorporation by Zonal Separated Ribosomes Using 


a Transfer System 
A. Ribosome concentration 


Figure 8 indicates the dependency of 40 phenylalanine 


© 























7 Ss * 
won! Yo noftecrin? & @P ness 21A9 Ya: ruts 
Hiyxodras bak elles. <pthipe geohaT vet -abingd ati ot ber 


sal: matin gat’, ‘eviction oh oint eg nos 


a 
"4 /P 10. sv'¢hent n! eat gaat ent f 7 


Ai ats tort 8 ah) 70 Hou oid 6 a 


; _ i 
“au Gf] ‘darn Y LaOSre ssnananen V 


‘T9Ad BG oP ethics} 7 aie Gry) O4 gy } baeued” cs iad aire 


' Jyowe (RAL oeipe ben 2kegW _ahthae eatte 
wool 
seyley coatt 2X76 ROA Getlnin Mott batatoat eae rod 
oqioon) bide On tne yey gh el ene ttadd 30) fou n feof 
v4 


asua basa tend “2 haage te tarts bebuf ona za 7 
VitMy’ pe) etn SeRIDe habanpe rw ve wotsatoet ; ot a) . 
doi vvaw 97 % “ont rei NOVO «| . (8a 

cleat 4 vasa _ssboaadh td 49 nba 

! . owt obs Mh Ste Hees vst 
nA ¥! Woovige oo =spiataen t eanwoz ovte oq 
maa a oped: yen Goahoe sort sonst. ow 

i tog, ASG ch sist sie net 

lio wep <0? dante iarsg ed db ttalsowse loner 0 8 

c Srggete Seow Re UP eames oun ot 


“— 7 


onf2 2emeadth bated Cuno) wa 


h 
ait! GINASE nD ' 1° 
2 


60 


incorporation upon the concentration of 80S ribosomes. The radio- 
activity of the washed protein-precipitate continued to increase as 
the concentration of ribosones increased. Concentrations of ribosomes 
greater that 70 ED 60 units were found to cause unmanagable amounts of 
precipitated protein to be formed when the reaction was stopped by the 
addition of trichloroacetic acid. Due to low yields of zonal separated 
wheat leaf ribosomes, it was not feasible to use concentrations greater 
that 45 ESE units in each 7 ml assay. 

Using the conversion factors suggested by Parthier (1971)-- 


1 mg ribosomal RNA = 25 E units and 1 mg ribosone = 13 E560 units-- 


260 
it was calculated that amino acid incorporations, which had been 


reported in wheat systems, contained the following concentrations of 
ribosomes per ml incorporation mixture: Allende and Bravo (1966), 


4e units; Mehta et al. (1969), 11 E units; Legocki and Marcus 


260 
Units eschulcz eteal e972) eee Fogg units. App (1969) 


260 


(1970), 10 E60 


used 8 E units of rice embryo ribosones per ml assay and Parthier 


260 
(1971) found maximum activity of pea seedling ribosomes system to 
contain 2] ED 69 units per ml. Beevers and Poulson (1972) reported 
pea ribosome concentration as milligrams of protein, They added 1 mg 
protein (the added ribosomes) per 0.5 ml incubation mixture, which 
may be calculated as 50 E560 units of ribosomes per ml incubation 
mixture, 

To attain levels of activity in the described wheat leaf 


system high enough to draw reliable conclusions, the higher amount 


of ribosomes (45 E560 units) was used, 


>. ae 

























- a 
Paks. 


“arose aff w mame addin 200 to so dona 


S2eo oti of bent hoe stadtytort-atote ah * 


te 5 
re 


bodes ah 

peodiy te anchtestgahed Giiieersn? senqeadinl Je nots 
IT suits o2 pct ne ia 0 

vd bequot2 esw autiaiey SA srt bomo’ od ad te an 

tsipqee | ) abisty oer my “A ae >1aa900r 3 
neon. ona? ahdiens) ton con 2h eam aie 

News fad ee m8 fie ggg 3h: = 

mina] Wl be eSB aa oe noteayno att orto 

fil oom sortie Get Bie ahihige ial as. am Tay “ 

shiv .cnepiayagiepat bios aay ts ditt betel 

tat Horiisens ara aaa 

(aa8T) auveré: tne ertA. > saudi xtev nobee exoareant Mi va 

, ogo) 1¢S tou geet A) ABORT) Je te sso cad ay | 

el Hy (2 tana sat tm) dyna? 20 ar: OT 

Svat Wes yaees Ln sot -eeieedis cuca ooh to ste. ¢ ash . be 

podt | Alfteer pom Vb “arutios miuin seen re 1g? 


1% bes avavead: tm epg arta y 

iv OF ang The Gare 21 tae a gotdersnosone : 
cin "QERRAMORR ima 0 tag ampere bobSayart 

jativad?. Ta van ennesrots % TP, esha * nl © 63 at q ben hae i* 

eo! thud bodrrpegt att at hig % ii 


tnuons sotlotd aay anatautonoa ohud toy 


dpm x 1057 


Figure. 8, 


61 


20 40 60 75 
ribosome conc. (Eogq) 


Activity versus concentration of 80S wheat leaf ribosomes. 
Zonal separated 80S ribosomes were suspended and appropriately 
diluted with Buffer II before being added to the prepared 
incorporation media. Tubes were incubated at 37°C for 

60 minutes before the reaction was stopped. Activity was 
measured in a scintillation counter, and dpm was calculated 


from cpm by the channels ratio method, 


Lee 


i vohyidoA .beqeete gow cotiogey st evo 
jaloutow enw mgb- hee iain roast fsa 23 





— EE SS SS 
' 


i. © i} oc , a 


rr 3) sno>a ‘Somftceo0dit — 


ry ‘ol seatw 208° Yo notte tnaaAO> 2urisy huts 


stg sal oF botbe gnled arated If vette & hatw be tb 
Ce on haledboal sen enduT ethan a oont 


a, . 


7" 


— 
= a a 


a 
he 
a 
ye tA gene aiew tedzodh 208 pesereqea nS 


>= 


be 










a 


iJ 
Ja 


62 


B. Incubation time for incorporation 


Figure 9 indicates the dependency of My phenylalanine 
incorporation upon time. Incorporation increased rapidly with time for 
the first 30 minutes, and then leveled off to reach a plateau at 45 
minutes, Although maximum incorporation was acquired at 30 to 45 
minutes, it did not decrease if the incubation time was extended. To 
assure that optimum activity had been attained, the incorporation 


mixture was incubated for 60 minutes. 
C. Buffer effect on incorporation 


Figure 10 indicates the effect that the concentration of 
the tris-HC] buffer pH 7.5, had upon Mae phenylalanine incorporation. 
It was noted that the lower concentrations of tris--5, 10 and 20 mM-- 
stimulated higher amounts of incorporation. Likewise, Bewley and 
Marcus (1970) reported similar observations in a wheat embryo amino acid 
incorporating system and they stated that the tris buffer acted as a 
cation in the system, not as an inactive controller of pH. When they 
used an incorporating mixture containing 5 mM tris-HCl pH 8.1, optimum 
KCl concentration was at 50 to 60 m™ KCl. As is indicated in Figure 10 
when the wheat leaf system was used (containing 25 mM KCl and pH veo) 
the optimum tris concentration was 10 mM. It must be remembered that 
the wheat leaf ribosomes, used in the incorporation system, had been 
resuspended in a buffer containing 10 mM tricine pH 7.6, 10 mM MgCl, and 
5 mM 2-mercaptoethanol, 

As described in "Methods", the ribosones were prepared 


in tricine buffer pH 7.6, but the incorporation reaction mixture 


; \s — © . SS 
28 30d 10) boy T nel A YG 2 WoW’ baw eee aad sattud fou 


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dy tie ql bigsy. boeseH? notsermqwonl . =o oqu nots AOGIO 


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26 balan sotted sige off Je sists vod bho neteeR entaoqees 



















ge 
a" 7 ae — s 
, nobisres ooreh vot. ats Oo} on 


ca 7 : oie 
Mf ‘ 2 7 a ¢ 
nine talymted Yo _sasbimqeb acd enteotint @ ¢ ae 


te weotalq & Hoses of The baleval av? Bag stunt a 
3} of O& to bertupes Bee Ralderoqrozn cra oth 
,baboaadx® cow eff? aalisAgony of) >! seessaed fon bb 3 tu ti 
notisvoqioodt sd? ,bentatin nesd oon ud iyiios atl $40 tet? s V2 2 


sr.) oh 40? bevaduseh 20w 3 wut xEm 
noriosoaeott no 1201s “ott - a i’ 
at} tad? Postio «tt 2e%eaTbol OF eqwplt, =, 


as — * 


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* : Pic 
bis ysived ,satwet) ,metteyooi0sa! So etnuhe «pdighd DARREe 


n ‘Ha Vo valTovtqas svtionn? a6 ep: Son are ong nt a ol 
Hq TOH-2isd Mn @ ontiutotnee awlsla enitesqqrosnt ns 
retosthn? 2t an .—4 Me 03.03 6h te een nottert _ 

ws TI3 Mo 2% gnlitatoos) beew ee ceeve Besl Sa 9 


Kt bore at od fopm 21. , in. OT 26 tat beeasomnn, pacha pen 


he 


wen bat ,meteye eolieinqwnont! edt nt fen, era sof 3 
aa: Oe OF 8.1 He enbotad 5,06 con oh f a iad 









: on sain wd it 


Thin) Pigy cxiaeeddny, Arig P eich ‘ 
“WW? apn nol Saat sciadeainel dl are 


7), 


sit S 








. ainine Coad yp ao notiaroyiosal onteat sTunstiq-d 2 =yget 
fata notisvomaiat af? wort .4anty evrvey zoimeodty 
le Im | , 2sMeod?4 208 bsrereqse Tends pnin)ssaos 
1 Ot .t9ntY gvciysy e713 2a ADT ofa? betpegts wiow 
“pedurlin Of ot “OF te botetuont orew golqued betes tqhoerg 


,botaves tre bavstilt anied svotsa 


Figure 9. 


M40 phenylalanine incorporation by cytoplasmic wheat leaf 
ribosomes versus time. Fron the incorporation mixture 
containing zonal separated 80S ribosomes, 1 ml aliquots 
were pipetted into TCA at the various times. The TCA 
precipitated samples were incubated at 80° for 30 minutes 


before being filtered and counted, 


dpm xX 10m 


12 


naan 
© 


ves) 


45 
time (minutes) 


6 


¢) 


64 


$a 


cnet? ue ey cytoplasnic whea 
ee ee | 


~~, werieweee 1, ri 


e ry cA a 
a Gree 


we a 


‘pe pabaees Yimes. The T 145 


i 












ton atxt w 7 
0, md alig 


oy 


orpoted at BI for 36: 


65 


10 20 30 40 50 60 
tris conc, (mM) 
Figure 10. Effect of tris concentration on the phenylalanine 
incorporation by cytoplasmic wheat leaf ribosones. The 
zonal separated 80S ribosomes were resuspended in Buffer II 
before they were added to the samples containing the various 
concentrations of tris-HC] buffer pH 7.5. 


THAM[ tris(hydroxymethy1)aminoethane], 


Li 


Pid} 


7] .2omeeediy Teal dagen na 
Vtvil at hatensega its rp apngeonty 5b 










Seen We ae} 
Y _ 
- ’ a 
—f\ 


— ——SE = © — —emineean \ail 


£ it ae ort 
\ 


Bains 200 ae 


sitnsisl yada S02 no no bteetine 


> 
7 
stim 


ay ot, patatstned eohyteg ans ot sae . nh 


“ie 
aes ia) aud falta a) To 


' nid tao ws {' . 
oh satin olen 


66 


contained tris buffer pH 7.6. Personal conmunications with Dr. Jones 
indicated ribosome preparations in tricine buffer, unlike those is tris 
buffer, provided ribosomes which when analyzed on sucrose gradients 
acted in a way typical of those reported in the literature. An 
incorporation experiment was performed to compare the incorporating 
ability of ribosones prepared in tricine, by incubating them in three 
different buffer reaction mixtures, All three contained buffer con- 
centrations of 10 mM. As noted in Table 1, there was no difference 
between the three types of buffers, and it was concluded that tricine 
could be used to prepare the ribosomes which would be assayed for 


their activity in a tris incorporation reaction mixture, 


Table 1. Effect of buffer on incorporation of 140 phenylalanine by 
cytoplasmic wheat leaf ribosones, Ribosones, prepared in 
tricine buffer, were added to the transfer system and made 
up in 10 mM of the respective buffers. In this and all 
subsequent tables, incorporation is expressed in dpm 


calculated from cpm of the hot TCA insoluble protein. 


Buffer 
tris tricine hepes 
dpm 2419 2189 ZOZ5 


D. Dependency of incorporation upon added energy factors. 


Table 2 indicates that the incorporation activity was 
decreased only slightly with the removal of the individual energy 


factors fron the incorporation reaction mixture, 


he 


ne 7 4 
; ' - / —. 




























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» 9 AD snot ecthn Hee cae. q q 9 
ewta ait tae bots at iit ) : fon 
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vudevedht oad ab Badsaqey ay pipe oo 
Savnqranih aly mnie ay bee Tag ere ont io nt 


4 “i rf 


1 vi Shed ot ud gbiffaid ni bavantmndy de eodty 


tort Hewhedris aptkp OTA .eotugatm hall ee 
b at aw SYSMT QR It@RT nt beton ah tor OF 0 enc ty van 

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Warn ia Monte ao tenogrodat r99°6 nt pees 


i 7 
rp 


| sites tel yaad’ We aero: nt “oud Re pend 
2 ‘ .permendh) tiof fmeanw oteratgedp 

tion wetaye, “Ate, T° oNg ow tebhe evew ,valtod ano 
dy nl , sv Wid oviyonqess of Yo Ma OF a 

a0 wl Ssararge al ereqroont ,zefded Sin pozdua 
ed | WT 204 eff Yo mo wort nstatusteg § 
1h hell a i 


uytael piterss ety? 


a i) ae 
i<O% - ays eres wy 
7 
io 


,2rosoet Yrians bathe Mega gil tei Dry of 
i : a - 

Zou VP pec the arin went aii, iil ‘oy ¢ 
epi Tete RAT BME %o revere ‘on 
oust mage 
none (vi : 


67 


Table 2. Requirement of energy factors for incorporation by cytoplasmic 
wheat leaf ribosomes. Ribosomes were added to the prepared 
incorporation mixture minus the energy factor. Two controls 
were used: incorporation mixture minus ribosomes, and 


complete incorporation mixture removed at zero time. 








System dpm % incorp'n 
Complete 2045 100 
~ATP 1783 87 
-GTP 1982 97 
-creatine phosphate 

and cre'n phosphokin. 2051 100 
-ribosomes 146 7 


+ribosones at zero 
time 142 7 
This small requirement of energy was not entirely 

unexpected as Boulter (1970) in his review, had stated that the ATP 
generating system was not required with the transfer system. The tRNA 
is already charged, and thus the only energy requirenent in the wheat 
ribosone system.would be in the binding and translocation step of 
protein synthesis, It was concluded that the small amount of energy 
factor needed for this transfer system had been carried into the 


incorporation system on the ribosomes. 
E, Bacterial contamination 


As seen by Table 2, one of the controls (incorporation 


mixture minus the ribosomes) proved that none of the incorporation 























y 


“Oo fi) wc! Mit. eum set more0t " 
ss a bed | a 
Viewer -OnF OV saasteial =n othe meee 1 OE Fae 
; ” ier an - 
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, 257neSr 2 TM sui xter nay TSM mont 


rey O8aS Je teyoney reas rm aw So eres, 


A'qiosmt J ae 


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va ihe 
Ve See i 
apnanea ha eniteon~ 
OT . bees uft iaonn fe 49. bie ue 
\ j 7 #¢ i 
Ab | 7 essai i Mt 
Vien So bbe moauitens ve 
t shh aed 


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in ON iia BAG Je Dee . wer: ; bir eis (O0Wi) ned tuoe hart ba9saqi 


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i - rs : 


' 


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+r devon ffawe 683.3607 bobubuies aw i i¢ cates a ¥ 
ty aint bulreo pend Dad epee Us vovanws ani 4 sh 


jesmmanere: wat ne mote - 


\> ia ay 


ein rue 2 6 


mia tHQSAT } aioe ay % a s sh _ saz 


mi invoquoadr ag +0. donner saad. ever rh ladle 








68 


observed by the treatment was due to the added aliquots of the 

dissolved ingredients. This indicated no bacterial contamination of the 
aliquots; however, this control does not assay for possible bacterial 
contamination of the ribosones. The other control (conplete incorpora- 
tion mixture withdrawn at zero time) indicated that the trichloro- 


acetic acid precipitation step does not trap We 


C-phenylalanine and 
that the activity observed was due to incorporation. 

Bacterial contamination of ribosanes was checked regularly 
by plate counts, and results established that less that ile bacteria 
per ml ribosonal solution were obtained if the solution of suspended 


ribosomes was clarified by a centrifugation of 30,000 xg for 15 minutes 


before plating. 
F. Rate of incorporation as a function of magnesium concentration 


Spirin and Gavrilova (1969, pp29-33) have reviewed the 
role of the divalent cation magnesium in the structural stability of the 
ribosanes. With the gradual removal of magnesium, the monosanes will 
dissociate into its subunits; and with further removal of magnesium, 
the structure of the subunits will irreversibly break down, Values 
calculated for magnesium concentration indicated 1600 to 1800 atoms 
of magnesium per 80S ribosone, and 2500 atoms of magnesium per 70S 
ribosome. It was assumed that the ribosonal RNA is in the form of 
magnesium salt. After reaching the "first critical level of magnesium 
content in ribosanal particles" as Spirin and Gavrilova called it, the 
ribosame dissociates. Because the 70S ribosome reached this critical 


level at a higher concentration of magnesium, it is said to be less 


1 ye 

| ho oe | 
Yn 2 i soe et ian am EL 

ty no bhenteectyias yaiane ee borat 


od- shete ety He shower eadb for 


tai m9 ens eit 

























fate 
( tg 





i on? ted ae (oat ; 
if aint. don reat qa ne 29te 
halaman of «uo caw bovrraedo 

Luv EaeeOwhS Yo no tientmedans a} 


» We. 


Ww 2 ine Weds! dates tof wase bap «2 
am 


Lmithiee aia 9 bs Hietde saw no tute Fama 


Ch 4s hod tay rindass 4 Xd seth be it m2 se 
> se stg 9 ovo te 






= 
Paws "1 
aqine pilteates, i poRFieyt « ta nol tsvonrosnt to atea, 3 
. : 7 
> y ; 
eit? ae 4. were Leatiee oy VOR | vel iived bie wiih. 


imovauns nid AY mPa nettes sooth 
nom why . neheang ." voit Lhubsip aie meee) Sal 

leven “ety? iw bow yee tnudbe est wy: 
we dead Yieiavevert! OTe esbaudwe oan: ob “ioe ae 
lot Q8f peeppin? aotteyd waned mut ean gna i ba | 


i 7 
=) 4 i eri: an tye dade iit) ae bith < anazodty 200 A | ie ’ 


A Compendia ‘ony ‘ey Somers cam 3 
mtzanena Yo levof (aotorya se oy iter wai 7 i toe 


a0 ’ 


wid Ht bvliag aval Pian Geis mivtge rs "ae . 8G: 
leaks = zh boul tinn enaaaail er, elle: 
Tar - 4 
onal ad ob B , fat stint 

wt od biwe ‘ai nj Shed his 


it) eae 
‘200 ant 








69 


stable than the 80S ribosome. At the second critical level of magnesium, 
where unfolding occurs, the 70S is again more unstable. 

Initiation in a template polynucleotide containing an 
initiation codon, takes place as was described in the "Literature 
review". However, if this initiation codon is not present, as in the 
situation where poly U is required, initiation is stimulated by magnesium 
concentrations much higher than is required when natural messenger is 
present. This phenonenon is called non-specific binding and does not 
require initiation factors. Spirin and Gavrilova (1969, pp112-114) 
described a model for non-specific initiation, in their review. 

In the present study a series of magnesium concentrations 
was used in the reaction mixture in order to determine its effect on 
incorporation. As is observed fron Figure 1], there was a lag in 
incorporation at magnesium concentrations below 4mM, This was followed 
by a steep increase up to about 14 to 16 mM and the level of incorpora- 
tion continued to increase up to a concentration of 30 mM, but at 
a lower rate. 

To canpare the chloroplast ribosonal raté of incorporation 
as a function of magnesium concentration with that of the surrounding 
cytoplasmic ribosones, it was imperative to extract and use the two 
species of monosones fron the same preparation. The incorporation 
mixture (minus the ribosomes and magnesium) was prepared as one batch 
and divided into two parts. The identical appropriate concentration 
of the respective ribosanal species was added to each half before 
the aliquots were pipetted into the tubes containing the range of 


concentrations of magnesium, Even though the centrifuged ribosones 






é an Ay. 

vo tevel fasttiee ie ots 20 une 
shine rrdutlite 2f 20k wd Fo: i mM 

und ted pars ees 90 Mami pie Ps 6 Pere i sg 
: hd at weal nests 20w 2f wnt 0 

oq fon & rt fot tsttiat an 


> @) Hobie lptnt , per ey na \ 





























nad | Teed Ai aR zi ant? vedgtt 
he Gaya 1 atte kaon bul !as ppt, stay 
ear) av beled bie nixtoe roiaaleith 

miven eat of. near Tat 27} ioede-nes Wo? Tabom @ Badly 
tedaneg. bard pelt ly 2M ke bs sty de Pree oii at | 
it shed ah Geta mt ota tn iam 

ob tet » cow $teds-) TT giawetd per? bervaado af eh nots c 
tut eT ‘Ss KOR TES eoncd | ww jem 8m coun 
lSvet wi? nl Mi ot 0) Juode oF qu seexiont qgade s 


A io; wenCS « OF qv Seagvon? oF seopae o> 
| Te . 
; . * = — > ; 
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Se 
io J6ky Aw no Prertqpanes mitésngew We * ' 7 vi 


Ls 
aha 
Ase 


- it aay fy era Me of otter noni al st re 
notievoqwaant eft ,AveaMTG aube way font en Na . 
dad am fe bAvaqetg tow [rei entpen ai cers | 
tio PS wees ab pin yertiy rf fant ioe t aft si 
opted Vis 4 iNSpa wd. hagtibe 28H aot gage: wmodts 


to nena avg ee eonsts me I 7 


» . = 
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i nat 
oe 
_ i 2 


Figure 11. 


70 














Tae: ai 20. 24 £426 
magnesium conc. (mM) 


Effect of magnesium concentration on M10 phenylalanine 
incorporation by the 80S wheat leaf ribosomes. The 

reaction mixture contained 45 E560 units of ribosomes, 

8-9000 cpm 140 _phenylalanyt tRNA, the indicated concentration 
of magnesium as MgCl., 80 ug poly U and the other constit- 
uents in mM were KCl, 25; 2-mercaptoethanol, 12; tris-HCl 
p75, l0;sAIP, 1¢ GIP..0.5: creatine phosphate, 5.05 and 


creatine phosphokinase, 5 ug. The incubation was at S72G 


for 60 minutes. 


bine Oe _Stennneny angen 18,9 8 yeaa 






















ie 
ak ' 
rh) b be a 
t: 
oom: as ’ 
Pre ~ CS 
; 
~ a ror 4 Seat oo 
aes arid sere, 
(ti? cones mui este 
Pe *. Apet 15 ‘ cs — See 
. i ‘ it rae 
z . . bay 
E ; ‘t : as 
athd-3 i per Sy9 110° no mst gangen ¥0 tos’ wa a 


eareendty Yas shall 205 @nt ya roiieroqeont 


ia 4p 
aseodty d yet .2 @ bontnd neo anus xt nobssee" at 


sone atastn aif? fia Reratsrenonteg ais Oe 6 
rapa vortio an ne U “hg ou 09 gh oe a motzeng ad 


[DH~wivs SF fonsntvorenantS oF te raw | 


b 


. ca Lay ~*~ i 
pre do he agen ol ea 2 aa ent lodgaary idl | 


: 
abhi ie 


71 


were resuspended in 10 mM tris-HCl] pH 7.5--containing no magnesium-- 
for these experiments, it can be assumed that the ribosomes will have 
retained sone of their endogenous magnesium, 

During this study, the yield of 70S was only sufficient 
to perform the experiment at five levels of magnesium. As is shown in 
Figure 12 it was discovered that there was little difference between 
the chloroplast and cytoplasmic ribosomes in their response to magnesium. 
Because each incorporation sample had the same concentration (En 69 units) 
of ribosomal preparation, it was further concluded that there was not 
a notable difference between the rates of protein synthesis of the 
chloroplast and cytoplasmic ribosomes. Boardnan et al. (1966) compared 
the incorporation of “Ae cvaline into protein, by the supernatant of a 
17,000 xg centrifugation of chloroplasts with the supernatant of a 
17,000 xg centrifugation of a cell-free homogenate. They concluded 
that the chloroplast ribosones were 10 to 20 times more active than 
the cytoplasmic ribosomes. However, if the chloroplast had been dis- 
rupted by homogenization so that the 70S ribosomes bacame mixed with 
the cytoplasm, or if the chloroplast ribosones had been pelleted, then 
their incorporating ability was markedly reduced. Ina concluding 
stateanent Boardman et al. (1966) remarked, "the present results do not 
permit conclusions to be drawn about the contributions of the different 
classes of leaf ribosomes to protein synthesis in the intact leaf". 
Hadziyev and Zalik (1970) using a method similar to Boardman, 
reported chloroplast polysomes to be 20 times more active than cytoplasmic 
polysanes. However, recent attempts in this laboratory to isolate 


wheat leaf chloroplasts which could be used to prepare the required 





























af = 
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aol 


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tad sanetetehb ANE em afailt tert harswe4 »D: 

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(2th qed Wo Peay Segoe ome eit hen ol quiz no! tevagos a . euess 
a enw seer fang bobul tea” 4pittwt can D1, nots ’ ods q 

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to Joe tacmasite ade vd . weeote sin mae -o" Mm a) ~teeiagag mit oft, 

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ehh wood fad Jay t oneal } aft Tt , oteno! , eompendty at mest ge 7eaN 
(i tw were eiejed comisodty 20% oto Pedt o2 witienientahal aa d bo ’ 


nell? ,beteilsqa aaed bat 2oqnzedin4 Jaalageaids ots +t 7. mest nos ey 


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Jon oF giTp2eey- there one” poet eme'y (aser)} me ne as “im 
wnt ae ret 


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‘eal toadnt ait nt efeitos aragerrg a2. aaa = pi 
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hare QeyS DAY Setdoe avon rowitd OS ad of “eamevlog 2 
4 ainfoed ay yrodsvedel aie at nei soo Pa 


ide ber: 
7 woo fal fe an 


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' : om a 

7 a Ae 7 
Fahey S on er ~~ : a! - oe i nN 


: 








i 


hs 


165 





4 12 1 
Vigt2 conc (mM) 


Figure 12, Comparison of the chloroplast ribosomal rate of incorporation 


as a function of magnesium concentration with that of the 
Surrounding cytoplasmic ribosomes of wheat. Ribosomes were 
resuspended in 10 mM tris-HCl buffer, pH 7.5 and were added 
to the incorporation mixture as described under Figure 11. 


O-ribosones of cytoplasmic origin; *-ribosomes of chloro- 


plast origin. 


h 


4 canstoodt® duane to Sonenedin, often fgets, pa tbruawa, 












i 
j= oa ey =@ serena <4 66-7 pn nd 5 
GT cf 
[ ij } o c+ * : De 
‘Mw saps *" oN 


wer Teaoeadi, tewlt qo lde ait. Fo soxtnagmed 


sith corioyiiwon swuTeongan to watson 8 20 


bn syow hap 2.5 tae eed (Meise Me OF At, habree re 
‘5. 
| grup?) ebay baditgedb 26 srudatng motairagseant a ew, 


wvwald2. To sei agli prigiae stoantqntee cy Sin ost 


ben nail 





Ta lea 


73 


uncontaninated 70S ribosomes were unsuccessful, as were the attempts. 
to isolate uncontaminated 80S ribosomes by sedimenting out the 
chloroplasts. Therefore, it was concluded that the honogeneity of the 
ribosone preparations employed by them was not adequate to make 
reliable comparisons between the two classes of ribosomes. 

An incorporation lag at low concentrations of magnesium 
is evident fron the studies by: App (1969) on rice enbryo; Igarashi 
(1969) on E. coli; Nicholls et al. (1970) for rat kidney; Murty and 
Tamaoki (1972) for mouse lymphoma. However, the data of Legocki and 
Marcus (1970) using wheat embryo, and Parthier (1971) using pea seed- 
ling did not show a pronounced lag. Curiously, in the present study, 
the wheat leaf ribosomes often exhibited an unexplainable decrease of 
incorporation at 2 to 4 mM magnesium rather than a lag. 

In his review, Boulter (1970) states: "In order to establish 
that the observed amino acid incorporation is due to the ribosomes 
themselves you should demonstrate incorporation showing a sharp mag- 
nesium optimum, the position of which will depend upon the particular 
systen being investigated". As indicated in the "Literature review", 
optimum magnesium concentrations for polyphenylalanine synthesis ranged 
fron 7 to 13 mM. However, Nicholls et al. (1970), when studying 
4e-leucine incorporation by rat kidney ribosoanes observed a plateau 
rather than a sharp optimum concentration of magnesium, When Murty 
and Tanaoki (1972) plotted the effect of magnesium on polyphenyl- 
alanine synthesis by ribosomes fron mouse cancer cells, the curve 


indicated slight continued stimulation of incorporation past the highest 


concentration reported (12 mM). 


s 
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inn of Stange OGn Rie mtd yd baxote 18 

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vhute note ,“ARep ta gs teas woh fi of 
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' » pul? nalseqogqesal soqxtenousd biuolz WON | vow 


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1% wifi (ie TU Sega Ai’) his a vassal ( ver) tao 
aA 


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1 






74 


Similarly, in the present study of wheat leaf ribosones, there was not 
a sharp magnesium optimum concentration in the range tested, It was 
thought that high concentrations of magnesium may cause unwanted side 
reactions, and thus incorporation studies were performed in a buffer 


containing 16 mM magnesium, 
G. Effect of inhibitors of protein synthesis on incorporation 


The response of chloroplast and cytoplasmic ribosomes to 
certain inhibitors has been used as a criterion to their origin. 
Whereas chloramphenicol inhibits chloroplast ribosones about 70%, it 
has been reported to cause only slight inhibition of cytoplasmic 
ribosomes (Ellis, 1969). Three inhibitors , chloramphenicol, cyclo- 
heximide and puromycin were used in this study, It may be seen from 
Table 3 that the 80S ribosomes were not signigicantly inhibited by 
chloramphenicol and cycloheximide, but they were inhibited by 
puronycin. This is in agreenent with the results reported by Parisi 
and Ciferri (1966) who used Castor bean seedling ribosomes. The 70S 
ribosomes were not inhibited by cycloheximide but they were inhibited 
by chloramphenicol and puromycin. These findings confirmed that 
the 70S were different from the 80S, and was taken as an indication 


that the 70S ribosomes were of chloroplast origin. 




















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saw yas tud pbtatnedoloys ud Dettdidal ton att 7 . m2 a 
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sobbat os 26 saded-eaw ban , 208 odd) mig? dnava Vis we 
tpi fastqorolds to oraw somavadty e 


1h yom 


vil 





75 


Table 3. Effects of chloramphenicol, cycloheximide and puromycin on 
incorporation by 70S and 80S ribosomes from wheat leaves. 
Ribosomes were incubated in the presence of the following 
inhibitors: 23 ug chloramphenicol, 10 ug cycloheximide or 


250 ug puronycin. 


Inhibitor added and percentage inhibition 


Ribosome None _ Chloramphenicol Cycloheximide Puromycin 
dpm a dpm % dpm % dpm % 

80S 1288 0 173 9 1134 We 91359850 
70S 1072 0 bzs 5] 1228 0 589 46 


As will be further discussed in Section IV (C) "Dissociation 
and incorporation of ribosones from plants of differing ages", protein 
synthesis in the cytoplasm of eukaryotes is known to take place on both 
free and membrane-bound polyribosomes and workers have provided evidence 
that separate functions exist for each. Glazer and Sartorelli (1972) 
have recently reported that the membrane-bound polyribosomes of rat 
liver were more sensitive to inhibitors of protein synthesis than were 
the free polyribosomes. Cycloheximide inhibited in vivo protein 
synthesis of membrane-bound polyribosomes 40 to 60% but it had no effect 
on free polyribosomes, Puromycin inhibited protein synthesis by bound 
polyribosanes 60% and free polyribosomes by 80%. These experiments 
have indicated the need for caution in the interpretation of results 
obtained by inhibitors of protein synthesis and have pointed out that 
the noted inhibition by puromycin and not by cycloheximide in this 


wheat cytoplasmic system could be due to the presence of a high amount 

























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an ve va 7 


76 


of free polyribosomes, Triton X-100 was used to release ribosanes in 
all cases. 

Marcus and Feeley (1966) reported that the formation of an 
activation complex, in the wheat embryo system, required the presence 
of three cellular fractions in addition to ATP and magnesium, and was 
inhibited by cycloheximide. Once this activation complex had formed, 
the addition of cycloheximide to the protein synthetic medium did 
not inhibit incorporation. These results obtained by Marcus and Feeley 
suggested that the 80S ribosomes used for the present study were in an 
active complex because cycloheximide would not inhibit M40 phenylalanine 


incorporation. 
H, Species specificity of tRNA and synthetase enzyme 


As mentioned in the review by Boulter, Ellis and Yarwood 
(1972) species specificity in the synthetases has been reported to be 
negligible by some tRNA's, while completely specific by others. 
Erokhima eGalhe(1965)4 studying pea, yeast and algae, found little 
specificity for the trnapne and synthetases; however, the trnanet and 
enzyme from pea and algae, though themselves conpletely interchangeable, 
could not replace the yeast component, Tarrago et al. (1970) reported 
the trnaret used in elongation of protein synthesis in wheat germ is 
specifically charged by the hanologous enzyme, while the trnamet used 
in initiation of protein synthesis, can be charged by the E. coli 
enzyme. In the present study, wheat germ aminoacyl tRNA synthetases 
were used to charge stripped yeast tRNA with 4c -phenylalanine. The 


purification steps of the charged tRNA involved deproteination with 


ot aomodhy, seaetey O28 “em DOT - x nod NT i 





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phenol, 3 washings by the ethanol precipation steps, and chromatography 
through Sephadex G-25; and thus would not allow free M40 phenylalanine 

to be carried through into the charged tRNA fraction. The tRNA was 
indeed charged, for it carried radioactive phenylalanine which would be 
incorporated into the peptides which were precipitated by trichloroacetic 


acid. 


III. Dissociation Characteristics and Incorporation with Zonal 


Separated Ribosomes 
Ase High) salt 
1. Dissociation 


When the zonal separated 80S ribosones were sedimented 
through and resuspended in Buffer IV, dissociation was observed on the 
analytical ultracentrifuge (Figure 13). The sedimentation values 
calculated for the observed peaks were 76S, 56S, 46S, and 37S. Only 
about 50% of the 80S ribosomes had dissociated and the 46S peak which 
had appeared was unexpected, Similar results were observed when the 
isolated 80S ribosomes were centrifuged through a zonal gradient 


containing Buffer IV (Figure 14). 
2, Incorporation with recanbined monomers, 


When the subunit fractions were isolated and mixed in a 


60:40 ratio of 2:1 (E units) they readily recombined to form the 


260 
parent species when the magnesium concentration was increased (Jones, 
Nagabhushan and Zalik, 1972). As noted in table 4 only the 40S subunit 


was used because Jones et al. (1972) had described the 40S and 50S 












, © 


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Figure 13, 


Figure 14, 


Analytical ultracentrifugation pattern of 80S wheat leaf 
ribosomes suspended in Buffer IV. Zonal separated 80S 
ribosomes were resuspended in Buffer IV and were then 
analyzed. This picture was taken 12 minutes after a speed 


of 39,460 rev/min was reached. 


High salt dissociation of 80S wheat leaf ribosomes. Zonal 
separated 80S ribosomes were centrifuged through a 7 to 38% 
sucrose gradient in Buffer IV. After centrifuging for 

7 hours at fetes the gradient was analyzed via a flow cell 
at 290 nm and fractions I, II, III, and IV were collected 
and were assumed to be respectively 40S, 50S, 60S, and 


80S particles. 





1.0 





Lu 


U0 6Z e2ueqiosqe 


sedimentation 


> 
* 


= 





25 ‘Ss. 
oe, 


= — — 
- 7 


= 
a 7 
7 





noite! mnemibs 


ri 
7 


7 






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80 


material to be two forms--differing only in secondary or tertiary 
structure--of the same subunit. They had reported that when the two 

peaks were combined and centrifuged through 10 mM tricine, pH 7.5 

(a buffer devoid of MgCl, KC1, and 2-mercaptoethanol) only one peak 
corresponding to the 40S peak appeared. And further they found that 

both subunit forms contained virtually identical proteins and RNA, In 

the present study, when the subunits were recombined by mixing them in the 
16 mM MgCl, incorporation buffer, they did not support phenylalanine 


incorporation above that of the subunits, as shown in Table 4. 


Table 4, Incorporation by subunits of high salt dissociated 80 ribosomes. 
The 40S and 60S subunits were isolated from zonal fractions. 
The ribosomes which would not dissociate by high salt in the 
zonal separation were isolated and used as an undissociated 


80S control. All samples had a final concentration of 


45 E560 units, 
Ribosomal Material 
Undissoc't 40S 60S  40S+60S Ho0 
80S : 
dpm 4364 1175 772 1164 148 
% 100 27 18 2/7 3 


B. Puromycin 
1. Dissociation 


As is shown in Figure 15 A, B, and C; puronycin proved to 
be useful in the dissociation of the 80S particles. When the 80S 


ribosomes were preincubated with puronycin at a concentration of 


0g 





yvioly9ed Ww emapeees 







Owd odd tare dete bag roar 
ur f 
2.0 Hq jankoret forge aa 

AB igh 
desq ono yino (Fu sss moron big | “ee nian 
Joni? biwet yord vaya bial porssam 404 20%. pit “9 BF 


nt AWA bow eabedowa Te cme wMTsussty bent , naz be 


















‘9 patatw vd b Sra ida ed trudue ota oe | 
owns 

lyeity Jragqyie ton bib vomit ” vviud nord 

dsT at aqae es. se btiidiie any wade ' 

7 MN 1 


. is z es 
dt OF basetuoae hh Seog Ae. bth “4 104 


7. Aik A, <7 ann * : ! 9) foat aor ry) made Eye 
er | * letnore ra ton | pi use det a aa 
) se bat the fotiloc! -4eewe nore 
) por : aii & ti BO: 2a] one ti A patie 
f i i ny Ayal 2 
% a 
: _ & 
’ te « ae 
fe \ ff Daina rani _ : 
' a r) 
Oni aNarehh § AOR oth» = taeaenbi 4 | 
, Ny ae ee 


a | 


— he on 
Bb Aayt” of) Sake weil Ree i by 





oT bawenq movers tine: iF A a ba 


208 ad mei vate scohnah: 
- 


: os ' : Ww mn 
Vo. notiautasaapp 4 allie moti: 
7 7 : 2A 





: i BP... 
<= i 


Figure 15. 


|eareme vanes 


81 


_ 40-60 80 — 
fg J 








Puronycin stimulated dissociation of wheat leaf cytoplasmic 


ribosomes. 20E units (A and B) or 80 E units (C) 


260 260 
of zonal separated ribosomes in a volume of 0.2 ml, was 
added to 0.25 ml ice cold double concentration Buffer IV. 
0.05 ml of 0.01 M puranycin pH 7.0 (B and C) or 0.05 ml H,0 
(A) was added and this solution was held on ice for 15 min, 
transfered to a 37°C water bath for 10 minutes, and then 
aliquotS containing 5 E560 units of ribosomal material was 
centrifuged 4% hours at 85,000 xg at 25° through a 5 to 20% 
linear sucrose gradient in Buffer IV. The bottom of the 
centrifuge tube was punctured and the gradient was pumped at 
a rate of 1 ml/min through a flow cell and the absorbance 


at 260 nm was recorded. (P) indicates UV absorbing puronycin. 













(yore Tesi Seow Yo noltafaoaerh betel umize ntaymany aa 
(0) ep tnw gyed OF 40 (# baw A) ettnu o50d OS amwzodhy. : , 


a a > 





.fm $.0 % stfov 6 AT eoamzonht badeveqse fomws 40° i" 
Vi er lu noftsvinzynes efduob bloo sat tm eh.0 os babs 


¢ 


im ¢0.0 4o ‘e Dns 4) o.¥! iq 1 Wiis Tug TO. 0 te fm 29, 0 7 


oa 


niin @f vot aot no bled caw notvufoe 2ldt tne Bebb ean ia 
nolt bas ,2asualm Of yO? Aided vOtew .ve 8 09 beret ws a 


saw Istvotem famzodty Yo 2ttnu 0B 5? é printaines ete a 
Ss ik 
20% of @ w dquo? 3°88 ge pe OOO, 28 te eruon a be ths % 


\ ol 
oo 


g3 to wodtod sit Vi sgh a rm 71 


te baytmg eaw thethorpe add tne borisonud 6 aa i a thir on 
: ie j ij 
gonsdierds ant tue vies wn f quod At 
i a 


_ntoworwq ontdroeds Wi estat Zz ) bate iat 


a ae 






_ 


82 


0.25 umole puronycin/Ej¢q unit ribosome, the monomer peak (seen in 
Figure 15A) disappeared (Figure 15B). Further studies indicated that 
when the concentration of ribosomes was 4 times relative to that 
concentration of puromycin, dissociation was still complete (Figure 15C) 
and thus when large concentrations of ribosomes needed to be dissociated 
--as for zonal dissociation--the concentration of puronycin in the 
preincubation was only 0,016 umoles/En¢q unit. In all cases the peak 
representing the 40S subunits was much larger than was expected, as 
other research workers had indicated that the peak area of the 40S 
fraction should be approximately half that of the 60S fraction. In this 
study it was thought that besides the smaller subunit, the 40S peak 
contained partially-degraded or unfolded ribosomal material which had 
been formed when the high KCl (400 mM) was used in the dissociation, In 
order that this could be examined, an experiment in which various con- 
centrations of KCl were used in dissociation and separation was 
performed. It may be seen fron Figure 16 A to E that measurable dis- 
sociation did not commence until the buffer contained a minimum of 200 mM 
KC1; and at this concentration of KC1, the 50S band was relatively 
large. However, at KC] concentrations of 400 mM or higher, this peak 
transformed into the 40S peak which attained an area as great or greater 


than the 60S peak, 
2. Incorporation with recombined monomers, 


A zonal separation of 80S ribosomes pretreated with puromycin 
in the buffer containing 400 mM KCl (Figure 17) indicated that the 80S 


species almost completely dissociated into 40, 50, and 605 subunits, 


















SE 7 
nf aes) W69qt on. nr ar 3\0 
a Me ee 
jeti haigarin’ 2arhuie: 1a (eet owe Pi o 
tart-ot evidal st wit sts anozodht, 9 [ vn 
( 1) efetamo flr sw 6tetoon atge iq ¥c 
h SU GF Alewsian esaeoilty To zn tieve =e me 
fa tt nips To fs vin ae F308 Y b ‘ent os YO" 
T 2 1s a OSTA gacd\23/omy FO 4 “ima i 4 fi0 A} 
26 , bags > BAW WBAT apret fisum 2ew abinay 502 ” 
. 4) To goes Abe etd SHAT betsotbnl bavi mt pow Jetiat a 
rig ob wehtony? 209 aff Wo Deny Tier ylatem sarQn8 8 2 nots: 
dadg 200 old ,rhawdue vothqm edd esbresd ist — 
ts tri | 1 laraendty Sepfotm a9 pobiapabagitet 
nd sishaxth aft ot boww ame (ie 008) (24) Agha een 
» wotveyv folhw a! teniegse ne .borfmene of blues an. 
zy itevedse ton. antdebsetsth ab bee etsw f93 No ae ried 
ci ofdenizene tatt Sod AST spt? chaapse Sdynenst fbeene 
ma OOS 4 ale iattud ait Thies estamos sof btb notte v2 
favitntes aap Wed SOR SE ak te neiseetananos: eft 7 1. 


699 ZiND sranets vo Sr OM to enetiy vengaines ics te Dit 


arr Res aban” tite codti4 ana: i 
cok sone snipe (nt sigh) im 0 on . 
22 Hinde cg. brit it 0 wink bags hove 









, 1 


beh Hove Me) hen ta bel au cous ott ote 


mD di gal 





ah ; 







a 
. 2aenpnen? baithinnsat tat vate r 


ie 


ys 










| i LS 7 _ | | 
ltceecae rT 
1] | | | ea 
ie } 
¥i ; ‘ . 
7 7 
| . . 
¥ 4 ' {-@% : i» \ a) f ‘ Ji i) 
ve " sit fo [34 Ya an " 3 pongo eu P18 a Jo a 3 -af syuprt 
j i . 
T ie (ec J Bhs f dang \ i. itp 7 niger) to 


Pn 
AT Tav es Ist seat digl Py es | 


ro. ae @P ae : —w te 
ee ee Tie Gintsdnes Ne Pas © a seonil 
‘ , | J 
> 7 ‘ P 
Ines ore Gaiqne wi |. Wruh of TIA 
4 ; ‘1 


oo © 
amnennenies > 9 ee 


4 { Ia ew 
oF Sompewecs Ins ety St? > <5 
: | 

w 1 to / ano tg sHesan2 Prt witty Yolhdpery cows" 
: ; i 3 : é } ' e AVA 
oy ) i ~~ ae \ OO > ef 0 OOF a 20 VA 
- 


_ 


~ 


EE 





Figure 16. Effect of various concentrations of KCl on the dissociation 
of puromycin treated 80S wheat leaf ribosomes. The 
puronycin pretreatment buffer as well as the 5 to 20% 
linear sucrose gradients contained the various concentrations 
of KCl in Buffer IV. The samples were centrifuged 4% hours 
ateoosO00TxG Fat 25°c and the gradient absorbance at 260 nm 
was recorded, The buffer concentrations of KCl were: 
A, 0; B, 100 mM KC1; C, 200 mM KC1; D, 400 mM KCI; 


E, 500 mM KCl. (P) indicates U.V. absorbing puromycin. 


84 


ih 
we 


ace made aS ee ee oman le eS 


» 
a 

ay 

“ 

0 

' 


Lay hoe O | CH 
TT yer eer ri] 


| 


——— 


opt tf 





Mass Fy ab ton 
nei fe | ; pe 


“24 
a if j ma) 


aa 


~. 

— _— 
ee 
—_—— 

: 


bs elenarts tea Ni dn . 


nemepen TH 


Ts eeblel 


rt: 
ae 


_ 
















a 


fries z? all 


—, Fan 
ST Feat 208 Jo PoFteraqes by vad nat tet 3082 16 ‘Trek .\f evugit 
~)" ey | bods brio tsmozodhy 












badeneqer ri y Sy ica hi a 4 » Zhan ony = 
ecee, 17 fy. ‘ of nf bet T nave aompradhy ; 
10 brows | ahiats ried \" bern 4 One ni ayo, “< . - 
jon T 40? .patgurls aa ’ \ polbere p08 Vi voted : 
sym 02g 30 ay 16> 2¢ biased taeda Pp 2 
dest sc i ian | Batosifa> 3 he 10 bas gt ‘ : 
& \ Vi 2599 .20e bak cd ,c0b yD a 
on q fe ./ ay betextink *@" offaw 





Figure 17. Zonal dissociation and separation of 80S wheat leaf 
ribosomes pretreated with puromycin, As is described in 
the "Methods", 3000 Eo¢, units of zonal separated 80S 
ribosomes suspended in Buffer I1, were pretreated with 
puromycin and Buffer IV before being layered on top of the 
Buffer IV zonal gradient. After centrifuging for 7 hours 
at ace the gradient was analyzed at 290 nm and Fraction I, 
II, and III were collected and assumed to be respectively | 
40S, 50S, and Bi Peak IV was assumed to be undissociated 80S 


while "P" indicated the U.V. absorbing puromycin. 


86 





wu O6Ze2URequosSde 


Sedimentation 





‘ | , 
‘ . ' 





Y din separa 
| ‘iresrented pe th 


4 tanta ai 


#4 at 

is ui vad\ te be roof 
4 ayeoned to ba Fe 
f ‘ 


4 





ab4e: sing af wa j 


Dug 








ax heparan, 2 9 of the 


| 


ie 

es 7 
acti t 
= = 

i" 


87 


The sedimented subunits would completely reassociate to form the parent 
species when they were centrifuged through a buffer containing the 

same concentration of tris-HCl, pH 7.5, MgCl, and KCl as the incorpora- 
tion mixture (Figure 18), but as is indicated by Table 5, the 40S and 

60S reassociated species would not support polyphenylalanine synthesis. 
It was also noted from Figure 18 that the high concentration of magnesium 
had caused the sedimentation value of the subunits to change. This 

shift was again noted in subunits fron NH,CI treated ribosomes and will 


be discussed in the next section of the thesis. 


Table 5. Incorporation by subunits of high salt dissociated 80S wheat 
leaf ribosomes which had been pretreated with puromycin, The 
40S and 60S subunits were isolated from zonal fractions but 
the undissociated 80S control was taken directly from the 
ribosome suspension and was untreated with puromycin or high 
salt. The respective samples contained in E560 Hnitss. 005.4% 


AOS 57800530) 40 6054157 4+.30;, 


Ribosonal Material 


Undissoc't 40S 60S 40S+60S H,0 
dpm 2423 396 334 B21 43 
% 100 16 14 14 2 


Lye NH, C1 washed Ribosomes 


A, Required factors for incorporation 


Because of damage to the zonal rotor which would prevent its 


use for sane time, other methods of pursuing the ribosomal dissociation 


. _ Pix . 
ay on mot ag osu 2eRaKe Meret gros Bim Oy un 
ot patntedinos wo tad & one ona 
: ag: NI i) 


wong bihow Hahn WON Kans ott OF wi 
ion sett see wit dniwevng to. abo 


pT) 


_ fabvatal Fenmzedts f' 


vantqytog srg ton stan tat 


























: ae 


— 


"AMGEN Ha (iad 
rt ‘lo et et wud (8 


Le a 


wer pti" ak Tot 8 viet or > baton 


the 
(ia: 


F rage ‘AD Bre ‘9 oF sated = La 
— 4 #) a 
“3 roth a? eon rue inh, bate 


pieseP ott > tae ned ae ‘be 2022 
, ¥ Le 


i ae es 
: Mee Muh Yo etinw dine ud ssi 


ips a is oi 


balmy eeig thet bar aardw 3 omeadl vost 
4) beratee? eisy SFnudoe 8 bas 
Holey] Oa Tetirnoy @0F basa tvteliny he 
. tis at 


ene’ ene bas folenadene : thew 


ft } } re PE ye sv} hey pet any dt 


Nh “/ aay 10 a 


4 


7 
—— ’ ar ar ? i 
ae . ESS f+ ' yb | 
' air 
ni 7%: 1 ] 
; of oy - fr - a 


| at 


ba OT 
. 


'. “Hd. . ee Hapeateail. } 


‘_— bene 6 


dotdrrerwont 70h, 


os 





a er 








: | 4 wi mak a “4 (9 et i 
| 
| 
| 


88 






1 ial ee 
= i 
Bee EE EE ee BE 
i ce r | 
poss: 1 
i ! — H i 
= eae { 
1 














) BSE be eee 






wohie nebae i peas 








Figure 18. 





| 1 
} ' \ 
Sas PE > 


i x i ‘ 4 “] 1 1 4 
Se Nai chives qimoanny selec 


Reassociation of subunits of wheat leaf cytoplasmic 
ribosomes. Two E560 units of zonal separated 40S and 60S 
subunits suspended in Buffer II were layered separately 

A, 40S; B, 60S and together C, 40%+° 605) on top: Of ao" LO 
20% linear sucrose gradient buffer containing 10 mM tris-HCl 
Deyo m1 Onin MgCl,, and 25 mM KCl. After centrifugating 

4; hours at 85,000 xg at 25°C, the absorbance at 260 nm 


was recorded. 










- + 
raat as & 
a & 
a Aw me. * 


id 
7 


oe 
aay, 


| § 
ot? 


Lad 


5 cme 


= 
= fe 
a 7 2s e* ae: s 
a tbe oy ve 
q .- ‘ 
sa] 


| 


P< 
& 
ese 
. 

n= 





“i 
e: 
5 
ol 


Ect 


stemalaode> taol ted Yo eshewdag % mitvivoeet 
"7 h sotevedae finek Bo 2p teu r yh wt ,aemnodty 
> botsys! ovew IT ee ltee oF bebmagene edt sal 


ta wae Se wee 
re wm «202 (2 Varttupnd baa. 208 ft saad 
(Heche Mw Ot oulabednoy wath taste onoroue sasat i 

Gnlingutindets woth ON Mn OS! kee gg ae, Vat ax 
wm G28 ta sonmehoedia GH? . 7°28 Fe pe C00, 2 a I conte ) 


Hn ~ 


et tape eee 8 
a bebie xe" 26 
ry - a 


89 


studies had to be used. Research performed in this laboratory had 
indicated that Buffer III, a buffer containing 100 mM KCl, would 
selectively dissociate and disrupt the 70S ribosomes while leaving the 
80S ribosones intact. By employing this procedure the time and rigorous 
treatment of ribosomes, needed for zonal separation, would be decreased 
and it was thought that the ribosomes might remain more biologically 
active. Since the objective of this part of the research was to measure 
incorporating activity of recombined cytoplasmic ribosomal subunits, the 
small contamination of chloroplast ribosomes was tolerated. 

Gulyas and Parthier (1971) had washed etiolated pea seedling 


ribosomes with 0.5 M NH,Cl to remove nucleases which decreased poly- 


4 
phenylalanine synthesis. In the present study wheat leaf ribosomes were 
prepared with Buffer III before being washed with 0.5 M NHC. 
Analytical ultracentrifugation indicated that the NH,C1 washed ribosomes 
did not differ in sedimentation characteristics from the unwashed pre- 
parations, and that these preparations were not devoid of 70S ribosomes 
(Figure 19 A and B--73S, 74S peaks). An experiment was performed to 
determine which of the salt and energy factors were required for phenyl- 
alanine incorporation in this systen and the results demonstrated that 
only MgCl, had a significant effect (Table 6). The results observed 

in Table 6 were derived from two separate experiments, so a comparison 
of the effect that the removal of each individual factor had on the rate 


of incorporation between the washed and unwashed ribosomes was not 


possible. However, comparisons of each system with its control demon- 


strated that except for the small dependency on poly U by washed ribosomes, 


the washing with NH,Cl did not result in the requirement of any factor 


other than magnesium. In fact, when the incorporation system contained 


yraty foadabo ef as wltiw here date a anoa! 


hactrrs? ve 1 rg he \ortetid vic tf an : 


i ; e" : : 
wt anata iesea s-rtuahll ae 
MO * é tvG6 2 a? n i ae fk >of } 7 be 7 
1 7 : Pies bt : r 


tet é 


sdirnx tael how yiludz year st oy Ade ',eF inv 


Owl) Hg iy ‘i Terreigiyeqo aul diodes ioae ww an 


Lave ere ealet: 4 Lsalenwiry HAH ini 




















128 Me OOF ent tadne nstty pb 4oty 
Py alain tit hi apr 
itd 6h) oyehaaony ts nto | 
LT un » fo eetan ke 70 sdiakt + amend 
Larot atehae honcet gauozod)y ' sn hg r 
Pat . 
jvvanesd sf De 2104 2 id Yo ovtia 0 id 9 mre 
isimeadi4 sii et qos ben | dase “es . vt: 08 
-badoxelos 26 somata b> yori to 
pafslotes bodemw bad GP yer) sahara b oe pan 
oh Antdy eezeolgut wena of np itty 
ei Cee ns 
igh [yi y ta) bone on) rotsepo redial 


i fi s fon Ww ety a var’ 4 ery sat? b o 4 ae Ort: z 
>¢ wn a4 nh ‘ my. Say s2Eh=98 wie 7 { awe 
wooy Shey PORIAT: ye Sis | ie attsa gag Yo afi ait ul thin 


chadoaiet, 2°) wpe SAP bee mpd ogee nevi ty rage a . 


. 
: erivass «f] (3 eS cn! r} oo Braeden 


Ly reviiN . wT Aca ya Vey ee anid » vow 


tot yotoa? Tenn: tee mer 


mS 


worokt oie VS sade pai Ee nt tye ton bi 


a co : bg a? { 


90 





A 


Figure 19, 


Sedimentation values of ribosomes prepared by homogenizing 
wheat leaves in Buffer III. After homogenization, the 
ribosones were centrifuged through Buffer II containing IM 
sucrose. Part of the ribosane preparation was suspended in 
0.5 M NH,CT, pH 7.0 before being recentrifuged through 


Buffer II with 1M sucrose. Both NH,Cl washed (B) and 


4 
unwashed (A) ribosomes were suspended in Buffer II for analy- 
sis. These pictures were taken 8 minutes after a speed of 
39,460 rev/min was reached and sedimentation values were 


calculated as is described in the "Methods". 


ey ab ee eng unin 8 nates aise 2 ugar 


fd even dite 10 2uifay no ties cemihee 


ehfushivn ett «-Ydhar eoveot 10 
cfe@s {2 s0°%00 Ape rea ioe em. 
ian : re 
tsw mor ied ~ ag old ve T ,s20 
: 





oer ke opps 0, on 
4): Been ra tine seme K ' it ee 
srt) ri baie 20 eomode bist ad 1. ) bor bie 


v4 


only tris, MgCl., NH C1 washed ribosomes, and phenylalany] tRNA, at 


least 80% activity was still obtained. 


Table 6. Factors required for incorporation by washed wheat leaf 
ribosomes. Ribosomes prepared with Buffer III were not 
washed or were washed with 0.5 M NH,Cl before they were 


added to the prepared incorporation mixture minus the factor. 


Incorporating Factors 


Comp- -KC] MgCl, -2 ETSH -ATP,CP, -GTP -ATP,CP, -polyU 
lete CPK GPs eGRh 








dpm 3530 B77 328 3460 3442 3247 346] 3439 


% 100 90 9 98 98 92 98 97 
+wash 

dpm 2739 2131 216 25/3 2445 1987 

% 100 100 8 94 90 73 


Yarwood, Payne, Yarwood and Boulter (1971) reported the 
requirement of elongation factors for poly U-directed enzymatic 
binding of phenylalany] tRNA to bean ribosomes. Unlike the transfer 
factors reported for the wheat germ system by Legocki and Marcus (1970), 
these factors were very unstable if they had been highly purified. An 
experiment to examine a transfer factor requirement in the wheat leaf 
systen indicated that washing the ribosomes with 0.5 M NH,Cl decreased 
incorporation which could not be regained by the addition of semi- 
purified transfer factors (Table 7). The fact that addition of the 
factor preparation decreased incorporation rather than increased it, 


and that nucleases had been reported absent in wheat germ Supernatant, 





















_ 


peot> bistent 


whonterda T ‘tk . a 
le) 
ool taodw borteme yd wen nf wt t reer 0987 


Jon evew 11t anThud —" Ronni ox: dt 






te AMA | wen 


a) 


aa 
Siaw Xs ) Ss (ated (2, 4 at Fw Sots oy ‘ parten 


cx 


J 
ey a2 cunt a owwdite nORegi£ont Belen alte as yd hebbs 


evos se Bates yiconl 


NOG DATA ATO Sd HET? S- _fdoMe © Tate 
4 at) i a 





- ————— —=— © —— ST Sateen d 
_ 
cent «Orbe SME SRE ee se CE 
a a 
ae P =& te 
(eel anne = ES ars reXs ENS: a 
t a oof 
i 
add nadvaqen (ter) -teefeathene hoowre? jeangeh anita: x 
"t4 wre Bed thet] yoy 407 ¢ at ey) ere to | 
tom? GAE-edP AL «eae zedr raed oe abet 
({ ai biw Moos! vd ied sys ered ei ede wa 


Lara Pitty. Ye Mt aed hen hadi i arbi hay sve 
sul thom ail wt sasnertipes 98a arene ie oat ms a 


Poa Mt. itw somaedry wilt. et Fa Ww Sard bate 
“ 
tite Bae nse andi 


=o 


- _ 
els y ere rit 1A TeK2 IS5) ot oF 10s. } 20 toa 4 


3) ps Bo“ ni “pny < a erate r ont haa y ad 7 101d 


- nararyaie. rap — 1 the 458 oe 


~ i—— | 


92 


indicated that the added protein was passively diluting out and 


interfering with the binding of phenylalanyl tRNA to the ribosomes. 


Table 7. Effect of addition of partially purified supernatant factor 
on the incorporation by NHAC] washed wheat leaf ribosomes. 
Ribosomes were prepared in Buffer III and were not washed or 
were washed with 0.5 M NaCl. Aliquots of each ribosone type 
were added to the incorporation mixture containing various 


quantities (given as ug protein) crude transfer factor. 


Incorporating System 


No wash No wash + No wash +~ Wash Wash + Wash + 
250 ug ~=s-:« 1000 ug 250 ug 1000 ug 

dpm 4710 4579 3954 3443 3078 2991 
% 100 97 84 73 65 64 


Tests performed to estimate the RNase activity associated 
with the ribosones, gave evidence that all the RNase had been removed 
after two washings through 0.5 M NH,Cl. 

It was concluded that further incorporation studies with 


0.5 M NH,Cl washed whole ribosomes would be conducted in an incorporation 


4 
medium containing only tris-HCl, pH 7.5, MgCl.s phenylalanyl tRNA, 


and ribosomes. 
B. Incubation time for NH,C] washed ribosames. 


Figure 20 indicates the dependency of M40 phenylalanine 
incorporation upon time. Similar to unwashed zonal-separated ribosones 


(Figure 9), incorporation increased for the first 30 minutes, and then 

































cv p 7 
y 
a 
is THO vl Me ails ea ato C best iA od 


o ,eevozedie oft od, NS foot (ute ‘0 wi 4 Aa 
S SR ne: 
7 — ae 
TAETENVOQHE ho PPE ulq yttot: la Rake dient r “oe at3 af 
an, § 


mizod’s Yeo! Inadw weal M yd a ll 
for svow tae TEP oath eI G oem an 

| 1369 Yo Aoupth RY, iit 20° Mar beens ws 

wotisy printetncs sd xteaebgeorooat ‘ott og btn _ i 

“\ veteness store (atatove ov 26 navi) eatsttnaup 


ae 


i? goegawdiooal 





dra! Av el * dean ot + Hinw of izew ait 
| Gy O25 a py wT Ou Des 
“24 i 
trot pave. ova Oren. : 
eX as {2 oof . 
a OPV PED ov afsniscan 03 Naarery tess a 2izaT 
7 » . 
vy oged bed ecat? ot Te feds const h¥goewng .eatbeadhy ease 


a 
aft 7%), tigi meine” 


2 aera Ww) 4 Pa eG ard pass} Se 765) PtwW a 
AY AeA} patovhoge ge Wieder weds sfoww badaaw Be Hi 
Mis | Wer Lar it Dar at si bg chia ya oni 


o : Sree 


=. 


aadl; Seteme TD aon | noe 


oahantePyrtenip od ta Yori thay athe , 


zommerod 2) batppeeasa4 [BGs y Daprtennie wa ver innz 
_ 4% a 

Hon? boo peetwhn G6 dat? eek 60? peepetont 
’ ras si 


30- 


26 


a SEE, TS RRS ESS SSS IL 
15 30 


Figure 20. 


45 60 
time (minutes) 


C-phenylalanine incorporation by washed wheat leaf 


ribosomes versus time. The incorporation mixture contained 


10 mM tris-HCl pH 7.5, 16 mM MgCl, '"C-phenylalany tRNA 


(10,000 cpm), 0.5 M NH,C1 washed ribosomes (45 Engg units 


per ml). From the incorporation mixture, 1 ml aliquotes 


were pipetted into TCA at the various times. The TCA 


precipitated samples were incubated at 80°C for 30 minutes 


before being filtered and counted. 


7 


93 


— ————— amici lf 










a SS : 
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5 





94 


leveled off to reach a plateau at 45 minutes, 


C. Dissociation and incorporation of ribosomes fron plants 


of differing ages, 


Earlier experiments had shown that ribosomes obtained fron 
wheat germ, would easily dissociate into the reported amounts of 40S 
and 60S subunits. Hadziyev and Zalik (1970) had reported that chloro- 
plast ribosones fron leaves of 4 to 5 day-old wheat plants were very 
active in protein synthesis and these were assumed to be mostly in the 
form of polysones. It was therefore hypothesized that the fraction 
of nonce obtained in the present study, was actually fragmented 
polysomes and thus containing mRNA and nascent protein which were not 
allowing the ribosomes to dissociate readily. The finding by App et al. 
(1971) that monosones prepared from polysomes would not dissociate under 
conditions which allowed complete dissociation of free monosomes 
supports this view, 

Experiments were performed to compare the ribosones fron 
wheat seedlings of different ages for their dissociation and incorpora- 
tion abilities. When an equal weight of leaves was homogenized, the 
yield of ribosomes from the four ages of wheat plants, varied. The 
tough yellowing leaves of the 5.5 and 6,5 day plants gave the lowest 
yield of ribosones, while the large number of tender young plants 
required to give the 25 gm of 3.5 day plant leaves gave the highest 
yield. As seen on the analytical ultracentrifuge (Figure 21 A) the 
younger plants did not appear to have more polysomes, and it was 


concluded that the method of preparation had cleaved any polysones 


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95 





giver Gie,2F Ye ve 
2 


Figure 21. 


Analytical ultracentrifugation comparison of ribosanes 
prepared fron wheat leaves of different ages. The leaves 
of the 6.5 (A), 4.5 (B), and 3.5 day-old wheat plants (C) 
were homogenized in Buffer III before they were washed with 
0.5 ™M NHC] and before they were centrifuged twice through 
Buffer II with 1 M sucrose. The ribosomes were suspended 
in Buffer II for analytical ultracentrifugation. These 


pictures were taken 8 minutes after a speed of 39,460 rev/min 


was reached, 














97 


into 80S particles. Figure 22 A, B, C, and D illustrates the dissociation 
of these ribosomes (prepared with Buffer III, washed with 0.5 M NH,C1, 

not pretreated with puronycin) when centrifuged through dissociation 
buffer containing 400 mM KCl. The ribosomes of the plants of all four 
ages dissociated into 40S, 50S, and 60S; however, only in the 3.5 day 
plants was the 40S peak area smaller than that of the 60S peak. When 

the ribosanes were conpared for their ability to incorporate pheny]- 
alanine, maximum activity was observed for the ribosanes extracted fran 


the 4.5 day-old plants (Table 8). 


Table 8. Incorporation by wheat leaf ribosomes fron seedlings of 
varying ages. Fiats of wheat were planted on consecutive 
days so that the ribosomes could be isolated on the same day. 
Ribosanes were isolated with Buffer III and were washed 
twice with 0.5 M NH,CI before being added to the prepared 
incorporation system which contained only MgCl., tris, and 


eceenenmialany| tRNA. 


Age of Plant (days) 


S25 4.5 SnD 6y5 
dpm 4071 5194 3807 4514 
7 78 100 73 87 


Plant ribosomes have been classed as two types--membrane- 
bound or free--which specifically synthesize different types of protein 
(review by Boulter et al., 1972). Radioactive tracer experiments on 
the developing bean seed by Payne and Boulter (1969) demonstrated that 


the increase of membrane-bound ribosomes during synthesis of storage 


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Figure 22. A comparison of the ability of ribosomes, fron different 
ages of wheat seedlings, to dissociate in high salt. Five 
E560 units of ribosomes (prepared with Buffer III, washed 
with 0.5 M NH,Cl and suspended in Buffer II) were layered 
on top of a 5 to 20% linear sucrose gradient in Buffer IV. 
After centrifuging 4% hours at 85,000 xg at 25°C, the 
absorbance at 260 nm was recorded. A, 3.5; B, ALLE Foro; 


and D, 6.5 day-old seedlings. 


—— 


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protein was due to the synthesis of new manbrane-bound ribosomes rather 
than attachment of the previously-free ribosomes, As the seeds reached 
dormancy, the menbrane-bound ribosomes detached and were found free in 
the cytoplasm. The acceptable dissociation of ribosomes fron younger 
leaves (3.5 day-old) and wheat germ, when compared to the dissociation 
of ribosomes fron older seedlings, could be explained by different 
types of ribosomes--respectively free and membrane bound. 

As seen by Figure 23 A, B, and C pretreatment of the 
ribosomes from 3.5 day-old plants with puromycin aided dissociation. If 
the ribosomes were spun through a 10 to 30% sucrose gradient containing 
the dissociation buffer, dimers were revealed (Figure 23B) which other- 
wise sedimented to the botton. However, these gradients could not be 
used for preparation of subunits because the subunits were not as 
widely separated as when 5 to 20% sucrose gradient had been used. It 
was concluded that the 3.5 day-old wheat plants could be used to render 
a yield of ribosomes which would incorporate phenylalanine and which 


would dissociate into the acceptable ratio of subunits. 
D. Conditions for dissociation of ribosomes from 3.5 day-old plants 


The 3.5 day-old plants had given promising results and a 
set of experiments was conducted to establish the best conditions for 
the required dissociation. Sedimentation of ribosomes from 3.5 day-old 
plants through dissociation buffers indicated that pretreatment with 
puromycin and centrifugation through a buffer containing 300 mM KCI 
would give the optimum desired dissociation (Figure 24 A to D). With 


the removal of nascent protein by the puromycin pretreatment, the 

























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Figurescoe 


Puronycin stimulated dissociation of ribosomes prepared 
from 3.5 day-old wheat seedlings, 20 E560 Units aA Cy por 
ribosones (prepared with Buffer III, washed with 0,5 M NH,C1 
and suspended in Buffer II), in a volume of 0,2 ml, was 
added to 0.25 ml ice cold double concentration Buffer IV. 
0.05 ml of 0.01 M puromycin pH 7.0 (C) or 0.05 ml H,0 (A) 
was added and this solution was held on ice for 15 minutes, 
and then aliquots containing 5 E560 units of ribosonal 
material was layered on top of a 5 to 20% linear sucrose 


gradient in Buffer IV. (B) 5 E units of the water- 


260 
treated ribosomes was also layered on top of a 10 to 30% 
linear sucrose gradient in Buffer IV before they were all 
centrifuged 4; hours at 85,000 xg. The absorbance at 


260 nm of the gradient was recorded. 


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102 


ribosones dissociated into the expected ratio of 40S and 60S subunits, 
while without this pretreatment the 40-50S absorbing area was much too 
large. Increasing the KCl] concentration above 300 m™ did not further 
dissociate ribosomes that had not been pretreated, but led to a build-up 
of the 40S peak. Increasing the KC] concentration above 300 mM with 
puronycin pretreated ribosomes caused conplete dissociation; however, at 
higher concentrations of KCl, the 40S peak appeared to increase while 
the 50S peak decreased. The high amounts of 40S observed in Figure 24 
B, C, and D was contradictory to the results previously obtained fron 
3.5 day-old seedlings (Figure 22A). Thus this phenomenon cannot be 
explained as entirely due to the age of the plants. However, these 
large amounts of 40S were regularly observed when dissociating ribosomes 
obtained from 4.5 day-old plants. A suggestion, that sone of the larger 
subunits had dimerized, was put forth. In the present research, there 
was not a notable peak sedimenting in front of the 80S parent species. 
However, it is possible that particles more dense than the 80S monomer 
would sediment through the 5 to 20% linear sucrose gradient during 

the centrifugation (Figure 23B). 

An analytical ultracentrifugation (Figure 25A) verified 
that a sample of ribosomes washed with NHC] had a major peak of 81S, 
while when treated with puronycin suspended in the dissociation 
buffer containing 300 mM KCl, it dissociated to give particles which 


had sedimentation values of 35, 45, and 60S (Figure 25B). 




















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Figure 24, 


The dissociation effect of various concentrations of KC] on 
the ribosomes fron 3.5 day-old wheat seedlings, treated or 
untreated with puromycin. Ribosomes were prepared with 
Buffer III, washed with 0.5 M NH,C1 and suspended in 

Buffer Il. The puromycin treatment (P) as well as the 

5 to 20% linear sucrose gradient contained the various 
concentrations of KCl in Buffer IV, The samples were 
centrifuged 44 hours at 85,000 xg at 25°c and the gradient 
absorbance at 260 nm was recorded, A, O KCl; A. puromycin 
treated + 0 KC1; B, 300 mM KCl; B. P + 300 mM KC1; 

C, 400 mM KCI; C.> P + 400 mM KC1; D, 600 mM KCl; D.; P + 
600 mM KCl, 


104 


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105 





Figure 25, 


B 


Sedimentation values of NH, C1 washed ribosomes and their 
subunits from 3.5 day-old wheat seedlings. From analytical 
ultracentrifugation the sedimentation values of ribosomes 
and their subunits was calculated as is described in the 
"Methods". These pictures were taken 8 minutes after a 
speed of 39,460 rev/min was reached. A. 3.5 day-old 

wheat seedling ribosomes; B. these ribosomes dissociated 


by puromycin and Buffer IV containing 300 mM KCl. 


aor 








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106 


E. Recombination and phenylalanine incorporation of subunits 


Ribosones from 3.5 day-old leaves were pretreated with 
puromycin before being centrifuged through a dissociation buffer, 
containing 300 m™ KCl, in a zonal rotor. Figure 26 indicates that 
the dissociation was almost 100%, and that the area of the 40-50S peak 
was close to the expected, The fractions separated fron this zonal 
separation were pure 60S or 40-50S, as was seen by a sucrose gradient 
analysis through the dissociation gradient (Figure 22 A, and B). If 
the subunits were sedimented through the gradient containing the incor- 
poration buffer (high MgCl.) > the sedimentation values of the 60S 
particles increased to 68S, while the 40-50S particles increased to 
55-62S. As has been mentioned before in this thesis, the increased 
sedimentation value can be explained as a compacting or an aggregation 
of the subunits caused by the high magnesium concentration. Shifts 
of sedimentation value caused by increasing magnesium concentration 
have receintly been reported by Reboud et al. (1972). Rat liver 
ribosomes in a buffer containing 0.3 M KCl, dissociated into 40S and 
60S subunits which when centrifuged through a gradient containing a 
higher concentration of magnesium, would partially reform respectively 
into 55S and 90S particles. Delinas et al. (1972) dissociated euglena 
cytoplasmic ribosomes in buffers containing various concentrations of 
KCl and they noted that at 0.3 M KCl an additional component (46S) 
appeared which they attributed to a transition of the larger subunit. 

In the present study, when the two subunits were mixed in 


a ratio of 2:1 (60:40S) and centrifuged through the incorporation 


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Figure 26. 


Zonal dissociation and separation of ribosomes from 3.5 day 


old seedlings. 3000 E units of ribosomes suspended in 


260 
buffer II, were pretreated with puromycin and Buffer IV 

(containing 300 mM KCl) before being layered on the same 
Buffer IV zonal gradient. After centrifuging for 4 hours 
at 25°C, the gradient was analyzed at 290 nm and fraction 


I and II were collected and assumed to be respectively 


40-50S and 60S. 


108 


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Figure 27. 


Reassociation of and cation effect on ribosome subunits 
fron 3,5 day-old wheat seedlings. 4 E560 units of zonal 
separated 40-50S and 60S subunits suspended in Buffer IV 
were layered respectively (A. B.) and together in a ratio 
of 1:2 (C), on top of a 5 to 20% linear sucrose gradient 
buffer containing 10 mM tris-HC] pH 7.5, 16 mM MgCl. and 
25 mM KCl. Similarly the 40-50S and 60S subunits were 
layered (respectively A, B) on top of a 5 to 20% linear 
sucrose gradient containing Buffer IV. After centrifuging 
4%; hours at 85,000 xg at 25°C the absorbance at 260 nm 


was recorded, 


110 








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111 


buffer, they did reconbine as is shown in Figure 27 C; the 50S and 
58S peaks representing respectively compacted 40S and 60S, and the 77S 
representing the reformed Parent species. However, as shown in Table 9 


the reformed monomers were not active in poly U-directed phenylalanine 


incorporation. 


Table 9. Incorporation by subunits of high salt dissociated (300 m™ 
KC1), 80S ribosomes obtained from 3.5 day-old wheat seedlings 
and pretreated with puromycin. The 40-50S and 60S subunits 
were isolated from zonal fractions, but the undissociated 
80S control was taken directly from the ribosane suspension 
and was untreated with puromycin or high salt. The 
respective samples contained in E560 UNT CS 20055 e405 


AO=505 522505570 ee 0-05 ete O0s.m loatmoUe 


Ribosanal Material 


Undissoc't 40S 60S 40+60S H50 

80S 
dpm 654 129 157 143 105 
% 100 20 24 clu. 16 


A possible reason for the inability of the reformed monomers 
to synthesize polyphenylalanine might be that among other things 
elongation factors needed for incorporation could have been removed 
during the dissociation. If these were not replaced in the incorpora- 


tion mixtures, incorporation could not proceed. 


eg 
200 ott bas ,208 bane 200 bat zaqiw> wlaveaade al oe 


cd no 
‘ ofdel nf mwone « 


‘innfelynedq botserib-0 Yieg m avit2e ton os 

















bas cOe one ’ co ste hid nwt aha + macy 


a 
' 


26 , TaveWGn <ephoone ra 


) | p22tb i 2 Nth Yo <ttwwdue ee ie de: hl 
‘ibse2 Jsodw blo-yeb 3.0 Gay 7 han tarde ssnaeodhy 8 pu 

ie~(t ott ,Ahov! aia tt tw sereento inn 
ststooeztbal ott god ,shekdees? {poe ma spanlaes 
lenageut-emeadiy abt aa) itso! modes ee toranco 2 
i doteé ve Aigynowua 48 hw -sbeendiw naw b ot 
j2b 200 s2Stnl need OE Gumi Noe. eat Mba ovtiosceen : 
| 06. OL BDO r> 202 -0a 9g 08 et 20% D: 


| * 
< ; a - 
li r'¢y fal cin south ae ’ = : 
' ‘ 


of  2heeO Mina eo)  ahaenk syont 
208 


i) rdtsnl etd wet a 

prom rat et ddgtw yaa 
toy quad sve blued Wefeanog presen able 
i? iid al taoelqey den sian ane ow 


basset dun biveo ao on 


SUMMARY 


4c ohenylalanine incorporation was induced by hanogen- 
eous preparations of wheat-seedling cytoplasmic and chloroplast 
ribosomes which had been isolated by zonal centrifugation. The sed- 
imentation values of these two major ribosome species, calculated fron 
analytical ultracentrifugation were 79S and 68S respectively, The 
final suspensions were centrifuged at 30,000 xg for 20 minutes to 
yield a preparation containing less that 10‘bacteria per ml. Wheat 
embryo synthetase enzymes in the presence of yeast stripped tRNA and 
M40 phenylalanine, produced 40 phenylalanyl tRNA which was used in the 
incorporation systen. The incorporation by cytoplasmic 79S ribosomes 
required high concentrations of ribosomes, high concentrations of mag- 
nesium, low concentrations of tris-HCl buffer pH 7.6, and was coanplete 
after 45 minutes incubation at 37°C. This transfer of 140 phenylalanine 
Fron M40 phenylalany1 tRNA into protein was not dependent upon added 
energy factors. Inhibitor studies showed: incorporation by both species 
to be inhibited by puranycin, incorporation by chloroplast species to 
be inhibited by chloramphenicol, and incorporation by neither species 
of wheat-seedling ribosomes to be inhibited by cycloheximide. The 
sedimentation values, calculated from analytical ultracentrifugation, 
of the cytoplasmic species and its subunits in a high K"- low mg*? 
buffer were respectively 76, 56, 46, and 37S, The parent species 
produced by the recanbination of 37S and 56S subunits would not induce 
polyphenylalanine synthesis, The poor dissociation by the high-salt 
treatment was overcone by pretreating the cytoplasmic ribosomes with 


puranycin. However, the reconbined monomers stil] would not induce 


















if ek 


‘me gs “> 


-neponod yd beoubal moo oat ‘ 

Senlqorolita bas shes gate partly 9s id w bene Tiovsqetg ¢ 

-baz2 sdT ,notis oy thats 99 Tens vd vrata a od be ] ‘th sts 

ort bedefuoles 2otoage simmeedry soln n owt sear aes cout 
ait ,yfeviivagest- G80 bie Bet Siow | antenna eeasbntt re 

oy zotuatw OS so? px OOD, OE Ic bs ate ore ng qeve 

‘W .fm 159 atvatsed On Sand zrut “nee ma bist 

bis ANAt baggtwte S2opsy 86 oneeevg sat at zemvene “peste 

aw Hott AMAT Tynatalynariq-) set banubong , anime st ne in ; 

zodty 22T stowelgotys ye nohiitoqeant sat wimaey2 i eee? a yIOSM 

rtavinnodos “ptt ,eatmecci 7 + Snibdas ana nh i pe 

wou 2ow One .d.. Ng were Toh-2l4? We ani ure 

wfalynadg-9” to wsteait ait. .0°%S a8 narnia eaduatn 2 96 

babbs noqu Insbaadel Jon aaw Wtednnid obnt Atte tysotst ye , 

vid GOR’ wont shewole 2ahi *: spin xo at Yer 

Hoag? trsfqovotds qd netidseqrogn a “ sate stn 5 

leone vad?ian y€ aalgaoepoet oR taal « dbs ian 


ata? xadgious 4d fet pidnl af of — 4 pat ibosens 













_ 
~ > aan 
‘ $ - 


yreeool ia fqeoew Tu ipo heyhagn wert tyacrs Tact agvlev nottete ; 
arm — : 


a vl - doth ont eftandar eh ig ae eal tad 


9) 29q2 snows alt DYE they ke xe af feviie 
int ton Sfdaw af haudws pena RAY bap ee 
Siez-teld od yd solsatodesth ee 
fiviw comeodis olmgsiqutys odt vem q 

me oy 


‘povhal Jon Giuaw rive oe | 


113 


polyphenylalanine synthesis. 

Preparations of ribosomes produced by homogenizing wheat 
leaves in a buffer containing 100 m™ KC], contained both types of 
ribosomes, Washing these ribosomes twice with 0.5 M NHAC] removed 
nucleases, but did not alter the sedimentation characteristics of the 
ribosomes, Furthermore, washing the ribosomes with 0,5 M NH,C1 did 
not change the incorporation factor requirement and, in fact, when the 
incorporation system contained only tris buffer, MgCl., NHACI washed 
ribosones and 4_ohenylalany] tRNA; 80% activity was still obtained. 
Incorporation could not be further stimulated by adding isolated 
translocational factors to the incubation mixture. Unlike the ribosomes 
fron 4.5 day-old wheat seedlings, those from 3.5 day-old wheat plants 
dissociated in high-salt to give the required ratio of smaller to 
larger subunit. A pretreatment with puronycin produced total dissocia- 
tion into subunits with a calculated S value of 60, 45, and 35S. When 
the isolated 35-45S fraction was mixed with the isolated 60S fraction, 
the parent species was reformed. However, this reformed monomer 
would not induce polyphenylalanine synthesis, ; 

This incorporation system, for wheat seedling ribosomes, 
was not dependent upon added messenger RNA, energy factors, nor 
supernatant factors. Cycloheximide, an inhibitor which specifically 
inhibits the formation of an initiation complex, had no effect. 
Ultracentrifugation analysis of preparations fron seedlings of various 
ages were the same and established high proportions of monosomes 
relative to polysones. The difficulties encountered in the dissociation 


of these monosones could be overcome by a pretreatment with puromycin. 


et | 


7 taht 
cOUy, 


mocornan Ve 
j Palsq2e th igi ay 


Hiwiet 


/ OATST 


ii4 wf 5 


rat 
| 


al 


fod banistnes , TX fe Gol 


(I,H4 MH 2.0 cttw aofed ¢ muro add 2 am tae 2 


S957 al... tne Ine “t2RY rot soromne ems onerty 
| of ot .1s¥tad ela vfoo ban tad no metry pot p10Q102 
gw vo ivigse RON ; Aas I voista'yngiqg) 2 smeod 


' ahtny Sevfapes at? avip of eet nt b ae 


ee 
Atiw Suetewitere # wt 



















- Poe hay 
.¢” y ”~ ! ~ ih 


iw — 
a hud 61 


tne 0 omord vd bosub ony peak, an pia Bt jan 


ryt seve ng Sedmgmth ie oid aot on p2 ie 


ii tw eommaodis ety pobigew, 9100 varia vv 43 


:c beselwals: sadsaw? of don bivor wr re git) 
a sim apefeduont edd a? eratoe rand 220 of ens 
2.) oert® prot ted (hese ieedw bioyyae D2 oe 


ery 42! tnomteeiseig A prow : V9Ris 


fae lusts « AS hy 23 tnudue 0 nf nots 
city Peatm enw nosey 220-26 batefoet sh 


ae aes ae 
t weedwatl Doma ken ai eatosqe. HS15Y Sf 
eteorniawm switig at enadelog avert, of b nn 
wily «0? , eho ce ey HeregNaD han - 


me yeyn: soeen hobs not am sta 


iotetw “meyataak. ae sstatatola@, he iz tnd: 
uw bed ,xetome. noteshttnl as Yo, no phrss mest oat (2sT6 


Ps 
reatiiser nyt anots a 5govg to. gh a Ten as rete otha 


smimoqetq dotd. beset deg 29 bite ai » ol: 


bas? su0one colt 


- 
ae 


os _ 
"Vibead 


114 


These facts have contributed to the conclusion that the homogenization 
and separation procedures had cleaved the polysanes into monosones 
which contained not only mRNA fragments, but nascent protein as well. 
This cleavage is probably mechanical, although the observed presence of 
RNase on the ribosomes likely fragments the polysomes to some degree. 
The observed large ratio of the smaller subunit compared to the larger 
Subunit and the unexpected occurence of the 50S particles, demonstrates 


the need for further research into the eukaryotic ribosome. 


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om 2RUTAAGT 


HO208 .J bas., AIGASIIT MAY wk aAPROG 
rfoa .3 Yo norsetoozeth pAreMee oloct notiets ia joo 
; 1OE-SeS $a ran mn “3 iat 


% norsst0"9 rOoNT bio e ont’ ,daef 1a 
ma .u .me? Xe ove: = 6 ut be ws Ty 
“OVVE Tyrie ib to 3948 tts ont overt <a! rr bne * 
esmozodtyloq tastq Yq Rotsteloet oat no 4 sae ™ 
03 5a sdronsl aqua .Totey at. me te 

evotost yetannys AMAT “Peasontne to 0 Soames AR 
2amo2od oye st Me aes aninals rg 

at feat “eh Toke Te 
hitor A one -ARDAAD a iA 






nt Jnemery? ups D + ohm 
ov sit ud 2  nisterg Yo nottoson setenery ata 7 
iL - ; if sth . bate un 4 nai4 eemenat 7 = 


y noljatoogeia Joa ae aw pre ef. ce 

a~ PS och stovd? dealt ie ~ se t o howe big aommeodiy © 
son! baa oat eet iS OOWIDAL At eu" 
yt we 


at 7 forte tael4 steel qh Avi hl 


fing 204 ertzoe to eobteteqeys . 11 9t Ree at bra 3 +2 Aa ArT 
pore " s] ; pba 7 eonosenit ret osy mon? ee Rath i? 
a yi im: a 



















wtinegs wt fov4 
a fh. 

Tesh egg tarball ‘tie se 

Sb-EUb 20h Petal" Stage 


: ¥ ‘ fy ry ?2 OTe I ast IRA ; 
Grit Hivcneyp res if bith an titty Oy dete * 3 
tet —tkor car 


ol i Brent th ? noire rears! d -PSer é 
Sof .co14) .ntawoseg ve en taeden cout 3 zat 12 
, bE ~ORE 188, 


atotov? 200% HAMLIN 1.2 bap. , bran AS 
an -isvant ossadgt $n 22 2ettxe sett] 
baddmyst she ‘ety bos eatiys tiem fortes 
ear gary 208 bas deol qeiatin. x 4a) 
«223-008 aM fare 


a: 


ie y 
ee 


116 


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Phystolwe i2irge9 lal 142 


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Dc&1. Akad. Nauk. SSSR. 164: 688-691. 


AAW 


{ Mia) 


4 
J 


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> Viz ere 


“4 4 
C1} E74 


{apt 
7: 


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a 


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‘taaden to calre) .TUet ae 


aA yi 
& Tf 


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ernove’ se nee 2970 trol tuk nae 


: f, DAG ivy 3< T LAS a jt) vhval 




























Li i Te, 


f uank .2danty oF son vietord 
= 7 = ip z: robes 
doors, SCR! QBOWANY Ay 1sa3 isa | 
‘Vk Won Joni einer ar a nye n = | 


—— 
DEAD 5 4 bas TAM RS Tea wAg 4 o.2 99K 
sFisnep Siz wn Yalgis 2 thie — 
y svyitsisqm? Seer , AvoaTHIgO ee bos 5 gue Di 
Ivo bee Seefabwal dy to rt f 7 2012 ot 
ns) Ate "4 OF ; o> t 
oo Ly 
a 6 VE 


_ i 0eh-2b 






“16 
midimn pra “Seyi ..0TeT WAIT 
ya boane? -23aebo" we siuten tn 

(2 "ietovey VOT, Dar eduon Rt 

OFS -K08 005 pier “UOT ASHE 


olaotts shag ey ya Q roe wh atatond: er 8 
Abioida mareesiog Aitw Joan oad. see ; 
H8- 1828 Hint 2a | ; 


heatimge nese? tee! AADROO- 08 bie Ai cee 
70 noliosnqmed?l = ,eueslt rata shuit=oFmod 

zi van tf Fey orb ve absdong anat 7 
HOE 





er 
‘ 


—_ 


om?+ ang at adhtpuetwe Ww afof ” eet + 
Sap Eat ihe) svireN 


cshtseqerd 490). it rows Wn 9886 

Sine nh sammgeds ota cian atl a 
feteere sa sian Tai abet 

— 


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A, 4 pr 

















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ae 

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0-088 (1 h0T) et 





117 


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Biochen. Biophys. Res. Commun. 46: 1418-1424, 


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' Gee nc 






























. a ) une 
Ove WIRASO: , 2). one AON A 8 ,AUg oT A a fie) 
rateve poislzerninve Afasowg ait 40 ok see nay Ai lrdicxe og 
fi tion?) aes-XeS Be Ene “BT ge eral he . 


. Vi = ue 
‘ AMA a) ae 3 LAS q eV. WTYARIG A 
Hi fio le rsoe2 ot iG eatbuse set. i WAS ‘ 
-2atioldtda Yo Postt? + omieodty Tart st 38¢ 
“des. eS 20RE 530A atm sMittao a 


viarznes int rere? rh. yet over  ' YIROTMA? : ‘ ‘8 bn ; 
» VOSTAIMAT oF 2umMey iinylog brvod: ouerdnsin Dm 
eres 


pear-earar 4a » LATE iy -2oh a 


etesloovelad © 1 LARS tM bas , AOUNAOAL oT A 
i) 2 vou? A * hte? be em 198) vise onthie: tab Tk 
ULS-208 0BF .otok  eyigotd: oe 


ninda abltga® .OvOR QPRRBAD .T cht nate: a aw ERE. 

2 Yo noltxe to offe bee qotbesr Vv 2Y pe Nort . 
Voges Ge UL tse hh . 28 

‘200 } pitt aL eb te eo Ph fo ttelaoae sets sit fer “ai ABN 


Ag ‘TS!  & .eeebore cel HDG ov oe OF ot 7 


' a bre trsi gore! io tr 4 5 ond 7°) Fr) viz 
, eeygs) % a V0. agn dt+ ol 
avt $+oer elon *: eysinaTl TONE 


Pee ae en ee ae 
sal wut ; ” ina 


havcnaontoels evidersqmd ee! Oi AOS ee. 4 - 1597 Alle 


ftaPiataetats | rr ee ateon ; 
ih] Joanne -QneF thi) ¥ 

a eS Re Takei er a 
; a 
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vars 
cleare LL bAg E svpel a Beri mrt bt es 
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n 





bon ehtos telat .e0er . aAS ei 
‘ cea "Old tm event decd 


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et 19 3h L409 
2tebslyjos stad AT? eeeraye aovt a 
AN <tat sear a seh an a 


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118 


HALL, T. C. and E. C. COCKING. 1966b. Studies on protein synthesis in 
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FEDsoLet bamelaDress 


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Bit ; 












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119 


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120 


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121 


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123 


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nOCeeot | aehCad..oCl.gaUo. e01¢ee7 01-70%, 


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Biochem. Physiol Pflanzen, 162: 220-224, 





























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PED ise, ‘mana ni ings 
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124 


YARWOOD, A., D. BOULTER, and J. N. YARWOOD. 1971. Methiony1-tRNAs 
and initiation of protein synthesis in Vicia faba. 
Biochem. Biophys. Res. Commun, 44: 353-360. 








YARWOOD, A., E. S. PAYNE, J. N. YARWOOD, and D. BOULTER. 1971. 
Aminoacyl-tRNA binding and peptide chain elongation on 
80S plant ribosomes. Phytochemistry. 10: 2305-2311. 







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