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POMEIB6#dmOWlbB ' ' OTSHBSIS' ' by ÔWÔBMSMIO M2Y1BSto y J . P v . W Y ÎŒ 8
OJhe poBBibility that the cell c rtoplaBBi contains .engjjiaee eataljrsing BIA M o synthesis was investigated in Iiandschuti:â aaoites tumour cells# Enzyme fractions were obtained by differential centrifugation of disrupted cell homogenates# Wie incorporation of ( P) DTP into polyribonucleotides catalysed by such fractions was investigated in reaction mixtures.containing primer EM A , ATVf aCOP and, CT3? to stimulate conditions for Bynthesis of HMA# !he miorobomal fraction was most active in incorporating (o<^^P) b%DP into polyribonuoleoti Expérimente in which' the miorosoHial .fraction ?/ae analysed by eitorose density gradient centrifugation confl3?med the particulate, nature 03? the ensyme#
An acetone dried powder* of the mierosomal fraction was prt?epa:t?ed and its. properties further investigated#%he incorporation of ( '=><,'‘*'P) IW? was stimulated by an ASP regenerating systcBi but not by ASP, 0SP and CSP in the preesence of the latter#. She incorporation of “PÏÏÔ?P showed a 5?equlrement f03? EMA as a primer and an
' g L - ■absolute requirement for Mgv ions while IM ions,spermine and. putrescene inhibited, the reaction#Alkaline hydi'Olysis of the reaction products after ;
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■’P)' OTB .inooiqpcreatioH-fBliowecl maluly HEP rcBldueB to be labelled Irrespeqtlve..'of-ûyhethe'%* • ASP,• Ct?P and. OTP v/ere present' :ln the reaqtlcm mixture Indloating eyhtlieBis of poly ÏÏ ohainBé. ■'? % ;/ ' ■ . " ■■■ '■•;''■ .;v \V
ATP# OTP and OTP were ,foimd to be Incorporated':. Into polyribonucleotides : under, the same. coMitione < ao,
) IJTP, though ihdividual micleotidea .were ;-inoorporated to different oxte.ntBt The iBoorporatidn of • H :ATP at inereaslng subotrat e oohoehtratloh was inhibit eel by equal conoentro/fcipne of ÜTP, ; OTP. and TO)? added to the réaction mixture a hTër:.the,:rahgé^ of:, eono entrât ions tested# These expéniment8 /indicate homopqlyribonuql^btide oyiithesi
than, the myht :BMA#. , ' ,.Extraction of ElA- from- the reaction mixtiires 'after
ribonucleoBide 5T--tr:lpb:oBphate. inooi'poratlon and. analysis by suorose density, gradient centrlfiigation ’ ..: showed no labelling of the .ribosoBial hllA# Tho incorporât radioactivity appeared between the;48 region and thè . HipniacuB of the BtioroBO gradient # , determination of the ohain length of the homoxbolyribonuolQoticle. product showed only short; Bequonees to; bo; ByhtheBloed ivhioh is: in agreement with, the :position of radloaotiye: HMA on sucrose density graxlients#, Oonoidefabl.o .phoaphodi,eaterase -activity was. foimcl to be associated v/ith the iïiicrDsomal
fraction wMoîi eoùlcl aoeomit for the laok of labelling of fibooomal 1#A and the short chain lengths of homopolyribbmicleotide synthesised#
An attempt made to demonstrate homopolyribomioleotidc oaeurrenoe in vivo proved negative though the poSBibility could not be ruled out that shoi?t chain homopolyribo^ , nucleotides were present#
The lack of ability of çytoplasmie fractions to catalyse/in vitro the net synthesis of RHA ie in.-. , agreement with in vivo experiments on the kinetics of. W . . . # #,wtv,T9 ■ . P.V > ;
nucleotide uptake into, .cells.in the presenoe or,absence of a.otinomyoin 10 w M c h /indloate, that synthesis of RIA . ..occurs primarily in the nucleus on a IDEA template# . lïov/evçr limited hpmopoly3?ibonualeotlde synthesis, does appear to be e at aly à e d by the mi oro s omal fraction, the possible Bignifieance of which,is discussed#
Polyribomcleotido eynthesis by oytoplasnilQ enzymes •# m #fim M m r # #»* ### #**%»**#fn#% mMiM##5W#aiK:'ii itttvfm t \nm i r* ? #4* ;# -
: :•'■■' ■'■ ■'. ' : ; by ,' .
,A v . \ y # ' E ë \ % k 0 à x : \ ' : - T : y: '
Tliesie presented, for the. degxree of , 3)00tor of Bliilooophy,
. ' / The ÏÏHivoraity of (l-Xaogow
. ■ ■ ' V-
■■■ : - ,
y.
y'; T h a n k s due ito/ Prof osqpr J #. Davidson : : 'top ■ providing / the 1: qppbrtwlty asid -f acilltiOB. to h oar3?y ;out:'tKia/wQrk*;..' '■ I .am.moat gràteful,to/ ■■'■;?,.■ v
for ’ ihvaXuabXa -guiaanoo. and . -Gncouragement durliigthoy"oourüo -,'of this ;;foaoarohV' :■ I'." should; also. \Llko; to thank 'Burdoh ■ and; BrV •E*.'EasoHyfbr; ueeful ,àdv:toé# : Miss' Bf .Dovms' for : officient' teohniqal- .ÿooiWtmieo#"Mr# pT#B.*' Bhopherd.;' for proof readihg mïd' l a r s f ;iv#Av;:Haopherooh: for •' : '■
; ;;. ' 1 ■; ■'■ ..Thia work \v/as y carried/bmty%^fxng; the ; tomtiro" of %;;Hedi.ea3...EGseaj)ch; /y
I AT TOMS
A -, . Adenine V-.
0 , ' ■ . ' 0-ttanihe # 'I f y u y t ô B x n e *
ÏÏ . , . U r a c i l 'y/y. -AdènoBiney'5i*-m6nopliOBp!hàté#'y;, , ;.o ;.
Oytldine U^"-monophoëphatê$/'GuanoBine ySÎ^-mohophôBÿhate # .
: . Uridine 5 ^-monophb ephate *
k W Adenosine 5-^dipliosphate#UUP Uridinp 5 *-diph6ephate # '
. ; ■■■AyAdenoeine' Ù ♦.•-triphosphate#'' ' ' Deoxyaderioeine 5 ♦ 1ripho Ophate #
'- . Ôÿtidine 5 '^triphosphate,' VGuanoeino 5’-trix3hO0phate*
■TÏÏP fhyiuiclino;- '♦• tripko.spha.te* -ÏÏSP Uridine 5♦-tripho sphate #
(^^P) ’ ÎÎMP . Uridine jl^monophoephate ' labelled with'•■ ■ ■ a'radioactive.phosphorus atom#''.
" T O ' / / y y . ' . \= - ' : V / / " /(o6/f''p) UTP ,ui/ldinè 5 ♦--triphosphato labelled with aratlioaetivo phoephorue atom in thepoBitlon*
Thymidine 5 ♦•^triphosphate labelled with a radioactive phosphorus atom in the gL position#,
(iip . 2-oyanoethyl phosphate labelled with àradioactive phosphorus atom*
2«*aaninO“-2-hydroxymo thyXpropane-X, 3-diol tris-HOl buffer trio buffer- adjusted to the required pH
with Gonoentrated HOI* '
DMA. Deoxyribonucleic aoid*' ■ " >. /: .Jlibonuelelo ■ aeicU =
\ BolUbio (or,-transfer) ENA,poly A g etc# Eolyriboimoleo tides containing - only
: adenylate residuesimilarly for the' . ' other polyribônüelGotides.mentioned in
the text* ' ' poly (dAT) A polydeoxyribonucleotide containing
alteamating deoxyadenyla/te and .thymidilate residues* ''
pCpOpA The terminal trinuoleoticle sequenceof SÉIA* ■
Methylated serim albumin on Iriesolguhr^
Eicotinamide mononucieotide
G O H a 'B iæ s
Introdixctionj=>MyÆZMgu*Mrra<tar^Ti»/'<ifi T »
1
2
Physical and. ohOBiieal structure, of .the nuclei G acids* , ■ -
Oeil Btructufe and. the intraceli.ulm? dl8t3?ihutiom of the nucleic acids#
3« In vivo studies on Bï A metahollsm#A* Metabolic ??elationsMps .between ' species of HHA*
B* BueXear origin and. relation of DMA to BMA synthesis*
0* The transeription of the DMA . helix by IlHA.*
The in vxt:eo synthesis of nucleic acids##»=3ra ewFKetiXimaeneW “ .
5 1
■ A, The, hioBynthesiSYof: ]' B* The ■biosynthesis of HIA
'" Aims, of present ; worlc* ■■■;■ , .
15
16 2 5
2 9
3 2
5 536Q'
Methods,T,
2 *
,3'i
r @
5 o
6
Propagation of liàndschùtgs'aBcltea; ;. carcinoma*/.Préparation of extracte from Bandschut^
carcinoma cells#^Preparation of an acetone dried powder . of the miorpsomes* -
Assay proceduresoWashing procedures#Measurement of. radioactivity.
50
50
5 .)
555457
Hetho^''(dont tdr) . ■ ' 'w'Z^aâ'To Analysis of ' ribohuoleptldçBV ' .
. - - A#''Bleetrophore8is*/'‘/f\'v ^ '58''\ B. Chroma oography * ■-. - %' - . /: ' " ■" : : .. 58
■■80 : Preparation of iWA#/ . ; //v/y';:-.. ' ■ ■ A * Prepa3?ation/of aàoiteë bmnour :: '\ // . y V : / , : W h b i0 : - G G l l : m A # ^ 6 0 /Préparation :of--.i40’-Mik« .' - - . gg -,■ ' Oo/Extraction of •;BHA/'from ireaction ,7,/- v; ''/,
’ , ; ■ /■• ' miztureo*././ ' / ■ ■■ -//:/ ' /•-':■-■ 63 ;;9* .■■.Bucrose'/donsity ' g3?adient;\contrifugatioB*-: . /' / ,
', A * " Analysis 'of / REA #/:///='?,;//%; /,..// '/\:/ '/,/>/;/:/64: - B* Analysis :0f -microsomal ..preparations;// :/. :;b5 ’
10 # Bpec t]?opho t ome trio est 0. " '/. ;/66 ■11 # - ' Protein-..e s t i m a t i o n s ; * ^ / ■'t /;'//■//.66 ^12 o Preparation-;of.;MAk;èolûms #/'/;; ., •13 c EucleaGe assaÿ-Q / . . - ; .,/ ' . -, ■ 68.
Materials ■ '■ '/,■• ■■■'■•'.'. / ■ ;■ ' ■ . ''. 1, : Ohomicals and ensîymes* . 702# . .The. .preeparatlon ' of bentonite # ■ 713». The prepa3?atlon;0.,f '(o<i'' "P) 'OTP# 71
A* The pj?epa3?ation of (3^P) OEP# 72B*. The preparation of UMP* 73; 0* The preparation of (cxL p) tfTP# . 75
Results ■ . : ' .rw «m » 'I A n«#iviw.Ttrt»73*
1> Preliminary investigations*; , ' ; ,782 b Further charaotor 1 satipp of the ■?
incorporation : of (oC^^P) D'TP * 80
R e s u l t s ( o o n t ^ d • )
gMNfcW Mil 4 w n
8 #
DiscussionDi
3 • . The Ineorporatiqh of. /If ■ ribbnuclooBide 5 ♦ "triphosphates ' Of: adomihe, guajilne, cytosine and urid:Uio. into pbXyriho-- nuolGOtides# . ' ; 87
4, The natiire of the hoiiibpolyrlhonucieotide' ' product * -, 895. Invéstlgation of the ; s^éGlflcity of'the.
reaction# ,• 946# Reinvestigation .of the taloroeoml . ■
location . of ■ the. j?e,actlon# . ■■ .957# The oxistenee; of homopblyrihoniàolèotides
in vivo# ■■ ' ' 98Nuclease activity of the microsomal
fraction# 99
1# The ohareicteristics of the'microsomal enzyme # : . . ,
- .A* The nature of the polyribonucleotides,
synthesised# . . , . 101• ' B* The. intracellular location of the
. engsyme # , 1092# Oomparison of the microsomal enjsyme with
other enzymes of polynucleotide hlosyntheelB# . Ill
/■ ■ ■■ ' ' ■ ■■5,* Reactionè interfering with, the microsomal iiioo3?poration of rihonuoleoside* .
--triphosphates. . , ' , ‘ . 1184, Bignifieanee,of the miorqeomal „
incorporation of rihonuoleotides into hombpolyrihonucleotidea # 122
5*# Conclusions # ' 129
Summary ■ ' ' ■ 132
B i b l i o g r a p h y 1 3 6— ■ — - — — X/.« ' . . .
■: : 7 '■ - --
T- ,
Kv;;
..r.',: ' - ï f ï . g ; s K,,-v. ■:,
I N T R O/D ÏÏ 0 T I 0 m: .:
à: combinatioix of blodhcmloWL»; eytoio'gioal. .and ■•• * . .geneticai s uudleo havo-Xead to the realloatlcm that two nuolqio..aoiciBÿ--ïïïfÀ- and IDEA aë, woil as protora are ooscmtial oomponeata of all llv:lag celle beeiaqe eziating singly la combinat ion with protein :m vlruaoe. The bloohomioal relationahlpa between these tîiroa- saaoroHioled'uloe are of fimdamental Importance In controlling the me.taboliom,' growth and reproduction of. làvâjxg oeXla# The-' claoaioal ■ demonetratlone (Avery, MoJjoocI .and Mcparty, 1944) that IWA la the ■ hereditary material/ Aya^Kohet^ o-. otoervationa that EWA la aoBOoiated with the. eyntheala of protolna, whloh .in turn 60 en^ykoB.’ initiate oeXXular. metabolic prooeBBea lead to the one. geno, one ' hypotheala of Beadle and Tatum*This hypotheëlB In expanded., .form. is the ’central- hypotheoio of molecular biology# Within euoh a fraraework tho médiation of : ENA in . tJ:ie; genetic Gkprooslon-of protein ayntheaifj is clieouBaed^ . . '
The two nucleic aoidB Hhâ and MÏA as found in natm.?e are high molecular weight p o l y m è r e although ohemloally similar they exhibit .cllfferont playaioal propertlea which reflect their different mode a of eyntheaio and their
different -bioXogioaX ;.•■• 'Ilydrolysia • of nucleio aoids/ ehemioaXly or ■ •
enzymic ally, baa allowed iclentification of the nucleotide a ■BMii bases.; wMoli- fbrm- the ■ repeating milts; of the polymer#The imolootltieB are i'-glycoaidoB ,of a nitrogenoi^B ' base and ■a'phoBphorylated; péhtdëe dUgâr# The Xàttër is 'D» ?ibo«- furanoBG IdîA ànd D-2-deoscyribofurmiose in DMA# The nitrôgenouB baeeo present in.REA are' the - purinea,' adenine- • and guemine* the pyrimidineb oytoaine anih uracil, the latter being "replaced In DBA'-.by thymine*. Bo sides, those commonly poGurr;lng hases trace amoimte ' of • methylated nuriHGD have been idoirbifiod, an both 11#. and 1)MA(Aider p Wolemman. and Gutman, 1958; • Bmm^ ' Smith and - Bpahhp I960; Dmm; 1959; 1961). ■■ In addition.g-mothyXOytoeine or !h4iydro]sy aothyl oytoaine replace some ■ or all of thé oytoùihe reuidwo 1%DMA from a variety of,8ourcoe*- (Wyatt^ 1950? Wyatt and 'Cohen/ 1952) # ■ -.Whilst pseudouridine: mid a 3%um))or of methylated purine dorlvatlvos obour in sMA* ■ ■ (Oolm^'I960;' IlaXX,- 196;;)# . . ./■ ■: ■ With the development; of ion exchange, and paper
- chromatography f/aa ‘XiothodS'for the separation of nuoleotlde .matures obtained on hydroXyala ' of nucleic aoidm^ it beoame possible; to''.'identify .-tW. mode of ;i:Wmge between the nucleotide monomers*, Thus^ IMA treated with snake venom
pWaphodlGatGZtmo'. gave a ..mixture - of rlboawclooôlde . '5^-:n%onophoGphaios which were mopafatod and ' Identztfiod '.•. '
.chromâtographioally*■- ;.(Oolm and Volkln». 1 9 5 5 ) > The ■
2 2' 3 '-vmo.lODBide, phosphate mixture obtained on alkaline ?. hydrolysis of Bffi. was elBiilarly sepa;/aied« \ Bncli •GXpelaments indloabod the'poeelhllity of a. or - \ ' -5 S 3’ phoBphôdlôBtor, Itoh:# • - ; The ; -probleBi was ’•’ rôsolyeû In, : ; .- -favora* of the latter'poBSuhility by hydrolysis of BIA with - Bploon.; phosphodlqaterase.'which- prociuOOB ■ a-- quantitative . •-- - yield of :3^~3auolqotldoD# /.Tlxla latter fact, qllmlnatee the poBBi'blllty of aiteraàtlhg gh, = 2 ,g hfh 9 '3 =..or 5 t>-3* j 3 ,731 I W m # ■ (Whitfald^ neppol mid 1955)'/ Inthe ;Oaee-of?D^A,'tho-"ihternuolootide-,linkage problem'was - . "more readily .soIWed, /Ihere being, no .2 -hydroxyl gjzonp; ,.B.tkilar-.expérimenta with'spoeiflc nucleasee have' lead to the Identification of either 3, or 5 - '^deozyrlbommleotides :m .quantitative yield from which • it " is, deduced that a . . 3^r*S’ phoephodiester iink is agean :Involved* (Oarber^ 19515 Koerner and Binsheimer» 1957) #. 8uoh. a structure la ; -supported by electromotriq - titration ezporiRieiits which show the hydroxyl- groups' df '-ùraoil •and---guaniîiê- are misubstitutèd, ' and .Upamztoation-: Gscperimente with IpIOg which - remove' .the amno groups of wytosine^ adenosme and guanine without depolymerlsing- the nucleic acid#;,//. . _ ;■? •■ ,
; // '././Apart from, oliemloal differenoes 'nBturally'Qocurrlrig ' •■-."PMA/aM M â iâifférn- An that the: latter/laB' -a .qug/-s.,one -ratio. % ..Of. purimes to ' pyrlmidliies* This.-causes .profound differences
, '/in,'tJie, .pliyèiôal-pfopertieB of %'hn two n a t u r a l l y ; ■'■■/;' mioleio::àolda#., ;?<C>-rây -analysis.':phowâ} that' 3)114 '•is a double.• stranded helioe/L; pqlykor»: the" 8 btWrnds beli//. held . ■■' ' -- ..together/by- hydrogon 'bonds,'bqtween'G#.0#- pairs, àûâ Â 4T4'• ’ ■■ paire on pppoBlUp/phaino# / .. Thé planes of adjacent '■.base .: pairs .are ''parallel ' to one another and perpondiGular to . -•:. -
the'long aisisVof :;thà. hqli iV' ' Thé sugar mid phosphate gsroups ■/■; -1 -:are';-butesmoot;' b;m- the- ’ûàckhWê'. ùf ; the 'moîôbulë-ï : • ; The.; ; ■•' lirbeznuôlèotide ..links are :: oppositely polàrieeâ in ..
.::pompl0menta%y .ohWnk^ , (Watson .mid;Oriçkÿ, 1955)# ' The . -.m'';; '/'■ validity, of. this-.: Btr%ybu#-/waû.::cohzGirmed Ullkino and . . ■. ' ;. 'oo-wopkerB.'ÿ'-.viMi;. were âlé0."abletoz:add some refinements/.in ,./•/. M t 0rpretetion/(BeG Wilkino/1956 f03?%a. review)/: : /3Wmy . - - ' ;■ 7 analyoie, of MIA = on thO' other ,;h#id Bhowo limited /double . ■, ' •'•;■;• ' ■ helical '•..régions. ' with a DMA .' like atraoture/. - '...(hich ;and ..../■■ / ,Wat'Bon$1954:•ç?vÿb/• 'Bubay -■and WilkiuPf/1960)//.,• .ShiB• io, due .: % 7 to ..hydrogen b(MdlBg-/hatweonh)aeo-paire.;'formed by/thé-. M A ./ ."ohoih : folding, .habit' bn itself #. - : Yeast has recently . ■
i . / V i i . 7 - i i . - e v / / : . : -; , .heen. oryBtallBGd whioh'.tîien-z'BhciîB ,.a;oloa5/-doid5le-helioaX - ;structure eimila.%z,;tp M A -(Bpehoor#..'3.962)# -' ■ The. precise ■
■' natufe of. the. folding of/.the. EÈA chain of .the hydrogen^■.;. bonding arrangementd?-oannot'he ;asoertainôd'from existing
X^ray. dmba.. ®oid:)lo s1n:mided native recently /
"boen chtainéci from, wound tumour and Roo vlmmes# ■• Tliepc ' qhow cllffraotlon patto^mb similar to DI# and 0IIE&* ‘ * - . - . ’(nangridge- and GOmatoe 9 -196$)'#- - The double : stranded • •. •BtOTotroeriB .oonfimod by -electroh' mrlerogz'âphs' of tlio • -fibroB'#' • ' i (iCloinsteridt- mid 3)minobaolm^ 1964) # .///-Similarly the replicative foz-m • of tlie ■ IMA vlrUBëB ' TI# ' and IE!2 , hàfj bèen demonstrated, to have a dbnbls ■Btrmuieti-'helieal /' configuration# • • • "(langzadgo i-i-Billeter# * Borst»- Burden - and -.. • •V/ëlBGüiàma> 1964) * ■ ïIowever ' difforenceB ozibt bctwoeii the doublehelical- ;otrubtm’ob of -OTA .and' BHA#.- It • id laioi-m• that actiuoEycin-J) o'omploxos ' w i t h e n d ' prevents tlio
A'GonforBational'Qliaagc on'loworlBg tlio huàiiidity of the fy;broB#'V ■. (Hémilton#; Fuller ■■and Holohy; 1963)/ ' Thus it apgearq ' that ' aotlnomyoln D binds only, with the B' oonfig/:- uratlon of the BNA. helijs# The ■ actinomyoin X) DMA oompless also shows.' increased Tm values' compared' with untreated JXhA*.; .In ,the case of 1MÀy "actinomyoim Ï) treated aMA? ■•• (which has been'shown to exist in- the A oonflgurEition) Ty oonvfplGiaent- àry .MAp or a hybrid of phage - o4M A ' and HEA show no . .difforoncc’ in Tm veiluoB compared \7ith untreated contrôla# - ., Eenoe .any hollqal régions in IMA are likely to be in the■ =A configuration#/; .'(î-îaselkom- -1965);# ; ' ' . - '
•■ She - ■Gxistonoe ' of- hydrogen-'- bonds ihr -maintainxiig-.tho '. aeccndaxy-structure in RI?A aiid DMA. is- confirmed by 'bhe ’ -,
■decreaBe■.in ’optical- rptatlon mid In .ultraviolet ... ,êfbBorpuion with, toiapemturo# ■ .Tiie. tranBrizions .aro. gradual ; , ; and. reversible ;m the Gape, of; DMA whilst with DliA feliby are ■ oMrp' and irrevefeiblo/' though in both oadeo the. Tm is .. proportional/to the G.O. oontent# .Tho latter oaoo : ’■repreoeiits the transition of à doiMle helioal .rod like - y - molecule to a .Single stranded’, rahdmnly/coileil molecule* ./In tho‘oase of BMÀ tlie .'tranBitibh ropfesehts.: tlie ohmigo, of . a:OOïïipact flessible/molecule/folded back on itself to fozm . helical. hydfqgen. hondod reglpnO to a: rmidiomly bqilOd ■ " ■molecule# . ghea,e oouclusions are . supported by .sedimentationcoefficient'and/ihtrinaio viscosity.! moasurmnente whioli ihdioate that in/ contrast to'-.thé;/figid structure "of XflA ■ present in solution MiA behaves .Eis . a flexible molecule . that gradually and . reversibly, c.hmiges its ehapo/ahd / . . .
' dimensions with .the ionic . strength ' of the splutiom#' ‘ " (Bee Bpinm? .1963 for a zeeviow)* The helical content of #2/
. forming hydrogen bonded regions lias boon’oBtimabed to '‘ involve 70' 80 'per cent " of/ the oha'in-' length*. ’ (Dbtyÿ 1961) .
Two broad divisions of cellula]? orgcmisatioii are . recognisedé . Irocaryotio cells/eopresentoci.-by haoteria and.
/ ' -'blue green algae and .euoaryotlc-.oelle* .’repreponted.by all- : other ;typoB ■ of pl&mt ■ and-.antear. 'oells-#'' : Both typôô oontalh ■'
.. a nucleuo ombodded :m /bho oytoDlasm- but b.o -nuolear mozabrano .:lo 'pfe,sont in the former group.!- • / -.u p.- ,'.•/,■-:■> • ■ ■. '
;•• : • The zmolous of;, the enoàryo.tio O0II; oontatos dlstiàùt■/;. bhromôBbmos- readily'■boob at/mitosis/and/pocaelcmallÿ "a. • ioed- a s Mbilod/Ilihieads.by; certain fixing'-proooMzreB#'- .••
Usually) y however^ /the densely -àtalzilng regiona-of the , . . '•■ -.• nucleuB fpi muig: tho ' ohfpmatl# and%ooiybai3#ng::thp D M A as y. : ,
' ; 'mîGléopmtelnyoan/ be-divlded into: 'Wo'.types;.; ■hQteroohromAIzân.j • ■•densely staining'' oondêneod .• material'• and. ouqhrpmatln which;
'. : is loss densely •.otalhing and- more/ diffuco.# lucleoll# • y ;;/ ' small" doheely; otàining■ sphes^lqal bodies: are. often aBsooiatod / -with .the chromatin» . ■ In-, oohtraatg• -mtolol of prooaiyotio■ pells ■■'appear .'tq-'' contain; a Bblid w b b b: of DMA fibres ohoW.; ;
no difforentiation into ohromooomeB and contain no / ■'■■' nuoleoli#' ; ' ■ ' / ■ ; ‘ ■ . ' ' : ■ • ' -'. - \!• : The-• oy*!;oplasm .of,ouqaiyotio'.-belle oontaino mmioroua ■
InolUB.ions 9 the/typo and nimbex■ deponcl^lg • on..the bpeoies ,’/of'•coll# ■ Most/common'asuong''the BO are mitochondria ): oiiiall.
; ///rods 03? filament8..between 0#S 8*0 \i -long otàlnlng with .greehÿ. lysopymOB and ohlprqplaBtÈg, tlie .latter, . /-; .
/ 'oocmrring-only;ân plant.,oeXlB/' : Buch incXuBlonB'a3?o '■: ;/;embédded--'in the .basophilic /groiind! BUbbtmioe • or .ergaatoplasm#
ExteiiBive bleotron-rniicroBCopy . of. many types• 'ôf..oelX. shpw •■ thé : oytoplasm: tq/W' : traversed': bÿ/a • three • iizuaeaielcmal -•■"••’• network of cavities Tby : b system - of oontzmuoua !
• 'membraviés#- (kaXaclë 1956 . ' Bsiall • electron • dense-; granules are • fbmiâ;-aB’s delated with the ;• outer - surf ado of :• the- /membranes#..' ;•■; Tîïobg’•'BtrvioturGS»- • whioli also ooour. free
•• ..:m the ■ cytoplasm g /QBpeciaXXjr aln timoûr ooXls.. are known,as ■ ..' - ribosomes • and the system of mOiiibfanes /and, ribosomes la ■. . knoym ps thq endbplaêmlq ! rotioulW# . Hot all the cytoplasmic. ;;mombranoB' ■ carryz^gnanalep»'/ ’/Rough' or smooth : sm^Çacod . , ' ' : .
'. ., membraneB .may‘ bo /disttogulahed /acoprdtog'to the. 'presence. /' • 'or. .absenoè ' of iribesomee#' •/ ; Mo • membranes appear , to be - ■ present in the eytopiasm: of / prooaryotlo cells though ' ; ribosomes Xmve. been .obtained from; such cells* ■ Bartloles' ' ;;, larger than the ribosome a - the - ehromtoplioros . analogous .to -ohloroblasts have .been identified* ÎT6 m'ItochoMs^ia/ • / -
1/ or vacùolèë appear .! to b^ proBonlr ;in; siuch oollBi' / • . ...." '•• ■ The/dis.tril:^ution of .nHolcio, aoidB'in ;intact-. cells .
. has he ell studied by ■ cytoXogioaX, toclmiqiie.B# ' Opecif lo ■ / . ; ■.: stains ShCh. as: the Feuglen ntain, ui’0Ugle;a.;,and Eosenheck^/""a. 1924} show .that ' IONA is ; entirely ..located within ■ the -rauolétis#’' - ‘ Baeio; dyes ouch as -toluidkae -hlhe' wlii . stains DMA 'mià ,ïMA ? ;the nmia-3?app0ïihelm stain g .à.mikturo of methyl green' (which :
: ataina..])MA.: gre.en) and-pyronin. (wMch/stainB JMA red) .-have .; •
• boom noüâ: i n . lo c a tin g - m iü lô iô , âlfeoS p tclna . sliotv.:'- / '
basopM .llë g:m ni&G a'ovo'r'# i0 \whole ' ('M:):l%w\'' '-TW'/nàmÿ/' à/Z - v '' '-
liâppon lie i# G ta in M b ?)©o:a. iisod l : a \ çanju n c tio n ' w 'itb B%o0 ... ; •
,. ' . ' f ^ z o d ç 'o i l l ' ; ' (iD-ils r o m o v o a ,û h o
;. 'pyron iù GtatoJsig - m tw ia l from tïip. e y t o ÿ X a t M r ’tte v ;.lo c a tin g
Mià‘ la tto/!cytûiÿla.oa-Guid ïMA.àhiHMoAuvÆemu • ''(/WapolKyby' ;... '
. 8 & ( ) l ï a r l y g X#%\may''bo..Üoï#m
nuolobluo à^%- Gm\logouü\-"G%porlYae5iyI;'' unlBB/DEaoo. pro A ... / / ’■
-... . -Woatmeùt; M e bhovm.-IWA .to %)o :lqoatoü /
'.(Kcm:5mnag -HcDo&mld-Wd iZagv . 1951)#'. ' '■'' ■•'■■■,./'■■ ■'■ ■''’ Z .'
::://' - " 'BooMoB' .ataM W gt' inioro-upùotropW^tômb^^^^^
•• Defôro ehck çÆtc-rê, l^arie /orf/D IaBo/tiw ^Auiont ' givoa- a .olm lXar •
;aia%2?lüiVkiqa' p:A aoM p; ÇOaVlüeom miû -Wpymoiruhp l'% G)
■.-:' Doth •riiothoClü-' Imve; boom ùbocI' ao.- tUo baelB' • o f /'a-; q u m itlta tiv e ' •
'''éG tiœ tlom o f / t W 'U Z ltxw lo ïe t .Diaioroy.' Z'Z "
■■ ' ; npeotropM iioM try-. in^ïasuyca ■ tlio t o t a l ’ an^omit o f ■ ïMA
' / m :iâ -fro o 'M o lo o tM o a . W /m e ü In .ocmjunotîlovi v /ith #.o-■■■■. ■ ■ ■ ■■. ' z ■ . ' . ■..: .- z^ - - , -, . ., . ;■ ■ /
'raaotlpnu- MTaoq (UaepovBpap'
X94Û2 X947)u .■,;-.:MovO” 0pecrlïrop]aotaz4d.t2;9‘ o iZFougM i'o ta iB ocl
nuo le i/hae &O.XoTmü: the o e tlm M lo a /u f
imolôua# (c lx 'a lg o i i û .Ria'g 'ZlSPl?) • . D oX liâ to rÿ --'1955 ) » '
- ' mothoiie 0how 'thiVü''#ze-aw i#ü 'o:W3)I# 'T)e% '.:'oolX'.:.: / \'.: '. .. ' - ' : ' / Z p / .- : ' ; - . ' / . . ' ^ -
- ■ ' o f its-', ahro/iopomo bompXpmcmti- ■ .; -Time 'iiip lo iil. ' m X l n ' çonta lu-
. 'ddüblG tW c o llo ^ tbtrcrqXold'G olXe
1 0 ^
double ' theamamib ' 'in diploid oolla; and so on; ■ /Slio amount of JBA' in diploid'• bôllè ' of - amy-- given opopios is ô l i a r a c t e r l è t i C o f t h a t / s p o q i o s # • ( iC iG U c h t m ib o r g o r » ' "
Daûohteâherger)/■yeiidrey ‘and Yoiiitcejf 1952) * The cellular ENA Content however# 'tonds to'Vary from tiscme to tissue . àml;.dépends on- the 'nutritional state of the .organism# (Oaldwell mid Hinahelwqod^ 1950) i - .■■■'■''■
The distribution md. location of moleic acids lias also been • aided by the development of ••tocîmiquéè" allying, the separation of.cell components# ■ Claude g (1943; 1946)first' used differential centhifugatioh of lâver homogenised in'-'0*88 M Buonooe#- Ueirkrifugauloii at 600.z g yielded- a'nuclear' fraction'at 5)000 z g a' mitbohondzezial/fractlcm . and at 25 & 000 z g* a mioroBomal fraction# ;• The ‘tqolmiq.ue has Binoe been refined using; higher gravitational fields. (Hogeboom# Bolmoider and"'3?alaüê 1947y 1940)* BubfracW ■ ionation of the mltoohohcirial and mloroaomal fractions has also been achieved* lyso^iomes liave 'been separated f rom ' the mitochondria (do Dwe^ 1959)? and' ribosomee have ' been. purified from tM; micros omal.' fraction after ■solubilising tlie Hiem )rane component I' /Lth dcozycholate* ' (Petermamx and Hamilton#, 1961) # Similarly,' ribosoitee 'have boen isolated from the nude us (Rogoy^'àl*., 1962)> from the nucleolus (Birnstiel,. OhipohaBo and hydO) 1963)
1 1 / ^ '
and from oIi.lorop3.BGte # , (Olark, gathowa and. Ralph* '.'1964) i\. ii ootreon zmioroBOopie ètûdlè.© limè Jiqeu piiaceasfiil
'In qorrolàting the üëllular featuros. ohoorvotl by oytological Btalning proceciuroB with, the etruoturqe obaerved in eleotroir mlcrograpM and .with the siibcellular particles obtained on disruption end differential oontrift^gaticm of cell homog-=? . enatep* Tims the endonlasmie reticulum Boon In oloctroM micrographs corresponds to the basopliillc regions of.the cytoplasm# ' 'Similarly# ' .eleotroxwBîlçrographs of the. micros omal fraction show this to consist of ffagrmnbe of the endopiasmio retiouinm# 'tme- mi(i attached ribosomes, (Palade and Biokeyits# 1956 a#b), ; Analysis of the B'ObGollalar fractions has revealed the, pattern of distrlb-? utlon of the imcleio acid in relation to cell Btraotures* Cell fractionation expérimenta hàfo confirmed tlmt DMA is almost entirely Gonfinecl to the nuoleua and^tlaat the average amomit per cell la constant for the somatic cells of miy given fjpecloe, They have also shown that BIA occurs mainly în the o^toxilaemic. rlboaoraea and soluble fraction of the cytoplasm,, though smaAl araomta poour in the nuoloua, e.Bpeciaiiy in the mioleolùs and nuclear ribosomes.
It is now possible to obtain native BMà in an tmdegra( state largely ae a result of the introduction of phenol as - a deproteinising agent (IClrby, 1956) and the use of nuclease
V- , , ■' V',., ■ , ; /-■':■ ;■/■
Inhibitors during' 'the -oxtraotion procoSB'. Analysia ■. of ' such material; by ■''ultraqentrizSugàtioii h m leàcl to the :'identifioation of three.oompomento having sedimentation doeffioiente of 8$BV. 17S and 4B from bacterial pouf cob
... mid ■•30 2BB? IBS and 48 from plant and. animal. ooiif ces * . . , These Bpeoiee of. -EKA.! have molooulm%' weights 1.1 - 1*4 tc 10 ; 0#5..' ■?* 0,7:'% -10 end 2,5 r 3*0. % ’lQv respectively and \ -
ZoorroBpond to the/ REA 'in 508 $ ' 308. ribOBomes, and the, soluble. • •' of •mlBedamentabXo.-. fraotioh of the 'oell,; /' - Spifln, (1953) /■ ./ ■ . has • determined the 8 values of a variety , of rihoBoml IMAs• -under.iclehtioal -oonditloha 'e;dd calculated, thoif’moXeo-ular-. weights, -.It appears that the. total Mlk from a variety of ,badtofial 70S- rlboaomeO M e a molecular;Z?eight of .1*7-% 10
-Z: though BuMuhits show opho ideràbïo .variation - in molecular, .'•.weight, ■ Thie Buggeeta that .thb'/fiboBomal M A may be
Z-.:'formed,as one imit and then divided unto smaller .units#..• The species of IMA described above bIiow no -Z / ' •
- ydiàëçolâtion 'Oh;heating or. dialyBis to remove, metal ions' ;. though earlier prëparationB did, probably- due. to .coMamifZ ;.
-' i atioh wlth-moleaBCBi . . '(Bpirin,# 1961; z-Bogdanova? Gw.rilova?: X)voxkin.ÿ, KiaBolov and Bpirin> 'X9S2)> , Electron'micrographs,• 'Of the: high moleoular'.weight' •probaratidhB of ÈMA show long. .
- : - / n z : . " Z ' ; ■• ■ . . , . ' /oontmuous threads ? in, some .caeee ‘up to 18,000 A long
-, : GorrcBpondiBg to the•;éôntimxoÜB yxtonded ohain' of. the ,238z .
IllA*- ■ • •(kiSBeloT^;-OavriXova. and ,%irlnp. 1963.| Kioller and ■• Boodtker# 1962) Z- Eonco 'the .‘oollula?? XMA, appoara. to. be a - oontimuôùs. mibranohecl. pplyiluolootido ohain* The - chainlehgtinDoing 4000 4500# ' 1500 - 2000 and 80 -100 rauoXootidesfor -thè-. ttoao.-0p0oies .pf'.ÏIMA*.* Z .
Ribosomal î®A'forÉs.'.??- '85 per cent of the total*cellular BMAg sBlA 10 - 20 r>or cent and a- rapidly labelled fraction, known as. .mecaenger IMA having a DMA like base ratio# •' 1, 2 per,cent of the 'total* The latter finaction ishoterogoneoxia In-molecular weight*. • . S . vaXuêa of 10 508toaV0 toeea reooraed 1b laoMaeioM.^ ooll.jai.a up lo mmmmlian celle* , In --.général/the haee qpmpooltion of EM, is •■ different ’ from .DMA • except, for the' mesGeuger Hlà fraction* The "base ratios /of total- cellular. IMA _are nsimlly eimllas? for EM. from different tièeuos of’.the eame apeciee* (Kit#.
' *;. 0MhA , phowB a strict one s one ratio of purinee to ' pyrimdlneo and thas is. of ten#, though''not nooonsarlly the 'case with.rlbosômal- OTA' (ooq Magaéanlk# 1930#.and' Brown# • 1965# for reviews)# AhalyBio/of 2 5 8 and 168 rihosorml MA. from a variety .of bacteria • shèwod little différence in base oompoeition# ■ (Miclgloy# 1 9 6 2 ) # . however# hotorogéneityof the baae Sequence in. fibocomaX ENA is. indicated by fractionation of partially dogradcd.prepamtlono having Bodlmentation ooeffioientB. of .2*3 48 by ’ comtarcurrenfc
, ÿ : : . . ■■■ ^ v . v V ; /
■diatri'bU'iîxoB; -lato .fïï?ac1;lonB5 of. dfffà^^eht.} basO', oompo^ltlom# -, \'- . liClrby : 1962):# ■ :. Bvoa - Tfhon.' leoo /dogradoâ iiKA; fromfrat llver emâ .D3?è,B0j)ji3.;la-has ’'-been- ptucl5-êavtliero lë. .eiridènce of both - iïïtër 'aiict•• lmtrampWoü%rheforb^oneity.-,of-; the :emgbr Bp'eci.eB"- / "■ of: the* - total; (Kirbyg LHabtfagB a M O^.Ballf^’au^ .1962). ’ ' \
Imo l)èeiï eltenBlvoly fsmotloiiatGd by Goimtcar^ . . .
diët^^ibütloh*(A;pgafÿ Eolleÿ &M: Meiwllÿ* ':1962)- fmtâ: epoolfic" omiiio -aoiü sBMAb- -hâvei-beem; 'Obtained-’ vrtiich ehoiv...différent.: ':..... ' baào o5iapoBitio:aBë;a -(Sîâda’ ami faêa^ 1962) * ■ . :
• '■ •• ■ ■ "(Che-:moloonl'àr::'Wight*: of; DMA • i|i gs^'rpkarlne;.mioerbaln ' - ‘booaufiè--:-of-■ tto eaee wftli-ydii li'.elioar - degradation' OcàurD and . ; .. the diffionlty la àoceoBlng ito Biso by the methods .àu.^rentiy• availablei - - Ohro^œtogfaghio^ -autofadiograplilo; • ■• -; aiicl/' électroBXiiiorographla''- tooMfcp^eôâmdioate% maXecular •■ ' . '
weights- of more■■■ than 10^ for'the'..best!:p±epaz*ationB.#. : It '/. appoarB' that the DMA isolated from.' Sg'-baoterlppMge.--of' ; " moleqtClar- weight' 12- %- -.10/ ip oirffioieat to aoootmt - for . ■• - tile.' total DM/' of the- bacteriophBge -partieXe. whloh iff almost•,-• . certaihly a''‘Biaag3,o/mol0Ouléf ■ (De;v’iDOîiÿ■ IréifoMerj,fHode_ ' f ' .
and ;feyintlml$ •'•1901 f ÿ BemB- and-.Shomaef.;.,;1961);♦....•• %he DMA . . ,.;may also o,%niat as' a-.Binglo.oaolooüXe in, bacteria#: ' , Shio. .is- •. .’ /Buggested by; the..: aiagle' olrcnl a ? 'ohfpmosomo ob#rmd,.,l%i I . - ■.eleotrbnM.crographa ; of/DMA extracted.: f r qm B# ■ d^li (Oalrns, " / .; 196g.) # : .:>’B.imilar • Irigh.-molepnlar '\;eight''ï)fêpâratiohG <of:-DIA .■.
îiaTO. been reportod'.-freosî oourceof'--••.•• • (Dat*:lBon» , .
" ’ 'Heterogeneity of the baso- .tioiapopition of DMA. liasbooh shorn ■ .by--, fraetionatloh-’ on 'MML-oqlimns . (Oheiig- and . ■ f . • Bneokay 1 9 6 2 ^ - Btioofe cmcl-.Oheng» 1 9 6 'i? )> and b y . odmiuor^ ouvrent distribution-. (Eldsdn and Kirby-f 1 9 S 5 ) * -, Bince\it 'is not.' known: .who tiler' •• miclegraded DMA - is pro sont 'differoncos' in ' base composition 'Can , rofloot both differences between . ••;Individual - mole onlas and: diffeÿenoea in nucleotide- seqnenoeof; : OeOl density gradient■ oent3?if.ugatlon of - DBA,, however^, from most vprtebrato speoios .gives a single bandg thou^ÿï .-a .minor - or.'- satellite band has been detootcd-±*i several instances> . :tn mouse • and guinea pig DBA (Kit., 1 9 6 2 ) f . - fîiepQ may .be due to tliç mltociiondrial DEA rqoently Identi*^ fled in chlch • embryos' mid '{algae (Bass and Mass» 1965 a,b)#; :. üüiG eatolllto band'- appearing. on .• centrifugation of ■ crab • ■ ')))& - on' OsOX 'density • gradients, has been Identified fes- the/ ; naturally/occurring po.ly. -dAS oopolyme:u (Buéoka mid Ghm%, ’ :
.. 3#. . in vivo Btudles Oh IMA Motabollsm# ' ■ -BDA 'Oan be labelled both • i n maxmiallàn systems and
mioro-^o%'g6mismS by small -moleoular pracursdrs such as orthophosphate $ - mmonium .citrate glycine^ ' ■ '
';i40 * f ormatC'i- ^^0 purines-<or -^'6. pyrimidines (bgC .BiaelXie5■■ 1955p. for a review)#- . .. fhe pathvrnys of purine rlboolde ■ and pyrimid:lne ■ rlbooide blOByiitliosiB ■ are now voatàblxBlxod frôiïL:work' on ■botli’mcrdiçrgànxBïïîs mid■'ajaisml/ayotéma*• •.. Dniâjrme'-'eystème"àrê' also'- Imovm-- for 'Into3?convorelono ••'among oaoh of the/naturally oocmrtog'purine'Vand-pyrimicilno liüoléôtldea^ their'phosphorylation, to miolooBido di and triphOBphatoB and- their reduction: to de cscyrihohuo loot idea# The details of / euoh pathvmyd are without' theOaopo' of this theaiB (oeo Buohaiiaiiÿ ‘ I960 and OroBbie^ i960- :foz\,revievm)#
jionBhl^ h,è,ty/qen BpeGleB of EEA»..ÉumorouB attempts have been aiade to demonstrate
turnover of total B l à in mammallmi o c I I b and precuraor produot releitionships between individual spociea of HIIA* Qaro muet be taken in the interpretation of m vivo ozperimentB and several different ei cpoz'imental appzeoaohes are x eciuired to give a satiafaotory inclicsition of the :Ultmoellula:e events# •
ÏÏO show the metabolio stahility of EBA Is cells have been totally labelled with 1? ox’thô;phOBpiiate and then trauBferred to mi unlabelled medium. . In the following two generations 20 30 per cent of the labelling was lostfrom the HHA and none from DMA. Ho further lose of label from PJIA, occurred ill the next five geheratloha, (Bimiiiovitoli
.mid v 1956 ; ; Qvraham. aiid Bliâlnovltoh# : 1957).# In ;a.i sMilar;. es^perlmeat kxBdites timour oells grovm vivo . // ;. and totally:..labelled v/lth. glyeliio y of .. Gdealiie wore ' -tmnsferred':to. x;ailabelled mice# • Ddrihg'. tlio subsecmeat two/generati&ie-yO--per cent of tliô label wasMoet -from ■ '•BEA. a M - none' .nn the Buoooedlng generations # (EovesB,/' ■ ■ ■BprsBbërg and Elein, 1956 )'#■•■:,-BeoldGs IMiositlng me tab olio ■stability-of'y-fraction of/thé-'retentions of fabel-■ • .■oouM also, bo due. to efficient roinoorporatibn of mono- ; . .rmcléotMe lireakdomi pfoduoto# "npwever^^ a more detailed ■;. .re-^essa^nation: of•->bhe above: e^cporlmohta showed. that radio» .. . activity was conserved In: the: purines of Mâÿ that there was ho ■;change-■:in tl:ie ratio- of.r^'*'0 ■ adeniîio/riO' gnonino and i that HOtransfer'nf; radioaotiyity'. to'/DIA.- ooonrradj;/ • wteroaB : . .during. tlxe zlnitial: two. -generations af ter: :traiiofor wimh : " - ■ ■ ',30:‘;pef-bGht: of \ thb ; was d.egra#d''.tlfLs ratio showed a ■• î:\oonsideiablo: dooroaaoy,/;(Boott and faft# 1950g iloottf faft ' ' and. Ii3tbi2r;noaa>; '-1962) #-/■ -.. In.'osspefimenta.. of ■ a'- differont- . ' :type the: relative .'rate o. of è ino or nb fat ion ' of. preonradrs . into IMA .and:3)ÈA have iDodn deteimlhed in iapidly .growing ' Eolà.'CëïlSè.i;!,' #here 'ioio<msistehtly'more rapid' iiioorporation into DMA 'basés thWn DMA; baseo .suggesting a sighiflomit ' , .turnover ■ of IMA- under .conditions ' of. ceUular Haltiplioation*. (8al0m.ang' îilagie';àhd, Bebring#/ 1958; ■ .Balsman'and Bobring,' 1959)
S'.. ;s\ ,'-'n ' s / ' , s ,
’ /'Bimilar'HMA tmria<wor* haB: beon'bhôwLi to..oooiir .In non growing - ■ ■ ceXXs.*.. .Babbit ' saapropliagob:.which show no lue oreporatlord‘ of:
o . '• " '1 . - - ' . i f o i o / . ' *: " ' " - a i : " Ô - i,:yç thymidine dnto DMAs mid .bo nxrh synthpoie . of. IMà/did, - .' : however, show. :moorporatloB/of ■‘'‘'0 -adeBine-darîid.MA iidiiloh \ . / ...- was; IJmear with, time. ; ,.;fraaofer to. a laodlim oOBtainiBg . ; '. labelled udonlBo..:resuited:jln..à.'dGGreaoo am'; the” labelling ■-■ of,, the , BMA. indioating /that, .ahoiit - 5 per oent wob ooiatiBiiouBly - ' . ;bhmiiBg::;ov0r4 (Vfatte and .Hârriçy 1 .. ;.//'.. .. • s ;
. /... %ho klBetiôO/.of .BHA.:labe:.üiiig hâve .boeii. studied in detail ."with-. chilturèa' .'of/mawàlla&i •and plant oollo. uBingr.-
. ,- /auto:î?adiographiqsp3/o0edin;éB#:''. ? In./,général',,B'uoh otudieB /, ■■/: phpvf, incorporât ion :0f . radlpàètiye/cpreour^ .iriitially. to;.
. . the imolow:' trims : lag--in.stho/; cytoplaem#-'".-.'ttoo lag:variosiooBDicierably from dell-to coll *bht_ is-. • •/ ...
■■/."; roughly proportlbiial to 'too length of-,the/, Gell/eyole#: /".:./ After toitial. labelltog-mid trtoBfer'.to,:.a BOB' radioactive' .‘ smedtom the amoimt. .of label in, the BUoleuG often c ont tones to . . tocreaBO.ospooially-lïi.'growtogipollé where, it is usually s ■
. Bot' poBBihle to. wash out. oh. dilute; .labelled imoleotidoO ; ::. with;mlabe;iyey .BucleoBidQB/Bxxffioiontlfvrapidly#. . ....
Hell.-and Errera 19(51 j Perryj,,'..dtorora> .Hell. anCl Drnvmldv. igGiy/ PrôBoottiV.19G8.)\• '.Afte:P:furth(3r-.elhqi&atioB/to ah"
:. uhlabelled mediwa' nuclear.laboiltog decroaaeB. and. .:1b often-../: paralleled .hy cm/ tooreaBO' to labolltog in the oyioplasm# ;
f // y y/-
y S Earrlép (1959) hqxvGVer» foimcr./bliat thq mioloar. lose was - v;,/': not/great -Gilpugh to saao'DtMtifor ' toe^'oytoplàSïMcs'gain^ 'and/' ' that the; imcloar/fraotion wae turning over ' Independently#
■ '/;//: further, - experlmoùts ' vf 1th lieiy oélls/buggeated thàt at least ' ,
' / /; 3 different frdotlons ttitor pvo5X at différent ratea* ./'/;' (W#to^; 1964/a,b)#/.// f'vay':' -'/i - /- ' i.: / - - /% -, ' - %/: '
yMore detailed/àntohàdlographip - otûdleà involving /■ ' eliort penipdatof 'todxdjotlon .wlto à labelled ■pvGOurDOrj
■ rernqval /of :tl%e-r labelled' precursor and ite replacement by './)y unlabêlled -prepdz'sor# Ci#.e#;/pulao. and;oliaee, .œporliaentB) . y ' havà;: provided:: etldenbé of ; the: sequence of''ïabèlltog of : : mioleaf. IMAs $ mid ' of the relationships of theee tp/ oyto-
'/:t ' piaMic BEAe*: /: Autoradlogranliio at miles of the ■ Incorporationb y ' y-ii- ' .y - /y*. / of .% oytldlnp ' and adenoalnc into the llËà o'Z llela cello v'-'/Mhow: that'' the chromatin ■ end/micloolav ' regions • i:a the
vmoleuB -are. 'bqth rapidly laballedis/wMlcy the cytoplasm. Is ■ more slowly labelled# ■ Irradiation/of • the - nuclo pine. by. a ./ .
' /microbeam of ultraviolet.light provents incorporation '■.:/■..,'Into the imcleoluB'toü roduGGB cytoplasmic' incorporation-:y '
' by:'YO/per' cent# ■•■ "Muoloar inco3?poration oùtëide :the/ : - './;/' nucleolus/iB'feducedySo: per cent after 1; hour but la not , •: ' affected initially* / Blimlar.- ifl/hmvlolcvt irradiation- o f \ .
the ohrommuto'doGB, not ' affectvthe nucleolar .or cytoplaemia / '//labelling#' "(perry ËÈl&l ISGly .a)*./: ' Using, the/same ■,:;///._' ■ ByetoM^'tiio/ihaorppratlmi of : imcleoaldeo was shoim to oGOUr
. ' &aa0Txmûo'îTü2,;f g \oÀ:mam#:a riTxl siiicKIooÎHO.:', SuxIïîs' ■ .0, oîKOOyO soi? q g # a . #lq: ,3 # )a TM#:ïiWü :ayüo y%xs o#CM: : ,
' pl.QS/.;r f>w:3 , oyOp&GÜOMgX)'' àWOS POA ' Ôb/h '/S - -'-- ■
■■ : muoleai? l# e : /À&Bÿâ% g:o a # . & % ' . / ■ , -. .
• ' : h i g/Ei? (3;ÿücs%2a' mui aûi/C'm%;ÿ "Oo iiotsi/Zo y / l i :
i, i ' y.63/$ : l a / a g y i t m ü ÿ BmïïxSa&b'
y .. -oaü- v .^x w o /e ' '&fex?o g,8 G onp/y .(igmoDoiit ; ,
; , ' - ' . w l//..:;/» î»æ3.Goyp c M l ' o ü y s a t â i j ^ ' ï y ï ' / o i X 3 : l , l ô d .
' :j g (#@3# cKtiF %/là#ÿ\&!3ôp5 Mya.o/?oppo??§- ' ' -' //'
: ..; ' . î / ; io 'yî3lg.0Q/- ul¥y''yp?'vîK?s? ü e ta /io tl. -
' .:.; ;:',,&3æ/Mo,;Gsit:yyia.;OSl. y o y)l(% i#àb;.I'po ï)o#om% tà.CKSQp:?.ono;.
i', ; #0, ovo /:c /i /.iî/o z ? /# is/ion A a j i o i j .
’ ■ g08#:ll')lQ#. ;O2%go3?:lmo:K0:o'.ï.K)mi;:.l -G-':
' -' GuosQ 'üomi to G:'t mioIWo'i? i l ï ï k ' c i i ü ts tm o ï/i? '
' :■,' o:S à ,i?;ïaot5xs:?, ,to , tis ) - cïM - a ,3à'r tSzS; Wozi of- tiso -,
. -,': 3ttttos? at'ti/feoaft' ' Z/giiOsaoitlw/ o;t|y&';:yi;ia:iü imcClt-çii&g o-
■ 'tba po?:/3.feî/gÆÿ :o5? '/îcilbposiGost oyop^Bfm lo ù y i M x 3 G i > é - ( y - ? < y ' .
■ ■ poDplblo DPiog. to là fiaoeSDOoiy ûsï'ï/ioX c:go#;:!oi3tü,
; ' ': â'i!î,)âg«:l'ÿ: âo IgysJÿ- to c,y,so boCKtioo of/întoyQSvoWBtonD , ■
■■■■' 'i;;-feO'&vGitA,ty:mgso.' c®y («soisîoa oj/oM t/io /ù l - . '
- ; d/SêH.ou3;6;/ 3b é / s b i . O ' v A u g i ÿ O ÿ M ' û ' ^ ^ ^ i È i y a tho iî.ç:io3?oo'ü3.üo ' -
I , ;bgoo3/ .f/toB 'p t& oo: 3zM3o%I0i){t-Gt:p8&à;t%#èg . ::;■-
inhibit Ion 5 diffes/onoée-inyiie/mielear •;'./;ppolo''sises'; toy tesmdnal/labelling/of bDEA#/' /, . .. : / i:‘; ;■;■ ; "-.Èarthor :-évidm%de toetaboîio ' différèades. 'bèweèn;. y différent'IMA"fraotiôj:ië;.Mo;'oôï}K5 from 'e:a:pea?imento on thé
.:''''':fràétidh tiohy\ùf' puleo - lahelloâiçellsÿ','. '/Initial oxporlmohto With puisé laheliod \:mioïoi todléàted/ tho ps/esènoo; of . ■ , ,
'//édŸo'Vàltÿp'0%"'of;IMA''.tiiat.differ ih/e^AradtahiXlty;mid; , ' \..metabolio;-activity* ' / SitoGifractiomtion df ràt Ilivar nuGllei . : ■'laballed ■'iti'- vtoo/'Bhowed; the I#A .of/:,thé'' WôléoXar '-chromoBomal /'oomplek to ho mocrb ixeavil ^ iabollod followed to order hy '-'iowtooîodulàrhvéight'!l#A fs/qm thé hhcXear ,the'molear'/'mioro (56.V000 ':3e 'g po5/% 1 hour )' - and the huoléar ultra-.'/\ài '///yV: h-:\/':V/ Wa///i ./.-:/: (-/'i i/ :. '• / ïïiicrbéomëê : (XO5t GO0y^ 5 fùr.-,2 -hourB) ./y - Ail thèae ■ fraotiçuq ,' werè ' much ; moré 'heavily,' làhëllêd, ■ tïmn the ' oytoplasialo BEA*y (GoorgléV' àiid. /. '-\/Euither aimlyeiD - of the ■.■ huoJDotoxv chromoBôml oomnleim qhoWcl it to ooataia .thrôe '' dl0ttoot opèqiës of..'I#A*/ . .Kreqtimmt ivith/1,0 H ÉaOl .. - removed heax/Ly ' àll the: DMA and ..oomë ,'aesoàiatGd: IMA# ' .,'. KroEitmôàt of . thé roBldue ' ih : dllute; Bhlt édlutidh With/'phenol/released'- abdiit ‘75 per Oént lof•■• in':tho' ' -■■ aauëbue / h h a o o ' '' fhia. was.; termed '■ nuéleolar . and had- Eh high/0Cy content'Bhowi% Bimllaritioe to' rihosoaaal Bl#* .: too resldhaX 1#A appearing at 'the. .plxonoll water interphaao' w'àd'.aléo isolâtëd and termed-■moleolar. ïï-HMA* , . tois had . ■
a baèe ratio ; ' veuerobling DMA emâ ton. ttoee; tto;- Bpecif le y - ' aot.lTlty/pf HuQ.;ieoIàr, I.-JIM;# With short periods of ..- ; '.. labelling M A . aos.pplatod wlth.:/DM/ to/'the, XM EaOl osstraots ... hact still Mglior spe c If Ic activity * - ”(S:lbltanide KXoet ,•■/' ‘ ‘ ■'■■■ ■ . " y.' ' g " ■ ‘ ’' Allfroy ; andJîirsky % ,1962 Ife:pôrtoeritq /Ui-- which .the,.- total W A was,'GXtraoted-using' cietergenta from miolei 'of. puXoe labollod rat: Xlver; and: sepmmted by querose density ■gradient. ■ qenttofugation - ahoWed ' a polydlaperae, rapldlly . labelled MA. :V/itîi.' sedlmcmtatlôn coofflGlonto/from 6 308*"toe 20 ^ 30Bi IBB rtooaomBl/ BlA and'4B tranafor EE& wereimlabell#*' * (Biattp. ISGB-X % / l y... : ' ' / ; -,
• .Htoy o%perjj#htB have ahovm tîaat when, oytoplaamlc •fractions or whole collâ' âré extracted;.with, phonoD, in the .: : ; -y- " ' ■ \ % ■ ' ,; •' -y/'-- . - / . .' 'ahaenco o:f liotprgenta, l®â ip, .ôbtaihed/'that. pan - he ahovm .,by auoroae.denalty. gradient, opntrlfugation tO; oonaipt of '■ 30S? 1 8 9 and '.48 o.qmponent.a -.givtog .Virtaally conigletc : ' ■séparation-from any. rapidly labelled DMA# (Glhitanl, . • lamana^ ..Elmira and ,0kagaki-^i'1959^. ' Sibltani,, Yainaaa:# ... \ y ■/ Eimura and %akahaPhl’ 196 0 ;. Jimmn and : Blbitani, I960 ;• Barris and W’àtta ,•• 1962) * . ■ When the extraction." is performod ■■ with.-phenol/dont aiiiing: detergents ; • or.-with' hot phenol all./ ' .• ..species of Thnoloar /#'& are extraotod# Using this . ■ //. .• teahniguo followed by -Oeirurif.ûgation ,ln,pncfoce.l density / ■ gradients labelled EMâ; from Hola : cells/inqubated 'with ■ - . -
2 3 / -
: ■ uridine hoûte- 'ehmvs.' .ÿôlydl'spê'^G ' ' labelling rlii •• the/: ■■•; 10 308. ■region.' tmci 'additional .lcd.)oiloci peaks/ .of • 558 àaid
' ■' \ / 1 4 5 8 # / . i m . ' / d e f i h l t ë t o . d i s t r i b u t i o n .
- ' /'• ■ of : ' radioactivity ■ and /material, •aba.orbtog • in/the ultra-'■ -// violet: at ’ 260imu' at, Biioh'ohort: ttoe, totqrvaZls#/ (OoheiTor ,/y:-/tod/totoellj ■:1962')\ //■.//./.■■?, . /// /y ; ■•/.■■■■ ■ ■ ■ .• 1..' ’... ,-■ . Xhua. 4: fraotionB/.of' IIMA have been clearly- reoogaised,:., ■;/■'. "though btller::ftootiQh8./,ëre/:'nôt? excluded; - .. .(a)'-diHA' to the
' n u o l e a r oÿtoplaomid -rto with /sedimohtatlon; . obeffiolentëi■ gt-i 508 .imd" 108 (25 and -168 to .bacteria) # • •
, ■'- ' ':.'(b)v;a 48 _ bompoheiit^ ''both/to nucleus' tod* cybpplasm,:■ ' ' '•/ ' ■ ' (o) MâlBSBOGiatGd'with the* rauOleblar/chromosoiaal-'complex :: : tltot/iiaB-.a/hiëh/CI'i.Ov hotoGUtvato: to" eli'ailto.'hto./rihosornai'
■ ■.•.' ' B%Av/{dyto" mbôndxïMA aèàoclatèd/with ..the/imoldôlàr- -y-/ . •' ohtotoBotol"-botolto/itot:haato'‘baPeyrati0”yw>proxtoattog to ':'./y.-:’\ that'/'çf//PMAt iwfeiTed* to to i(c) tod; (d) 'are'' ■ • rapidly labéllocl' •àpqoieB*-' a:acl.-. oôrrèospond ■ to • the, rapidly. .. .
':/., ’. /' .sedtoehttog .-peaks.*, tod .;the\ polydiopGrqe' 'labelltog ' seen to ’ ■ '... Oucrapd: graditote# ' ■ DosBtoly a • fifth type, of IMA exists ,/, ‘ which. is ■ closely assoqiated. With .the chromâtto# » • fhe . :;"'/' '.-'.■itoetofelatlohshipB' hetwoto .those;‘species- .of BE& have ,. i."’/ /yheon todioatod/Dy/pulsqvltoolitog experimehte .followed ../-.', ' /yW'- trtosf er./to./to- imlabelled médium to . the pfestooe and-->;f'-y toeenoe/of.:aottoa3^otoMv • 'y é; r? ,■.-■/, /.
— - /'Pulse •Deen-extraotod-- from ;-=Hela ;oeXls and , aiialyood on énoroso .densi'!;y gradients -. / . ’ shows 358, and ,458 Gbmponehtèyof 'rapidly'dabellocl-UMâ*' :/■■■•Xf tb.0'”:cellB, wore; pretoGubateâ in .10 • Ii aotinoraybin .3) .'the , lahoXl:lng';in' M iob.o ' neëkê" was. ôonsidèrahXy •. reduced ; '•; though • labelling'•=i:p;- the'"-10 308 - region was ; imaf feat eel#.-’.In stollar .ezpoitoento i.n which dncorDox’ation, of :<il: ribontolQOB:idoB''WEB.,oboorved'acLtoradlographioally^'tho ' ' ,mamp .Eietii/nomyoln. D: treatment shows' - that moloolar / ■• . • .labelling -is roduoed without. affooting the extent'Af jfaholling in ■ the/ ohromatin*^ nurthesMoro g ; phenol I ■ ' -oxtraotiun of .theIpxilSG ItoeXled/MlI after a 4 hour.
showed:the,508. aY# 18S 'o(mcp6IîOIri/B :haâ■ .become:' labelled*; = • ' khio.. lab.elltog/ of ytobpsmml/%#&-* was- not / : /v " qbsértœd-•■•iîÆ . 'açtinbmyçih 53 xmà./préséht-' during'' .;tliô:yxhit:lâl'/'-'./ . puisé: .though it Mid oooun if aotimmyoto .D .was .aMpa., ■ ;, -/ . during the/^èhaeef 1 ' .:ShUB./-#'%- that 'the- .rapidly • labelled:'558'- and 458 B M e eœo- qmitheslsed. in .the implooluq ;md', dotoented-to/SOB and. IBB 3?ù/boeoiiml- 3#Ao» (Bbherrerg , '.
' tod Demièli f .1963).# ' - ' experimèhte.. p qnfina 'the -preTiouB' roBuXtO: of/Penry. (1952) .With n/strain: •' :;fibroblastB*' .■■■':/: • ' 'A- -.-- ' ■■■ ’■■,■/. " ...., • ’y ';•;. Additional evidence confirme the : mcleolaf origin; of -Tib.os'ômal'/MA*- * \ M A f rom/tîie two oouroee has Doen •.'ciemon-
/- //by:, / - / 251"^ - ,y. - -- ' Â. y \.y
■ stratod to/havB; a. qmilar base ôomposltlon# (Georglev and Ifmtiovap -1963;, . Ddstrom^ 1963) mû- a /riboimcleor
-:, / '.protem has- been/oxtraoted.-from- a amolGolàr: préparation freom' pea seedltoga with a Godlmc)mtat:lon coefficient',of
' •■•BOB#: V (Bitostiel, OhipbiWJe. eind'Hyde>.• 1965) h ■ Whether; such ;. ■/y rtooBomoB: aro/ transferred into the nucleus and.then into.- (the ; oytoplarm-has not .been- qônolûsively- demonstrated# . ; :
Evidence.':1ms-. been/ obtained that the rapidly lal>elled\:pL4 . in-'the .398• and;. 4’9S--’;pcake can W ’.converted into 308 and' SOB
-y rihoooiâàl ï#A# / / Borne of the ' S08=.BEA ' W t not the, :308 M A • ' - ■ appears in tto .cytoplasm#'* y .kliie. iaay of course,-bo a messengerfIMA'fractïo:ù#i,/ (Pcm/ick,... 1964). '• ;. î
y;, y - ■• *' ':;•" -y - ; Dittlc ..Inf ommtloh • ië: a.vailàblo-; cohéèrntog. the /' ' ;.• •;.•; synthesis ; of \ bW A or - its-'- stability - qncc" synthesised#.. ' y-/Hèlà--cells after 30 minutes: 'incubation- with ' ' ir'çyüidine
: . show only nuclear lahqlllngM ; -. ■'. .Extraction' .ànd; sucrose . // àydonBity .gradient. cetorifugatxpn. of the IMA, shoiv that the / ■• y;C48 peak, is lahellèâ#.' , iMlatively îiigli .oonoontrations of .. /aptinomycin B; iiihibit; this .luhpllingg: thus' suggesting the . :' auolear; origin Df; bHIjA*.-. iPera y,; i962-)i-■ ' '.
/fhe. majb^rity ‘ of ' the 'autorMiographlc -experiments /iv described above indicate thè nuclear origin 'of. IMA oyntheaia#
Shough they,fail- to show-. oonoluBiw* transfer/ of ,#iA to > the cytoplaBm tliey. also, provide little ovidenoe of the todepondeiit. oytoplapHilG syntWelq of IMA*. - . further evideBoo for the huolpar..origto of • 31% oomos from oxperimontsi on .- miolear tzsnaplmitatlon, imd^for DI& deiiemWnt. IMA eyathesia from oxpôriment0 -with totlnomyoiii D* - . •..••.
' ■ ' ••: Huolel.,'from labelled,Ame()ha proteuo were traneferrec to /aon-laholled rooipient oytoplaom ancl, vice. Ve:ma# ■ Iiabollod ■proci'aOte were deteotod, In the Bxm-labollcxl • oytoplaem but not’- in •non-labelled nuclei ih the roverae transplantation ■ indicating tranafer. of .3® .. from the nueleue to the.-cytoplaam*- However?- the rosuite could also be interpreted àa tranafor of degradation.products -of . nuclear- M A to the cytoplasm. and-not vice yeraa*^ ■ (Golds teto- and Plant * 1955) % . - •Xhoorporaticm of nualooaidea into EM, hàa boon.Inveatigated in enucleate: oolls:*. Of aix typop, of cell tested# ..two aneoleB A, nrotoua and; Aoetabulartoi meditorraïiea ahowed IMA- ayntheaie on onuclaatipn#. -, However# both of, thoao contain, extranuoleto DMA»'' in_;rlôkçttaial ondoaybiontB in the fo:i:iaer and in. oliloroplaBtB In the latto%\ Ho IMA ayntheaia could be Mpteoted to enucleate Hela cells y. amphibian fib%'obla0'ka , Qg à ^ m Ë M S S ^ ' # »(See I’resseo-k-bi 1364 f for a azoview) i In IHSâMâ.where cell division ocqura to give one cell oontatotog
a mioronuolouB^-end erne: .a maoronimloim IMA syntheBls . to , the former, is reduced by 97 per qënt# Similarly ;m order to oYpfoome the, eurgioal obgootions • • to,: eraueleation . expérimenta ÎMÀ âynthesis. has. been studied to maiiimallan • , re 61leulocytG• formation where natural onuoleat ion '’occurs ' on matoratlon of .the normçblaôt# I. At the ,time of emcXeation ®A. synthesis stopè- though pfotGin^synthosis. „ oontiîiuea- for ’ 1 ^a-2 daysf (Dtoiieiro? Deblond tod Dro^? . . ,
' \ %he off cot of : aottoomycln D on. eelluXar ;MA• synthesis can- be taken as. a. pototor .to . its,DBA ’ depondehcê* AotiBOiayoln I) has been shown to Inlilblt. DMA dependent 1® A •■••’pô.lymeraseb iii vit%' by ’speoif ioallÿ'Dfiùdiag ■•With '(%# : ; -residues to- the .primer DMA* . /(Goldberg and’• Eabtoowlte'?• . ' \ -
.:;1962; /Emvjit^pqEurth? McClamy a n d :■/A In vivo aotinomycto D has been /shovm :lîo Wilblt 99 per ooiit I -■ - of the BMA ' synthesis - to D stra-i;h..-’fi’broblaatS'' though some / '■- protein ahd DMA synthesis - vmo, still -able to '-occur.• ..(Wloh?-. •’. ErankXto?. ' Bhatkto' tod. Saturn#;- X'962). / ■ Similarly aqttoomyoto-’'• /-D/.'oompletely;.itolbitéd the .incorporâtion' of uraoil ."-■ and-/guanine- Into IMA' of aseitos’-/tumour cello (Harbers tod Muller# 1962) -and into .■ all ‘ species' of/MA synthesized to BactoiuB Buhtilis ? ~ Includtog that produced to. the
1/presence ..of ohlpramphenicol# .(Aos# ’Reich and Valanja#. X'965)-
oonoeatratlono of aottoomycixi D have W o n ehowa- . to . Inhibit progresstooi.y 28B, and 188 ' ribosomaX ' IBA ' then ■’bIMA ByntheBle in D oolCW# - Hosidiaal ” tooorporation v;as due to tùràovor. of the'tôrmiaaî-’- p O p O p A ' - to si®A* ' '• ■/ • ■...■ ^(totot:Xtoÿ-i963)é' . r . =/. ' ' ' ' . - -
%rther 1)1% dependonGo; of syB.tlie8is ip lllaqtrated by specif io hybridisation df all the. major oelluliir WlA components with homologous DMA# tîS '!)H4 was first nhovm ■ :.to fors^Bpeoiflo RH'ase résietant %1)rids with 52 Bpeoifio - , : EEàj todlcatiBg Begaonoe homology betoeen the haso .- ■■BëhaehceB to each* r (Hall mid SpiogglÈa^i#: Î9G1) # ■ fhlB'; observation has boon used to ahcw liybridlBation .between heated DHA and a rapidly labelled rnqsBGnger EIA obtatood from Btopdovm. oulturea of B* ^gli and SSMSiSSiâ»Blaoe résistant hybride are-only formed with BMA and DMA . from the Bame epeoloe# (liayaBhi aiid Bpiegelman#; 1951)# /,Similar expetomehte have shown that 230 and 16B ribosomal IMA of foime hybride BùecificaXXy atdifferent eltee with Its W A euggoeting two diet toot oletrone for Ite eyntheelB# (Yanofehy and Bptogelmanr 1962; 1963)#fôlMA has also been eliown to-form hybrldo; with its hoiiiologouB MA# (Goodman €md Eioh? 19GS)# • nybridtoation GXporlmonte have been need to show - hotology botooeu. ribosomal and nucleolar IMA which IB further, evidence for the nuclooXar ■ .
•iorigin- of • tile 'Æormer.. - V Sime toi? laballocl riboBOHial IMA * from .pea sootlltogs forms IMaBO -'roBistant • hybré&lB witu
/.its homologous.':])!#» ' m i d V t h o - / f o n - of - hyhricfe is oompetittooly. tolhibiteü .by xmoleoiaj? IMA* -•-•••(OMpoliaBe- ' to&,Èiâptiùl9..--1963) W :, A 'Gonoeiîtration of - the ;MA .-.. • - /comptomontary:.to 20B- ribQebmal ;lH#.,hap.boon:domonstrated -ih;the Wmcloolar .aeqqo:la/Ged. pteDmatln of-'poa. Boedltoge*//. .(ivïoOomkey and Êqpklmi?. 1964-)>■ ■ / / ' /, - '' . _ .
/Oil. - .
:/■; •;/ • Having eatabliphed the imclear origin- and DM. • ■ h'êepemionoo of. %®A ByaitlieZlB# to'. l%ortmit/.%watlom^ in 'the - fiMotioning of'BM in diheotiiig protein syntoosls is •; /v/hothor moDBGnger'-pJA. .is' a copy of one or both atrahcls of . the doable helif• --./It '1b; MiGwh from to escperimesito/with, purified' I®A 'polymotoaea -.'that both 'strtoda of D M -- - -IÙSÙ oopiecU (ÇoiduschGk'j Bakamotp and Wo is b? 1961#000 also eeotlon'-4B) Investigation of the' trenBcriptioii -vlS'SIS2 proviclGo evidence .that in some'.caries only one .: etrand of 'SMÀ "Ip '-réplioated" for" tho production, of mèssengo:
• Ihcamtoation uf the effect of fluorouraoil after-54 . inf GOti cm of. E.: 00^ , on- .the phonotypio-réversion of a?XI ,. mutants.showdd .about half of the 0>A pairs, estimated to be present in the/-mutant., phagé .MA-were fomd to roirort /
with fluorouraoxl* . /Shis'.would, be-’expêotqd if;-about half thé. A# /iéë# /ono Iztrtod- could.-pàir. with ; fluorouraoilL* '. \(Oîîaïïîpo and Bensor# 1962) * In thqèaaié. aye tom differoncoB '.have• been'-fpuM..botoéon-'-tlio haso.; composition;of mesoengor ■ IMA. and the 54'phage suggoBting 'tlie fo:ncier la hot a ....copy of both atmmda of .the I#A* .. (Bauts/and Hall#... 1962)." Â Bimilar oonoluBion was reached Arorn.. studies on. the . - traiiaforming ability of Bepaivated,-atraaids of pnoumpcoccal .. IMA* . ' One strand O2r.p:m00ed Its. genetic tof ormatlon. laore . - .rapidly thtm the others (Guild .and l.lohlqon# 1963)*- • . ... /
.. Mo'ro cosaorote OTidenoo■'hap'been'ohtainod hj .hyhridlBatlon of messenger BEA; from pmgo *^lnfeeted Sr. SUsSMzSiB and- OP*B i n f e o t e d - w i t h the phage IMA* She IMA from these phageq has .heen aeparatod .toto heavy end light, strands, the latter eiiriched in purines and the former ±n pyrimidlneB* (Oordee? Bpetoin and . Murmur# 1961) * . '. .Mepsengea? IMA has been tested for. its ability to .hybridise with the .heavy and light/.strands* EMeoiflc hybrids are only formed with the heavy otrcazido . of .-the. homologoueSDHA’-.to each 'caod* • .XSoechinto- •Valent toi. al# 1963$ Miarimr et al'? 1963 ) * - ; Blmllarlymeseonger IMA from 4-ss -174 ■ infected E*: not 'hybridise with single stranded. IMA of the .'mature phago but only with'the heated double ' stranded M 4 -of:.the .
replloative f m m and lionoo has an .idoiitloal SQtiuqmce to /;the mature'phage OTA* ’ - (HayasM# Hayashl and Bplogplman# 1963)*; In qtotrast to this# vn emperlmdnt in. which-B*
au5Cotrophlc for hiatidine and tryptophan wao 'trmiofozmod by a hybrid OTA made froiï atratop alngly àmco** •' tro:phiG for hiatlcliao and tj:yptôpblm, ahjjwed, bof.ora 3MA replication oopurrad onsyinea ayatheBletog both hiatidine and tryptophan appearod*. Shla Indioatea both etrancls of tho transforming -OTA,are .producing moeacngor • ESA# ■ ; (Breeelor # I '. Krone va? Kaahov and Mosoviteltil# 1964) • , '
. Aayiimotric ayhthoela • too also been obtained to vitro . using a crude , aupesmatant fraction of B* cmdphagec( OTA ae templatei (Geiduçohek# Socchtoitoalonttoi . and Bamat p .1964) * '.. She ability of OTA to; eervo ac a template for aeyiaBBtrla/cyht^^^ic-is dependent on the native cohfig- prat ion of DBA but not on the continuity, of the phage ' > chromosome* iln contrast to.this the asymmetrio,OTA. - /eynthealB oocinrcing with nuoXeaBe free : B* iiali polymerarjo .and double stranded '0 x 174, OTA. is dependent on-the circular a nature of the'latter OTA. Breakage of the oiroular DBA. template : allovm-. replication ; of ; both. strands nf. OTÂ# indicating strand colection i.e a feature of 'Idie circular chroi#Bome* ■..■.■■•(Hayaslii#' HayaoM. ,aiid BplogGlmto#' 1964).* 'She details .of: what oauDOB, .otrand selection.for.to'vjvo
' -
tranéoxiption. -rèmato unkiiown* • - She/ miomalous .results . , • Obtained, In.. vitro may. ‘be duo, to tho .cilfforcmcos be tween.: the ‘ rrcpurifiod and highly punliCled .Bl# poly^îie/moeç iisod,? the . . latter being partially separated into BiibnnitB* (kiichs#.giXIig# HofBOimeicier■ and Breiae# 1954).*' , ' ' / , • ’
: SeBldeB.. asytiTaotrlo transcription of OTA the liybrid- iaation tectaicpa has been .uôoâ to ehow-timt non random , ■ reading,of the genome occurs# On 52 infection of B. oqli , clifferonfc meepenger’peaks, oould be identified at different time intervalB indicating seaiiehtial non random reading of the gehome*. .(Sueoka and'Spiegetoan,; 1962) * , similarly#Ih B# undergoing sporulation ruination ato . -stepdown. trmisitzwhs ohazao.tqristlc. OTAs are formed which co:mpe titive hybridisation ■ ozperimenUs show ; are, dorived' from distinct genetic -loci#, indicating sequential transcription of the , genome during morphogoneeiB*. . (Doi andIgarashi#, 1964) * ‘ - '.
5hG first ensymo isolated catalysing a .net iByaithesis of micleie acid was polyauolGOtide phosphorylase (BO 2* 7*7*'6)‘ ■ from#sotob^ao,(^,©r ' _(Grcard:)erg-“Manago ?Ortis. and Ooiioa# 1956)* - Riboimcleoside diphosphatos are required as s'ubstrates and are tocorporatecl into polyribo-
q / ; ■■ :: :
■ . xmoloo’liMee from réaction^ mtotures ooâtaintog single • /ribonucleoeide dipliOB’phates or laixturqo of ribonuoXeopide dlphOBplmtoB# :,‘. In the latter case,the. base .Composition.’of - ; the..product reflects tho base composition of -tho -reaction .•■
: mtoture ? with highly purified■ enq^iio#4/ Sho reaction-ocourB , '■after a lag period in : the abqenee of widdod ■polyribomicXGotidoq
.; ■ .ae,:primer •. but •’ this lag i8 abollr:hiod by. the addition ■ of ipriHor / polÿribojiucleotMee:*; .(ëeo''GrtobOrg4%hago#•vliüôS;fér ;alreview);»
• - Although the .-eimynie occurs \videly isi/bacteria?, it, lo probably . ; not Involved in net BHA eynthe oiS em vtoo* / /■ //, //
, . ■ Since 1956 • .enjsymeB oatalysiaig .tto net .-synthesis-, of I/- : ' DMA and; EÉA have been .iaolatedqfrom- a - variety/ of/plant 1/ ' ■ -
,/ ./, animal; and baotérlaisourçes#:. . /Dhese . ai'O DhA.'polyiaeiegBo; : //,;:■ ■ or DMA imo loot idyl transferase, (ïlO ;2* 7*7 *• 7) and RHA; -, /;
■ ' polymerase .or. liltA .nucleotidyl -transforase (BO 2#7*7#6)7 --... ;.Both. enzymes* replicate - the . base /secjiienoe of .a primer -or /•;template DMA ih_the'I.-formation of DBA q:if.MA products*// ;.-;. / /,■;
,/ Other onsyiacs'/are\'imowh,whioh/-iâeo3?pprât^--deozyrlbonuoledr;'-• ;’ -tides. or, ribomioleotidoB onto the ' 3 _-hydrm;yi-. end'- groups I
;' ' of - oxistiBg/polydeOKyriboimoleoticlo or; polyribonucleotide . . .. chains* . .... /-.' f; -y l./A-l-I'/'-./■'■/■■.,, ". \/; ' . '
; i.: # m ^ j s
■ . : IWA 'BOl'sRaercaBs v;aa £lrs-k'cllsoove5?etl ia.es-feacos of'; .I : ■ ■■ :k.- . ''/'%%/ ' ..
R* Q Q M (Edmbergp Dehimn/and. 8imm # 1956). and subsequently-.;
.. -■■ ■■.. ■■ - ■■■.■-. ; .A' : - : - ; . :, .1 , i;
/ ' to' mitoui'' tiasuoB*/; ].957j'''/:-Davidson .'' . Amollie# l/eir/ànd/Mtotolog':X95B) # / /51ie 'E#. ■c.oXi- polyaeraso<
. ,/. Y/as at -first wmrlftod two tlioueaatl. timee and itsl uropertiéB
■ ;•- ,.5iié/ oiisymo' -o'àtalyêes: ;bW simultanéoim ;llioorpo;ratioh of - .•/::••.■ adontoo # • guanine^/'cytoDtoe,.• tod\ urvaoil- dooxyrlbônuolèoolde: / ■:/■■/-/' I'iriphosphBtoq/iuiiO' OTA, to thé/prbsoiiCe/'of template: DEA#- ;; i// ■•;■ : , .Ommlsalcm -of: any'/ohO' of ■ toe/trlphoBpIMteo; oanaoB a hundred - '. foXcl MéoreaDe to the roaotion# 5ho prodtiot DMA is doizbXo
■'• stranded#- one atrmid- betog' derived- from. #%o',= prtoer .amd • too ,C ';otoer.--bètog/'noiYly;• Byhtoesiséçli. hae.a high moleouXar weight
: ;£mxi‘ semhed.-tO' be-. '-todiettoguiohablo from • native .OTA4 '■’• to 8.toiàé-'a%'hl:hE ):# - ; Who. molar - - ' -V :. "/• propoztiono -of •'too-vbasos -to; .the - primer : mid • product- are/'/.. -, ' :y atoayi3 eloaely xkhmlar mid.-Wre detailed analyaiq'of tho, ., , . êaaçteen'--mareet. Neighbour •• f réquencioa- ' the ;produot;./‘DM- best .'.fits a/'modOl. to whieli .tile ohalno of the double - . holto • have opposite polarity as predicted- from X-ray data* ;.
..'. • ■ -Iteoently • M A polyiÂeràses have- been .-further .piirlfrted from E 4:• coll and - (hioliardBon/; Elchlldkrantg-
, . Apoahian'!%%&- 'I/ormberg?; - -1964'#. / - Okasaki /and- Koimberg 1954) *‘ ., Ifepemments ■ wltla tooee-highly purified, ensymoq.have ''thrown : .further.light on the. mode of 3MA replication* ... Uqtog
vu
native DMA. eiB, a . template it wee fomid ilmt the .product , M A? ualiko, native D M ' readily remtures after heating and BllQxm a,htonohed, structure/to ,eIectr0îiiEicrogré:iph0* . , Bo eviclehce. oouXcl bo foimd for covalent linkage of product to primer and it .Id suggeatod that replication of the/ product DBA oeowa boforce the primer DBA 1b completely roplioated* .. (Sohilctoaut# Eichardoonwmd Koroberg? 1964) $ If mat toe DBA ooOTorted to a^partiaily a ingle stranded form is used as. template # . DMA polymerase activity first repairs tho single .stranded regions reoonvertlng the template to à doubla strand, with t he same . properties as, mat toe DM# . ■fliG.product strand is ,Qovalently.boimd to, primer and any further eynthosis, results In the production, of DBA showing ricn-dcraaturabiliky emd . a branched struotm?o in elootron^ miorograpto# ■' (HialmrclBOn^ Jnmm-and. .Korhl:)Grg , 1954) * ■Buch GxperiïaentB indioato the lack of control mGChanismrj for. IllA synthesis %itro and-; are further evideuoo for a' Bero'loonB.ei'raattoo mode of ropXioatlon#
. ' DM. pplymerasoa frpm .moAcmalihh .systems have-Deen lesscxtonstoely purified# though to .general. Bliowrthe same charaoteristlco as ...tho bacterial polyaerases# .A partially purlfled .DBA poI,yiïierase has boon obtained from.oalf tliymuB which shows £in absolute requirement for heat denatured DBA* (Dollum? i959)* Whis oufaym has beon obtained nucleas.e free on further mirifioation#, (Dollum# 1963) * DBA poly- ,
36
have, also. been pa3/tially purified, from Storlioh ascites tuioour- cells and boab marrow (SmoXlio# Keir and Davidson# -X939)# from regenerating rat -liver (Mantsevanos,. .X964) and mouse-fibroblasts (Gold and EelXetoor, X9G4)*
• Besides repXloativo synthesto of template D M engymes have been itlontif xod VvMcb incorporâto : single deoosynucXeotidop onto the. .3- gliÿdroxyl ends of lOHA* Btioli a system was firstpurified from calf thymus moXoi* (Krakow# OouregeorgopouXoDand Oanellakis# X9GX) * • Wheso engrymeB. have been further tovestigated and Bhovm to be disttoot from the DMA poXymeraBe* (Keir and Smith# 1363? BolXimi, (Troenigerhmd Yonoda? X964)*
Considerable evidence from, ezcperiments jgi yrgo shows that Eià synthesis is DMA clependentf It is therefore not surpristog to find timt most ensymes from oelXiilar souroes catalyzing the. net synthesis of BMA are DMA dépendent. ,■
DMA dependent ElA poXymerabGS recognieed by sensitivity of incorporation to Hfese and DMaso have been found widely distributed in nature and have been;partially purified from several sources. . ilumelts, BroEiXer and Diringer (I960); Ochoa# 'Bimoa# Kroger and Weill:(lyGX);Burma, Kroger? ' Oelioa? Warner and Weill' (X96X); Burbli,Hurcwits xirid (h)Mnmn .(1961a# b) ; h w e described bacterial systems simultimebUBly incorporating tho four rlbomicleosido .
■ 37 ' ^
. teiphOBpiiàtos# • /. /Similar-- systems, have boen doaorlbed from.. ; -.' plant: eouroeq. by IEho ' and- Boimpf ' (1961) $ 'liumig-#:.JmMsïmàrl - : \ and BouBor-'(i960) liions-todiHovelli/(1954)?■■/-SoB)al,?-. .
SpeiiGGr#/ Kira ‘ ahcl Wrllcten (1964) ÿ. and .ffom/anto#! oourcad - y by-W'eiBB/md- O-Mdstbne; (1959)l/’Wëisa (i960)§: "Burdon and ■ Sïïiùllio' (1961)./.,: BtirniBî/àmd Goldwassoi? {1961 ) *; ..-anC-, .
■Goldberg (1961) w- I n , rmay of,. the so preparations; the ensymo;;is -parljiGUlate'; and "appears; to be lasBooiated witJi'nuoleic ■ ,
" 'aold#r ■■ In the qoll .froe: preparation;thé; eh ymo,. is uopreeipto itatocl with' mmleio: aoid by .streptomycin- or"protamine- sulphate •;' (Burth at ol, • 1961# b.) and on gradient oentrifugatioh
'"'tile enzyme oediménts v/lth In';"rat liver preparations '■■ the ensymo;- appears to- he . o'peoifioally.'.looated in the meleuql a.■ (Wei0B;B I960) ? 'to'.plant preparations/it-is associated with .: - the /ohrpmat-to(Huang .and;'Bonnor,rA962): and in; manmmiian. •. cells a’-nucleolar .-origin- d.q■,enggeote'd : (Eolaiid Busoh# 1964) * Eeoently# /a • -lOlA Iprtoocl ‘ -BMA pplyiaeraoe has been found aOBOolated with the mitochondrial, I)HA; of.-Hoiiropj ora oraqoa./ --■
;-.-(ïtok:b: iâ'B:fe:lçh# 1964)*; - //i//'' -'-’0/11'- 'i'f. .. I; h-- -, . , Slïo W A -polg^aerase f rbm .B$ (^11 was. origtoally ; .- ...///b.
■ purifiocl goo times, and its properties ox'tonoively . - , - ■.' /1 ': " - fh-y/:-:;: -,/:h --- . . - '//.'/•Vinvestigated#•• /to'B-:eth;#whurvïits and - Anders# 1962) -# . Similar- énsymès.• hwo' vhp^hvpurif iô.ô-.. lÆMSÉàÊÈSM. - .(Hakemoto and Weisèh 1968)-.': and-' A#- -vtoç]x#dto., -(Krakov/ and.
■ ' ' 'f ' • ■«'I , pu
, .ûülioay X95S)' . %'Êiüh: - nliow-. a f or G % # . .
/ ' , ,'. 'pflmoriWhlqhiaoto'loatalÿt^ - %--to/ a- atoty .foM-.okaoao':■- ■ . of produo'u. over o^rlmor Ima been ' obtelmed, ' (Ohamoerlto 'Q2&& ••
Amlor'a.fmdlW /
.. ■■■Ho dogzmwïon or. a ltw à M w , to ‘tho-.trk^mtum of BHA oooitro
\ . awi;%'/the..:roao ' %%dg0d by/tko.cl#^ of. buoywt■■ . (lonqlty' of: SB /# A w d ' toteiruifm of treaimforAing. ao tiv 'fty .
: ' ■ bofoxm and. Gftur* lye-fdr-'urfmiig the- ■polyi'iioraco roaatfoa*- :: .(GqldimoIiOk'f 3.951$ Üïflliu.4 tbo
• })M -polymoraoe • reaction natlVQ -WA lu. a- bettor- primer' thanObmpoqltzmr.of protoq;vH% Iq. -.
:•/.',.■■ dlotàtod - b y . too ' DM-, .rxaocl to nrtoo- tho roaotloaiovor a . .thoimaml folAy&/x%e:-:.6f cg.atbnaWxa/'/lW:)0'.#alogm^^^^ of f
' ' ■thcanx)#m?l/:zl3wiioleosto(f irc?;phoq foÿ- tho i
H'-.o/o' to -à -kàïmeia t l i a t i f o m i d - t o . . ■
' ■/;.,.' ,- D % :àmitoezto* • r'%)Mi'hpmorc^ :tho-ncaroat/neighbour. ' ■'' ffaquënoleo; M. ENA: procWt bvo .-to qlooo agroomoht 'With %
êjlglÿ ZlSGS^a)# /. . - ' ■■; ; .. ;;.; fhlo? _ t o con junction w ith toe formation -of ' bybriclo
, : I . ;W#mô& p#xmzx:' (aelâhBOhük' ' '’■ ■• ; ■'ot --Big : 1963.)#'...-nrovMon goWr ovMonoo- thetrthe-base üeqxionoô
. ; Qt.wk- ;iB. îe$èo QXièlylvo»iiefl.to,’l»îKî wmi#*)#)# o',s.îtlA* Siie .
,' ■ '.; BWK go;;.y,B3%w()::iu.,±;iW.bl'WA; a'OtiROjayp;!a; {D;:'Wtloh ,
' . : ' - ' - " : ' \ \ : ' . ' - ' ^ , , : . . - ' ^ . : - - : \
reapeot oj/mthGBiB .10 abotii; ve&i ttlméa. laora >rje:asitlve‘ . : ' .to the', mtlbiotlq -than AMA'/%^thê0lo. &t' #31$... " ' •"1962p KahQ5.j . Kaheh'- a z i â ' : 9 ' 1963) # „ ., .' ÏB.-'addition to ÏJlÆ^Byir&etic polMeoiiCpmcleotMoo aiitl .
. : . .■■■■',- V, -:,:,.:.V.;;.;, i''.-.,. ' z" ^ ; V' /■■.;.pplyribphuolèùtlâoB cajn aot ap primera# - \ l olf A? n mid .G/ - , .
have been' Bhown to -•■ fseparat.elj;' prime • the : :Lnoorporation .of - ‘thé' .0omplementary'-nugleptlcieB#’ ' Üoïïtpotltiv’o inhibition , ■ , "•OGOurB -between a mKturo-of polmuolootMoB and .their V\ . ;: .; aUbBtrates $. BUggea ting . .-that . a single ensymo is Involved# •
OohOB) 1963). ■ .".Similarly , .poly aote as,.â ' ■ ■ :-primer, .for ■ the inoot^poration.’':ôf A W . anâ/üiP as the only } substrates# ; Ho-,’reaction ooours^with one Bùbatrato and _ the ' pi'odnot ' "as ; shoi#/:by/alUi'allne.....hy ^ : te'r-'X- >•• ' p./ .Bucleotlcle. inoorporatloh.•• conslsts-''-of• -.altorhatIng A, and Ih :/ residnes ,-'£n‘ -a'- double helloal" oônfigurât 16n; '. (Ohamberliif: and' : ;Berg,./l965):# ' .n.':'.:/'
’.Purther' purificatioh; of a,%#A %)rimed ]#A polyiiienase from. M#'- - %)oëiilted%£:ii: a propagation froe oi. . ...
.pyrophosphatases, 'phosphatases,Masoÿ polynûolôotide -. ' - .phosphoz^ylaSG: and. %MA : pqlym # (Eakemoto g ; Pon an.d ; /Weiss? .1964* ) - -W.lth./Uhie,.purified enzyme two reactions, are ;: .dêteoted) : one. involving the Complementary synthesis of .BM as described above Atho- ■•.other involving homopolymer : r *•'. •.. formation at a single higher, ribonûoleoBidè.. t.riphOBphate. • - . :
A ' : A " : t ; '. - - =" \ .
f ; oonoontmtloB.il ’ Khe. lattês? roEiotlon Igi InhibitedVby the. . \y.,;. : proeénoô,';Of ■ tho otlier ■ribpniK5lGpsidé/ tr:IpliOüphat0S and by,v ■;■ poXyaïïiines# ■. : Both: raaotlons ‘ooii ' be• .-primed,\by niatiiml 'and ...',.
'y,,.:-.8ÿht|aeti0; polyrlbom#3.é ot Idee /as. - well as DBA.# . %V She ;; o.omple.œntary =.ByiitWBle": reciiiirea Jix'" and- tlie,-riboBUoXeoBide- ;g; trlphosplmt'ë:)QpmplWmitaryytp . the . p r i m e r # a r e ,' :.Inhihitorytthough sti-imlatiOB booürs with DMA primera.
.. : . ' ' ' ' : V : ' - I :!/ ' ' : ' ' . : . - / - '••••:. With.;,hplyr:U)OhnoléotldeaVaëlprüaer-heating' 'pf -the:-, priîiier$•••■'• .. ■ : or aotihdWclh D liâve no offëot. •-•■DMA- ‘ competitively •. -• :■ 8..tlip'/;rêâptiph 'oiià It appoars. that. only .one ensymo- / ..'''1b ' lOTplveê • to- Both-'EM : and DMA, primed 3?e act ions ; i (Post .,. : ,■.-l.Oiia WeisS ÿ. 1964; . EbblBBon^ Eaeelkorm and WolsB, .1964}., A : furbhêr.\Phrlfibàtlw.. of-...thç-'.E#'- •.qqH.üMA ; ? > o l ? m ô r â d e ' , ■
y":' \ ■•beon- -bfotainod.' by-. BtevenB.'.and.'Penry Ci964X. i ■ Blinilar ; 4.. Gomplemehtary: synthOBla with, natural; or s^ithetlcr.:goly- . .
. ;•••• cieoxyriboiiucle6tide's or pplyr-lbonupleptIdèb• anti iiomopolymer-./,/ syntheolB has: hoen deiaonstratod#. (81ovens -.1964),.. .'Ihe ' •■I';;,.’'’ .'Glsêypf theproduct-IfflA appears, to depend' oh the .sIse of - ■..'•■. . : the. primer# tlie en&iyme. .px^paratlon .a:hcl the method, of ...-■,6 lÈblatlon of .product#-' .•■ Sodlaentatloh’coefflclonts..of ,; : '4 V ' ' " - : " ' . ' ' : ' ' : ' ' / v/ ; ' ' ' , ' ' ' ' - ' ' ' - ,V : ' .4 ■ •7; S have Peoai-reported#...4"/(Geiduaohelc.al» 1961;, ^4^ .Ohamberlm- asad ;Borg?;,196H)..* t;,End"group. .amXysia Indloates4 - -'Vchains :;of ,3.00._ r.Gs:idu.@B’.. ,(3?.urth e t- al $ ; 1962):* • \ ghose 'o:hal3i' r leagths /md ..ëedlmeirbÀtlo ;aré m c h Slower than- ,
for:naturally ,ooçnrrl%,Ï^%B ..and. are probably, due ,to '' : ' miolo.aBO 'contamination of the 'oxm'jftm .preparations» . However^
. ' -..using the purified m. ^.polymerase : and . ah .gw poll ■. ' DMA nrlmer^, product 4iliâ ±b ' obtained 'which-, forms, .hybrids •'• ' ;
. / : ' - ' : - ' f ; : -' ' \ -,3 ';; 'p - . ' ' . - -..4 , BpepifiÇà^iy;•y/ith.Iil*. ©oil labelled(238^..-IGB,'.and 4B EE& r -y ''■ indicating Bpep if io Byiitho Bis of .-.high .molecular ..weight■' ■-speciep,, oi'Elât;., , ■ .(EoblnBpBt:À&Uj^y.gœ, and Woios .41954) » - :
■ ; . .. .fhoughjreplioatioB p f l D M . appears to .occur-.eemi-../ :Gpûservàt Ively gmd .\in. : v i j ^ " (Me 803.8 on ...and' Btahl ,:. ■"'■ ;.’. 1958 § ' • .Wake . and; Baldwin,. • 196.2)-,;-. this is .not . so with the ' . .
pplÿmerasq, reaction* . ■ : .jh vitro; both ' Btranda-,:of, a native ; primer a#p0ar to bo 'szqpliôatod.-.(Huzwlta et, el?.-1962 a)*
. ..Identical': E#':prgduàt‘;..I)ït4 primer heareet neighbour, .frequoraciep. ' .are obtained, whothèr4bingle. stranded, or double .Btraadod :. ■fihâ.'-Bota.. aa priWhf ,.:.4iAVith ..the,: làtto.r. priunes?.. thefrequency.bf .diiruoleotide paire, indicates ayntheeiB. of. each strand - ;
, proceeds,.,in;thà;\oppoDlto. direb1h,Dr'to.;:thah.':pf the ,comple- ■' ;4 mentary'^üM Btrand# . f he Œ&: product. 1b time .a replica. .of
■ the DhA template à .the.: twO' strands .being self complementary 'r 4 .and of opposite polarity. ;.Üs:mg poly. dAli as a. primer the 4 product; pply, AÈ - has s ipxilar ' physic^,! 'properties..; to... the -, 4 .y.‘
■ primer .indicating a .rigid rod--like double:-'heXical structure., Ho evidence, could, bo obtained for the. .formation of a; . ... hybrid d  M ^ A M ôppolymer. during the reaction though this. ' ;
. may be diio4to the -Instability of .qnoh à hybrid^ or ito ■ d0Btruoticm:.durii^g Isolation or oent:elfi;igat:lon. m .ObGI# : . ■ (Ôhambêrlln 'and..33o3y|;\ 19630%?": :- ;'Eén6o,_In' 'the, absence ■ of : \
4. y deteotabie • ; hybrid • •£ omatlon'.a CoonsGivatlTp mode of. replie- ■ 'atlpn waalpropoBedy^.l^Hpwovexjliaeihg'BiBgie^Btrandod.';-’ .' ., p %■ 174: JOM ■ as, template a DSl-lUEA : hybrid Is, first : formed ■\mà-.pay- further z^pllctlulon .10eomlpoonaezTatlve,^ i' ' .
growing. strand ;3^eplacing the oxlctlng, 8t]#nci before any .../ : freo' RËA/ and'Bei'gg .1964):*- -r / : ' ' 4r' '
:•',•■ : Gentlyÿ a .hybrid hM-lilEA'/polynieraso has' been.//'. • ■ isolateti••.•..£rom\ E-,=:£oll ; which. Inoprpprateo riboimoleptIdea
and dedisyriboimcleotides, into .tlae Bame palyivior.'oîiàibasrf;Tlio -•roaotlpn’. Xb ..prlmpd by. ciouble s'Wmded polyrlbonuoleb™ ■ tidoop polydopKyrlbohuoleotlclery ana^;naimral43MA# ..Both ohainB ,-of‘ the prodnot iaus.t ,.bo ■ ;Syhtho0lped.. simultanéouoly. •. suggesting thé, hybrid polymarase.ds' bifxmotional.or:cliiaerio#
„. (Huang and..Oavallorlt -1964)* ,, - She. :en^ymçyimy• be, an artifact ,. ’ of the isolation, prooeduro. duo to combination of : submilts
: of .;both B M and BMA polymorasos ancL honco .Is .evldohce for the subunitnature, of. thea.e polymerases# üîhat; aubimits . make, up the. polymeraoe ris - eupportecl by the, appea5zanoe =
/ .. of a, hoxagonal'structure. m'Olectroh .micrographs' of ...the .M f -âSâ polyiiierase when prepared. by. methods avo.idlng protamine: or .etroptdmyczm 'sulphate precipitation*'. , (EuohSp
'W
; BOB-idep. DMA;, primed . 'MA , polymeraâob-' pnotîier - Imporkant; • Glass of Gnâymè'ê Qatalyslng the net pynthople of .IW occur ■during intraoeXluïai^ virus, .growth# Possibly BMA\v:lruÇeB ' . iiidUGG a DM- primed IWA polymeraBO \o:e utiliso the 'host . polymerase but M A -virueee prooeasing either single or double stx^amled HBIA ludUQO a RiiA primed H M polymorase# ■ Buoh an emsyme has been detected , in mongo virus infected Il celiB# (Baltimore and Eranl liUjs 1963)# 0he eyàtem inOorporateB the four rib onucle os Ida triphoapho.tos in the présence of qt is inhibited by Mase- and not byDlasê'or aottoomycin B# . lilie activity is %)reseht in the mioroaomal fraction of virus infected cells- but not in a \ similar preparation from miinfected cells. -A sixûilar ensymo has boon detected in Hela colls infected with polio virus' (Baltimore> X'fggcrs,, Branklin and fmma* 196g), and In
.-SjS M infected with M ^ 2 bacteriophage (Harona,, HoKiUj ' , ■Shtafe and Bplegelman# 1963)• Kho'latter ensyme has been . partially purified mid the preparation contains viral Ehà , in a • double. atnanded- replioat.iy.e ' formé EWA synthesis occur ill the, absence of expgmioua .primer by an asymétrie rjoiiii-, conservativo ; replication : of tlk) natural primer#-g (Weiscmmm* Borstÿ Burdon^ Bllleto:e and Ochoa,. 1964), fhe same ens miehas been' obtained free of. the natural template, which then
shows-, a- Bpocif iOccreqiilrçment for ; the, homologoue MA, ' ,■■:•■’ , :, / :} A /(Eammh: ct ,'6X965;) , Ochoa : and % oôivçrkora g ixcn/everr are'., '' .-v ,;\.mabiLe--'to- ropeat tlieaa results and oonslcler tile' viral. HïïAi-- ,, -4.XA,. /pplÿmoraGG.''Ophmlox" â8/&: :'/'. ' ■ '"'-n . ■ ■
: . ■ : 4' flie .above systems are 'pxamploa..of. ensymo.B catalysing : ,the.. met. syiithesIs ; of /# A ; fianyv émmÿme f ractlohs^ liav'e also.
; I ■■;: : ; been-; qbtaiaed which . Incorporate • çinglo;. rlboimolooBido 4' ••' ,,.kA: tr|.plxpsphateB ’forming homopplyWrs* . ': ;Buoh:by8tomB .usually
4X ,: repiiirp. MA/.as. : a 'priiapr axid\àréginhibit0d.'by''-Bioré'4jliûn one . ' ' :,.'. ‘ ribohupleoeide. triphQspbatE .4.:/.\ ..' A. cystem iupo5?porutMg' Aa!? ..4 li'ooidûcs ' f roHi: Aff ; into. 1ÆA: has ; boon described in. the ;■
; •. : oolublo'.fraotiou of. chlck-'OmbryoG# v (Ohuag, l&lflGr and4-:;/r /., %U'lohù)4l960) i':.; .pH optlBîunr of- 94' ropuirec• .-■;. ;4%^4 .cmd EMvàë.vprimer# . SerMnal incorporation onto the 4.
'4% .primer'oliaJne;; ocqura^i .% similar.-eyateiir-hae- boon, partially ■ • 1- •;; \ ■■•■ purified 'froii'afehe •, q.hprioàlltetoiô. momhranos of eleven day . :’4'- •■•■•:•=• old (Milck-emhryoak' ' -.-Ohaina : of. 8 11 reelcWo.; are 'formed* ’' • 444,4 ;.-(y enl#karaimn/aW 1953) ÿ 4.Ac bye tern ;'fv?oi\i:-tho pHg.
-.4:4-precipitàtp. of. :the 105?000 i>r g _supernatant fraction of ■ .. ' .:4-rat 'I’lye'r., has4,hocn.ipartially ^ .. ■ (kXeB^perori;4t963a)
■ : In the xpreocmoe pI4 Tîi# reeldueo aice ■terai.nally:• ■ ;..ihqorporatqdilnto'\'th0. 'primefe,- g'zlving ;-chains- .5 \T. 5 rWldues ,: 4
;■ long#!. feraminal GI# . or B # groxipe in the .primer act as . • ■ .4 aooeptorBf,'- 'If :thp4métal. ioii, uSdd, io then A % , . /
reoidmm freoiu -AW az'o tonimally % InciorpoDzated giving oliaind , 20--rop:ldueB. Xoîig#'..,- (IîXeiïiperer ':--1965I)) . -%ho''pDrWer / .y
,:.ro<pixœmont is oatisfiod by ribosomaX-^ 0 4poly 40. and dmall^oligoBUçiGotidoSf especially tri and totra oligopoleotidoB.with frae.-.3 -liydrbxÿl; groups^' -\ HeatodBHA and poly A .lhbiblt .thc. , (ElempereiC' 1964) # ,Oytoplasmio fractlphs -haveibepa obtained ïroia aeciteB - .
: tmiour cells, whioli luiqpipprate D%3? ihpbd- pply[ If# - ■ 4(Burdon ' and SniGllio 1961;', .1962)4 " 4 Èüèlc^r.'.ribosbmes ■ from' tiuoh' • cells4appear 4vO inoorporato AfP . ;(Burdon |41963a) # .An "enayiae, synthesising /poly.; A •qhàiné.iiii-tq; lpÔ4;cepMue0,4., 4long, has ' W e n . p u r l f nom;:oâlf î-'thÿiiiiis* nuolGi# 4... Old? is 4. ,
- Incorporated- to one tenth' the -.extent’ ' of •. Aîffî.-whilst 4GMP 4 -- 9 mid .##'''are. hot -''iho'ôirporatod at;, all'# •- Initial observations showed .apparently no.-primer , reqpiiroBient- for this réaction# . "(Edmonds, and Abramoy ••1960)'...--.4‘ 3%rther-worlc has ' Identified a' .naturally’•.ooourrlng.• ppiy. A in -oalf -•thyrAUs:-;huoic i .'and' 'in,:,
. the purified .o W ÿxiê-. .préparâtIbny •;• (Bdàèhclè;. -and- .Abramsÿ'-19625
' ’ . " . Besiâos the above I#À primed inco3?poratioh:of single " ' : nucleotiâea and elHilar BEA p3?:med incorporatloa .by the ; hlghlyrpurifled haoterlal/polymeraBoeÿ; ■ an çn^yao .has been ; 'found 'In; thd-..particulate fraotion-.of--.?hmr liver■ which - : iîiQOï'porà'üés Aïïp- .into -poly A 1% 1;3ae, p.t'esenee ; of ,1% _ ... PÏA'
; , . v : 4. v.
and/ïMV-.-' -:• M A .does_not -ropl'aoe/DMA- is not 4 .:lnh:U)il:Gcl' by :a;Gtinoriiye:U%4D .t;he ribaiiucXeoside , 4triphéephatOB• ■■'4'(0bambGn,'jVéill and/Mabdel,-;X953)-4A. '. ■; ■ .4 similar . enb)yi#;: from'!thé miclol of .asolteo: cells'. . .-• .4'■régu:ire0'-BEA .4!or thebincorpoiatacm- of-XïMib- repiduea.linto -.' '■' -polÿ IT* (Bpjzdoùy 19654b)# ; y:;.; ; : - : ; : 4:'rr4> 4-..-, ■ ,' '4'4'
4; Also, of. noto.-is. mb. eiiBymo/ systeia tlmt inoorepoxcates . . :3?iboimôlèoslde. - triphosphate s.T into. polyûe oxyrlbomioleot Iclob*• - . '• (MTakow^ . 'Eammeh'.• and ' UaimçllaM,s, .. 1961; 4/amielIakia and . , . - ,■EràAoWÿ : 1958) & -, vhiy.>onG. ‘of :th0..;-4ï'rihoiûtcleoside.. -tniphop--, ■ ' ' '■ phâtOB, are: • Moorp'p3?dte.cl,'termtoaXly■;■ intoLDllA#. :is, .dietinot^.frpmiHA.4polyao:xaBOxànd;'adtl8- only4oW- irosiduo ;'4; .'onto ;;the4,ancle ;/pf ;yWie.\4)3;%\.olm3n*:4-:fbb-.'.ribotidyl4i)Ei; ■ '4)'î ‘• then, act ias • a^.primor In. thC' rat;i l:brpr system; which/-'inoor" •.. -h;- nonatesàlE'O? residues.Into po3y. 0*:, i 4.\v-: / r ';, 4 .4: ... .A...;> ■ : .Whilst, .the- fmiotion. of the db.qve,.hqmppolyi'mr ; formingsystems:, aro; not. lmowii" and;may even reprès<mt4,ârt#aots• •.. the te:mina3. ..'formation •qfa'a.BOpteAVsecffionoe: .for,,id'B3A .•■; 'mbleoAloe'le , of'biological liap.qitxmoe for the latter^ e •’ fimotiOB-aB'“eâi^ainMo-Eoid '.aco0iiton in protein:.eyrbhoele.,' 4'‘ ‘ ' Heidelbergèr,;-;Hax5)03?‘B.9 lalbm Xg:': f akagi, aaid, Eotter-(1956)'j?. -x , ahWedathat^-'a-cytoplpemiO; .fraction of /rat- liver.- hompgonate ' ' :. whloh nrotainod ;a,.oaprsnityx'fAr; c>sldatSvo-4phOBphorylatioB . .■ - .. oould4cb:ioo3?porat0; %4 A®, into ■EM.*, .I)igaetion of/the.,; :
product, with' 5/' phom^hocliésteraBe - results tUi* rooovGry of.'V-the. radioactivity as : . wheroae liydrolyeiB with .alkalirésulta :ln recovery of labelled : 2 3 -01®.#' ; lUhic ouggeutd ’ A W -±B Incorporated-:-tezmlm&l'ly 'adjacent to-’OI®, roaiduGs# ' More .detailed inveatigatioms ue:hig pasxîîially purified ;|xreparatlonosfroa tiie,vpH5> precipitates :'of. 105î?000.2. g ' 'eupematent fractions .have ahœii that. 01®4rosiduos are also Incorporated# ■ If the onsyme is first. p37einoubated to the;. .. absence of substrate Its oapaoity süb8e(iuêhtly to' :moo:cporate AT© residue a is reduced^ Jîhis tocqrpoiratioii. can be. rootoieed by . the presence of OÏÏP and it appears the two nucleotides ;are,ihporporatod to a ratio-of one to-two rçBpectivelÿ^ to a terminal G^equenoe* , Ho other ribonucleoaide triphosphates xtoa .toô03;i)orated and it .appears that s M A is a Bpeoifio . acceptor ...for these en^yBas#., ..(See Siaellie 1963 for a review) #
. ; fho above onsyme systems are auffiolent to-acoount for the not synthesis of. thè imclelo; acid components of ' the cell# ' • However, both I#A and IMA contain methylated v; ; bams,and some .baotariophage^lilAs--’.oontato ' 0*-glyoosyl' . -residues * 8üoh..•modifioatloW of: the nueloio acids probably occur after synthesis, of .the polynucleotide olmto . oven.though methylated bapea'cto be tooorpprated bybthe = ensmms. to vitro# fhis hypothesis gains support from the• iclèntifioatioB-gf eimytios mothylàttog sIEA and DEA# ;
(ElexBBraer and Borek, 1962; - Gold and linzwlts, 1964? a), fheso envyiaes have been partially purified and oho%m to be specifio for a partIonian base of the homologous aEEâ or DBA# S^adonoojl methionine- aats an : the methyl donor and the reaction lo irreveroible# (Euxwlte;,Gold and Anders, 1964, ix, b; Gold and Eumlts, 1964, b),
: 5«-
In vivo : experiments on the- klnetloo of inooarooration of ribonuoieotideB or pulse labelling experlmento followed by a ^ chaBe * in.the preBonqe or abaonoe of aotinomyoto 1) indicate è while not definitely pa?oving^ that all lATA oynthOBlB ia directed by a DMA. template in. the nucleus# harria (1959) ?-flas ris and Watt© (1962), liowever?. intîerpret their experlTmnta a© ehowi:og no ' transfer- of nuoloar BhA to , the. cyto'plaem* ' 'fhu© oytoplaomlo BWA ©yixtheai© may occur . wlrloh reduiro© a. HEA template o,r prime:e#g . Burcion'and Smellie (1962) liave deeoribed an ensyme system from the cytoplasm of' Bhflioh- aaoitcB.txmour cell© whioh incorporate© (od P%), BÏ© inoBiduea into El# - utHieing üf3? a© a ©ubatrate. 'Jihe prçfâont "work 1b -coacomed with thé further''investigaroion mad oharaoteriBation of oiioh a ©ystem to OBttVblish if the
:Ai
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. above and; the. microsomal pellet was rosuspendod : by gentle ..homogénisation in' 0*01M tris^HGl buffer pH ‘8*0 'to give a /
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homogenised to a pmoL 1.volume.■• of •0*GlM.ts?iB î01'pll 8*0 to .. • •-: give' s thick lEmëpënsioà^which juat :3fïO#oS4 on pouring, --'. bDually the miorobomal peXlet from 200 ml of lliBsupted coll toomogemte .¥/aa liomogenisoda-to 'S pal-of biaffer# / ■1 ml' of the ;mioroBQBiBl• .ouoponalcm' was• poured into 80,m l ... of acétone cooled - to = 509-' Xn 'a - aolid O.Oo ,= etha-iiol.- imhctmsa# fho BuspensloB was rapidly dispersed with.a Heleo blender at full' apeed for 1*5 .mlxmteB.' ■ /She Buspehaion. so = obtained ' ' was polleoted by^Contrifugatloîi at 600. % *g .for 5 -minutes. ■ ht -15' to and ■-washed :,' 3--.times by .resueponsion in,- 4 . . .. ' 80 ^ 100 ml of acetone -at; the. same temperature#' : * .the sediment .was . dried ' imdor vaouùm giving, a 'white -••powder - ' . ' ; which was. ■Btored at-.-/rl5 # assay'.:purposes' it .was . - 'suspended by hômpgonisatioù In,. 0*OIM ti'ls-EOl buffer pU B.O^
a*-- _ ,'
, • ■ , All àssaye ,weÿe' 'ipo3?3Co33,eci:'M ,5%%: 0.5 üa. or65 % 0*25. ia.
4 : 4 ' 4 / : ^ ' 4 ' " 4 " : ..../ , , , . y
.■■ 4, te a t ; t# e à .',a h d ..In oW ate d ' w i t h ;©W%:Ing at:, ^^^b/toto-^conatant'' ,
' - - to inpe ru tu re ;w a te r/fe a th * ,' . Where, p o s s ib le 4a apeClal;,3iax*vure
/ vms ; made .up. t o . toeXuap: a l i* .. reoàgènta,: oomaap.; a. g lvexi 4 . , 4 , . :■
' • ; BBsayf:.i:...0MiS)Wa©;:-tlvto''Pipctted4tobo\'iCe ■'oooXeditubeD ;-/,.4' a... ,:•' fa llo w e d by any ÿ à r la b le / repgeiite" and .the exxaymo' ,4 .
• f ra o t io a . to g ive . a. t o t a l volume o f / 0*5 ml*-- . A f to r - to c n b to / .' '.4 .... ...... .. ; . ' :‘4 . : -, ■-' 4-4:.'; ev-4 ■4'0-';;'; ',;."4 ' ' - .(.à / " i" , '
4 :4 a tlo n ^ . the--'©ample©/ impq..-e ith e r -proooabed :k # ç â : la te ly .. o r : 4- 4 the • tube's;wero r a p id ly f ro z e n : to a;m ixtu re - .of ;..splto--GOg‘.to; . C'iEmmql.aml s to re c i/a i;- --15^ .u n t i l- ro g u ire d f / .4 / :'4 ;. \ \ i : 4 '
'4 4,. -4: 4-' ' ■!. V- 6 !4:--ç;;.v..;=\-':;-.'. 4- . , ; ..;\;9':4 •' ,4 "
-.4 ' ;'4- 4- ' / '/ 44-5,; '>, ,4.:-' - : -4 ,", 4 ;. ,, : Xn i n i t i a l ; : exnorimmxtS; HIilA vm\ e x tra c te d from the" •'
4. ■ '.rp,action m ixture© w ith .H a O l b y . t h e , prpoedmZG :;Q:k Welsa . ( I 960) #■
. ..toe , reao tiph© werp ;e l t l ie r ©topped; ,hy adding '4: -ml. w f .. cold-
: 5 .pe r . oe$%t (w /v ) t^ lq h lo ro a o e t io , ‘a c id -opn ta in llig .--10. p a r -''cen t ■ B atura tëçi „la^E g0o::or f ro te a - tubop w ore ' thawed -'
. In to th e tame mixtujzO;- , -1 - mg ’’ of. :,o a r r lo r .MA, wad added' ■ , 4 and. -the. ip re o ip ita to p -v/ere. o o lie p te d by o e i i t r i fu g a t lo n a t
.600 X. g f o r , 10 m lim tep# - fh o /p ro o ip lta te . was. rqeuGpexided 4
. '•• i n -.4 m l: of. the. tr iQ ix lo ro a o e t io aqid-- Hh;jj;PgOry\m:br.ture and ’
.the prooodiB;©-.reported: .2 :ü1bîg© a f t e r w h ich I t waa d r ie d b y . ;
. , ' vfaphlmg w ith : 3 . m l/ô f e th a iio l fo llo w b d by 3. m l b f;-e the r# 4 ' . ■./
•4EWA: Waà, b x trao te d - from the - p r e c ip i ta te ly - add ing .-2 ml- o f 4-4 . 10 p e r Gent, Od/v) EaOl. adgaated' to. pH.'8 *0y m xd.hoattog to 4
://■' ■' , , " ■ 4 ■' ' ' . t o 55'^ " , ■ ..,v-4; .
a .water-.bath' at 90 , for KO/mlmitos»:- , After cexxtrifngat 1 on ;■; . the. pugojmatont fluid was:• deoanteci. and a. .furtWr. l,.i%. of ■■'
:'GBp?idef RM/àddedï beforo théeoxtraôidon'wacttopeatodw: ', file oo3Àib:uièd' oxtraoto wore treated ■with ;2-voIUtooa of-
- ■ • otiiànôl? cooled fortgo mtoutbs : at -15S .to- preGipltato the.■ ' : EB4‘ which : was: odXleotod by oentrifugationtot 600 x g for ’ ' ■ : 30 minutes*. ;. flie 'precipitate' wbo; dissolved In- 2 ml. of water ancl toepfeoipitatocl with 2' volumes of ethaaqlg ooqlod and: :eeGcmtrifugod- ae before*. •••. fhie. was- repoatod twice . to .. QZÏBXWO: .o.ompXete removal of imtooorpoxBtod rihoxiucleptides *
, : 4 • In :,later experlmonts the follovjlng vaehlng procedurebaaed on ' the' method- of, Baltimore- and'Pranklto-- - (1963) was •
:•' • uoodi . ' All: operation© were performed'%t-,1 - 3#:.- - 1 mg- of ■'■ oarzi'er proto to- (0*5--ml- 0*£-;-per'.'Oent w/v) -bovine -©druni/ ' -
labumto ito-B-added to. the reaction .m&tmi'o. followed by ..i '..■'"'3 iaX;iof/3/'psr cent/ , ) /trioliloroaoetic'acid to dntatoin w- '.
: 10/per cent/ (v/v) saturate d';;ie p20Y. - neutralised • to ,pH5 — -G ;■ , .with ^(Bplùtioh -A) Amd- thé ' prGÔipitatç-'wào collected ' /
. bj/ooirlîrifugation àt 600 A A 10 ndnuWa# - fho oeaimezit.4 ; wae .dleoolved- to .0,5 mil(Agn laOIi and.,:l#%ediately roprocip- A itated.BVito FK lutiQU' A*; Subsequent .WacExlng was 'pe foto .
by'euBpencitog 'the -'sediment to i- ml of 'solution'' A with a .Vortex mtoer- iBoientifio Inptrimohtotoüd. )4addtog-a further 4 ml.- - of -, a olùt lou/ A aaà; allowtog-to ' equilibrate to.
9l>.: *■“
. xj'G&r loo bsdÆi 2o%ï M ■tsytoM' MooM£lfiëi9'üiài&MoïZ;o::mool!ia#ü # ülem IBI? .
■'. 1i7au Vio jâQ Mümvosoà' 'Uî3- vfâGlîâag'vteo 2£iDe#0ü34 “91» ;■■ - . lülà' prooüïi'ü &K /W%) f -f h g ü G o l ÿ o e i l , â% .■: . 4 # oA 5W'‘c)M5Ïi3,;S2iîi:6-1-1)6 escaOB ICuB tsâ pî ûlcBri'
■ 1 MtKze' p#ü;Mi3B48(y Al ' .
';; i ,.8èü;94#') -#3x4 4 # %aay#. gisoMl##: by ■; : :. .£ ' 'oo;%9Mlw;yùl(%o ü-. !k2Aidti% % ';m%30£sï4c3'#. ' - : ;6 1; allô - ’
- ÿ"l \ ' ::%f mOQ?%ü3àt:9m al ,i/MmmilGotlAoD '■■ 1 '. - ; vkk):bo&ïS' Wc\oi&3&’ # 10 Vimi iMas s:M)oe@i#p‘Vixr u;>i€oa,asd ' '
vM: 9bo !%%; m W '33yiWi)%#ei of - G poy ,-
. :i.:', '. ' ' - oesl :-üMWüa%?oWotiax) çioM-i®:- 3to46% 'e'S-9S. 3>dO® dOî , ■ ’ ■. \' ' : .180 maigMüE; 13%' -B/tK*'#!' bc#;ü,f S # :,.61. ' bÿ eosilMS3i48,paii4' Ofe4^4'01-' :ülo oWai- mipomKMlM. .o. ' ■' 3..l::'MtiOu;iai ’i;4aâ;M333?3üïl3 ilàlo ïîos? ;aifliQiolM;ïdÿ' ' il- i:' 6' ll%w(Sg%%,)»:)a'ügp-; U0SO .:; ‘6 .. . .. ; .. 1 B3iooMo%:y310;l '#6i,'ÆKA:&C R W ..qoaÿuïiM:# do nioh: 'Bxi oMMtmii . l'I ' S'viliÊüo-kM- Saïïm3,ÿ :âlSBO!â;M ..llvàs 11- fallüïï'-ciœio&wlj ifel- ■:, : ■. .. ■ ËMMM. h@%£a oÿiMïlGGB #006 / , .. 1'1 ' p:i.i;3iOliü'oloB tù'û ie&lQâG'(K#l'4' mbo'i:i/o,oi,':a'va» ••’ BWüi ’ : . 1 , -
mo'üloilo, 0:M) loi: bçaoM'iiMil:üoü^iôo: W Ü020 -ü:l:io.cia#%)l0 ' , . . : ';. %o. bo -'i;D-iBoMaip - pBOioluMù Koôaag ' 'll:l;;.;.llHi%ll)i$Ë.iW' og a imollco .
4.6/- : l4i; 6. ; ; . - '■ 1
,j, ,of,''’bile g a a o ' b i p m a oa filtoi? mtér,.'flisosfollw/oâ l’. b ÿ ; .oWmgoe of aoludion A% oi 'wasîiiag,: oa. Kvi3.1ipors?e- filferB'v/îricïh. tos.iaetl bo 'olOf<.';-: witîi the larfio
i:';:/!'. ;■ , .V.Ba:-,-::,, : .,0 , bi,; V l i . : ■. -prpte-iii present;:.%/the, réaction .mixtures# / .
tly-lty#6. ;'vîâ)aeUhidago :ga©". flow -' •.
Aoomtor UBmlly-fittQçl wiôîi a-toorOitol.:wiMow#.;. \toio "44. ;gave (m. ciflolonoy of nearly 50 per" cent vûMi a bàokgrorsid■ '
: " " ' /: 4 4::.". 4, M' 4 - .4: 4 4 . V 4 ’ : ,.4: ';:. ,of. 15 to/ 3.8 oir'cliromtograiB© w a s v . ^
-■ doteciod/UBinÀ a luoXcar OhidagçvàotXgrapli fitted; with a. wtodowXee© : gas flow detector* ; . A chart -recorder .moved . : '4 synohroBously withntoe' ohroBiatograii baing'©çamed#-- ■ '. , / 4. '■ -ami. -3*4;q; xm:ce ■ measured Iti a 5 ohamiel Hueloar .,
. Chicago ; iiiodal .725 liauld aototlllatioa ooimter .with ■offiolonCiea'of XOto X5 par cent.and 75 w# 60 per cent; 43?eBpeotivoly-as oaXoulated from the : obaimele ratios# ,■.BampXos for eomiting ware prepared from"! volume of test .: .
4 solutionusimliy '0# 5, ml9 ' and _ 10 volamea of ooihtlltotorh': y ., .'fluid*:- toe" latter ooiisistod of odlnstant. W^5?2 (Imclear. Bnterprisasg Edinburgh). diOsolvad in sointillâtion grade ./' ; : '. /' \. : . r ' '4/-/-. / ; - ' - : - : ' / / ' /. / ,./fdioxane# , Double : oountlBg^ofj to:'and'.: 40-'waB’'also. peicf ommcl; in the , aointillatioh’ oomiter9.:.the ayaoimts, of. each isotope -• .-beink calculated from the ..ohaHuels ratio©*:/ ., . ;
7 .
A#. :// ,4-fixG éoid toehed.'iixisôtoblG Matotoal dGBoribeâ ', :. t o dëqtiôn g-'wa© hydroXÿoW to KOIAyneUtrallDod^ as ’ ,4- : ' t o ■ ■■, before • mid applied at a- narrow.■ bancV .to • etripB of Whatman'/ ... 4E0#//A 'oh%t'mat(Ig:eap%'';paper /70- om©: 1#:.2- : oma% . '%ually.' . 44.4;/ • ‘•0* 15/ .'0*2.'ml .'was dried, on the paper to. a stream of. warm // air from a .hair Airier*'' •, fhe iiapdr4,ms moiBtenod: to 0#0541, '/ /citrate? bitriô adid buffor p$ig*4 (2 litre©'6f 0*0SM citric, •■ acid tod YOO-to;. of ; 0*03H BQ&tof ditrato) Amd olootroplioroniB 'WaB; carried' out..at ■ 4!#./for-- 80 mtoAtoë#/ 43 Btrip©/drawtoS''■ a bürront 'of app^roximat0ly 20 mAc oould bb-toun simultmaeouoly toi''a ’Bhtodon voldïago'- Qleotrptooreoto-apparatus* fhe ' ,ultraviolet totorhing ApotB were locatod with tho aid of .'h ^ Ohromatolite-\ Ibw./pros.ôurG. tiorohry ..vapour lamp, the /'; ■ , . zradiàtioh f3?om. which was filtered bo that/%0Bt of the / -/oüiisBion was at 253#g. . fhe uXtraviolet/ahBorhihg spotowere .out out and'tfe^radiptotivity was.'meaBured 'directly. 4 / to a luclear .Ohioago gas flow oototer# ; / Alternatively the strip, was: somméd' to a. Hue leaf. .Ohioago AètÊgraph (see., section ;.6)# , ' 4 I'■;»• '4 '.,, .. . . 4 ■
.Acid solhblc mmleotâ.dGp tho supeîsaa‘baîrG. fraoi'loa .. af tor' bhe. i:a:l-blal proolpMàtioa of ■:êeàe,'bibB vaMihiiœs .woree , ; ■
y . : : ' . Myy:**-,./:- - _ - % \ 6: ,%
■ /Boparated-by âosoeuâtog ohro%tography ' -toV.an ;amvaosiium \ isoWtyratp oystem/oonplstii% -pf yXOQ ml •rlsbbutyrio- -aoidp • ' '55(^8'.ml water 4*2 ml 0#88 M^OII ancll«6 ml / Of IK yeroenë * ' • ;.0#Q2ySiil4of/the;-,BaMcaoliable, niialobtiâè-,fraoti6n2 'dropB of à intoturo of . UDE and 'ÜÏ® as marker© wol'o 'dried on
•; hhabmaai Hot 85'-ohromatogrsiphy paper in a stroam of: cold . ■- .;:; air from; a'hair. drier* - ' ®he cM/omatogram warn dovélopeâ for 20 hours?"and ultraviolet absorbing spots wore located as : desprlbod In sootlon 7A* :y. %ho chromatogram . was. out . intOh’Strlps md-pcaxmocl , i p . an Actigrapk. ( b o b .section 6 ) #
fho:total radioactivity'-coan^espoBdlng'to -tlio various peaks was . detesmlnod by weighing : the chart paper ;imdor. tho poaks*
,y-‘' ■ ■. •- SonaratioB of nuoleooltlca .from .imq loot ides obtainedIn noutralleed EOH hydrolysates of the -acid washed: insolublo. sediments was achieved by desooiadlng chrqamtography to ah y,
:. aqueous isopropanol ammmla syetom :contatoing .131 ml water - 05 ml isopropanol and 1 ml ,0#Bü ;El'L01î# - - .%ho nputralisod hydrolyaate was dried on.Whatmfm-Eo*-. 3' ohromyüqgraphy .. ..paper with the. .appropriate/nuolooslde mafere.md tho
\.chromatogram.was .developed .for•• 16 hours# » . fhe ultraviolet /absorbing spots, wore. located us;, dosefibed to section 7A ,/and the-paper-: out .into, strips to a/llow o to tlpn of y tho: spots .; with. '0* OlWi. fofmlc = held/ dimectly into '.-bo tot illation-" vials * About 1 #l..üiyelù#e/waB . collectod. and 10,mX.,of Bcintillator
3 v.; x - 'fluid Wore: : added- fortotoi^8Gttoity\toasurementB'4'-'>,-/'- . 4:-:4
/;.4v. .0*5 AagYaàïapleé./'ôfy k'fO'-^ hyâroiyaeâ /with: _0.è5/'.ml-:?8' por cent ; (w/w-) -IlOld y byAieàting to/'à Aolitog. :watèr/bath- Aoh/X.Aiour- fôXlofed//bÿ\,pàrtiaX;neutralisation' '/v/ iiltix -lipE . and/.dilution; to; 3/iul / ihé pilrine- and'pyrimidtoe .. basea were/Beparats^d toy /deBcënütog/cîxxamatogryipby ' toni ■',: / / Bystom^ooniatoing.'ydj'ml lBopiopanol/37 ml vmter 33/ml , / . A onoéhuratédyaùàlar-HO 1L / Ô# 05/ial 'of the hydroly^too vms .dried; on ...Wha'tem ifo•;'••; 1 ' chromatography, papertoith .the ' .. appropriate purtoe- aiid' pyrimliUne aaarkero rmd the ohromatO'-; gram:=waB',heveioPecl for,/18= -h'oura#' :; Vüitmviolcrb- absérbtog 4 " /epois,wore loocted. and eluted-into >ooim).ting, vials-aB / ^(lef5Gribed\ in '©èotlon ;?Aw- ' : 4 • ■■ - - ■ .y . ■ '
& * ! • :Em i m m A ï m t 0Ê6aâolia3j■ i' .. , : %he ;ms.-bliQd. of Baaonj Gl:lp„e mid'Bw.elixe' (1965) iMs -: followed* '6 10' ml .of paoltecl- aoi-boBitmiio-aj?' cells were■ siispeadéd :ui■ ;2, ml of g po;? oen-y .(-ïj/v ) bon-boirl-bo 'M'-O.Olîl oo.aixim aoeta-bo Wffeg i.gE 6;/0j;;:;6 i ml ..,0.5, pos?' 'obia-b; soflitsii ':docleoyifriiliiha-co ' ai'ûd - ôO .ml: Oi.OlM soclima acefate boffe:c .: >■, -
-pH/5*2 ..'/Who, -blirt*ougli ajÿa'WM ; - .hqmogeAlBo8. o w e ràl' -blsleo, '5Q6 eiiBiùra) d:lB:/i:ipti ' of -bho oallov -V . 60 ml of 90 :Dol. 08D.-b.' e y e 'acimo#6p^^ Waa-
.. - . : ' " : : -y, /;. ;. : ; - eii. : / - - - - ;.. /...,,. . ' :' . - • 4 - - :.
; added and the mixturo -ehaken-,meolimiloally for .XOhmmtee; /■ . at r o W .tè^apëratWG# -/ tohe.re^ulttogto^malsion-wà©-' ? 4 '-.; \ / :; oentrifuged at- 10? 000 4s g. for 10 mtoutes -'cmdy'-tho aquoquo ,';;■ phaaq/was erred w i t h paçteür pipGtte- ihtq/% ; 'a'- - '
' ■ ohillad -■flask ' oohtatomg 0*05 /ml - of. '3 -.'per., -otot - (w/y) ■ ■6 ■ ■b'Ghtqmi'W ' auaponalqnX/; toe (phmaqi- to .réaldw was 4 ,i'’";re-‘extracted wlth/pO ml; of ■,0*'0XB4 aodlim açotato pE '5*2; ■- vtoxtor phaktog/f or 1Ô .mlnuto©,centihfugatiom ; oiid 'removal of ' '■ : the ' aqtiooua , pEaae aa ;.}/df ore-, ; tko ' oomblhod ato^50ua ' oxtraeta..- ‘ were-'AHtoen for furthor 10jaiâùteà with .ah/oauaX'volume, ■•- ■■ of 90 per cent (w/A)4:ptoEo34tod\A'otoflfugad4agam-' at6''10?000/.X g4for 10-3amutea;v ytoo' aqueoua ' pha?ye waa/removed-and ■' . 2,TOlutea of .chilled ' othOmol/- q.onualnlmg/.l- ' Do;è cent po taaalim' aootsAa/were'adtled* . . ,fhe precxpitatG;l/ac: oollootod by :demtrlfaga/uloE at, 600'x. g fof vK)'lalmitoa., aty:0 tmd dlosolved
■ 50 îxg' -of qryBtalXtoe-bovtoo .paiioreablo ÉEàaé.avere added- . / ' - - -,... - '..-.., ' " : 4 • ;. " . - 4' ’ ■ .. 3,;" %/: .' - !4:. .: . • - / ' -. ' - - - 4. ' '4 ■ and- thb mtoture was .Inculcated' at 37''";4 or 15 mtoutee; , fhe ■’ mtoture was'doolod^ on .ice" and : M A / préclpll^ated with , 2 voluiâes
-"■■of- étHanoltoonbatolhg'l.pen.-’oent'pofeèéilm ■toetatè-and ■ ■■ , 4-' ■bolleoted by èentrlfugatlùn^.as bef ore; ■ fhe HMA wae then/dlBéôlVéd to '20'ml^of (0»\031il ©odlim'acetate ? - - '■/-.0;o5mi%m44Ofooo5n:;Mgiiig/i)E;^ 4
■4'2' cdiehgea of 7 11 tree of the / same' buffo?? 'for 18 hours* 4
'■ '■ gîië âiyiÿdeâ.' ooiitr ifugatidii'. at ■ : , ' 3,:; ' 3.0%000 S :M to X’ôBiot’o ■: , #aA the #îA: waë - 6,': ' \
'pi^ëGipitatecV’és, ; She v îm .. y /yiûOoÎTQâ iau.% Sfflall6 ciU£%atltÿ b:C bmCf ey g*, the 6 >: V-, ,
/, ;-‘vfàe (ÿ#raotd#. Wioë with an : oiiüaï' voiump : of .ethess/ana'-thQ’ ■.■. r , ■ ' ', last.' tï'aoee, of ïëtheh , îjemoye.d yith à 6et#à#.., ot jhitx'ogoa;. '. ■ :3.), 'Siie :î#A:6GoàoeiitMtlOîï \w ,a màaem'eà .oipéôMypho ,,., ■., ■.; .iaad. atljihetecl, to 10 x iig /m t , gseifiaiEg '"aû.losctMytiôii:6£>f 1;:. to':! ' ■, ,: : . ôonrQBpoml. 50 W 256,'»3(.u'.'v ;àA:ç.fâlys'Æ , '-,■:; 1.. 0ù8r.qs0.. aëâsitT ' oOatz'ityse/üipa- ' a ] ï 0 \ 7 0 à y 3';. -..;'..to :ï>&' of hli# molè oiilar - vm Iglit ' 'opyrbaiai'hs ■' thp •. oiià;>yetorMs tiâ' ï ; ■ ■ ■
' .,, ... ;, ,.g.;i%8..' of.'solid:. 80di%% t'qiaiatè; qOatadiWïg iO,,:.\' ■ W0;ç0 . aiasol'VGâ:"' Im, 2 ml' , of a fat z". si#: 0*4 .'Kil' alfQuiq.tb .l yç:' -). ;
. ; Imjeotèâ.IdntO: 'g 'mlod,' yitli'., 6 ' dàyTolü- tmiqkf ' hë?/9'G8tQâ'.i8 hoüs'B' -latoî'î ;iO.,ml'''of yEiolœ(y Qollsi\vèrë
obtM.ao&.: the MfA .%g;,:e3:tfa#Wïl dO'sGi'j.bod;'above. # :.. . I...,,:. 'S.. : 'gOlO^.Wy6pODyytiOa.''i 6.,. , )
. : of : theypûriaoB. 'aity pyi'lbiiûihOS . 8 # .: '6:, . ::.' .the '. pÿhialditfè' :bâGeB, to. :b$ labOllgd,ltd .leaB.I'tMmy.' 'par " .. ;;
C0aat : of ' the pwhiW6b#eo » '; .ïAhalÿbisa.by aiyz'ose .yea@itÿ ) 1.. ' .:' •, : gï'elixâat üehtyifagyüiom. B.%iow#d: thÔ;.:o3:ia.%<apte:el .1.88: \
'- '.'arad 48, .peeks.: whioh: 8h6«/eci, a hpas'Wat 'latlo::,# ;
, 'Iïr\q%p0i?:ü%Gnt8/v:in; 4% ' : ': .
:'v':./;:''''KV-:'''- from%%!0ào,tibîL ini:%tnrea.- a . wüf0 .oi0%ï'U. amomit o.f miei'oopmeG, ; '''■ ■y--','-,to' give .1. 3^M-'per\b'Bbby '.'-t%1)'6''';'wa8'.yua.ea:5;;ml-:"-of -';v;; "/y.
; (-''"''O #lM..apdimi%40^ta^ ;pH/'Bv2//:76 /oent _-;. y- '/ /(,v;/v);': ëoâiim doâeGÿië%%Iphate : qiid; Q\s05 Oil par oèn%
■■' 'bbà.(#À^ÿv'])onlOnilè/'ausp©BBlbB'^Wové'"6É^ed'- tob'tixe 'lmüü1)Elteâ ■ :\V% '/-T:".x,):::' pb; - 2: - V.:::2..::\: 'y: :
'■;■.• ..- ... :3?eaetibn mixture0 whloh were tlien' .mixed ,wlth ,a.voriex'mixes?;.;y - s h a k e m " 0^ ofï- p'lpor;ô (w/^:-phe# :'-fo'r " _ ;\ -4;
?.;■ ■ > ;at -r.0OH:;tempor ; %ho' 'reëuïtdmg/' ë ^ ^ ..; was \tho62 b3?okeu 000 -x; g.'fbr -y%
: y : b5' mihutee ahd- the aqueoüa: laver tvm'BtùTVB&' with; à':pasteur -yyb ; % \yy;:::yyy:' \y-ÿ.. 2;/ y..yi;/:\/-2yy. .- y - ' ': " ':.. pipette rüiio a ohillecl 15 ml centrifuge . tubé e.ontalniBgy: . y 'y :ÿ i:yy." v :y:y-y:vy\;' : y-.:yy'";::: '.-yy/ y. y./'..y/y,:.,:'.„,y/.'•.; •■'•• • 50 g&g bentonite* '-. $lxo; phénol = interpbaoé reaidue■ wao ' 'r - y ;;,, /--yy:/'.: .:-: y^rv'.,''\\-yy-y:yy:'";:;-yy%y . rb'^oxtracted '-twice'more bÿ addlngbOy? rnl OyOlH, sodium ■ '-y/''.'' lyy/àéetatë.'buff ér- 'pË:': 5'*2 . shàkihg f03? 10. mlïmt.çs' ohÂeéntrl'^ \"y-;y:'y -yyy-'-yyvyy: y y\;y& ' ,
:; yy fugiiig at /.lO;pOO % 'g fô:e ..5,miiuutëa;= îchô-ycombinecl,:.; =, -'\'y-y .' : : /:èx{UQùUB iaycrs v;erey extracted;, with::1; mi :;of ' éther ' a&id. R M - . ,.''"t: -'.y'-y"':':' /"y-y ' y-yy- y -y'2%' y''y \''yyyy'' %' ':y--ty'»yy:y/ '-y -'/t'. 'yi aty:-■' ■ ’ ';y.was preeipitatod'with 2 vo lxmëfô o f.,' e thànol / o ont aiming -i - 'yy-:' "y' 'yy'::yy.yy-y yyy- --y-y ^.y-. ' 1 ‘për..-oènt'“potaB:sitxxav:aoô.tate--o: : /After:' ooollhg -at. ‘* 15 .' for —-y:'- ■ '. ' 3 0 mimùtes ' the:.:prë,eipltat0- -'was ,ool.lGcted - centr 1 fugat 1 on y , at 600 % g’-for 30 -mihutoe and washed. t wl C ê '. : by rcdissolving-y yyy':'-y:y. :- y/yvyy/y /yy'yyy:;yyy:>' ^;/-:..;-;V;'y 'lh':buffer\\%andy%'epré ^ ,withy'0thano yy:' y' -.
Éxo'ptatèA f t e r " \: teplioato. tubes' W ê ‘'UBually'oàxab'îùedé.;•’•:. lIîliovMïâ, waa thorn
. \y\Cy./yêy% :yyy-;yy:Vyyy !y.A y;/y-y ay/'%%": y :': ;X' dissolved'" :t%i -ymllybuffë:e 3 yyacl'^dfalvaèd'againet''A':GliâugeB ..' .
ÿyb:y:ya y' ' -yyy:': -:"' yy-, /'/- /yy'yyx .t.yyyay-:'y'^yy:•; bf î;:7-‘- lltrbü of-"thé .ibaMa lxizff f or yiO-hourBà.-y.: ■ (Exe.-âialybyd;: : eolutlpxi'waEi olarifled bv-'oentrlfugatiou ,ut- 600 y .g yfor ■ ; 10' 'miuùtoÈ:ymê théyR^^'rpprpc ',the; eupeim/ùànt,: ae-heforoi-' vvv‘fhoy:fiuaXbpredipltat0y^asycll0aolVéd; lu a of buffer B’ aud Axtraotod/wicoewlth aii iecmal VolLumo: of :y .- other* \ tBio Àaxstyrtraoea ûez'o ycemoved with- a . ;•/" ,•' etrèâm 'Of> iiltrpgbui = .;■ • $hq^' q.olutIqhs : Whloh:'coutatoecl;= • /, : -OiSxy 0%75' mg of .B3\fÂ:WGfo aimlysod,by euorobe yionaity ; ;■ . .'\gZ'adlGiït yoeDtrzUfugatldu^' y.'y ' ■■y;; a - -, a ■ . \ . -ya..
radx tm'o
..A*a - , , r B âdliitôu ta tf cm .asm ljoeb/of v ^ M A r ^OGlWtlous-afe^/e'-. "' ,
■ ù o rf oriued bv ooù trifuga t:l,ou ■la lin e a r : oucreoOe, ciexxoity. yry:yy:Aayy-\ /:y- .yy:: ."■:/yyyyy::-yyy- :y:: :yya.y.,,'yà/.:ygfaaiêifitpy y A mlzlng. aevloo: wao UBOd to (Wlivory ygG àml - o f BUdrÔBG/ihybuffer'B.. d/mèarly ■g:caded.;fro®;20,yer bou t , y
■ (w /v) to 5 ,\pë r - G 03# -.. (vj/v: ) ••lia à 0$ g: :y y2'aaie* - o e llu lo s e , '. '
!âoe ta t 6y ë g ii t r l f uget tube (Book' eu(llom g f - ‘I954 ) ()*4 #1 • •.
o f a ao lu tida^ ()CKata:mli3g;.- 0*5';,T.:'.0#75y%; havimg''.,
am e x tiu o ticm o f 10 - 15 ayly SgG, mp:) wae _ roâ ou , the ■. .
auoi'oao g rad iou te and thQ'/tùbôs;;were‘;bem trlfugod lu a u ..
■;■: .‘r
y;::..' 65 -- ' f;-
;.,- iaiplæt ;>otô3i: .(Bf 59)'::()ÿ- S' Sp;t:aoo aoâel :& ,■. ’ :■ çoî tï’xft'^-e',,, ;',usv-a3.1 y. cèatxdfttglvfcUm. was ■-psro"oxTaed a-ü ■ ■. ' :
; : ,4 3.2 b.ô , { -. ./r ;Y':'. :' : ; ; : 1 . : 9 B f - w b # - oolle.Gted 9>j/- gmo'ijttpâng. -Glie ' '
; d)88e : of " ##: '"k##: with a. hÿpodezmîq aceûle and' allowing-, '■:.'.'tîie-\oo:iiiaa'(is .tp : W tnboB, , 8 di'ops pes?/ ■ , - .-t#e wé3?o ■f0011oo'’iecl igiviag a :V'olU3ftè'Of-al36ut-''0«2-nil, : . ' ■ ■
/, gakf# àné.;t.h@, nltravlolot, al)8o: ption:-■ ÿ of ; eae|i' gyrbion wap, aieasui’ecl*, ,, ' : 0. !3 -, ml : cgf r oaeh-: fraction v/às . I l f - f o s ? \
/' ' / y■ ■,. ; moÿoÊ 03î6l pnopaNtiüns ' in' yaÿi.üns wayci ' , -
;■ Ptex’Q,. f**aotioîxated, by .-sediiaoatation 'inaûènosé:.clQ:dsityy ■ '.
;.■, gradients*. ; A. gradient,,'waa,-preparod in 1 - ÿV5 ' .ins* ceilwlp.sb -.' -'aoetate-; Gentï?i,fyg8. ' add:big'. 5 e?1 of. ■50., por'Oesit c':
: (w/t )':.sndroaci‘ '.y '.tris-EOl.bnifes? ,pH 8*0''end .l&gerl'ng.'.' .-. en snecosBl’y'elj S.;;8il of ' 85 # ■ 20» .15,,.. 10, 5, 'per ooïi'îî r(v;/v) .., ■:' BixcKoso aolùtiopei-'M tho-,. same. Wffor * - \ ; ixf ter eg,nilibrating . -, : };at. 8 ,4.': 'i'os’-.12 honva'i v£ .lai’ of ,a. mioroooinal ,fraotioxi "
ôontàûaxing. 80 ^VSS .i'ag.'of pro'le.ln 'xte.o layered.'oil .the. aixprose . .£aid-;'tîie.:gradiontB'-Wero-'ô % a swinging bnoket
• ••■rotor.' '(W 85) In.d'Oplxioo -lAodol:; S' p^rOparatiVQ ultz^ooAt#- '•;.■ :.fVigo 'for'-6 üonrÉ).'# '87*000'% g# .::' : - .. -. •'-. -.' y:' -.'' .
î -■ . À . : . %bo be,8o' Of : .t pmioidrod wl'tli ,a hyijoderiaio
■ao9dle;.s»4-1 ,m:L -fraotltma.woro .OQllept ■rin'.-ia's'-.QOO.Iefl \:'\::::y ■tec-itytuMti.». . Dïîvally ,11 Ilg .dropà gawp' a fzmot of.' appro^mOately r 1;'hiI* ■, ,■. .'0*89 ïal;'of ■ eacE; f î;aè-bioa ,waè dilütpçl \ td „5 . i\llra9’±ole-t .abBpa piKlQii '.maasiWmeatB 0,t 260-'Htian#/ ,E80': #l ahd 0^58 .:ml..*8a- uspcl. tO'-mosBfeOytiis-y..;':W.oo3/po.ratl(m, üf, :% - P.à ((X.?%), ,# : , y - - , ' y;' :.., /
10.* -D'Ci
" - ' ; ; i?he Ultsmviblet- abBorptioui of: 1 ml. BWA -f#ctionb' ■ ;. were - obtaiüieâ :at 1$.$ y#wup:ihg. B.llidav/ r ;.,',//"ïïuArQOeilsyih $ .IMlOam'8p egeotÿoghotpïâètçm^ ■":;; y\-/:U ' y./yy,v.y ' \y,\ \
y She .ultr^yriolet oi ptiçxï ôÿ ,5 m l ; m . i 0 3 ? p G ^-.'fraatiohe- were-, obtained K k : b f -86Q or 280 .mii' . . -'either■ a, Ijfeiioam .81?--500 ' ;
U --/. ' ..-: : y ÿ: / /. '.% ' .y" . y.'-;. .. y: : -a./-, y . .-'-..-.y , ..,/..,. '..V'.,.;-", V y ;;@ro-,U8la\:ÿi#@. ,e8tl!%at .ittellioâ of..IiOïft?y, .,.--.
$'azT çmçl Roadall. .(1953-) « .... . ' ■ '.: ■:■: ' . 'r ooîita'Meâ 8 .pe%' :pc.:a-b .(w/v) WdpüO* to Oil JJ' ,■ y ;,,. y ,. y:\y. V .
'■. lèOHi. '. ' Seàfsbîat,-B. ao:atato.ecl.-,..0i5:■ pé:e ceat. ('w/ÿi. .Ong% , ;, .y -y-:. ; '.V .'-.' 'yyyy:.'y-\,- ,■-; y...' , .x.B'" y.;;:':,- v- -,. :.-..: 5HqO: to :1 -pere.-p.pat Bodtom o,r.,..go-kaB0l-%my-i3&rU3?at0* Roagent ,.0 was ,a-.îtotoitoo of 1,00 ;h1 of .-peagent: .1, aaâ 2, ml of . s oagoat,-,';
,' ,:8i'. .y.'l.m troaleâ with, 5 'wl.-.of 'V:--, --. reàgéaiî 0 aa# .afl9%' 10 toimtos* 0*5 ..sil of. a aitoto. S'ol'to y;
; : , _ y i y 'i-Wf W t o d . - .plpoalt0iiyp,%toôi', #âsP:àt. - y
;, '. : "' y;:'y ..wasy-lîitratpctyt'o' àyplXQ:aQlphth8/lQto_ pïM p (mâ .'dltotod v’y. .' y ’ yy/: \ ■ ' '1 'Il :m.yaGià) *y y ; Bhpi Wj%t#'0'yitos : cdml&iy :: y'. '- ;■ yy,:-and; àUbweff;-H3o;: Bjkmd' fpÿ-at-y leiipt: 50 xalxmtee ;befofoythe ,y .. y": - y’:' ,'y oolowry''âe>'0lGpedywasvtoady ptotostya'^'reagôiat/Blaiy 'a -y''-,/:-: y y ': y / y - yy -wWelèhgt&ypf ; 75Q' mt' toy a ■ttototo,BB''-SO'0'’VB;dGBttopKot '. y/y
. y:;: yyyyv; \y:&ypal'' 'U%'toito' ptopajyed. 'datos^a-y atôy.'idaï’d sdtotioa y :
.y: ': : ' 'yyyy.f#toAW ;■ :yy ■ :y: -
-y--yig.yyy&ggpa c«
y , y '' ,. y,:,y ' ■y.SîéiJhytot'éa- alterato ito;Eieb©lgiair-.t?os-- •D.t’eptoed yby, tfee' '' yyy3 --y-yyyyyyyy'y. y-yy:;./.:, %' '.yyyyy-i::y/yy/:y-y yyyyy ' "yyy.yy . y: yy'y', '. y '. .y; -y metoçiyypf'BneqtoytodyQEpàs ya 'f 5 'grafflSy df. a lb p iiïi .y ''- ■;■
y,:;.; y. wQjyo'suBptoàtoytov.'Sop'.îùi'of toûto^-pl/.todyvli pf 180 -ïïOlyyy yy.'
•' . y wëfe'B^Sded» ', -ffilie '.mxtotato :'(faéy..ailox#d'. tô. o '9 ytl'ayâ':. .yy
:.yy .y mïdyébtoma oppaèltoaltoXy; y. .; ',/.''
,' ■' ■ 'bjf yqOï%trito8 atioâ,ytodyi'fa@hQd ' to y/fit'ii a p tto l 'alOpIiol . aî’iû - - • y
■■ y, iiwipë;w ith toiiydx'oiio'.''e,tiifôx*.y' . '
,'y' . ïKi?0Otoitate yy'àB y'dtosd tonder ;a .%»açdum axid .Btoro'd.às.ya pov/dey, -
y,y y. .y y ;iycpyl*etoy .ôoated.rlc:lèBplgi0 îfy.ywafâ. .ptopared % as.': f o l l # 8 .'yy.y 2 0 ;•.■ -.. '. y
y -y y'gï’àffià' of ' k ieaèig tor 'were boiled to. ezcno.l::@,ir''in..lÜ'O .niil Of. .. y.: '■'■y'y. .y;. ' y'yyy.'y.-yyy:y .-yÿ.y.y y . - '' yy . - '-yy'; ;:y.yfyy:yy'yy.yyyyyyyyy.y yy'fy.yy;y.y
y.. - y.y.O, ipylaO Î .to' 0. pii yé« f..,toÿ atooy y pôo.T.;mg y' .;.
; yy.;: .toïfi' pf afiypenr: 0ëto.ysotottou .6 f, PèyWiytoiody .albWto l'^s . y.... ; .:
y'' y y... 'totod'y/ëlîowP&.ybF' -a yfà^ïtw^i 8 py-:mB'of . ..ttoyy . -.yy .
y y-y;v.yb to f è f •:' y : IW im.toÿltoPd atouïüto': pœ -KieBplgtdxr.y,;(M&K) y y ■ y. '. yy y, y' y
suBpeuBloiiispf'ObtalUGd etmreû-'.mt-1 . i:" :.
•- ; ‘A; oolùto was .g3zegare&.--frô.m; lOmiii/of ' t W ;%E % . ,••. '■-■■ ,'."suepeuBiou' là, a-l/.QBi-glàéb ;tu5o#;■.:■■■ She-/oolûim-: was * paokéâ : - .and' .oxoesB' bufferi.wàB/â:nlVezi .doW':\tdivW'\tq^:r material' 'imdcr: air- pZBOGUrë - IMé/d'ci,* lii§..)* .v.,’ ®hovtop. of •- thé eolmmi waBpOQVered wi'bhi'a 2'3'.sm'a'layerV.of /Icleaelguhr - àud itheû with ' a pleoe ét[ tVl%0T: pax>03* r .. ■ After waeliing 'with. •. OiXM-iaOl :m\0*05M' phospha;i3O/bMffer;pH'6#.7AO*5 'BIÂ -
.W03?8.\ applied - to the: columi/at : à GoùG.eûtratioà of 20 |xg/ml ■m-'tfey,'Baffîé buffer whioli.^ao, again tiriÿçh’’!(ührôiigh 'imüùr a:lr Tî3?e Bsurç * ‘ '. (rrraciicut olutlùii w b b thon çporf oacaexd using a - \, :'mixing vessel .'Oontainin-g/ItSO -ml • of. OiÎH'laOl- iirvO* OgM ' -, ', phosphaté buffer and. a robo3:" ()lr: oontà^mlng OM MaCl in v ' . 0# 05M phosphate\buff03? .pH; 6*7* ■ ' ■ UJhlp; was,, puàpeâ: through, '• ;■ .
the qolimn givlng.,% .'gradient -' from -0# 1.-r'-lw211 HaOl /. • • ' ;aùd-pO^, S ml fraotionftMoreioollepted.using an.Aimer g: f raotlon. .oolXeetof # ■ :.: lUïio ' éKtinot ionVof o. paoh.. fraction. v/as '■• roéorded .-ami ? -. after; • adding ■ 0# 5: .mg-'of ' carrier-■ El# to,. eaoh^ • - ; 3B#-was p3:ecipitat0d with'S voltuiioh-'pf ■moXdvethcmol* ■'■■
The procipltato : 'ima :&iâeol*hred ( in 'Î ' 1: of water,.mid -ferréd ■ to • aointillator ■ vialo' fo:r-'3?adioaotivity; meaBareméâto*
Eue lease; actilvitÿ’: .was dètormlaéd using tiie samo
f ' " ' " - .. / ■
■*; • reaction '-oqhaitiôw "a# fo r .miéioQtidô-; dnoo'ri^oÿàtiôii' ëxôopt/yùé A:; / u - ' ( è m ' / / / ' ' \ g .
" • '■'•■■ ' /.mbotmdlB'èeido';-% 'VtèrG\jMittëd* /: :V;BMbgoHDUë'-/--■yy-..:;.: - : y ' / ; y y y : y : y ; y - - y y - : - / ' :
, ;:: :Ëî)Ayliïy.t(iQy mloto aêileE ■ y yy
y: y-:' ''yyyyrkto'-tocto# ' -y; y y yy' y .; ; , : :'gMsptodie %#%% ü0Gily.# , Gnb#3?at0B.., -'y ' y-
yy,':y,,, jy-y ; 959»;, JSSl) (.y,;;; ,Wilâïl '#% '('/#: .%80dyy%y/'tto ybho y ! :, y
yy: : : ,yy' ■ - ■ i':qWyblo:a .toé yë'Wpgeii :% ytlxé aMi'Uloa 6f ' ' S.Sylaï 1 .0M‘ HOIO '
yy'- ;■ y yi..tii©.,; y W cîtow' at-:SoQ.ya yg foz-yio ,-,yy ■ y. y yy
yyyy. / . y y mtoitWBy a'iàâykhq y GÉtlao'bloà ' # : they'stoeitotoat ' clQtqxrmMea - a t . y
,. y/y,E6gyim*'yy,y aW) àmqmit. of: miGlâQtidë yimteolm'iëâ wés ' oqyxeyatotoft , '
y -y y y y#toiyto3^%l'(;to;phon.yiytoÿ^^ 9t-yyy;y-y ■
i ' y yy. y . phob'iil'^bo'/yoy. t t o t o d t o Q .' .y
y. yy , ■' :' ■ y. y #.e& Iqêj 'Bubst'to'bQS-. tW ■ s'oaoIjiôïi.-vmB.y ë'bop-qo& 'w i# 3i 5 ■ ml -1. : '■ ■ ' ■y-y:'-y : ' ■': -yyy ÿ y y •■' y'^yy:-. : : y/yy-toy-yy- ëÿ'v-.y y - y
yyyy- y. g..iE., BaOH$ tmy. xasotosl® :i;Bteyeiàl .was ...ooxxti'ito dorm. at. . y .
yyy :6Q9...'%yg'yfo3?yyi8yi:atototo 63!t'too,'W.q:%:yof'y 'ühG yoüBë^aattot'.-'
.; y.. ' ■: yy-y’yyywao yrëeÆ- to-WO: tot.y, '- ■ '
-.y . . y.yyyy v#y Q§lüto#eÀ:'to . yVyi^l9:toQ%yy . .y.'.' , -y. -..- y
. yy.. y ' y-., y y ,
1 0 ' * :
■ ' y ÂfS '■ ©æ® d GB?®y ÊSid ; WTS:XiQT& .pTiz’qiiGeea, %(m':Eabèt',. ; ■the Sigma Gheiœtoai.- Ooto®W, o yyyBjjitish' 3totîg
Htoe#.: Mmi#â*y.%'y#io 1 ■ :: •■ ':0al'bloehëmd:'8tre#^ 8ulpiatè'.;to/i';<oP '-lijisliecls i;. highly';' 'pelymeKlsei-y yeâéi" l A uee#■aâéûÿs frdmyB%?ltieh D iigyiïdhèeeagemilÈP : hyto'oélilpz icieoi .iBeBphqoyeattoe ; kixxaeq ,y, toapP '■.' -■tod''lïfesèy.iî30ia tte GheiaiWl' Oe::%mn,y:,:; . ■v-yS y : ,
y:' ; ':., • ' - ' tottooWGto ll Ëae : a 'g;btt ■ fi‘Oà Hex’oK,j sixl'toy 'ato- '' y ' ■. l'iQîiffiè,, yiho. ;: ;■. Buïified yeaet qïM&hme :.a l)r«; :D, ■..
. : y - A
;: ". y ' ■:?H',réiho»'woleoside triphoepMtee tmd to- M S ® '.weto :■- : •■'• ,' '"''/'" y /■ - ;■ ■ : ■-' .; '. -y y' . '---y :': ';.';': :''-/'y ybûhtaiiip# .fx’OM.: Spieara.Bieregècytoh ytooi,. - /-.'G foaqoatej ,
.'éarx-ièr - free# ?%? .-oa?tho;to9s:i?ItoG ; Gytopptoylphosphayüe ;Phtatoed '.toom .the -JtoüiüOheatoàl ■0,eiitto v.'Me2?aha®s , :;
:togl'à3:ia:* . . :' g' glf'h 3?% !#*.y y -, y: ; “ loO"c)ot'Si.ïe,yoÿ :8,2^4,* / 'phtatoed y ,'■
ftom j3ritiéh 'Byp% Heneee .'ttoA:£Wd:ym yliweeh -80 o», .polyosiy-', ethylene wap y ohtsi^aoû Bight e;ad y ,,Qemga-ay: :ijtoi'Uea. y'" 'y; '■ 'y 'y- ', yy-y y--.: '/y y ' "', y '. J' y-y
I I-
MyA-iO?:® È ,.X A I, S
I 1 I
-y ; yy' ■ ■ ; . ■t3to'84S.ihG' wae obtainedyyftom:GàibioQhem^ây# ptoitzbBhWto^lythyialâime:T'y %ÿaB , à.. glft -- ■ :y;. y .
, , SGie bèh/ijoaita euapexaBioa,# tréio.'pnepayyQCl toom -GOBanox-ûial beiitoiiite obtatoed. Stotish .Bïug Honsoe : Ïiim;l1:eclf, : ' 2 grcimp. of ç;<?Wcls beatohitos vîbtq suapeadeâ - ia.40 3fl'i of wàto ilïàtl çeatMfugQ# #t 750 s g. f o%\ 15 mtoatop*:ïïîièyOôdMsiit'was disoarcd'e'd and tîis 'Buijetotvlsàïit frnxotioa 'was, oéïrfelfugôd- at 8,700 % g. foà 20 nO.aate'0* Shis y. seâlnieat v)as iE'èsaspoxïüod la 0* veœexio pS 7,0 aà.â stofody : at z'ôom ytempe%tiirq f O):'' 48' hoarp* , DJho aasgoasioa wsw - ; toôsntoifùgod ' at 750. -S g, the' esdiaeilt 'disGasjdetl. and' the . y
siipèïaatasrlj agaiiî Gaatnlfiiged at-8,700.25 g for 20 ïiiiimtes»' Bhp : .é.èdlmeàt .waa rosùBpëaded la 0* 01M .'sodia-ùi' .àQètàtG...baffer pB 6*0 at a ooaoeatratloa yof 8.« Oy per ' - oeat y' % y 'y v y . ' . . t: yy ■■...... i y ' ...
3*yoisB?'wçi.s .-prëîîas’sâ from. |-%)' oxthoahoaplîate by.
.: themethod of Seaeîz (1961) md.waByph.os'phorylated bÿ the'y:.':''.'', y ;yy:yy:';..yy yyy y y y ■■'-i t - yy■,...' y yyy ' -■y,,.m,ethody pf IMith aad iOiprsaa (1958) to yield ÜS®,
. ' . . . . V
'■ ;■ -X'v,,;/.-.. • à aolu'bibn of,* A%-JabqlXod.;'phoopîxp3?iG’- acrid'-:(1Ô0 ■ /
-, . ' ■ ' ' ' ' •:■'■■ ■ : . . • . ■ ' / - ' y - .V . mcuries narrior free mid {Imiolq of ooKli .
,pàospîior:î.ô acid . In- aip-ooua- solution) was oomoentràteâ Aio . / lu; at 40^ to, remove t2?acoB 'of HOI* XO-ïtCL■■•■ -- /of--aîüxyclroua-vpyrid;uie ; (prepared, 'by:.storing; over .‘baXoiuH' , •. ‘■-■•îiytoiaë) > 'ana•.'i--nil'•• oyanoatbanoX' vmre' added end tlia' aolarbion ■
■ ' V- was oonoeut3?gte.d. to:':'à% oil at \ seoond... .5; -portion of, anliydrons pyriclihp-- i m n added and, tiae -edliitlon• was .;; ■ again.-ooncontrated to.a#- olX*/':. 5 'ml of, anli^rouB pjridlnD ;'' ■;■■■' ■: ...and .2*1 -g. ml)..= of; .(^oÿq.iGliexyXoa^^^bdllmlde (300) were-■'
added and. tb#- réaction, was left dvosmigiit .at •.room tempor^, t o ; - i n . a wéll’ Btoppêred, fXasfe V - / - , / .'i:, - -.- . "...5 ml water;,were added to stop, the reaction .and' tlio .y ... resulting solution, was ' heated. in' ;a -..boiling water bâtii for:
.. '30- minutes " '.• iiie.'.- solution ; was' • 0 oneentratqd • to- ..'-■ '■■ in, vacmo.- and 10 rnXuTator and 10 ml of saturated Ba(<I£)p .'wore added .to. the residue*- iAfter 5 mltiùtefa at room -, ./tempe37ature the solution was adjusted to pH 7*5 with, glacial
. ■• -/acetic• aoid---mid .filtered/.to 'remote dlcvolohezylurea (DOU)■••■/.. ami bàrium phoaphato.B: Swo> vqîiiîiies - of ethanol were, added./:. : to preoipitate the .barium :8-«oy£aiootî XpïiQSpliat.e'.v hioli'waB ■.
collected after'1 hour at 0^ by centrifugatioii, - She , -■;/. .' She orystalë'.wcro arodiaoolved -in 5. lal water.--by. adding a ..
/ to - A to- - - ' '
;,gS -glacial, aoetio- aclclÿ/né'utraliseâ' with '.%(OE)gg "coHtrifiigéü to remove=:a .trace' .q:&. inBoliible’ .• ■■■ ; ..■ 'mato5?;la3.'.‘ and-, reoryetalliseé -by •••adding--..lO-ml- ethanol# She product.wae oplleoted'.by • oentrifugatloh' in ayprmvqighed... ; tube 9 .washed .with bthmiol ..• 'acétone • "and"finally.- ether#She..- .yield. • at this • stage, ivao usually abpirb. 60-, per oent of ■. . .the otartliig material#' - BJheCproduct wao dissolved' in-’.SO ml 20 per oeiit .(vyV) Àoetio- acid and passod. through a lOoweZ'-SO-'BA col'um (B. x 2 om)# .She effluent was talæii to ; drgheSB-.and’.dissolved :ui 10/lal’ anhydrouB-’ pyridine: to which ioopropylideaie.nrldino ,had. heon addod in the ratio of 1 mmole Isopropylldene- laidihie to 0*5 moloB- cyasioetliyl- ’■• phosphate# - . / ■ - . ’
The above ■ solution was- ooncentratod to an - oil in vacuo, at 40^# 10 ml anhydrouri pyridine web added and thesolution again ooïioentrated to dj^ynees* . Slié ' prooess was . repeated onoe more and the residue was dioeolved in 5 ml ..anhydrous pyridine and 300 . added'in the proportion .2*0 mmole 1)00 to 1 mmole ioopropylidene uridine* .. ‘
After 20 hours at room/.• températurethe welVetoppored flask was,'opened and 10 ml of. water were added* . After 1 hour,, the mature w m oonGenbrateci - to drynesa vamio#She residue was .hydrolysed for 90 minutes in, 10 per cent i.\
/ ' C - y / v ) (40 to), to- ::toQP 0P2toclG{toa':MQàphoca4(âe;:‘GotoGto?' /top îâoôto/:
/ : : M ’GÆtoauc'a?»’' ' !P%r toqto#; \f,. ,: :.witli 40 ml ol' 9M am''iioali.ua li vtiroïGlûc (to.TQmoVe' oyemooGW'l-
A'rou-03'} a t .60 fox- 90 jaimitoo csiâ tto::##oMo. »toaoV0.g, ;W.G . . QcàiOQntst'.t'SsxQ to ûjoatf'ïr-tolLOi-ifflL''' of
'toto'v/'Sciçieô to the ,roitoM:toaU tototo''ftottottoa.: unGos-. ï?sGiwoû■ m^esqutoi'
‘.Olio;.-protoÉtoMo vm waëW&yvAth/a; pœXl ■ tolÈto-'of ':::.-■ 5 .to&'ÿtotoÿtoV ttlisxfi to: to?'
Æ 'ühitoic/üôgtoMÿc: pto - )%o#%#&%%/ '
,wq,o, ■ tïitm. À%8W4,yoto' r ilioroufjWly -q ':/{WàgMüto%': urns
(ooe i,"G-ü.hoâSj .ueotlOii 7P>)* r ul'uim'iolot al)00i’b:ta.g «pota ■. : 'wofo oemi&#%fok:totoqa##l%to4%
. ' / t o / : ;;:! . 25G/mi a'.'.0iWtotok' 001#^' # % /
-tho 'ozct'Wtiom
She :t.otal .ex'banotxon of / the \U#? ,fadaption- wao/ .doteraixxed at ; '/thih-btage ■ and" the - yield : of àlR®?/ oalomlatoc/*. . \ She ‘oluato
•;■ ’ was-:Gbmxmt3;at(3d:.\tq--an; o i l m . v a g ^ / a t / 4 0 p : * v . ‘
DSP*/to .;•,; '/ : She .reaotion:;mlkture''for - #P%'prepa3%itïpn' contained' '/
toto/toé/; to'/to '-'D' tov.:w/to''-: /:.:, • r the f pll.owtog.• oompmlehtq -for/each 3.00 • tmolee;; of ■ ;.; . 1*2 ml'. ■ 6 .ml pyridine j., 0.2 ml. 85. per cent/ to (v/v) plioSjiliorie: adib’;'ahd;5 'g A3G0'7toto' 'Sho,.'ïftKtu3?a'-‘WaB '/to;-, -■ allowed to stand at: room temperature, for 40 hours * ’ At /. : ' /
-/ 'X 'toy::'\ -Pto, /to.:::;:'to -Ar:/to/;::;toto.to,;/to;to\\/./to/ the end; of the • reaotion a thick preéàpltatê. of 300 . had ■. ;/
:to-!/to../- t o "A ' . ' to" -: " v/:/ : '/f / - '■■ -.formed* ' About 2, Volimes of/water, were adclod-to -preGipxtate : -:to; : - ; - / v , : : : - - ' - t o : , ' : ë t o ' ' v - : ' %:'to'' , to-/::/:::';, to/ J)Op .from.mreaouod.hOU*.. • She flask- was -shaken and left for/ ' _./:l.. hpuh/at "0*///to/'.Slie DOII :waa ' filtered/ off :imder ' re.diiOed.'/, presBUfe aaid tom water*. Slie oXuato/^aB e^ctracted :;■:/:.'■ /With/four'. 50 1# /portions : of ether to remove-/,pyridine ■ and., -to •■to the etherameheà/were :oftraoted w:rbh; i;wo 10 ml portiona of . -./■/ " water/;and-/ these water washes Were'= added to the -main': aqueous,?::-toto//: to.:. .--to/::to :/to.to/to/to'toto/toto./":.-/"//- ^// t o / p u a S G * . . ■-.■ ,./ ■ ■■ to ■..'■• -■ ■■/'. / /=- - ; to|]he eomblned Wuboua .phase wab ooiieehtrated at 40^ toé'.h:::/:m:/;to-://///' ;to.-to: . to'Xm toto/to;;\:/:to;:/to'/toto/'':' t:' ih yapuo* She toaaberial waa applied ,to,. a/%)6wo:c-50»"ha';- coXujvm (4 %' 10 cmB*) to remove" tri-h butÿlsfiiiié.* , She' / uridine • derivatives ■were.’'olut0d//,l)y ;waahihg with water ',, / / until. the mctinotion of the eluato at 262 mi^/was less than ./ . 0*05*; i'/’Slxo/total'■:eKbiBctlbutooftotlie>::.Dluatetoat/262-.mh-WaB to.
;-,7Ê
dâteumiinüü and t W maoimt of uridine dcr&atives calculated;* Shô effluent Was diluted to 1 litre and absorbed <m'3mim^ '1-pi' '•coliimn.-.(2-x SO cm*.)*. She column vitm washed with water, until the . extinct ion": at 262 h m was lees than 0* 05# -Hradiemt elution from the Dowesc l- Ol" ooluom was then carried out; with 1*3 litres of 0* Oil Wl'-.in tlio,missing vessel-and 2; litres of 0*3M 1101 in 0#GIffi. IIOÎ.In-/the reservoir* - She elimte was oolleoted In 20 ml. fractions ■ which, were scanned automatically, for a^adloaotrvity# , She. > . fractions .containing (^?%)/OT3 were ;pooled and the total, yield'-detemiiied* to' AjiCl> H01-. 'ahd-/isiorgaliic phosphate were ' •removed on an active oharooal-oo3ouiin which was prepared . . - in the following, inamer# : ■ A .eharooal colmm (1 %'10 cms# ) ■ viras washed with othanpl^aimonia (70 per cent v/v aqueous •ethanol containing 10tnl of..ooncentxBtod' ammonia per litre) imtll the extinction at 262" m\i fell to loss than 0*1;The ôpluim was then washed with water^ 0&01M MaKOOg and finally lilth 5 column, volimies of 11 HOI; tDho colimm was then rosuspendèd in water^ fine particles were decanted off and washing was ..continued until neutral; ühie oolmmi . was -then ronàçkod; ': ' • ; ffihe; ('=A2%?) UffP fraction from- the 3owo2C l-01' " columnwas absorbed onto the•• eliarcoal. oolusm which was. washed with water until no chloride ions could be detected in
,77-A./
thé /effXueiit# . : Washing .was /!c6ixtimiôd small volumes . . .•pftoOèOlM SàBODto. to /remove'. iùqrgaâiô .-'phpspbate, until uXtra^Yiolot/aboôibing material, bogan to be plutod# /fhe bolmm.'waB/.alXowotl.; to drain aiid: then 'washed with\2 \oolumi 'Voiimofâ .;;/of - water* / >. ‘ W B was fluted with .about X- lltre/ofe tlianol^ammnla and, wae coho ant rated in 'vacuo at 25 *. "fhe. material -'was- .rodipeplyêd/ water.#/'paqeed.ythroiiglf £vemail.'Dowe3: 50-ha'°' oolmm'/iànd cteckéC for, purity b y . '. / / ■ .■ehromatogmphÿ : 1b the xBohutyrio' ■ acid; mmonia water eyotom.(see. Methods;..:seoticm-,73); .. !Dho amoimt of IhH? ■ • ,'lB;/tho filBal/.eluato /was ' OBtlmated. spectrophot,oMatrical3,y, .aesimiiBg a molecular eztlBotioB coof f ioioBt. at, 262 m|i of' 10% :tho .oohdontratioB of the :BolutioB ;was. aciguated ' to '/ ■'
■■10 imolOB -perc/.xal and'stored -'in 2/h 1 aliqaotB*;-’:
■. . 1
H E S W I S S
.78
- . to '^^/BolyriboMoXcotide' syn tiia o is . was ■ moaeiirod. by .,
' ■".' v followmg-tke^ inoo3?poratiOB' pf/radioactive; ribo;aaGlootidee ' ■ ■;•; ■ Into .aoiâ/preaipluable: material* - 'In Initial. oEporimeats . ...to; tq.detéot met oyathoaiB of' I#Ap % the réaction 'mixture . ;/to" ' oomtaiabd equal. aimmrW. of (c.6 T) WIW amê milabelled . ..
. ASB) GSB and OS?, which should ,Gtimalate./(o&^^?) USÉ / imoprporation if ' heals of a tetero-q/olyribomiolooticle
- . ! ' "/ooctlra* ;.. Highly polymerised.. IMA. .iaoliiâed as a' prime a?* to = '. \ .to .'fulfil' any. motaX- ' ion .raquiremeat;. and bemtomite as
.' 'em llfaBOxinliibitor*/: .vShe../rQaotioB'mtetwe was buffered .
.. . W ith t r ls -& X l .emd stad leo on the e ffoo t,-o f .b u ffe r coïioen- •■
■/■ ;to tration showed this to be. without effect, on' the reactipu# • ’.■'to;; ' She tohidorporation of. US3? by subco toLular-.. -, ■
fractione .of ..XipkieototB aocitoq timour cells was -first .- . toâçtermliiëa, toShe time, course of incorporât ion "of %3to'Ul#
reeiduee by broken miolelg /à mitochondrlêl fraction, .a;-.,'■/■■' jiXorospmai. ; fraction .'and .’a 105 9000 x g... supernatant fraction, to
■ ..■:■//-'.is shovm-.'in kiguro'''!:*. ..' Only the miGrosamal ; fraction shows /'■ • ■to.app ' ciable aptJvltytotouid; tlXQ reaction; appears to be ..., .complete ■ after Ig ixmutes « ;■ ...Shla fraction,v/as- chaî/actorisod
;V ■ .toto.in- fu.rthe%to/cAp'P.:toi ^ ■ " ./ _toto' to/.:- 'to'-'"-to'-" .
■ - to/to'Mgixfe '2' ohowQ:. the topB of . uiao/.BÿBtom* / ' /. ■furth.er; aBsàyé- .were'-'-pereformed, at/, the • optimum;• pH -..of. 8V5#.. '- Sable 1 Bli<Xm-'tlxé /offeç't0toüf ....omieBi'oai :of ./;elbonuo3-e'0'sMo’ •
/' 5/-trlphoaphat08 'f:œm;tho fQactlon->mixturo»/ A,' deoreasé ■ ■ ; . /: . in/the'''tocK)r0oratlonvof■ 'observed' on romovlng, ■ ;• AIO?. and/GSP fz om/ithe .Byeteia# . V She removal . of 'Off ? ho?/evorj 'to.pauBocl■• ëomo ptlmilatlohp' pofâsibly/by■■-allowing.eoiae- cosivorBion/ /toqf //WP' to .OSl? and / aimuXkmeouB 'tooorporation of both; . A ■■"■■;'■/ vfurtlier/dGcrGaBO, ih-.'tlie/ inoorporatlon. o f / U S X was
■to oboerved ohtoeeWrlng.AS3ÿ. GSp.and .OSiH//toSiiQ. Byotem was... aloo shown to be/atlmlatecl by added ;1#A .and -inhibited by.: ., •’ ■ Xt/wae. not lûbxbitëd: : by/aot inomyoixi ' 3 whloh, ■-•..;
to/. /BUggeato. that the obnervod.' inco:top6;mtiori caxmot/be aceouated ///for ' by utilisation ' of an endogenous 3)DÀ ' primer i ' and this » .'together'with.'.the etiiaiulation-by /IMA,..' Buggeots/ a-.;HHA ; :' \:/. / '. template 6r primer may be aeed, ' .. . Eoàupnitc) .appearo to have no. off eot over the Incubation tiiïie.: employod;*-. \ , Sable 2 Bhowo. the stimulating of feet of . adding, cm ÀU5? fegenefatihg., pystem;, . to 'the. rc3acticm mxtureB.;.to - .PhospliqoreatiBO; and phospho-
:. / oreatiiie kinasq'-'(EO S*7*.3*-S).-Wô:eè. tiê'od . in .the.approzimâte '. :/,. ratios given by. Krakow et ' al ■ (igGI) #, '. , Phoàphooraatine ' -
.. ; . kinaBo catalysep/the transfer, of the phosphate < group of .' G : phoanhoorêatino .épocifioally to "MB' ad an hoooptor* ■ , (Eubyp : .totokbd6:'mid; %rdyf Ogstoh, : :. ,
//-to to EIGWEE 1 : M to- -'-to', to; to. to '//to:She time oouree of.the incorporation of ( P)
US3P by suhcellular fractions of Iiahdeohùtg asoites tumour, ,
She reaotion mixture contained :50 fxraolee. tfiô--îîÔipH 8*5, 2 pmoles MgGlto, 0*1 jimoleo each of HTp(5 X 10 coimte/min/imole) OTP/ ASP, GSH,, 20ppg SHA,:20 pg bentonite,; and 0,25 1,0 jag of protein in a,total .volume of 0*5hnl*. : :\.to"/, /'" /"' %.'to\to- '// ':\/-/to //; / / :
, Incubatloiia were/perfqrHied at i37 for: 15 Minutes;; V toHMA v/aa extractod from the reaction mixture by the
WeiOB procedure. Â t o r . ./'/, ' ■ '/'■' ■■'■/;' ' -i Miorôeomal /fraction *
'■ : :;ë; y ëA. -/to. :Hitochondria#-o/ -L.::' i05,00P/x g supernatant /; ’;.é" / fraction. y - /" to-'-
" to : ' PIGHEE - H .-'/:-:■//' //i/toto: ■ / 'to! ':"to;to '' 'She effect of pH on . the .incorporatioh ■ of ( USP '
hy 5tomicrosomal fraction of .Lahdachutg ascites tumour'cells,
, She reaction mixture contained 50 limoles tris-HCl at the pH indicated* 2 praoles/MgCl^» 0*1/ pnioies each of C'cto’to:’) UÏF (9 X 10^'counts/iJiin/fimole.) OÏP, iTP; GTP,20 pg RNAÿ 20 hg bentonite and 1*3 mg xaicfdsomal protein. in a total volume of 0.5 ml*
Incubations v;efe performed at 37 fpr-ZlS minutes.HHA Y/as extracted from the ■ reaction mixture by the
V/eisB •procedure. ■ 'to //- - \ - /’-,./,, /•/■•■://
Figure 1100
60*
40
CO. 20*
I— {
Incubation tim e (mins. )
Figure 2120
100'
•5 80'<D
601
401 8.0 9.0pH units
TABlE i':;. ■32’ AffoGt the incorporatibn of ( oc %) .'
IHDP in to a mlcroBcmal fraction o :tiimpur cellB* . ' ' - . \ ■' .■■< ; ;
%ie reaction mixture contained 50 - Mmoléé trie^-HOi .buffer'.pH 8,5# 2. ixmolea MgOlg# 0^1 ^moleo each: of ; ^ 7 ( OL^^S), ïïl’P.: (5 X 10^ ooiints/mïîi/pmo.le) ATP,-'GTP, OTP20 iig 20 |xg bentonite and 1,65 mg. miordBomai ' ' tprotein in a total volùme of 0*5 ml# ; . ' v ; ■
V/iiero indicated 5 ng aotinomyein D and = 0^5 p,g ! % \ HNaae were inoludcd, , . :: \ . i , v '
idle incubations, v/ere performed .at 37^ for 15 ' .minutes, . . y ■ 1 ' - %
; IUÎA was oztraoted froni the reaction mixture by : the V/oIbs procedure, / ''"t,:
Table 1
Incubationm ixture
/ijumoles (oi P)UM P incorporated /m g .
protein
Complete system 95
""RNA. 65
- A T P 74
-G T P 87
- C T P 117
- ATP, GTP, CTP 55
+RNase 7
4-Actinomycin D 122
-Bentonite 95
/. liîho effect of pîioBphocreeatinG. and pliosphogrea,tine.-■ ■ ■■■ ■ '■■ ■'•■■ - • •■ ■• " • ■■•■ ■ V'--- '5P-N- ■kxnasc on the inpornoration of (be. P) HIT by a. \ mlpfoBomal. fraction of LaBdhohuts aecltes tumour oeXlsïa " ■
The réaction mixture contained 50 pmoles trib-EGl buffer pH 0,5j . 2 pmolea MgOlm # .0,lyrjLmmles ( oc -3?) UiT .f (5 X 10 coimts/min/umole) 20 pg iWA, .-20 pg W and ]. * 3 mg of micro a omal .protein, vf: / t :: v -
,V/horo indieated 0,1: pmoleB of ea'clf\uf.AÎPt:-dfP: and CifPy 2,0 pmbles of. phosphoorfeatine (PC)and 50 - Hg f -of pho 8 pho G re at ine lAnase (PCK) were included in .a total] Yélume- of vO.5 mle .'ai ;-1.' m
InctxhatioiiB were performed at 37 for 15 minute'swi.;. HHA wao extracted frora the reaction mixture by
the Weiao p3?ocedure. . ; , .I/--’ ■ ’ 'a
Table 2
Incubationm ixture
ju/imoles (o^^P)UMP
incorporated /m g . protein
(a^^P)UTP . 49
(a P)UTP+ATP, GTP, CTP 114
(CK P) U TP+PC /P C K 262
(a P)UTP+ATP, GTÇ CTP, P C /P C K 213
/ . , ' ' , .y: - ' ..- IK) ,. - y - ' - -
' 1961): # 3h0 z'eeulte aüovr that of ,. W É VBB Gtzümlateü by plioBpbocrGa1;lnè;:aàd, phoephocroa'uiiiîS îc:lnase#V • %Imo 9 ei'cîier-bP? ,oan àct ae. a plxonpha'fo acooptor
■ \a'6'..the much higher'.mayme 'oosiooirtrâtlokiByuaed. la the , aaohy - : ôoiaparecl with- the' Emomita UBocl. by. ICiiby :al - (1954) or
. 'trace •• ’amomato • of ..AD3 -: 'la tlie,prep0aoo‘ of :riboHUGlpoBiclo' ; .. ' ■ diphosphate ' ■ kinaeés (110 ,2 • 7iA^ 6 ), .• proBent, la.,the haicroeomal frae ulp3i Eiot catalytic ally'. iap ropliosphorylat ihg I0)h* / She phosphate-, regenerating system cauBes. .a> .-Bprkod.. lùprp.aoe la - the IhQorporatlon ot ' DTP alone g : aiid while lt\ also ;'•Btinvalatee the iaoorpoi'atiom of (oC' 3 ) .'uOh?; In.' the %)i?G8eiiae of AEBg ODD? and OïïPÿ. the overall Incorporâtlo<;i of • UfJ? in the latter case Is lower than In the. former.-.Hencevit ie* - 'poBalhlè • that stimulation- of -incorporation of ijfp, byAiTj fHOP a:ad ■ O'iiXS. previously obsoivod -in the ..absence of a . phosphate / regenerating eyotem^ ' is due to a higher concen*-., -t rat ion • of (oC 'l?) : M ]?. in the reaction' mibstxire. when Af 3? § ■
à G'5D? and ,.0K? • are .present- than when .(oC^%)- Ufh alone is- .•/;---preôentt'":---' a ' - - -.f - /;/ : ' ’.Af-y-’-a'-■■'■ "t
' ' .. S# % /
' -'further oharaoterieatlon-’''of.. the /iilcorporation of ‘ . V (ohr h?) TOd? W 6s . perf o.mod using an acetone dried powder of .
\ ,: they.mlcrqéomal frâotlfm wliloli .could-beyetored:'for-.up to -y ,r2. weokB at : ;4,5 ; : v/lthout Ipasi of ao'tlvltyv ' ': -lOhê • acetone. -, /'''
■;■ dried .powder had; about BO '•*-. :,90- pqr ;céht. of the. activity of y'y yythQ.: original':mlprdeoa'aa -'frao dopqadlng: cm tte/preparëv-; ■ - ■ -■ atlon y ■ though the . ovérali hotlvity varied. from - preuaratloïv, /.:.\y-.to -preparation# •■ ■ in; the .following crcpeK lmmte ;Uho second .'\,%vaBh:mg:,prpcedùrQ/wap, Wod . ., yyy.
■f myrihoïiucieosido.'5: rm rij?hdophâte“y(o0é: yiethodSj .oeotion 5). x \ '• •y"x.yv.;.y. -y SafolO: 5 Olyqwa, the_;BuhE3t3;ai;o z'eqtilroments, in the ....' y presehcoyand■phoaphoo:roattoh\yaid^jhc>Bphoorea^^^
' kinase for! the: .adetone dried powdor* .iiiooiporatium-of■ / w.ya./' r.;: X ;-vy-\;-:V-:e ■ ' ' V Z x .-.U r. .:v(cgy r) #?l*y-lsynot\:: 8tlimlatod.. bÿ. Off or . OSiy-.and ' Oiiiid 9.
' but ' Id /êtlïmlatedzÿhÿ' phoephocreatlhoy-ahd phosphQo'rOàtrtoe'y ;’ kinase# ;, In the latter oaéoy - addition of 'CWi'.of- M T CfSP aacl...OffiP- inhibit. thé ' lïicbfuoratioxl*..’ - IPimme. 3 ' ehovm the .y'
. : - V y , . . / : : y r y y . p . ' ^ y ' y . " -V':. ,-pE' recgrlrameiTu.yf or .the- :lnoorp6rO;vlon of g). by the. yy acetone'• dried"' -pot/'der - in‘ the .preSonce of - Q-fPp XHüPy =
■. V phosphooreatine and : phosphoereatino. .k:üiasoOihe'-- pH curve.- ; ■ is sharper’ than for them#treatêd r:iioroeomeà and the ;■ ; . .. ' , Tmiclmum appears to;, haveyalrlf.ted..; slightly to pE fhZh • . ■ ■ . ■- pOBBlbly beoauoo,- pfy removal .'••of % Interfering .'oîi ymep ,pr .the. ypreBenco'.-,of phosphooreatlao am%,..phou#iocr0atinoy; ^ ^ ^ # ' - ' ": ' ;• y However^ both the elmilarlty of tim naubctrate • requirexaentp ■'.' and theyÿi optimtmi'.;i3;x&lcatG GBBontially .no . change :hi the .
■ ypattem:/of:,,iïiaorpdratipîV'p f o h - p r e p a r i n g fané '. • * y--.- yP.- aoetone ■drioci ! poMer of- theItrlo-rosOmesV’ • " ; '• In the -i". ‘ absence of m regenerating System-AfPp 0%3? 'ancl OfP /y. .stimulate the incorporation of catalysed, by ■■ the mlGroGomal fraction but. not when catalysed' by..- the ..; ;aGatono; .dried pomcief, (see fables‘2 and 3). fho time
,•;■• / oourad of the reaction id ‘ ahp%m,\ln Pigure 43 incorporation .
, viB :etilX\oo;dtimlng after.40uiixxxutQep' though at’:about 25'---' ..per. Gont .of the rate at 10 .minutes# . , ,,
... .' , . iiîisporimexita.'were also.'performB.dyuBi:a.g-;a.'œaotion ;■ X • mixture• 0outaliilng' -''-GfP''smd.‘OS’i? and bentonite .as a .
- nuoloaee" inhibitor to;, study the effects of vaxyilng conoen-' . -,. yy tratlons, of ÊWÀ ,ï)iA?. i#asèÿ- M a n e -ÿactlnoiayciny-l) piitl ‘= -y_-y ;■
‘ ions :on'the .moorporatxoxi;,of\ P) ufP. ‘ y .Piguro 5 ‘ shows ;thG'effect .;of-.ixicreaBidg ccmoexitratlonD of EM-andy y■ ■•. MA#:y■' - ..stimulation: is observed.ivith the former up.'• to about
GO hg •■•per.; assay:, tube and thereafter,-thoy iuoorporatioxz- rGmairiB ■••...■ ■ .constant•X-. She .ma^lmmi.;\âctlÿlty In the ’ureaono.e of added ■\ .:,i#A,.l8 •only;.35v'. <-A0--per-.ooxit''above thatyobtaiued-la^'itB y': , .
"' a b b e n o e M A neither stimulates.^nor''Inhibits- thereaction#. ,fho: retmirement .f6.r ân.REA primer la .aJ.ëo: indioated .by the , V,-. : .of f oo‘f-‘ 'of-/‘adcilaig ■ ; liiqreas ing-'-y ç onq entrât Ions of EHaso- toy the y. . y.'reao.tidii'mature'ô. ' ;i?iguréy6;ysho?/è asyllttle;as‘',0#25 \ig
• ., .Ihaso. oauses a 70 per pent inhibition pf the Inoozmoration# :
y - y : b s è r -j . ' : $he effect of rlboBUolédslûe 0 ti'iiihonohateB
/' y/,-ymdyplioaphocreatlne'. klnasé- bm the Ihob rp or a t i on of'.y-vr:g/:y,\%2:' g.:/ ' ''.'-'/-y"'':'- ' h'./ 'yvy yy'yy'yivyv' yyyn.,. : ; :(. V': ), m'P \by % mi .acetone dried powa-er of a oBilorosojaal .
fraction from hancls olïut s an o 11 e o tumour cells «
y ' - .jtThe\T0adtlbïtYOOii&itloné v/ere as forgnable 2 except yy-yy 1#7 mg -pf■ aoet'orioypowdepi'y ppïi_tàlnlhg;-:Qt8G'ymg;;;y::..y'Pnd ; O^ipraolëo ck : ?)y iî$]? IX 2# 5:"%\yi
r/orc arMod.iy-yh 0?he ;Inoubatldns were partied out ut 57 for 15
y. . . _
,y : - ’ : : EMy was eAtraoted from the .reaction-mixture, byi' i f theyWeles/prodedure# yy^yÿlki' yxfiiy- .%'yi^
Table 3........................................................ ■”
Incubationm ixture
Wmoles (a^^P)UMP 1 incorporated / nag.
nrotein
( f^ P )U T P 115
(a^^P)UTP+CTP 114
(Q! P)UTP+ATP, GTP,CTP 120
32{a P )U TP +P C /P C K 257
(o:^^P)UTP+CTP,op P C /P C K
(a P)UTP+ATP, GTP,160
CTP, P C /P C K 184
piGimE 5 .
■ ' ■■■''' ■' : ■■ y y: ‘ ■■■ ' ' - : ^y--f ■■■■ y-y. i g :Pp- _ yyy-:Tho effects of nil on the incorporation of (çx* p) ,; WBJ by fin acetone dried pov/dex* from the raicrosomal : fraction of Lanclschutz aGcites tumour cells#
; The reaction laixture contaihed 50 jimoles :0f iy ' ; v- v tris-HOl buffer of the pll indicated » 5 pmolee y’; ;/y0.1 Mmoles of each of .OTP-.(.5. -10- f counte/miD/'yyy %; y ;fxmole ) Gl'P., ■ ATP, OTP , 20 imolep phohphocreatine, .50 jug phosphooreatine-kinase f lOO jxg BIA,, 20/|tg hentonlte and. .y i,; 1.5 mg of, acetone driedypowder. inyaytotal-volme.‘of yo..’5 mlV:
Incubations wore performed at 57^ for 20 . minutesi^^y ÿ
. ' '■ ' y ■ ■ -: - y PIGmtE \ 4 '-. -...-y/: y ■ • • iyyy:-:- ;V-yyy.ÿy'The time course .of the 1 no 6 r p or a t i oh of ("cK" vp) TJTP-
by £in acetone dried powder of :thé mlcrosomal fraotloh 'ofV'- lands chut s ascites .tumour cells. y .-gyh y py :■ yy>'iin iiiw. #1 I
10 reaction mixture was -the samey-hs.'.for 'P^igure y 0X0opt 50 pmoles of tris*-HCl pH 8.5 were used* , k
Incubations v/ere performed at 37^ for the timesindicated*. -'yyhkykhh'-hyy'4
Figure 3' 400
P h CDg g 200Ck ibflS
100,— I
10.09.08.07.0pH units
Figure 4
4001
300
oÎHA
Incubation tim e (mins. )
effect of Minore as in^ c onoent r at ion 8 of /,.i # m P f v mü - 1f m-,in w i#Minw »,# f,%i w < # # ; ) # K iw#M#*wmi»*##«##w#w#W4# # m # ' W e w w # * » # # '# « #k #.i#u u.# f . ' im M e # w # r n w #
and I)!IA on the inqorporatloh 6f ( P ) IÎTP i)ÿ an ; n-Jl' •/3;:.:aootone dried powüer of tiie mxcroBonial -fractionfof^ ' , ‘v .
1 — 1 " " ^ n 1i n ' T I t \ \ \ m I r m " f tT ir r m i i i a i r T r n i " r r - i m T n # n a n # m " T " ^ T - i — TT— i m - n r r T - T i r m m « n m # i ^ # i i w i ; , i 1 1 n f i i i m w m i / iii h i i.ii i .11 iw i . i m ^ w . u ;
IgmdÉplmt^,. aaoites- tumour dellav.-/)-/:/": _ 'v
' ■ './ !Dhe\reaotlpn:mixture/p 50 iimdpH - B'ép f 0 0: ;molee Mgpl2 / 0 * 1 {immoles /of;:ëâqh\:âf m p , (2V25, % 10 ,oOWt8/&lm/j#ole):; Q$p, ^pmolee phoephOGreatine^ 50 dg of phoophpore :20 pg; "bentonite and ,lv5 : mg: of aoetone' dried: powder « M A and DIA were added in the o p n o e n t r a t l o n e in -a tqtal'Vyolume ;of. 0 ' * ' 5 / - ':V ; ' r
1' ;1 Jnoubatione werè perfo3?med atl37^ 2Q;iaimteB*t m A + DMA,
"; The effect of IncraaBlng noncentratlone; of jlMaeef:(=: -:,> :: /;.;'and 'Masepn the% ihoorpgrat Ion of ( 3)173? : "by an : }' acetone • dried powder’ of - tli0imicro86mal":fraGtion/:off: ''f ' PandeohutE ascites :ttuiioiir cellsi:;.:' \ '":f ':U%
':' %he -réaction' mixture ''.was, -(th'e;'sême-as for Figure 5* HHase;.and DIase, \were( - added- 'in-'rthe: ■ C Q lioq ritra tiona ''’' ' in d iq a te d ;.v v l’l:'^:hp -
1 / : Incdbàtiôhs were i)crformed at 379: % r 20 :'minùtëa.: n 1
400i
300-
CD.
60 80 100 jUg. of added RNA or DNA
Figure 64001
Pi0)oPtAS
100“
1.0 3.02.0 4.0jug. of added RNase or DNase
'\'/\'. (ièG;î?qà8e&Ce, ^ ^ oomoeiitratloWi*- -.' >%:>"/ aOGtdhe-'^A^lGd nowaer;:#^
; : y âèg%^a&a#pK%#^^ midogemo%â' MA, qyiphpGphodleiatei^adoa' / . weœ]it lAlqrosômGe* ; ü)h0- ' '-V '-y/::'""'" -y..: ' - .:\y: ' \ W8àÿe(l}fo:e ^ # ia6o%*poTatloa ojT WosGhôô :
\\ ' xl'ymïâ:aWèaGG'\qf^^a .giigiœ,? ahowsvtüt# p%*a:imGiXb \. :: / àtiozi o:iï. the. v'ëm;ÿimèya%â = 0:88^ :lh tW' aWeïïoé' ofy.adâoâ Œ A . ':: / ' y r. oaiises a\gn^à6ual:%eG_#a#.\ih':,#0%p0%'^^^ . -. y yy '"' %hq: ' pze'eehOê. %Q$' hlgKë;e : levelEiy 6f-'. loos'gor'- y \:; C,ÿ/..;y/':'% :aïl\tl$ëa^:oÿ' j^m#qWatioh:büt' it''i'8'/aotlpëable /'A' -y.y':'/-'": .%at-''tW:A#èolhtè:/.;bïoreàeè :my a . ; :
..00Wtm#&yy\%hl8\;%dq8ym-^ - 'y ./'{"':'''y'-'':v'':y/'' '' -"' '-yyÿ/'/.-:X. '' y'y"y '''/-'yyyy'-y. ; yy ' - 'y- fy: D3?0:mo#atlôh^^:t ooüM W ê W to heat iaiaot of . :;c/"y^=:;r..-\.v 'r{y''y#/'/'\y ^ \% ; '.'y:: \.y-y ; . , ; : t #. y ::if It W03?G oaneod by ^eqtruqtlpii of -qâapgeiioizo
:\ ' ;. ]%Â pzlmor a ; greatêz? .âl#e:^on^ .bôoii. ezpèoteû: . :\ betweéh the oüTve^ afto5?g ëày: 5ymiîi%te prezÜïOhhàtioh tliati . . ' / . ' : ' ' .,.y yyyyÿTy/-' : -.y. -- -y' ' ' \ '' y -y ; ' at: .0èroy time^ ,whioh wae'hùt-.:,# : y y - - ' ' . - y■ ' '■ ' - '- - - ' , .. ' . ■'■■/- ’■ ■ ■ > : > -'' ' -, % ' ■■ ' ■ : " ■ ’■! ' ' ',. ■, • 'iffiîie . laeîc ,of lahlbltloa of iH.ooimoz'atloa. of P). : /.■■ B'JJP bÿ :i.ko:fë80Ïmg(/Gomoe6t#:ulQh8: oyryo'üimoiaÿGia IP /alao ■ ■ ‘ ;::-;'tocü?.égilæsÿ a .laék'.oÿ' )78g,%i#B|0àt . f or : a ' M A .px tme:c\(f%5U5?e 8) ■.;‘ simùe' thë # y .Wpëiïdeiit 8ÿm#.08i8 \a iB liigbltÿ simeopt- b
. Vy ■ '
: - ' y.; . /' ■ ; -/y
iblG' tO'7tlrlB./atitibiotio*' y Xlmwits- et 1962g jA) #. 'y . ' .■■'■ ■ . y -\'y ■■■ ■ - y ■ y - - , ‘ -y ■ . y '■■ , - y ■.. yy, An for- 'Hg-' ions is ' inciioatocl
'........ - ' ' ■ ■ y obj-lî igure 9* . Incorpoætlon of '3 ) uiup 3?eaühes a '' ' - : . y , : . ' PS» ..mœimùm' as the.. fM ' -Ion •.0ohContratlon 'ie .iaorpaaecl' ■ to •8 «molos nez* tnbe imâor tlie Pondit ions used ând sroiaazlcas constant-ai lilgter GoîiGentmtipnSf • IPurther faotos^s • affeotlng the Inoorpozation :.of y . ( c ^ ) were . iinrestlgatecU' Piguro'10 shows that:' ’ersehe; and 2‘-iïiQroaptoethanol stimulate the- réaction' ancl that this effect-le additive# In funtiier
, esspeniiiients these-reagents wore routinely Included' in the . réaction mixture-* ■ In • the • presence • of 1%-"*',. Ions ÿ • if'* lone - . ‘■ were fdmd- to inhibit the reaction slightly^ while: . y -V'-' ■ 2 4' ■ ■ ■ ' • ' ' . ■ • •.'iohsiwitli .the raabt'ion- mixture ; at pE showod a.marked inhibition of the reaction# A lov/er pE.was used
-■■■■" p4-y ' \ - - ' ■ -, ■'•.■-, in; the latteryoase as Ih’fy ions ;are precipitated at pH. 8*5* Figure .11 shows-.the react ion-is Irhlhltedyhy spermine and putreecenGÿ. the former ;being the .moi=e effective lolixoltor#- , .. D3ime curves showing - the effect of• rihonuolodruide 9^
triplxosphateB; on the incorporation of DfP iai tliey presence . aiid àhsehpe - of phosphocreatine" and phosphooreatine “ kinase: are shown in Figure 12* With (ol i?) phospho-'.creatine -'and, phosphoa'raatino kinase, only incorporation ■ ■ 'continues yup to 60 minutes t, removal of 'bentonite causes . only a slight losEi of activity dn the later stages of the
:■ y : - . ' / " Xïh'G efxeot of breiacubation of an acetone dried
the mieroBomai f raction of - handAchutz ascitestumour cells oh the ihcorporation of ?) TOP assayed
; ; ill the presonoe and a bsence of added v BWA *
.y;, ‘ fhe réactionÿïiiixtürey cbntaanoA^SO jimoles trls-hOl.
y ; (If4 X: 10 Oounts :aiii/|.uiiolb)y(/Q!P s AIÜP/ GîüPÿ 2#0 pmoleo : phosphoÇ170atinéy 50 pg phpaphqçÿêatino kinase > 20 pgy ; bèntbhite E%hd''/lt5': mg: ox ra e'ëtbhe/ydried powder* 100 pg y ■ ' of ; M A '-v/érewaddedyv/liere yihcli’dated'■ in à total, volume of
:' y% y;- y-yyy yÇheyinoubàtionpywerergpjerfoSaeci at 37 for 20 - minutes# h .?ho ace tone. drie<i .#bwder y in yOr* OlÉ tr is-] fül pïî 6*0 nas;ypreiïioubatedyat;:'37;. / for'/theyîtimë before adding
'' ;:4b 4 h è ; # e a e t i p h a n l ± t u r 0 B - ! .b : — &— -f loo jig h m * :
' :yy:y:'r.yyiT % - - y y : n : / y y y : : ' u o MIA added.
Figure 7
400I(D
bha300
Ph
r - i
preincubation tim e (mins.)
m t m m 8effect of încreaain^ 'actinbmÿoih'D .
coneontratlon on tlie inoorporatlon of ( cx? *P) tWP by an acetone drleO. powder of- thO' mlcz'ooomal fraction., of Lanclochutg aBciteo tnmour cello# ' ' : :
The reaction mixture contained bO . ptK^iéB trlB'- HOl pH 8*!i, 2 iimoloB Ufj Ql2 9 0.1 pmoleo each of (ck ' P) UTP , (2,5 % 10^ coimtei/«iiin/p.mole) OTPj, ATP» CrTP, 2' pmoles of phoephoei^eatine, 50 pg phosphocreàtine kinaoe, 100 \ig W Af 20 fig hentpnite and 1.5 mg of acetone dried powder* Actinomycin ’j) wao added as indicated in a total Yoldme :of 0,5 ml.' ' ' : . ,1 : " • ; V .f V . '
The ineühatiüho were perfbrmed at 57 ; .for ; 20.: y ■ minutes* '
2+ ■Tho effect of xncroasin^ ïfe .çpnGcntràtion on ’ . the incorporation of/X pÇ. ?),/UTP by -an aoetohe'-..driod’'■'/; ’ powder of the inicrosomal fraction' of LayvclBchutb ascites
:. . . ‘ ; ' ■ ■■ ■; ■ ; ■ %: . :
The reaction mixture is the same as for Pigiire 8.;■ ■ ' '■ p. . : X- ■ ;le ooneantj?ation of Mg ' is Yaried as indicated. -The inouhe.tione \7ore performed at 37^ for 20
minutes*
Figure 8
fab
100 •
jug, of added actinomycin D
Figure Q
CD"SU Oooa ^•i-j r j
^ %§ O b Sh
ÔCQCD
300
200
100
â 0 ^.0 4.0 6!ÏÏ 8.0jumoles of added Mg'2 +
: r ; PIGHW; 10: ; ;lühe ofxeot; pf MnOl » êroa'ptoothaiio 1 and: . .
Yor^eno on the ineornoratioa di* ( 3) TJTP W an aoel;onedriocl pov/der of the micro eomal fx»action of Lands chut ascites tumour belle# '/ ' -.'.Ov-/'.;- --
IChe reaction lairture contained 50 imoleg. trieWlOl:' ■ ' ■ ■ ■ ' ■ . ' . ; ■ ■ ■ ^pn 8#5ï 2,5 M-moleB MgOlg, 0,1 jimolea eaoh of ( ItUP(1,5 % 10^ oounte/min/jnnole) O0?I\ 2 pmoleB -;;pliOBi)hocreatine,, 50 j.ig phosphocreatine*-kinaae100 jig ;/Ï1WA» 20 jxg. bentonite- and :1#5' mg c>f- àcetoné: dr 1 e^powder* >KOI,.KnC.igI 2-mercaptoethanol and veraene were added; a.t ..the concentrationB indicated in a total volume of 0#5 ml# ,Heaction mixtures bontaining: imOlp alBo ■containech,- 50 jiraoleBtriü-HOl buffer■ pH 7*5: instead of- pit ''''.."ty
Ithe incubât ions were performed at for 20 V :.; t-minutes#-. . •■- . ' .
Figure 10
r-
C ontrol
+1 jumole KOI+2 •T 114 - 4 M f !
Control pH 7.5 4-0.5 jumoles MnCl^4 - 1 M î t
4-2 Tf t t
Control4-1 jLimole 2 -m ercap toe thano l^
4-2 +4
t t
I f
Control +0.05 jUmoles versene +0.1 +0.2'
I I I I
I I I !
4-0.05 jumoles versene & 2 (Jimoles mercapto -
ethanol100 200
.32
Zi
300 400 500/ijumoles (qî P) UMP incorporated/m g . protein
"■t
: -, : -• ■ ' \ 'y. % - ;' ; ' ■-...■ --y- ' 'V"'' %'' '.''' .eX.'-.r
and rmtroBCine on tlie iiicorporation. bf byacôtQX]>e dried powder bf the ndcrosomal .ffaciion of 1:%:' % /Lands c h u , tumour - cells ; - . ' ■ ' ■ ■ ■ ' ' Â %'\x: C'
liïG reaction mixture coxitaliiBr 50 pmplesy df ; trla^îOl n - ■ buffer pE ,8,5 ' 5 ^moles each .qf ; %01p';emcbj B-meroàptor''' n);\iethanol, 0,5 (mole a : of - voz'aeno 0,1, - pmelea:: of - Ih ÏÏITh, / ; ■ y ht 2 jjiiiolcG. phospho.creatinei 50 ' pg; %ihopifiooreatIhe ='Icinaae, tvh ,h> . 100. pg EîlA; and 1,5 of aoetone:ydfiedtpqwdqr,,:''"''.=ht-'h'; '-'sht hyf Spermine. and putrecoine v/era added in. the concentrât ion s : indicated, in a total volume of 0,5 ml* ' - f t' i ‘ \ \h! : h- - ' ' '..
; Incubationstwere ' norformecl ata57 - for 20 ; Biinutes>’fth .■ / a
■yy: y
■y: y..' ■ -f '
Figure 11
f —
Control +0.5 jumelés spermine+1.0 f I+2.0 TI
11Tt
+0,5 gmoles putrescine +1.0 +2.0
IfIf .
100 3
200 300 400 500p.jj,mol es H UMP incorporated / mg. protein
y 8 5 %". ■
' y .+ .y y . . ' : ' : v . y y + : y V ^\ the roaotloh -to.'! 8 low{.âovm '^%%piY' a ftë r. a
; aWemoëhpghDbOGphoorea^ \ i ..kihâéa:ytherë'))iÈhaYdra8 t:iG'\redUGtiW( ih h
. of ' ' hMaxl mal ..ill booii^a'
tlio rq l8:/à:'.deG iia0/:.i^\:.t^^
: d f y la W lio Ô pol3f%'lb%>6udidotMG.:'-\:h:/ ' ..f.- . ht:-: ':rh\/;/%.fh/^m'Tpf^eeenoe '0 f f:'AUJ3'ÿ GG3?. miê 09!ï? ôiiimulatG - the'-iilooimbration
. • ■ • - ■ ■ ■ --■■•y' ■•■'•'■■" , ' - ‘ ■■ y - \ .' ... ' ' :''.y..ÿ : - '■ . - " - y ■•■'.. y - ,
y s lig h tly , tmâ' loBâ:: to , a. ÊlüWèÿyâëGllh@. fz-om. the. .msÈ&mii ': y-yir :..yy: :;:y'
.:■ . Il’he ;'e3Stôilt:.i:&oy7?hlqh'. d:6gradBtiqhy''OfyaG±cl."'soltLblé jy-y y
, z'iWïi^wleotida'È ; liiy"tW.yÿ8GQtiozi' . podu^ : clüÿiîig. yy y r, yyyyyÿ;
y .:i6o#àtiQa..wà8:-'03:âiW.i#&yûÿyiàeàst'6lhgy:thé ;'& làtribht'i% Ëy:q.‘y-'-'.-y"' y-y- .yyyyyy y - y .. y "y ' %: ; yy; y yyvyyy '.yy-yy.. y y . - y y - y + y
./8 ,egare)yke,â'':oh%'6 imtogW3;hÈG'a] ^ ^lâ,'apmmèâ: A o tlg ^ ■.:'; y •;':
./:(àëoy%th6 â6 .9'y;#eoti6m:'6 )',y .4îhàtyp|iQàpfiq+hy' y .yyy
'G.i'eËtlme y8héyBhQ@]jhgGi'e0/bïiie\.kli'i8qéyyià%8 lly 'yy ./ly
. '& è g r k # t i6 m y . .6 3 f .yy:(o6 '%.%P ) i 'M’È : Wz-lag.' tîië'', e 0U3?së-; ô£ y th è y geéotlôh# . '\y.yyy:.y ..yy:yy<y^V\Vy;:.y 'yy^ :y: :yy.':ÿyy^yy+y'y:yyy- ■ Xia> th@y ahsëhGGi.oÆ-'.phOBgho'Greàtl ^ pliOsjjjîoça'oatiae. y;\y. ;. %
kiîiàse "addi'tioa.yi'df âïïî? G-SÎ’-" and Oil’3? càhsè'S’ à;,‘ èlowea.” dég:';ad~' ‘y '. yy : -yy., .;y;„_'y yyy-yyy y.yy^: '.y y .yy - 1 " ■ '■ - -y y-y"'-- -yy.y -y^y
'■' '■ ■' '-'-. y ." yy:yyyyyy''y y', y w - ' ' y^yy ' "..y y • y ; y. : ' . . y ' y - : - ]y:yy. y . ffilie-ldôËvtiün ù f '+ap :;UKP;':K‘è3MtiQé rhioosroo'+yited. iiitoy-. : y:y.y - .. +yyyyyy:yyvyyy- 'y yyy' yùy.yyy.: ::+.:y.yyÿyy y+/ .tb.e aexd-:lne6ltih.Ley.:. ûj:fOtliiôu wa& then-stvtdiaa.» .■' .AHseilisa©' '
y'^yy- .y " -^yyyyy-:y'; yy"y .-w '-- '^ y " y yyy-:.." " ; ' - yyy.y'y.yyy^yyy; ' h3rdi:oly8 ,i8 \::eol.ea , tlïë qÿ ■‘yS’- ,o£ sh : lnooï^pq%'ated:.z?ibomiG#0«..; :.:.-
ëiae -S'ytïdphospimtèy.ôB.' to :the: gy-hydi'i'BC fiy of .,tW . " y y ‘ : .■ ■ ■ .
pÛÔlëÔ8i#::.Të,B^ uh ^8hway/;lt.,48y.:/hy
'r/'' :po;a0lbleht^^ ;âe.tegj;hthqhe%#zrky#^^ -, f h+y
"y-;-' - '_W 8l&uep;% ro.,#dgaGèat\:$bh:o#i0ÿ
y- ''''\h ''''ribpnw% e9tiÀehpro&W tvpfh'|^hd,:rea^^ '%
y- , y o f ',:P% : (fro m mi#a y.or lesëyëq^^ the yy ; ;
y- .y. ,.' . 4. %'#%™A3.e:o80G,:.#OR#hO8plm'üë§^ ; : ; y;'- : --’
y)': " ' y y-;lB4 d»go_fey’oioa;yp£y.an, ,4'-'is:'l'b ^'--''livlphoBpbatë@ y fôitl; ■ y ■': ,
■ - - y , y'BÿiîtîièSis -of G /.n tp :c h à t lt 'e lF iy i% :P®yyy'yyy:
-y y" ■ : : ylS ; Ir ^ w l lë d ÿ iW m a t l# : 6:g;:ÿ d ly yù: elmzüis ; ls : . ■ ’ ’ '
'- y.. - y y y l%dlG#-Wd *;y', ' ■ y'I».y t|xQ, yi-attè^yease-» ■ys pm® y ■ iahpiy sppoaylïi'g . y - .y y.; y
y y y , - : GI#y - l M i p # # y M pfy pplg- .n +30Ciuenoesyy- - '-
y,’-- y y .. y ;.tp,,;.tfwy eïïâa- 'ofy'mclstlmgy d ïs t r lb i it lo m o f . ".yyyyyy,.y.;': y--y^p^yyy - , - y '4 y ■-■■y y.•.■:■■ - yy .yy-y-y-y/ ' "y yy‘y:yy:yy;- -yy:- 'yy/'-y^y
,:::;: y+'P. a$oi%Bt ;3,abBiyi@#, ÿlbomÙGlpooiâë :2y;;: (5 î : ) -ÿ ^
. r'yyyy yyy; Wàa ; Ozamiaoi :l6yyt%e, ppodwtbyyof- 3% iG tfbh yMaaWÿüs c o a ta in ln g ' ;
y- :. y y- y + 0 4 ? % ) usi?.; Apyi?yr:-Xpd>?y'A^ w%y ypi;iW; # ;A w i& y xhcp .p lus y y ;
y, ' ; ; : .■ g -Q-üB ® id OïSp. w ith bptl% _thp mlorôSQiml... f %Q.'kl ah.â. .'khe . ' ;
: ,, -.y. ; yy. ; .yaeétpae . àrlëay-.ppwaps? : p ï ’épa2:;ett. -f rpav' f t » ; '; '- yShe.- .yeSultsy yare. ' ■ ■ _ :
0Î:ïow.!l i ï i Salîlë'■4»y ' V&oth.e% . # ïO ;.ïA loro86W lyfm #-G i(m , 03? the . .
driedy ' d e tp o téé :m t h e ,d le t r ib u t lo a amPag8 la b e l le d :h-h- y ':h . y h : ; .. \'x:y:'h rlb o a W le W tl& e e ï3i th o y re a o tlp h prpdW'W,îf.W%ï (<^
' ,y y'hh : ' r ' h%p' ' f : - %, : :h ' :'/h:_.% \ ' y h
' - - ' ' '0\ ' -:(y%!\)TrmohophpGp3q ^ /rp im atlo ii. q f a : '-h:'
-yh:' '.. y /ypo lv nhpliâinÿ.. , ô.ëht è f '-i^hey radiè'^\. :/"\'y-
:, " ' aotlŸit'3T':wae reopvered yla the , o th e r rib pziùd lôôelâe 2-V (3®-)~.y; . . y
.. FiaÜRE 12Time course of the incorporation of ( tlTP
by a miorosomal fraction of Landscimtg ascites tumour ycelO f - showing the effect of bentonite ancl nucleoside trinhosr \ - phates in the nresence and absence of nliosphocreatineand nhoBphooreatine kinase, ’V : i ■ VA'
The reaction mixture contained gO tris+HCl / h :buffer pH 8,5*5 pmoles each of Mg0l2 ' and 2^meroaptO"^ ■ f-Vethanol, 0.5 hmoles versene, 0*1 jmoles ( W K -VV(3*0 X 10 count s/min/pnole ), 2 fimoles phpspho or patine V ; ;:A (PO ), 50 pg phospîiocreatlpe kinase (PGK), 100 : gVîUÏÂy v V;V;'20 pg .bontonite .and 0*76 mg of microsomal proteins;1'+V ; "Removal of bentonite, PC and PGK and addition of 0 .1 .pmoles each of.OTP, ATP, OTP in a total volume h f yAV/y VV'':.::AvV 0*5Vml' were made where Ihdicated, - ,\VA'''VyVv/V
Incubations were performed at 37 ;for the timeCindicated.
(,6c ;p)y+ip ;+;pc/+dK. yy y(oh®2p) trap + Pe/POK - bentonite, ( ot? P) ÜTP + PC/PCK + ATP, GTP,
OTP. y : y", : y( oc52p)' UTP + OTP.( o J ^ p ) w . ■ +;y-
Figure 12
600
PîCD
500
bhS400
Oort 300
CQCD*3 100 IZL
6020 40Incubation tim e (mins.)
PeroontaKe of ( cKT"]?) UTP remaining in the reaction iaixture after Ihoubàtlon \7ltl1 a microsomal , fraotlon of
- - - - - - - - - - - - - - - - - - - - - - - - .... — j f •■ ■ • n r r r ^ i T i T T i ï ï ' - ' r i ¥ ' ~ [n T ' - i r T ' n T _ ' ^ ' i ' T — 1— ' " i iT r l ' T 11 1 m i i r i i i i r ' i n 1 1 w i n ¥ i H > f ) i 11 < 1 1 m i i im u r f i— H " ' ' '
Igmé8ohüts asolté8 tumour .‘OellB for -iribreasdnM time 'intervals$ / ^
>eac^ ion ■■mixture vwab.;ijlie .ÏBame::;as>' în-- Figure 12. The aold soluble nucléb tidee were : analysed:; as i;deecribed: in -:the Methods $ \8eotibn;:f7B,::, :, : : SysnbolB as, for-::F±gurev:l2 . ; -r ; ;
. Figure 13
100
PhHP)Ph
CO,
CD
I•I-'a S
4-4 fnO O• r ^
S ’S• r-J CO
u
(Dapjoa
.3ICDfH
30 45 60Incubation tim e (m ins,)
ÏABX.B ■• ■ 4
. : I 'îhë fllstrlBution- of .■ P amonft'st thé rlfeoaücled-■ ■■ n , r., i r . n . - n r f . . ■• . . y ■ r, , — - - ^ " ' T ''' ( j l " 1 1 ’ ° f l T ~ Y 1 l Y
e.±de.-'M(g l )-monQ plio s dhat e b ' af t er : lncor p o rat don ■' o 4 rO'■ :by the ; mlcroBomal ; fractibh âv an nade-tbné : A ' '
dried^^pDwder'-tlieraéf- - from I*and0ohntg aêciteÈ : tumoiir :oa3,la vafte3? alkaline , hydrolysis df the proânot RI A %
; ' : •-, $hë 3?baotloii mlxjbWG contained 50 i#pie8 t ''>-ày\'y,C W f f en pE 8#5p 5 pmole a each o f Mgpig and 2; meroa p to-- :' y_e t3i€mol|d'0 *5 {iimole a vers eue $ 0 * 1 phOlcB of (oc ETPi;;
: i:.': (IQ? qounté/min/pmple) # 2*0 ^molea pliOBphbpreatirie ::; / 'r50 pg ; phôapbooreatine kiiiase y 100:, p,g .#ÎA;yëo) pg bentonitei^D
y/y%' and 1.2 mg protein» :: #ieréi 'iW ;: y :\ Of? ,we3?e added in a total volWe of 0.5 ml,:: yy’ ; ' ■ y ïncnbationovfore performed at g?. ■:- f of ■'■y,2ü;
.;; V üiie reaction mixtufasKwbre cubjeetod to tho; ; : y prdcedU3?08 described /iEdtEp Methods ; section TA •
Table 4Reaction ]Percentage P in each nucleotideconditions CMP 1 AM P GMP UMPM icrosom alfraction
(a^^P)UTP 3. H 8. 6 10.8 76. 7
(a^^P)UTP+CTP 10. 5 8. 0 11.2 70.2
(a^^P)UTP+ ATP, GTP, CTP
7 .0 8. 6 11. 7 72. 5
AcetoneIried powder
(a^^P)UTP 6. 9 12. 9 8. 8 71.2
32{OL P )ÜTP+CTP 8. 5 2. 4 4 .4 84. 6
Q 9(0! P)U TP+ ATP, GTP, CTP
7,1 7.2 6. 6 79. 0
pax't ' XeïïgthGîaing'i.ofi-ïjr-Qf-’e iïis 'tia g ■polÿ;ribôîmG
aïi$' ;ter3i:W-eyy/%al)8lTlRg' o&' the JMA-f'.- ; .ffiülsvpat'bom •■ ' ".
/ ^yabelM , iioi:'. g re a tly ;gîMi secl vfWa 0$P; ;o:i?iifi!lî?s GÆS ■' -' '
■y.&mâ’ 03ÿr m lstiiro-s; ■ • I t Avould/; . ;
-;:Be&myy:thefefd^e;g :ïm.:jor p?:'0&iiQt ;$i# -m'daotloiiB
w ith : the'm iioronoiaal fï?àd:yxon,^àiiü,,v/ith tii© 'ieetoae''d :cieS .:: ’t ryy-y-; ::yiryy:-y y y r y y , y yy .ypoWGr' :W; a\ polym or . llbw&ver'n
y thè.sWÿnç w itd (do ziot . : th é . f oAW blbh o f \ W a ll amoimte .'
; -o f - opht&i i i i i mg' all''!y4':\:';ribpad9 » - -L .In fa r th e r ozponim w ta' tW ' liïG dnporàtldhy.ù
. W olèoàl& e' 'w ene 'd tW lod ih 'the;/abAW oe o f .;
;. ai#:':othea? rlhonW ld.oa ldè Si--t3;iW qBphatèo# B o h to h lte - wàu.
;: 7él8b o m itte d . as . I t had' hb" e ffao t.: om 'the; iz io o rp p i'^ tlo ii 'o f
/ to Sp mlhüteB.^ " . '
/''- y/' /tvy'y';/■■''i ' /rÿ/ÿ'f and'.,. .'-
\;k)ro\dc:'jin'ctrily #.6dr|)P%^ Ineolàbloyj;: ; . / \iactorlal.nmdc3/.:th0 ,Bame-re#tyi6n;ooBdlt^' . / ' / ' Zi^nrth0r:'hhafa6torieàtlbh:/^hdiWd that ' 'of. - /.. " ' théBe::rlbonWiébtid8B ia\% faotpro -wd
.in the-;-BBMé - way as tho' :biâorporration of (oA^%) .IEÔ1?# Sable .. ‘S .ohowa the offeoto of Hhase^ Dhasog aotrhiomyoù.a :0g MA^.' ionB - phoepîiooreatWe and phosphopreatizie ' Icumsé- •..: and ribomiolooAlde 5’4-txdphoephatoB’ on’tlio : incorporat±011 of 'h: m u , %I 053? , %I om?' and ’K 1ET+ 'J’lie exteirl;:. of the inoorporatlon yarloe, being groatost for AfE, least for
■ pfj?; and roughly eghE l, f0%' and BIT# . fhis is also-.Blipwn': : by the. tiBie oafYes..,for the Incorjporation of each nucleotide
-(iri'igure 14) and the, dcypendenoe. of the inqorpoi'atlon of each on'protein oonx3entratlon .(]i iguro . 15) f fhe depehdenoe of
'■ rh rdhonuGleotide. incorx>oratioaa on protein e one entrât 1 on is ' ialmost; linear up to 1 mg protein per reaction mixture» :. .
heimlly X#5 .mg of; acetosae dried powder ; ooyrl;ainlng 0« 75 ^ '0*88 •ïïîg of'protein -,were Padded'per-;tube and':moat of the results .
".^presented were -pexfor^aèd at onBÿme conoonu5?ationB on the /.A ;i]hnear portion'nfy-tte, ourv'e#;' . . - -..' Although: all 4• ribbnuôleopide' ; 5 triphosphates couldhe ineorporatG,d. into polyrihonuoleotideB j, it is again ., striking: thau thbre Was ho atli^ulatioh. of the uptake of ■ any one rihonuclootide l>y the other 3 oomplementary riho*- ■ ■
, huoleotidee:/^- . Since this : might have hoon- related to the • aubatrate-'yconoontrationj'- the incorporation' of, .* 11-À.fP alone:' at WoroafôingiooncCntration end in the preaence-. of .(gîP ' Off and. BSP at, aiiailar inoreaeing 0 one entrât lone was inveotigatedi
f n't:: 'h;:;The effect of HHàse # SHaéë, aotlnbitybin P »
, DhoephoorèatIhe blue phosnhocï^eatlhé-klhaBëf;and hualéoside 5^— trlphosphates én the incorporation ofeach of 11 ATP. OTP. driednowder of hantdachutis ascites tmour cells » y.ityK'yX'.xr:;:
' ■ i-.-iThe;:reaction "mixture' oontalhed 50 i-hmolea';,trt "buffer pH 8,5, 5 pmoles, each of MgOl^ :bnethanol, 0* 5 p,moles yersene, 0.1 iimbles o f o r 0TP or OTP or UTP, 2,0 pmbles phosphooreatinei(PO),50 pg phosphocreatine kinase (P0K) ,1QC) pg 1*5 mg of acetone dried powder * . yv ;i;, ::
Where indicated MgOlg phosphbcreatin^^^^ ■.;■creatine fcinase and KÈA were omitted '^d 0,25 pg HMase, 2*5 i% DIMse, 20 pg actinqmycln P, 100 pg PNA and 0*1 pmolcB of each of the other unlahelled ribonucleoside. 5*“trlnho{3nhates ware added; xhi volume of 0,5 ml*"4 4 ;; Incubations ’ were performed at 37 for 20 minutes.
Table 5uumoles H ribonucleotide
Reaction incorporated / mg. pro Leinconditions A TP GTP CTP UTP
Complete system 518 97 313 204
+RNase 81 47 76 49
+DNase 539 80 300 172
-R N A 379 68 194 91
-R N A -f-DNA 398 53 154 80
2+” Mg 4 0 0 0
+Actinomycin D 593 83 306 212
+3 unlabelled 416 43 226 101ribonucleotides
-P C /P C K 214 51 226 96— P C /P C K +3 unlabellec
ribonucleotides ......... 29 208 73
f A s 'gib : - FiaUHk 14 '' / _
’; ; V ' The time oouroo of incorporation of ATP, C-TP*J , OTP aiid UTP by an àoetone dried powder of tho microsomal
:fraotion,;of laiidschutb ascltes tumour cells >
y;The /rGaotibny mixture; cbhtained 50 pmoleo tris-HOl ir Mfferipli 8;,5-iy,5.;jM3iol8B each of îfgOlg and 2-meroaptO'-*
ethariol,"; d.5;|imoles versene,: 0,l':pmole8 of % ATP, GTP,■b-.g'OTP ':or UTP, 2*0 UBiqlea phospho 99 Ug: phoopho-
:i\y'ycrGat ihe - (kimaee'^lÙO;ypgi,UMA-yand ' 1*5 mg of acetone dried' powder; in a totol yolume o f : 0 # 5 ml # ■ ‘ .:r f ; " : : " Inoühàt ion s'w# f e f performed /; at 31^ for the times indicates.
ÀSP\'. .■ ' V ;■ ;'■':;a: nTi'
CÏÏPGÎP
Figure 14
S 1000
r-(
O Q
I—I
20Incubation tim e (mins.)
üîixo effect Of enssyme concentration on thé '% ‘ -: ■ ' ■■ ' / , '■■■■,'*' ipcorporatiQn of A9i!P, GTP and CTP by mi àoetoné dried
ppv/dor f%'om the, microBomal fraction of lianclGohutz/-o.BcltoB tumour ooIIb * / : ;
fhe reaction mixture contained 50 :jimoleo trie-EOl buffer pH 8.5, 5 pmoleo each of IlgOlo 2--mercapto-^ . ; ethanol, 0*5 praoles yefBene, 0.1 pmoloB 6f - H ATE#03?' OtM?, 8 pmoles. .ph6ophooreatihe> ■ 50 pg phoaphpcfeatine ; kinapo, 100 pg IIHA and eiigyme ao indicated in a to Toluifiio of 0*5 ml * :'■'x ' - ' -■■k./'-k."'; /% ; ' ; v;-:k'-\'; f'';.i;
The incubations v/ere performed at 37^ for 20minutes# .. . ' % / ■> ■
H% ■■\ n OTPv % : = 1
Figure 15
-§ 10004->
500-
oo
1.51.00.5 2.0mg. prot-ein / tube
:eiG%IRW 16 ' ' .offaot of rmbntrntîo ooucantraMonontho
'lApo#pj::a#6m": o ' ii .fifCl- by <rn aootone dried wv/der of ' tiic wiorocdmal - fraction ' bf bclmts apoitem tùhb'ùr% 3 % " - - - - ^
vy/:;-. r,' %hO'/;'reaotlon miictur^ ooiitainou 50 junol.eo trin-HOl %ffbrv-pH’ 8#5.f•■’;5;<pmoles each of rmd £*-?ucrcaptcH
V ethanol# :0#5 imolcb iveï'ééhc 2*0 j»malca nhonrJiooroatlnu, ': 50: Mg rohobbhooroâtlhë : kish ,100 Mg ItMA and 1*5 mg of• ■:'; : . . , ' '■ '3 • " • acotpnc dried :pbYfder; ; \%'% of h;■ 'either. in. 'the} preacncb: .b of cciual ooncontr^itloDe:. of and I Î T P varied;;a8 incliouted in a total:lvpi™è-::of''0.5 ipl\. "
Inoabationô'^mirelimrforjacd at 37 fur 20
A— r % A'i'P+ c®r, cwr, mr.
Figure 16
' Ù ~
su• r-JCD
bû4 400-
CD"NShOAUOOSU
comQ)01UL
150 200omjumoles H A TP /tube
100
0Gt of pH on the incorporation of ATP^' GTT and 0Ü3P by an acetone dried powder of the-microBbmal \ /fraction of Landechutj^. aeciteB :;tumour cello; \ /- /f''
ü?ïie, reaction, mixture contained 50 ^mole8 tr 18TH01 imf fex at the pH, indicated ; ■ 5 pmoleo each of HgOln ' and/ ■ ■■ 'a-- ' C': \ ::ar . a ..2 -me r c ap t o a than o 1, 0#5 fmoles .veroene, 0#1 pmolea_ -H . ■ - nuclootide^ 2,0 pmoleo %)h o 8 pho c r e at in e 50 - |ig;/pl%oephO' / creatinetkinaoe100, pg RHA and 1 # 5 mg of n.eét6ne dried = powder in a. total volume of 0.5 ml, f •' '/'r
The inoubatione %vere performed at//37 for 20. / /minutoQ, _ ■ '.r ■' ‘ ■'/■/■-■ ;v\-v //-/.""f/:
- ":5,, A3?---é— ' % OTP. - 0 \_ .% :G# ■ ; ■..
■ / ‘.
A-
Figure 17
fajbaQ)C\j?HO&8Ü
400
oCD
200
fHWooCQCD01
10.07.0 8.0
pH units
. . :gABL15. 6 V\/: /v:_, fl'ie effect of -pliosphooreatlrie and phos'ohoGreatine '
kinapo _ln the prosezico end abeenee of . Qompléraentâry ribonucleoeide 5 — triphosphates on the Incorporâtion - - : . ’of ni ATI?* G-f]? or Off at pH 9#5 hy an acetone . dried . iiowder of the mi ero e omal - fraction o f . hand a chut g >f : ; hf ■ ■ ^aacitee-_ tumour cello# ■' ■ ' V; ■. "ri../,
53he reaction mixture ' çontaiïied-S^rtooXes-of ' tris-HCl- - buffer ,pîl 9.5 * 5 hmoleh:each of. : 2T%nercaptO"fT . f ■ethanol, 0.5 imolesf of vprsenefiumoles . of : ' f '•OIP or 05?P 100 hg '.'of/ IhiA a%id ^ acetone'; dried ’ -f > -powder# Additions » ydierp; ihdlcated included, {2* 0/^^ ; , / ::photphocreatino, 50 pg-plioBphocreatine; kinase and; ;0;.l- . f . ;p /pmoles each of the other imlahellecl rihonucleosidep; . ' ■ p. ; ; f:' " 5— triphosphates in a total volume of 0.5; ml.% / p ' / _./ - ; ' k. -
Incubations wofo perfomed at 57./. for ; 20 minutest a / ; v , ; ■
Table 6
Reaction jLijUmoles ribonucleotide incoroorated / me. orotein
conditions .. .. ATP. tjrT 'P CTP .
Complete system 181 79 213
+3 unlabelled ribonucleotides
171 67 223
+PC /P C K ■ 427 178 307
+P C /P C K +3 unlabellec ribonucleotides 414 79 239
p/cjOïiooa'î>rai;xoa at WM<p.;,Mw
'//7'alîfeibltlïti'éooiirij ; '77/7 -; 77"777-7'': ' ' 7---7---'" ,.SlîQ qgge# # 7j%':; %77&é7:Wÿ'#go,%01bm7:7 ?B/G®? cad - n <j@ÿ71 7-0ïp%;%:'lg':%tg
ATS ohov/O; & lio iuoov-bro?ÿi/i&x/afv7';;;-^, -%;.0KU üM % ü$#,\ qvür tho W- 9#-:7s7:i.:--'but ohovf DO oloaï* ïasKiîiàji. She clii’foroncoa iii. liptoîïô
.;:'7: bOtWOOÏi 'pii 0,5 #pB:,,#p777::-7,%' 03*3?: i#fes-7777;'’
;,7:7mM ,7^ ;èÀbu|#i7f o7MaÿMî|1î7-??(îî33fe
:ûl0to*'ûed7:M!:,'W.g.W;è.7#I :(W:,:W7 ^ 7 ' 77-7:7-;7/7;pboÔ^O0»éàtlàe,:-k#ïWà'}7À-7/o'&b S^fMlicapAolQb^ ; - 77'-',, '.Igiiiaooü, ' .;7--#iŸe#iË#W%% WP##,)!:' 7'7.,.. -. - 7.7' 7-; ■ / :pl#8B.W'k8, 'éÿstèaï''st'imil®7fi©ct- .tîie7:ân'obî?|5Qî?at :%, -, ;7:'-'7'7t677tW oztoat at ])li 9»5 ao at pil78'g77('#(''G ü'obïo-;6), ' '-- -7:7777/
77' 7'-/7'77-77 7778'l&oa" o o iia lâ o rh b lô ' o f -z 'ib 'o h w P O o tl# .' '7; -7/7 ;
7 ' " 7 7 ' o b o W & 7 ) # # ' ' ; i à m a b u o a o o o f a t l c l o d î M A c g f O p u s a a b l y d ü 0 7 % 0 . •.■■::7 / : b ; ÿ 7 è M o e o n b i i . n lii'JA l a t i i c a d O D O f j b s a G ^ i / i t ’. W u u o f t o ; . ;
9 0
’ ï : / * ' ; : - 7, f 3 e é ' . v Æ t ô ' i i l i ë r ’': € a e ' z d l ) o $ O m s l # ! & ; ù a ? o t h o z / - h l g h / B i o l G O Ü l a r ■, ' 7 7 7 7 7 7 ' : 7 ' ■ ' 7 ; , v - . . ; , - ; y . - 7 , ' . : . ^ 7 : - 7 . - 7 v : : 7 : 7 : ; ; : 7 - v : 7 , ^ , / . 7 / Y / 7 ' 7 7 v -7 - , ■ 7 7 7 - ; 7 \ 7 ' 7 v 7 7 7 7 /
; ' ' 7 ' 7 : 7 : - : 7 , ; , 7 W g i g M , ; I ï 0 À a s ; a , . p p $ i e y ; : , b p o a m i e , / W i l l e d : 7 : ' ; ; / '
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:r--f/ Y't:' bYv8#A foz’ Yliv : :.:y ::■■ :U>.dfeatop rsoriie oyoc3.floity in,the jJX':iraoz* for this lut'l'or ■
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" , S I k ï o U a l a l o j i i g t u o - f t i a o l i o m o ; i ? o l y r l ' b o n u o l o o t : . L c l o
y:Y/:::yyof. t&' YnM#%;:X'ëQWY#$ V- r yS-Y;Vit
. ^noptloa ®ror£iga;;o| kY3 fit’-
l Y l Y g Y . . . , , : : : I ' f . f ; :Yli:.ibaoeo ieam- tlmi:fo##®#, noi, A bolAg the , 1%m tho gll : ;
; y'yt "y:; XyA/'-y '
'■■-■"V \. A : ooiimeirlBom of thé ■ effect bfveasty 8 ^L yeast . highly polymerlsod HlA .-ahd -asGltee HtimbüÿÿMAkOn --/ , •y, ■ the Incogporatlon -bf YiyQgFyTtir: aymiafOscaa?fractionY ' : of JiaBcIspîiiitFY asoites timoiir- oeliLoi' .
A- Y, : ü^Ixeyréaotioh mixtÜTO îîmolaG trlG-3101Cy'y-buffer : p i iyB »5, .5;Mmci iqbYeaoiÏYpf yifeoif Yaud 2-morooy)tO'- .
, ethüuol^ 0*5 imolee veretme, 0*1 pmoloB .11 01 3?, 2*0AY'YYx <: YY:;:::YhX: y: :, fiaolëé' phoephpcreatIbcyj. ■ 50 aphoo3?oatine kinaeo-:::y; 0# 67 mg - of microsomal protein. .Yeast sHM, *ë pbmmG3?oiaï yqast %1A: and itîmour BîiA v/oro ailded .f -where:indioâted in a total volume of 0*5 ml#
Y/h'y \:‘y'ÿ Y ' : XnoiihationB Were pbrfprmed' /37 ' for 20 minâtes *
: hhh'jb:/-, '.. .. odÊmércial: ÿeast M^A. . t.
ii'Y'Y {r'yi/::'y% :::;\f# .... ' . . :'-A-- comnarisoh of "théeffect--y'Df-veàst s M A and :h'tvy.-: ’
' aBÔltee' timour R M 'àn ' the '/Incornoratloh ' bf " % --%? ' W - à I K d %.. % % '' - //;; microéornai fraction of landechütis aebltes tumour .éeXls. y . "y' Y
-i. - Y: À!he réaction mixture iwac / thb êame'Yas described..fbrh'hyt;/friC;; figure. 28 except 0*I;wwlbs;;r^H:Y%Piwerëiincprporated . Yeast bRMA and as cites . tumour ':Mhi-wo3?G. added Y.aa . ' 'Y'fhhaioatèü. y ; y y  ' y
' ■ jneubâtloho werb-iperfomed YàtiS?: for;'2QYmihut *yyy-y; Yy :, Y:y :Y:'Y:yYy..y-Y;Y'#^
COod
CDFh
ooLOooCO
oorHo
iT3CD
1
uio:^ojd *Sm/po:^'BJodJOOui d M l so\ovaTiri
CMOCf, CD fn
Oo
o ooyH
ooOL O
iTSCD
1
ux0: ojd \ ui/pa: 'BJodJOOiu jx tO H SQiomrirlS
The cHain: length of homoDolvribomuGleotiâeë : formed hv a micrQaomal frabtion from handhohtitz ; i aBCites tumour oelle# .
The réaction mixtures odhtalned 50 8 trie-HOlhùffer-'plï 8*5> 5 pmolee each oi 2-meroapto-ethanol, 0*5 pmolee hrersenei ^èd ^mol^ ,50 fxg phosphocreatine kinai^e\,%WA:::andj0#8:-ing I ; miaroBOmai protein* 0.1 Mmoles of % nucleotide and ÂTWt GTP and OTP were added v/here Indioated in a total volWë :of 0.5 nl.
Ihoubatiohe ,were' ::performed'{at' ^37^drthe' ^ ' timee ihdlcated, elkà^ and reparationof rihomicleotides from nucleosides %/as performed as deacrlhed in the Methodssection 7B*
Table 7
1 Chain lengthSubstrate L Incubation tim e (minutes)
15 1 30 60ATP 2 2 2 or 3
GTP 3 or 4 4 or 5 7
H CTP 2 3 3 or 4
H UTP 6 or 7 7 or 8 7 or 8
3 H UTP+ATP, GTP, CTP 3 or 4 4 or 5 4 or 5
À
. ■ „ . : AM;.*, Œ6B ükP ip s’Oiiglily.
i "%&& ))ïm8i)hpG3ïëa'biïie' k inase . - ' i'rXi;■ • ' ' ■ \ '.. '.: :‘a4';laiv:ia%e '# 8 '■'i’e a & iib a ’- mà&;.'üü iïOu: :im iT ib 'ït c it^ / i t à'onearo ' : ' > .\ v■':■ :■> ',,; ;■ : % r "" - 'cA.v.vi-'v.-
'A; ; ÏA i- A A Ï : ... r-AA A ■A ';-:AA ■ A ■, . ' - a,A-:a: AAA'- ' #bo%RQl@A^#ûeA5j—# 4 aa.t &l;pho8%)hate8 ai^é ■
, .' _ th e A g q tm il - '-. A
A'--', pî'ia.tès a:m 'bhe -t,m%e APfïopIxo'fApîfeto-À' 'AA-'
' '. , ■ Yxdüiil ' bé- oz'oeotsâ to;. z ü ïb ib it thë ' i'e a o tib ïï ..ràAlïe^? 'üüaa ' ■ A-"’ - ■■''-. - ' ' ; ' ':iâo.?sgaiQA 0;i’ t.Api)îipspM ité.*-: '2 4''-BpamB: t lié e f f e c t s Aôf A -) A A
îneyeasiag , eoseen^feiAatioas'û f A-àÿfOPîiôsak 'aîlû .ortliopiios-DM te!. ■.AAa - :■ .:A-:A: AA'':AAA;%AA*^^^^ ^lA^ÂA ^"AA/AAA.-A .A-AA.-v :; AA!.': ‘ ■■-■■.-.sa; .tho.-:lào0%*î)O30àtioa, o f - 'îî .OuD?# % %- O rthonhosphate '-ezCGi-tB' : - ' - -A - : AA: x;: . \ ., A.AAAAAAAAAA:%#AAA:A"AvA AAAA.aAA' A A - .A"-, : A, A. A-. AA^ : A. '. ' A'-'- Aa %-aA . .•■-.■.■ ■' : dm ly::a .v e rÿ :'.p lig h t%e f fë q t : on.' t W ; &3xoùz'-rooz^ation. ô f - %î IHDT-:'/:A'\ 'AAA.:A-^A;AA/AAAAA\A: AAA^\A;A'AAAAA::AA^ ' :AAAAA\' ■ ' - ' iv h lle pyi-onbosnbatc :Ca'ü808 s 80; pea:»,o^eati ia J iib it io 'n ; 9/0 t î i e - ■ -■ A’'-. 'AA': a A-.a 'Aa '- a -A.,; - AAA:a aaA'AAAAAaAaaA. .AAA.A'A. a 'a '-- %A- A-A- A- ■ A: , : a -^aA.; A ' ’■ '''MasiiïMà'- ccp^^ .A : 2&e Wâ?ophoB&6te' '■- - '.: ..-'AA'AA'AAA’'-' A-'AaA:.:À'-4:PA; aAA-' -A:.‘,P:;A::A:A:,.'A''''--AA' . A ’''A-'A--'-'-■g4i''A AA'*' ' - A - - ^
' AA A.Aift. |îs0.1g,vS'ûllxi*wx;onA;';tb-'p3-A ' ^Àà -rMxf .'MgA -i.io a - .. '’>. ■ =
'.'A: '- ■ A.-A .AAooxiodxït%atioxiA:WA*thcÀ4@àCti0àAi#zt%A8 .A ;■ A aA:
A A:. AaaA ' : -G.oaGënirà1Aiç:p':A%.a8âAA%A thë, .eiiséaÿ^ .-thÇA'iWzimn# 'ApoBsible. 'A: A'Aa A
‘"'. : -, .:" ih ë t: ' 0011I&: Ijé'Alisecl' wxiiio.p,t-‘. eax:s.’siîigA n fe ç ip tt 'a i'iO î'i'iô f ' '.■■"■ ■. , ' ' ' . ':,■ :A AA- /AAAA-AA.AAA'A.-: A;.-.;A\AAAAAA'A:AA.'.AA:AAA''-AAAM ■ 'AAP:- AA'a''"AA-"-A.,:.AA'A; * ■' A
.A ' -. , -, .'/pyfopîios|ÀiS‘';:aS--A*î '. 'A.A': - '\.:A'-:-A . ,■'. 'i :A ". ' a A A-’
, -A i: -, : : A/-' ' A fh e ' o a ta lyB e ê AimA tüe Aiâcrogomial- èmagxme : '■‘; .a;A- . A A,, A-A;;4A-AAA-AAA--A;AAA-A-:r'AA;A;;.;.-A' A‘ -* ';-AAA'i'fcAAAAfAAA-:' AA"-A - : ‘ "A ,; ; . A -:,--■'a '' AA?' A% A :A A #W ##;(t% be:A 8p0A G ifiG A foË A fibon iiG #d8i# :4 '-^ ti'ip W 8 p h a te 8 }' :- ' AA
- r ;,. The effeot of Inoreaslng CQndentrationsv-ofa - ;vffe;: ' Inorgahio orth6 ohosnhàtè amd pyrophosphatè om the incorporation of -H ITT? by an aoetone dried powder of the microBomai fraction of lahdaohutz ascites tumour
W v A : ■ ■ -- -..A-:
The reaction mixture contained^ 5 ^oleo tris-HCl m',a buffer pH 8#5, ' 3 (implëà ;Caoh of ;2-ti©rbapto-ethanol, 0.5 pmoleB of versene, 0.1 jmmolee % HTP,
; .2 fimole 8 pho spho c re at in ë, 50 fjtg phoBphodreatine kinase, 100 }ig RMA and 1*5 mgd- of acetone dried powder
: HagHEO^ and made up with ah equal doh o ^: of were added in the amounts indicated ihi ;èi total '/Avolume 'of "0*5 :'ml*AA- \ 'V; : h
Incubations were performed at 3% for, £0 minutee.
a ‘;;+AhrthophdBphate: + p^rrophosphat e.
Figure 24
.3S 400O5hÇUbi) '
^ 300-CD"NUOAo 200'F:
OQWCD
1.0jumoles orthophosphate or pyrophosphate /tube
■ 9,!hé = utilieatidn- of .-.rl'bOMbXeo.feiidé' 5 ? riphosbhàté8 :v;/'''''and: deoxyriboxmoleosida -5'?: 1iriplioaphdtés a$ eübatrdtës ■■• vddfor polymio 1 eotIde/âÿiïtheëls oatellÿaëd "by an acctona ;■•'
* & ,» ,, ! m Ç ##,44 Ij . m w m # ^ j P l f . , ' i i # l W « m # " # M W w f I M " M P H IM f , i # ; w I , , 1, | j j | l j i ^ a M W j j T T M r ^ I w y p . j i ^ W t P ^ / U . J i J l U l lllf r . W t iij . i^ l W i i ^ * # i .' à n l ,I |* # # # I 'j l# .. ,,
::; dried - powder of the mioroBomaXdfraotxQn of Iiandoohtits ' '1:::- '/: as cite a tumbnr ' cells-*' ; :■- ; : t;-; v?••' .? #w4!kAf i#*y rV' : r 4 * r<i'!jj.Mint&rTM>jgp tmm w m mi— if.aijj*i ■ ■ lij ..> f i T ' r '' ^ - ' /' •■ ‘ ■>■.....•> '* • i.:
- ;''d:/'':/Æhe'/.re%cti6n'\imi 50, fcnoles tris-HOl '8*5$.' 5' "'tmoles each of TagCl and 2-mercapto-
ethanol y 0 #5 Idoles of vérsbne * 2creatineÿ 50 jig.-phos^hoc'réatlne^ ' 100, jîgJ W A onû
' 1*5 Bg of acetone-drlêà,: powder'<;:ll/ H:'''MB*ltJ Î/-:di P anda'%Y - 1% . //y . p." / _ .were added In';'th -ÿamoWtb/:in
\ Ml*:; ;1.".;,:V-,;;, TBonhatlons^-weré'^^nerforM6dlàt/::37 for 20 milnütes#
Figure 25
400
2 2 300'
a 200co
oo 1001
40 80 120m/imoles substrate /tube
- æ « -./'.y' " .i
etiiloo l'iaoo2?r)orû'ô:loii o;C %i tWi’.f.-.mû (06 '.'ü-l' ooor.roto loDO tîus.1 oue. tV'JGJitlotli tlio cytiiot o" Jî imiàî#! jjiiJ
:'À' ■ ;.
;y
jy y .:,'..vi\.'
;,.y/.'yÿÿ- ±gaQt:Wii:/iAÉ%«!t;dÿbçeseaë' //ü-caof.'CfÇftoolî'iünôl, p:b.bKr6Wom#Éhe.'# ',■ ;;
■ ■ -
3<ina9o and 0hd/#r3:ag;,A%l * .êWB» , 0 # Àaâ bëï%tOï%#ëÿ: -# ^ability of "--y
.
:iwmopblÿrl1)O].WülQOtM%.{%0/ -y ■.;li'iiiure 26 o?oot/g tlio 'timo ootanq ':of;;;f&9 S?BPÿat
«.OOtl-à' g 8ëé&ièWë''#& "a";' ■;' ■ ' -■''' -" /■'■ 10!5»000 z ii of lADâeolmts ànoiltOEi
tm-iom' ooXlü. (Mlÿ tho : 10!j,000'% « «oûis'ent approolublo aotlÿilty» 10000. sr, 4 qugomatrüit .%#,
y;::
2 bouro fvlyiîJéï a 150,000 % 14 ood:üimi.t riicl y. 150,000 %: y' m’.3>eAV.,iatant fyaatioa. ■ '.dm xriOO'iVnmtiiK), by tuoso faïutio o
• '■ ■ ■ , ■ .., :yy:qy V: lé- é8mpâ%saâ ' ' 27*-q;;', .,Aga « \A lb<>Jiiçronpiiial fmqtiqm
wîio'v/0 iwüt oomo a#lvlty lu" rooovesW^'/# ■iîho ir>OiQÜ0'v3£;Çgiéé tïao .r.iipoxmvîsairlï i'raotionubot*, f t e lou ootlvity. '- ' . Y % : ' S:./:/:?'///., 7. i"7®SYi7YV/y;:■ ' ' AttoRpta \7e;eo r.iaüe to oubfx’aotiomto tbo laicroooisial
%:. y/ .i/Æ:::;;r: : : ' :0:/ % /- % :'...';%illïim%:XàW '('3,96&)p\ (boe \-'thl0,;.Èethqa;::lt 'v&a'--'])p80 :ü)M%tp%'#3ZQpa .1 %-.'\ %qe/ Z'iboomsq# 4'Oügi: aw:Eadeâ- ve^lpleK^ _o# mïé#33?aâo.'.w ^boimâ l'IboGome#, \A QOËDàTlBùm :bf l%ioôri)oratiom of -' : -.
% 3? Into po:j.yrlt)0maq%eb^^ by theoe ; fz^aotioms % .
. #id''-by rnlorpsomal: .lo. bhŒm:ln\ÿlgm?è'-28;:^}' : She y free: x^ibosomoB-. ; moèt}. aot_lÿe ' .liki: :moQr^)0%tiii:g -. :-l. ÜS? fqlloweÀ' '^ the'ïmtreateâ\%3?6ügh:ènrfâGëâ .voeiolè fraotioh m%&, tho'membz'ahe fraction.'-:' -Sheqe âlff erpnp00 in aotlvitÿ imÿ bà â W tb (&if foronoos liï : % /;' the êfsWibûtioh bf thê mnoieàobb intb%?f with the . : ; ...
'/ V ' "'' '. 7; V;/:': V v -' . ' ' ' ' . = / '.':lhGo%qratibh - (eGG^Beétiozi:8) "bray.aB,^Wii% -tWt\ thé': ' % .\''''.:;.iyeaôtipn la. patalyaè^ : hF./ howA, to. tho. ribôBomoq: ' _ 7 ;tO\,ôiffGi?ohtrWpi;^tB ofv%hPBomè0 -weBehtyi&i eaoli- bf / ) - .. .
::th< ::fraotibhb.L -\::T. " ':' .%''7 - - '-She. pài^iôtClàtb 'nàtühe ''bf thè ehsÿmo./ié 'alBO' ' i ..... 2'%:':'2' 6%âiQatë& by7G.eatrif%âtion qf t i^&qroboiml frabtioh bn ' .
BWroab. âeheitÿ gradio3it0#\. y l%gW?o' 2B. Bhoivo the êistribatlomof the:'Gxtlhctloh a t ..-'"'27Lfs^aotioh toge7khe%' .with zmaBhibm^ . ability tq\inGprp.onàte-\ '7':ShG'.;pWï: hf . inooi^ôratiba 'ie\..'"'r'''7-di$hlhobâ/to,/thG 'ilgïït\,#i(%0 of''thp.-.Ü0;3\ szibobomal pèalc*' '-."'7-':;,:''A' '8mall7ee.6imeht albo'/'^peahp' at the;hottqiâ/M7':the . W
'"7
; ,y E t é t i y i t ÿ ' a W ' i f p o m a l ' b l e .% o - 7 ': " - : - y 7 7 -' . y ' V . : ! ' ' " : : :y v y , : V- - -Y>V: . . y - yyY ,;.:; . : - , : y , : ' ; , y ' y\Y' : y \ :. . x ■.;-,]: YY X Y . y ,
i2?qsa / -q/;V7;:': ■ ' . y ■ ■ ■ 7 / A - 7 ; # i g a m 0 y @ # * 7 , # # / # # 7 3 \ é Ë # 7 4 W ' : : . è # # E & y 8 Î e ': ' 7 - ' 7 7 y - . 7 7 : 7 y 7 : 7 - ^ , : ' 7'V; :77. ; -7 7:777 7 7 7 :7 x 7 ' : 7 7 7 y 7 ; 7 7 / : 7 7 7 7 ' 7 : ; ÿ : 7 : : ; j Y y ; ' 7 7 " • ' y y - x : ■::7i77. - 7 ^ g # 5 # . Q , O M ' % M . # : * I # l 7 ' ÿ # 7 # % 7 D Ë ' # & 0 . / - ,- '7 i, . x
77;.;7 ' ' 7 7 'O * Q W 7 : '% % 8 # Ë 0 i^ ' 0 , : 6 ( ) % t 7 W % W R 7
7 7 ' 7'- x / x , ; 0 ' / p l M . € # 0 . p # l $ 7 : Y # ^ W & ' 3 7 % 7 # # % x i ^ f i i s J y 8 a o .T j i t t e i t f i ; ■ 7 YY7777
7 - : : ' : ' : ! * W : é e m # ' 6 a : W e 8 : p a # i ' a y W # a l c é # # 7 0 ! ; : % W \ # 0 8 : 3 ? i% 8 w m e 8 7 $ d. : , : : . ; / i 7 x : 7 7 y , / : : 7 y : < 7 :7 7 7 7 x '7 ;7 ^7:7: , . . - 7 : : 7 , y 7 % / q 7 7 7 Y 7 , 7 7 : ; . . ;.:y 7 7 7 7 7 .7 ::; , y . 7:7- ' 77: :vY: -7 7 .7 .7 ;-.:;7 ;:x ' ' 7 .777 :7 :6 0 9 , . # # 7 ' , # 0 8 . . 7 # ^ 7-‘ljjA e7 ,{iô7? î7 i7#^ s l i g h t l y ' - ; ' : 7 : 77 7 '
" - '7 ' ' ■ 'y- ■: 7 ' i : a 7 i B p i g o i ^ 7 t o ' 7 t b . e - l l g h t ? s y 7 i i 4 e x h £ . ; ^ f e ; - 4 0 S 3 ? i 'b o s O E © s - , 7 , ., 4 '■ ' ' , , ■;. :
7 -,-' 7. 7' ' : : ÿ a ï 8 t l ç w h # 7 i.a ù Ô 3 % Q 0 ;k ; } ; ; ig . '7 ;^ ^ ^ ^ # ï â ' 7 % 0 : 7.7
7.-' . .': .7:7 'Q h ü i.o à 7 1 ': \W h s l # ' - l o i m t l . 'w h e a ' I ' d ' . - - 7 , - ':77. x 7 d 7 . . : , 7 : 7 :7 7 7 .7 : ': :7 7 x .± Y 7 X 4 y ; /7 7 Y y 7 7 y 7 : :y 7 7 \# ^ ^ ^ ^ ^ ^ y , . - , x*■'■' . .--. .7.' ' p 2 ? é fe e a t . . . - l : a : t h è . . . d l 8 l y 8 l 8 . m e É l l i w , : ::- E h l 8 . i v o ' i i l â ' l â o x o s m ^ t o -v :,-;7 7 7. 'X- ' ' y . . : . , . 7 ' ; 7Xa : - . . : - . X - 7 . . : 77 ; - 7 X ,.7:77;.. : ' 77.7. . ' y 7VX.77;-7'':'-7'.x7:'..7'. 7 ' . ' ' .'■■.-.-'yx.r , 7 . .... ' . . : x ' . ' - ;'■'777.' ë W é 7 - i ' l S e 7 7 # 7 'ù 6 M # 7 W 7 % /o 7 ^ l7 c 7 6 % .h u :7 ' t # '0 7 X 1 ' j i i . t i ü 'J o i X i f X - î i r & 0 '7 .' .. '777.X.7' 7 . 7 7 - X ,
'-. ' . , : x 7 , . .7 tW i3 ÿ - @ 0 B : " 'a â ( l . : : : :4 G 8 '7 .^ ^ t l i o & g h . ' L ü ü ü ü \ ^ u r o . ; , . u u : a . . . ' : lé ; s o iv o c i7 ; x 7"-
...':''. ' ' ' ' 7:-'. : . 7 % y 7 7 ih c r b # h ' ( 3 a M ^ c u a g i t i c s i B 7 W im a . : 0 f O M 7'7x7: .\ 7.'77: :. ' 77' 7" . : : ' 7 : ' - . . . ' 77 77x: ' : 'Y -7y:X . . . x . % # e m - ' l e ' W è a :.m . t h e . : 3 j %Gd . l t # . : 7 ü W7 ; i 7 i ' ù Q i 0 m # . 7 a p p e 8 # . _.
77.' .- , '
■' ..:... '- . ' . : ' : t 'o : h é x # l 8 # # t @ ' d - : ' - e h d ; ' t h @ . : W o d % = p ù K s : ü # i g . : a a t l y # K ' . % '% m m é :-::: ■>'... -:7.-:x''. :- . x i " . 77 .7 :7 :7! .x' . ' :: ' . ' - : 7 :7.7 7 7 : " ..' ' ":'7":7.'77 .7x::x:.:X77:x7.x:7'7x7 7.'''7:.,:":.: : ' .7' /y7'77 77 :'7 ' x .7.x: ': 'y "7 . .x x : x.777
:x:" \ .'7 ' -X7 G s g m ë ' i à t ë d v n W h - : : .1 % :. . # l 8 A ip t g â : ; : w a t e % ' l% 7 L . ; ' 7 .y7-i':.% #ÎX77#^^
''-'.7.7.' : 7 7 : - ' È M i G # ë : 7 ' W i t ': . iA i7 8 7 7 Q ïig ÿ m e 8 I h ë d K o ï ' à t # ^ ^ - 7';:.,--:,xxx7 :- y"7'7:7 7 7 . . - . 7 . . . . - : ; 7 7 7 7 7 7 ' 7 :
7 'XX: x x x . x ' . - a , # x a o è b ô i u y t i o a . i i i l i 1 ; h x p a a t î q a l h ü e x 3 m t e 2 l a l ) i r ' B o s s l p j ^ t h m . i o a . : : : - : . - . . x . ,
%7K#77::7::7':::X;:-x:::7:'7::x:x :.■7 ";.;y '7 7. :■ .Sl!7#h#7g '..eVl#a46&7't%t774hqh.7M ^ X.-:7
7.77:7;:
yifiTOE 26 . ,/ 32 VtlBie-■ course of the: ino6: ï)oratd.on of ( P)
nSP W m ib 0 Gllulâr f r ao tiozis. of hands chut z ascites tumour cells #■ -v/m
; / She, reaction; mixture contained 50 (mmoles triB-JIOl ■bùffer pH 8# 5 r 5> - pMole.B 'ba6h:;hf :4%01g and fS^&ercapto-:-: 7' x,7y ,ethanol*:; 6 H^nbies pjâblea - - - - -P ' y;/': 7 :'- 7y ' V v - , ' 7/ x ' g ' , .(4;* 5 % 10 : oomits/mln/pmplie):, 2;/ ,2p; hg phbsphpdreatlne kiha^ mgprotein in -a total volume of 015''.ml'•■■xV:'!: : yhv 7%-"f; ':;;She incubations were %)erformed at 37 for the'timesllndioated*: . -
' x l ' i *----- lliorbsèmai ' fraction'.105 *000 X g supernatant fraction
Illtoahondria,
: : i She time courre of the Incorporation of { 10:,• ■ IÎS.B by Buhcellular fractions of landechutg ascites tumour ceiio $
: ^ was as desoriOsed for Piguro 26.tjlnoubatipns f x vior/ the times indicated,
: y. • • nicroBomal - fraction'\-yx ---•--- 150*000 z g sediment ■
o- 105*000 X g supernatant' yyyy/'x.y' y: /' - xracuron ■ i-:;7 .l yh y — .... 15(5,000 X g supernatant' y y :/h %ÿ ://h:.:Tyl ' . fraction.
Figure 26900-
a
30Incubation tim e (mins.)
Figure 2790ÜÎ
-+-J
600
â
Incubation tim e (mins.)
■FiaUEE 28
The - timo - eouree of tlae inoorporation of ,-H - UTP, 3 into nolyribonucXeotidoB catalysed by -a miorosomal- ; fraction anci onbmicroooiaal ffactions (prepared by the wotbod of'Hàllinan and Muhrb) from bande d m ta abciteo tumour ■ cello • • - -, - '* :3
reaction miximre contained 50 jimoleb of triB-HClbuffer pH 8.5^ 5 umoleo each of HgOlp and 2-mercapto-^, 33;ethanol, 0.5 pmolea of vorsene, 0.1. jumolos ‘H ÜTP, ;• ,2 pmoloe phosplioereatine, 50 pg phosphocreatlne klnasp,; r .100 pg B M and 0*7 mg of protein (0.3 mg in the case ofthe membrane fraction) in a total volume of 0*5 ml*
Xncubationo were performed at 57^, fox the timeeindicated * j .. ' it,3'-''-/_■ \ ; " ;3,
#— Free ribosomes .microoomal fraction 78 y 0003 X •g , sédiment : ; membrane frac tien. :.
Figure 28
.S 1500
“ 1200
CQ
20Incubation time (mins.)
The incorporation of -É Ü9!? vby frsiotiUnB of the ; m03?D8omea ' of lEmasohutz aecitea tumour cel3>s ohtainefl - by eudroae deholty gradient oentrifu^atlozi* - < : {/- '-
\ \ : A# : niitroated mloroàùmeà, were oentrlfuéed oh a h:5 30 per cent (w/y) miprooG gradléht at 27,000fox* & hours :ln a'; spinco DV25 rotor and fraotione.0 pile G ted v/ehe , a8sayeci':dh;/a,’ r e ao t % bh m 1% t m?e o out aiming -'$0 "pmolcB : plL'8*g.,':-5',,tmPleù each of IvigOlg and;2-m0roaptoethaBol>, p#5.;ji3Aolepi\Yêr8ehè,a y.'U2P, 2 ;molp8 phoBphdOreatih jphqsphoph ^kimaae , 100 pg EMA artd; 0, 3, ml; of : each Bomal fractionfromi;the gradient: in a total volume of 0# 5 ml # a r ;
' r Inbuhationa. were perforr/ted:; r : B , Thé microsomal fraction waa dialyoed againet - 6*Olîi trlp^HOl buffer pH 8#0, at 2 4' for 12 hoursbefore G eh t r i f uga/h 1 on and assay 'àà.lh^A' above. ■
: a : : : :;;vf%tinotion at^eomix •
^ — -;thf;haâldàctiy
o:CDU&
* r ^pLj
o
cc
orogo
totH
cc
COo o
. FIGURE 30' : ' y: TZ ■ ■ ■ : ■ ■ - . ,.The incorporation of ' II Ulff by fractiona of the
mlcroBomeB from LaJidschutz ascites tumour cellsobtained by suoroae density irradient centrifugation• ;
A The mcrosoBal fraction was dialysed ;against 0*01 M tris-HCl, 0*001 M yérséne pH 8*0 at 2 - 4^ for 12 hours before centrifugation and asaày as described in Figure 29A,
B The microsomal fraction was dialysedagainst 0 * 01 M tris^HOl 0 *OIM yersene pH 8*0 at 2 - 4 for 12 hours before centrifugation and assay as described in Figure 29À*
Extinction at 260 mu Extinction at 280 Radioactivity *
COo
oCO
0)&p4
CDQ COr r l
LO
OrH
Ot— i
O
LQ
^ œ03o ocço
CD
t
g4-îÎHk
. ■: J, ghe eiTect 6h the. inobrbbratloh bf %r HIPPV by ^ laigrosomàl fraction of Laftdadhütg ascites tümôur cells of repeated precipltatibn and résüepénsiori of the ialcroaomee.
She micrOobmàl ffaction àt à brqtein cohoehtfatldh of 2.5 ing/%1 was treated with 0.25 ml Qf 1 ger bent (w/v) Btfeptomyoln 8UlpK#i:e/^l« ; $he (P v/as resuspended in thé original volume of buffer,' V: A': , ; :either 0«1 M tris-HOl pH 8.0 or 0.01 M triéfïïOl pH H.O# Streptomycin sulphate and ;0.1 II tris-HOl were /removed^, toy dialysis against 0^01 M trls-HOllpH. 8,0 ^ .% Hours .-r
She réaction mixture contained >50 tris'-HOibuffer pH 8.5» 5 iimoles each of MgOlg and 2-ilercapto-: j ethanol. 0.5 uooles yersene» 0,1 umoles -H HfP»2 pmoles phosphocreatine» ;50 ug phosphocreatine; 100 ug KHA and 0.66 mg protein in a total yolp®
Inoutoations v/ére performed at 57 for; 20 minutes
Figure 31
Untreated 1® , precipitatef ,
4th1® supernatant
fraction
U^-^treated 1 precipitate
ord 4th
supernatantfraction
ftTT
IT
0
A m icrosom al fraction resuspended in “ O.OIM tr is -H C l pH 8.0
B m icrosom al fraction resuspended in ~ . O.IM tr is -H C l pH 8.0
100 200 300 4Ô0 500jUjUmoles H UMP incorporated / mg. protein
% i % ; o f 'Sic 'Ærabi%rà%\W^ n:eQGlDil%000^:!Ktk■ Dti?o:o-GOjiiÿ:c:ui ouipliat» cinû-,5?cov.opcîisio:a ■im ' hnf.-Iev of 2
:S ^ïimro euy J 1=004 no&tziag\ju,o ai. .h)iü#l
iÿÔos|5b0l50g.-.f" giybt wiiw%i6tao:1; ■''■'v'v;i'-' -%0'-;
?i» l ' î io î ta r i iv l ’o n o a ot rCo>iioT)Ol;y'ï’ib o n a c lo o t id e s '' , - :%
1 '.®i v:*-04-'0o0in£j voGult J do?'io;ïüt//â'ko'Wi#:-.#/ a
W Ÿf#'#e''Âao *Uuï%âdiiî;iry
■ ))olyriuoïmolooLi(L) ' Ho^aoauGO fxrihùotl ±n vlvjü \?:\kiX±b±21( tL\o ''üWb;'#4%: p u ly A'ooquogqo0,%diâü 'bo /jo o iâ ta a t cbd}''tW':/: ;- :
a0^Si«-d;Ê:::P^iQœ8.tlo XSlaao* K M ^ W a a ^ ::--: 0 b;b) p to a d'ol%m' ùf.
albüËÜI. #&\ : K ; l 9 n i i d ucparated lîito nj&IA - ùa!:&. ÿlb6#ô%' .AkIA '% gzaclloTit olrA’iiOïi T/:lth eaüAe )>horip.liri*uû . !VW \V
A . ' ' %%Môtlvi%;oaa..o#iüal ivâeauoimt of KlDi t roatccl at 3Y^ «or,43
: A ; ' '': R/# ; i# 'of) #miorc at le lAInoo xûvxoii x'mB D?azïn5:(lÿ ' -smaïÿao&X#-.%-: odK##':':of on, . <; % '
- I&tbOüu», œ o tlo ii l ü j , P rW 'tüë œ aixlta ,3-2\:::... :
. . m a m m . 32 '
Analyaia on a methylated aerum albumin colmm of ^^0 R1ÏA prepared from L^mdschutg ascites timouroellB before and after treatment with HNase#
!Dhe preparation of LîAK colimns imd the elution of HHA ie given in the Methodst Section 12*: \
Extinction of H MExtinction of BMase treated sample
■A—— Radioactivity of ENA *: "■ Radioactivity of Hîlase treated sample*
CDo
o
O
OCO
(XICO
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o o oo o
A'
V, -■ V;:'# ae%iw%g8'0\# ; vi'/ ;,i£' A ' PüW i48-: # - : W ) W o 3 6 % : ;■/£■■: " :ï-?’v
.; : 4#figvlt,$0.. ^y:;, .: :### \ thO;y■,;ÿy:y:i , % r J i C ! # ' f 1 îs Vyy.,;vyr■ Ï.,. ;. ■,0'i;ttl.ypQS©'ifelé-; ‘{ÿ5àÿ ;ë3l®t;-;S!Sqwéi&oêB''®ïi’s'tf4iii--t]ae-' f "vy;vaÿ£^taoîi6â # .7:\ :;:;.:Yyy , ,y .:.7:://7:%7yy7
?osoBa»rs#i
lërèatlW-$37:%8 - #0^8^ 7'iî&-7-:sàme ■ ■'■" \ .. o.oa,icl5liiiott@ yia : tWW ■ eiaployett: atMJ; .## ■ moasUreaeai. ' ,. aiSo» 7'-. ',.; -7 7
" ‘ .';>- 7:. ’auâXeot,idfâ7 ■:hab‘0K»o3?afioayittto: aG#/7ia 8p%1)lè yèat 7'\;;:-7--
' ■ 7 ''7 7 7%.- 7 esaept- tfie ;ifcllJoai'aoXeoaltM7#*-t*phpBphat#s 7were, oalttéct ■ (epo 7 7 %
777.7 ;- ;y 7 î# % h 0 â ,% 7 r8 0 p - ; % gm ?Q 7'g% 7@ hotm y k ilm e : Q m ir s e o f "-■■■’ 7 -7■ ' yy :7y a # i e s 80 a0a ë ia e $ a b le 77b M # l y 8i 0 7p f / # ^ 0&7- W 4 - / 7 .-'A'Ï
7777 ' ' 777:7'00o W s M i i é l i - i s y s o t j T i i i i i s i t e f t y f e / l i o a t o f t i f ^ y ’a M 7 ;:.:-yy77';y;y 7.
, 77 - yyy- ■■■'absipaQp; pf7:8###'7.#7& bycirsXysfe'7pi*7’ejidpg si-terosoBjal ’ • 7 7 7 7 7 7 7 7 7
77777'' 777'777#A:': G # '' 0QG# ' #P% gM 7# 7#''mWi7@l0#re7^r^^^yyiy-yy,::'' 77. ' : Y. . r / y - .y-'-zy- y yy yy; 7 77 'yy:./y\yy:yy77y:yy7-'''^ yy v-'': 7' -, 7'7y:7y.:yyy,7y:yy7; 7y , ,.7V7 777, ÿigiîre 7' . 3 4 Èh#ëytWyygyhitmpMeaylyit 3 ’ 'Tyfjfepsyy 7777-7'
:yy yyyyy 7p!m'b87 ; .I':77y77:yy7
:',. ' ■. y7'y78f#p#ye :8uftë1»0Æejè;7!7fa3y-yàa,ci|àîiôy4P1
7/''yv7.777yy ' 'bo p#0 #a#q6 -W%ii@G77 #tivi%:y7i8.7pz'es*it:#' . ' 7 'r 77"-yyyyy . ...y. 'y.7y'yy'yy:7777y 7y;y;yy7y:yv5777'77 7y?' .y,^.y ::% yy ..:7$ < : ^ ÿ y . y y y y 7 y . y .7 7:7 ,7 7 - 7
.. 7 y77'7 77y.,'é&7.of'imhlblti mag' m@&#r.7#mt'.ao enoaxioXpaeQ: ïL'77:yy77yyyf77#/77yyy:7m.7\y7'yg
-■ -y,-: ■■ y:-
' :-:y- y.yy■ Haolease- activity of a micgosoraal ' fraétlon of .
ïiàMHChuta aaoitea tumottr oélls v/lth RMA aa su'bstratet
y :y 7 reaction raixttir© contained bO wiàolos: tris-Hpiy 7 , buffer pîl 8.5♦£ '5 fimoleé each of 7B|gOl0:'7,aha=-■g-meresbio! ■ethanol,' 0.5 wmlee of yveraene, 2 paolêa .'.pheôphbbrëatiîi©,. 50 |.tg ph08Y)hooreEitine klhàae yanay 0«,?5 mg 0 lorçqpmBl :7 7; yprotein, y . y ■ " .y. 7y y' £. y "'y y, y' yy; y' yy7 :y y yy yyyyyyyyy b'yy; .Where in fliça te d y 100 wg EBb .çM 20/)ig:behtphltë77y^.b7.:
were added in n/total'volume of 0^5 ■ml./yy 7.7- ■ ■ y y-y/y!' :.,yy y.ïnoubatione Ÿferc performed at 37? -.for 'ythë.y.timCB. y 7
indicated, , ■ ■ \ y .,..;;. y7y,y77-;;y;;yy':7-7;7, .77 ■■ :+ ÏÛIA and .bentonite7;+ Emyy'y /yyy-'-yyy-yy;:. yy Ho HiïiA^àaaèa.'■- '■:%':
:Kuclea3e -activity of. a mldrooomal frâotion'' of 7 - y y y'y 7'handéchuta asdltèe tumour colle with n-hitronhenyl
— -'“ ”'y-yy:-;y.:yy,':'y'- .,y ..7, ■7.:.-:yyt.7y-7yyyyy-;y -:■■thymidine ^ -Bhoephatè and iwiitarbphenvl thymidine ■■.; ;. f; yaB’7 Bixfestràiesfc /'/ 'ï /■_
■:23he reaction mixture Ame., aa-■above;-.éx;çepty'2,0'.y'‘’77-7'y 7; yy imolee each of either p-nitroplmnyl :thymidihd 51 or 7 7 y . -y -y •.•S’.—phoqphate 'were added as oubotrate,.; y 77. • y 7 .• ; ■ y 777yy-‘ 'y'.7 '':■ 7;
: Inoubatione ' v/ere performed at 37 for ■ tho,'.,ti7megy'y7'' 7'v ' indicated. - y - ' ' ■ - ■' 777 :, . . . • . '777. y.7 - 7. .£7':7y7'/7 77 -yy '• '7:7'
. — .# '7..' 7' .+',"p-nitrophenyl thymidiney^tbphosphatey'-— + p-nitrophenÿl thymidine7 3 '-4phbtph#o.
Figure 33200
soi
Incubation tim e (mins.)
Figure 342001
•S %S oË » 150A .b£)s. SII'"I
i ia
100
50*
a 0 15 30 45 60
Incubation tim e (mins.)
.s '■
'' /I;-;;;;./ . - t hon#! ah -y) I:, ■>’/--f : /Bôï%i%e ' s»â.:ïloi;;:-isÈ.oÿd" '.■. -I ' /(J r
: ' V ■ îr^'iiàivv u.c.! ïÿ à r$ lb l0 ‘-lioiAaâsî;^' '
■Ç; • '-' . ';;. .ÆliÿBiictiiie ’pîaGspiiatè0.-’âsd.: si3;bËt.ï\?ctes':-a;te.i:.â:û/-'a|?^e« ; ■ % :
.4; ;: Æ h o à e of ÿ;;Ÿ&0 ; haf@/:i&ei#l ÿ h O ü g h o A l G ; ^ ' ' " ::. ■
' '4 %'f'- ' - 0 Æ W .'mi0s?68QË08\ li'RomiWt:: %&iP02
c'It; :î,s a-epa3zé#r.from'lflg;LD%a. :.ÿ%! 8ü(ïiy$ f liât: th# ;gatO: of. ; :■, .. ■ •.. VI-" s d i d ' 4 r ': ': ;VV%i:'V';fiV:VyV ' V ::;. :.'44 - Y4V4( ::f 'vy:: 4 4-4: . : . . v-■!; .. . : :b%'0&kÆoW ..6f:;4#40EBB.#84 A)#! OSOgeîgniif .1# ,.#.05# ■ ", ■
'•■ 4'" 41.4::ïit>;d,lsa0es. :i0r.V"fâS‘ÿ4-iattOl;i4.te aars:üatim-:-ÿa:l:o.'. ■', -V '
' o3)0o%^eâ oà-%#bm:iGle.O:W.@ %lüo'oz#of@,tlQa :\P%ufe^ 12 amâ. f 4. ' . ■:;v V I:/V . 'm v v V d V:,4C;:VkV:#V'4:a'V4:4 4V:.%ya44.V/!/' ' : ':4V:44,,::\:r,i%-\,: - ,.. V'/\ 5jb.e\v63R8#i%,Gi'0,f .,,8,:Wï'v : aot.l#çüy,: th o . . % =
.;,. ■ ;, ' ' , , , a 8 $ a y i o,paâl$foa844.o#AoÿoA .. ^ : ^ ;#iê,,.laGOi$dimt:loa,,.%eaG,*l0%.,.". : :4 44-4
. . ■ .4'-; :' %%\qb8Mgi e^gplalïWt 'l(W.4iqvoZ :4of : étost ■ " /■: ■ :'444"4-:':''\ V.4d.'.: \ ■ ' V : '-4/4VI/ . ■■■•■ ;.:'V,/'; ■■■ ; ■ ' " " . b A â l i ï % ' : L o i % t h { : i ' 8 m . $ h 8 e . l q 0 & . s m & , , # 1 0 : : l & i o k o Ê c i a b e l i l a g - . - o f ■ -'■: . '■ •c - ' •44:/;. :#v.4y4VVVV.V4 4 V ^ V v v / v ^ " / I V V V V V ;4 / \ : ' ' ' V . ; / . : / -
:. :\:4'\4 plthezp.pz'lme %':mo80@Bf ,I#A ' pe.Gii',.oa ' euôwse'., # K 0 ltÿ : ' . : '
-4 4’ -.•."■ g 3 ? a d i e a t a M à l y a i , 0 . ' 0 f - • . t ï . i t l ! . 4 ' i P 4 ^ : ! O î t f ? à o . ’t e c b d t V ' ' o a , : c e g e M
V- -
■\g-
vs. ’.'
- id ■
D I 8 0 y 8 8 10
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d'V.n,:v
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' ' i . — 101 — '
■ . ' , ..VBÏSGÏÏSSIOI
li üüie Oliaraotaristloa of the Microoomal B # ■
A 6 file mature of t1Initial exporiiiiente were tfeeignod to detect the
‘ ■ *?p ■■ '? - ■ . ' ■incorporation of ( p) n@p into poljrlhonuQleotldeB* ïhe incorporation of ( ocr g) yÿ? was at first ohsearred .to be Btimulatecl bÿ the preÉehoo of M Vf 6ÎB; aiid OIP*- l his effect was however • •aboilshed' ■ by ..an’; A3? ' regenerating â^çtem added to■the reaction mlirtureB and in these - . eircumetances the ,praB.encei,of g 0SB and Q%P slightly \Inhibited the utilisation of ( VTP* Hiese resultsGonld be related to the extent to which degradation of
U5ÎP to ypP and dip oconrred during inoubation of the reaction mixtures * Whilst the addition of ATP , GTP and OTP to the reaction siixturee to some extent prevented the degradation of IJTP this was much moreeffectively prevented by an ATP regenerating system (see Pigure 13) # ■
- Alkaline hydrolysis of the reaction products after the incorporation of ( cxp^p) IJTP followed by measurements of the distribution of radioactivity amongst the rlbo-* nucleoside 21(3* )^monophosphates shows that tB§P residues
are; mainly Incorporated adgaooat‘to .BMp .residues, whilst‘ ■ - 25 30 per oemt of the label appears In MIB, 6&D? ; and - GMP*..This -pattern ■ of labelling I s largely ùnohanged when- #( t(»ï> alones (cV^^P) n m plne OTP on ( AT3?, ,' '6TP mid GT? are,present In the reaction imlxture» Prom , such an experiment the following tentative oonoluslohamay hé (lra%m'.ia,8' tofthè matureihf .'the:'produet - and' the.' function, of'the primer# (1) Inoorporatiom of #KP ■ residues onto the ends of existing EM/ohalna, Biaj occur on which a further poly B sequence is synthesiaed# . This '. occurs whether BTl ailone or BTp plus AT?, GT3? and OTP 'are present - in the: -reaction mixture « . (S) Separate' poly ,IJ ' f ' chains may he synthesised# , . Since the four rihonuoXepBicle 5 tiziphpsphatea have ..heen .shown to he incorporated under the same conditions (see Tablé sj separate hoBiopolyrlho*» v ; nucleotide ohaiha. for each substrate may be formed# : To account for ’ the /labelling-'of - AMP, ’ BMP'- and OMP residues . '.c:when .C'^^^P) ÎÎTP, ATP, BTp and OTP are present in the V.., reaction mixture^ eiubh residues would have to be . inoorpoi^ated into the,’poly Ü sequence# (3) Alternatively, acme heteropolyribomoleotide chain synthesis màj take place whilst JBoriA\of ';the. Itabelling .in -BMP residues is . / -v due to formation of poly IJ synthesised by a separate ■/enssynie' or ..perhaps a, subunit .-.of % A polymerase * ' Eowévér,
' ■ 103 ‘
' " ' ./■' ■ y . ■ ■■ . ' . ■ ' .in the case.-'when TP,) BTP alone is present in , thereaction mixture,it would then be necessary to postulate the presence:of sufficient endogenous ATP, BTP and OTP in the, microsoBiàl. freiction ■ to account for the observed labelling of ■ AM?:, BMP and . OMP ' residuee # (This alsoapplies in (?) above # ) (.4) A combination of the abovepossibilities Biay, o o our. \ . .• .
The ..chain ■ lengths of homopolyrlbonu'cleotide synthesised on = the; incorporation of ribonucleoside 5 -triphosphates were calculatecl from the ratio of labell riboniiclaosicles, to ribonucleoside 2^(3^)-monophosphates « The results (see Table.7) show that the poly ÏÏ chain length is roughly halved when-ATP, BTP and OTP arc added . to the reaotion mixture # , This tends to eliminate possibility (2) .discussed above# .
; Xt is known that.the highly purified bacterial BHA primed HHA polyBierases exhibit horaopolyribonucleotido synthesis at a higher*, substrate coneehtmtion than is requij^ecV for synthesis of heteropolyribohucleotides #(Pox and Weiss, 1964) « In one .experiment (see IPigure 16) the incorporation of H ATP alone at increasing; oonoen- tratlons and in the'pfesenoëlof BTP, OTP and ÏÏTP at similar increasing eoncentrations was investigated#BTPj OTP and ÜTP inhibited the incorporation of ATP
: /y-, '
at all conceritratlohB- tested# experiment tendsto .eliBiinàte^pQesibilltÿ/-C3)tMsçuéeedv;at)0ve# ■ ' .’ 4 . The inioropomal / ë n ^ y m è - of stimulation
of incorporation' of one labelled: 5 * —ti'l -phosphaté . by- t W :bthof ; three' and in thecase of th8iiihqorpor6Ktibn\\o ^ ^ of/8timùlg%tionEit\ any eubstrate eohcentratidn *: 8uoh ooneideratlomBtogether with" % shbrt : ohainilehgthS of product fotmd . to be ; Byxitheals.ed and the ; iackyo f : 'r andoralbat ion ' of'.label-. frqti ;. ( 0$B makethe p08Blbility :qf hetehopg% - eynthooie - by ,;.the\microsomal fraction; extremely unlikely, ' Prbm the experiment on the distribution of radioactivity amongst the ; ribonnoleoeide 2i rmbziqphosphatëa i t ie. reasonabletb; oonolhde ; #D? : sequences : are: being incorporated -into; exl8ting.vHIA>'-ehains:-# • ' . ‘ . 't'- ' ■'. : Bixioe the %icr0Bomal fraction contains 40 50 per
cent of ': ehdqgènoue; #IA no absolute rebhirement for HIA or any other; polyriboxmoleotide , as ; primer oould be directly//demonBtratacU ' /Hgwpyer,; stimulation of the incorporation : Of ( c<^ p ) : BT?;: by. 'Bot- 'by ;inhibition by '’Elase /and not by 'Blase br aotlnomycin B ihdioatea;BIA rather than DFA'qan act as a primer# To .% obtain; .'iiiformatipxi;.- about the -role of I#A as ;.a primer in :
.4,
the ■ Inoorppmt ion of rlboimoleoslde 5 --t r ipho e pliât e a : \labelled'HIA'Y/ao. axtradted from the reaction i : . ,,.■and analysed'by. euoroee dehaity gradiéht oentrifugation# ■, The results' (figures IS? 19? 2Q) : show no .la.bèiliïig/bf^ 'primer' or piboDomal, EEA/oodura# / ' The-positionlof-' labelling of EHÀ in the gradient indicator/hyntheais of oligorihO'^ , micleptideB.'or. lahèlliîigr'Of .sSIiu - 'lYeast - ellMA has been shovm to stimulate : the /incorporation of H OTP but not- II ÏÏTP . This';in»dicates some ' oohtrihution ' to the labelling of the extracted EMA due to synthesis of the pGpGpA ' terminal sequence of ■ s B M ..when- ATP and %I :GTP are ■ /present in the: reaction laixtufee# It seems unlikely, . IiOY/ever, the inoorporatiqn is ' entirely, due to ineprpor- atlon into eKMA#;t ■ Determination of the chain lengths - of the homiopolyrlhohuqleotide products of the réaction, shows : ’ that;short seqUehGea are synthesised* This is in .■ agreement, with the position of lahellecV'material seen.' • ■in sucrose' -density gradients and suggests synthesis -of separate oligofihonuoleotide. chains * Bueh'ah interpretation disagrees with that suggested by the distrihu11 on ;/ ■" of "*3 amongst the ribonueleoside 2i{3’)-manophoBphateB ,. obtained by alkaline hydrolysis of the prociuet BHA which; indicates .attachment of .rIbbnucleoaide monophoephaté residues to .primer EMA chains#' The latter conclusion /
' . - . / , 1 0 6 /r?' ' '
±B-. supported by:; the; results yshgwB, in figure > 210! . flie ■ : .:opincldeBce .of the remaining^? ;0 ; primer, WA/ mid . 'II AM? ' ■ .residues- Incorporated .-In .-MA' extracted = fromt-reaction.- ■ : • : mixtures ; 'ooùtàining / 0 . highly .polymer 1 sad aeoites- RIA .•and AS?in'dioate .labelling;,of the. " 0 • primer‘.RMA#- •
' ' *1 A' " ' ' 'However j- the-.; remaining;'*'0; H M 'pould> hâ sHIA,; possibly-- 3 ' ' '■ ' ' ' 'labelled: with ’.E;AMP residues or. small oligoxmeleotidee. ■
imdegraded by aticleases an.d not hécèssarily labelled with ..3"‘. ’ ” ' ' ' , ' •' n AMP. residues # -■ ;, . ,.. ' . ■ :
. . The\REA extracted from the inoubated reactionmixtures ' shpws •.hb iiigreaae in ultraviolet abeorblhg , ‘ /material’ .in . the 4B, reglom oxi aziaiysis by sucrose • densitygradient OGiitrlfugatiom#, : \The imiomit of ElA • extractedfrom .tlie; reaction, mixtiires is; oohsiderably less in: .theincubated samples (figures'-18 eacl 19) /than; in ^ero time -contrôle , , Buoli observations: iudioato %ittle' or nodegraclationiof the REA By^ah endohuoloase 'present ’in ;the microsomal: fractiou m . The loss of ; HHA in the 'incubated reaction- mixtures [okm be aocohuted for by the .pre Béiice of a ' phosphodiesterase .in = the micro soraal fraction.3 and ; 5 ■ . pho spho d io s t eras es : have bean ' showh to be , presentby direct assay with- p*"nitrpphehyl •' thymidine' • 5♦ —phosphate ■ ,and p-nitrophenyl thymidine 5♦^phb sphate ; ; as substrates« -
.. ■; If there,.is terminal labelling of BIA it -might be
expected that: the ri'boBomal BIA peaks should be labelled^ especially In the case when no primer IHA,ie .added. The: ribosomal protein• prè#Wably protects-rihosomal HHA from degradation by the phOBpliotïiesteraBës# / Àéëùming that ;..r the incorporation' of ' riboimqleoaide 5 -mmiophoephate , residues, occurs.-on. the ends- of cxisting-fBHA .oliaine 'it, • , eeema reasonable to suppose that association of the incorporating onsyme with the- ends of the %)rlmer EhA chain ¥/onld prevent simnltanebns attack by a pliospho*» Tiles terape# In order to aô count for the; re suit b . ' ■obtained a..3* phosphodiesteraseworkingfrom the other ‘ end of the, aame chain .wonlcl have to remove at least 98 per cent of the, chain# This, is - unlikely as the-riboBomal BHA,;,will be aBBooiatedlwith protein and- in any case is , notcompletely degraded after incubation for one hour in the reaction.' mixture. '• .However, the amount, of degradation' •by the 5* phosphodiesterase may considerably’ exceed the, amount of synthesis by the incorporating enzyme and may 1 be able efficiently to remove newly synthesised seqiiehoes from the ribosomal EHâ. "clmlnB# This would aocoûnt for lack of labelling of the gOB and 188 riboBomal. BMA .peaks but hot for the appearance of - radioa.otiyity between '-the ■4B region.and the meniscus of the sucrose -density gradients # . IDhe phosphodiesterase mhy.have limited
; the absence of à #parate/ i, i.;•, J e m d p m u G l ç a a ë ’àmd\mày'\remoyo%.\the" hevAy-.ey n t h e s l È e d " 4 ëv;,;, ; . .
j '' ','èé4uéagé;..Wiïdi>;a_-'f0w ; r0s£âuèai-ot. t h e p r i m e r ; ^ B ^ E p h a i h 1; t'"h./ h t h o u g h t h e r e ,.l a :no;': e T i ^ e n e e q f ô r ;thih:;aiternative*-- '' , , -v-
by . '
V\:Buoroee;.40nsitÿVgradient/héhtrifugàtion indioateâ. i".{ \ eymthesls pf Béÿarate ôilgqnuolëo chains# - On the , ■ -
/other hand, the ; dlà trihution of. " 3 'among at the -rlhonuoXooV ; :/';. ;'eid© '21( 3'^:)"m#h'qphôéphatea/.-oh - on ' al&àiine hydrplyaia'///J/bf/the ■;prQdmot4,î^’-indigato0; incorporation' of W t reaiduee-/ /;" '-;omtO' exlating'EMATohalna#; '''T:':;/!;-/-.-., •/ / .-/' :/-'//:./' ;:' ;..'in.vleiv:,of ./the- 'ahprt -plmin 'lengths-;of homopoly-^ -. :/; - - , - rihohuolebtidea/formëâ.:in the.;' reabtlon;;i t- - ia. pbaaible. /
ihat''treatment;/of'■iheVreaotlon'/mixttirbB with a d d does ;.' not hrihg \about : bbmpléte :prëoipitatiôh/ef the bligorlbo*-
y\-,._.huGl.eotidea.;froDi;the.8 ifrabtion#: '' Oomplete ■ \ - '/-^/■'/"preoipitation-wouXd;^h©texpeotedt'hbwbver, ' if the imqleotide;
- ■>■■ - -- ' i n o p f po^rated:/!^ to: - e x i a t - ; ohalhé' iii'-the/'''/, -//// - readtIon mlktnr e #; ' of ; ithe-jWlnlmum. - chain Ibngtha' ; bÿ bligbribbnûoiw^ acid -aré not knbwh. - • -
;._v'-;/QhainB' :of.. 5 .reeibhea h bxpebtefi to /be inaoluhl©'; ..::///T.:.'lm:/tri.o .'acid;;d;epehdlng oh tWir ribonuoleoaide /•/:■.,;:,■ opmpoaitioht / The preaehhe^fo ■ and : REA in the ... : -y/;' ■'réaction■..mlx*tureay:ïïiày/ho/ékpeéted,..to hbprepipitate ;/.
' ' 1 0 9 ,
Bhoii o3-igonueleotides though there may not he suffioient copreci'pltating material to abeorh oligorihonueleotides
' completely# ; ït io poesihlG therefore, that only a proportion of the product may be measured and that the actual amount of polyribonucleotide synthesis is greater than that observed#
B* The In tracellular Bo eat ion of the Bnigjyme#fractionation of dierupted cell homogenatee by
differential oentrifugation suggests that the incorporation of ribonucleoside 5*—triphoephatee is almost entirely located in the mioroaomal fraction# Virtually no activity could bo detected in broken nuclei, the mitochondria and the 105?000 x g supernatant fraction*Eucleaee activity, ami reaction conditions designed to 'detect a HHA dependent OTA polymerase presumably prevents the detection of the PHA dependent RHA polymerase. Imovm to be present in the nucleus# (Eason and Bmellie, 1965). Mueleases may aJ,so prevent detection of ribonucleotide incorporation into po3,yribonueleotides in the 105,000 x g supernatant fraction especially as further centrifugation of this fraction at 150,000 7c g produces a sediment which is about one third as active as the microsomal fraction# .This also indicates the particulate nature of the incorporating ensyme vAiioh is supported by experiments
Involving oemtrlfugatiom of the ml'croBOml fraotiom la suorose density gradients.and 'pveoipitatiom of;the - mioroBomal' fraotipa with streptomypln siilphate, : ( see; . . ' " Résulta, eeetloii 6) # ‘ It .might be expeated that, the ^mltoohoMrial fraction would .show some IncorporatioB of ^rlhonueleotides into poiyrihontioleotidpa; as; a D % ; ' • *dependent SEA .polyxâef asp' has , been Iden tif lech in the ; ■..mitochondrial fraotipa of It* .oraasa* {Jmok and Reieh, ■ - 1964 ) # 'So reaction was' detected ..in Initial ' experiments / with a mitochondrial fraction from ascites timour cells, poBsibly, b.eoaiiBe of their ATDaee activity or lack of .■ .X)ermeahlllty of the rllmnuoleosidp 5*-—triplioepha/taa tO' the mitochondrial memhranês#, The reaction conditions employed are not, however, appropriate for the ; détection of a DIA primed R M polymerase which in any ease may not he present in the mitoohondria from Iiandsohnt^ asbltcs 'tumour colls# . /- / •
Although the microsomal fraction appears/to he / - - - the . only cytoplasmic subfraction incoxq)Drating rlhoimoleo- tides into polyrlbonuoléotidës, there is a possibility , ;that disruption of the ascites cells In five volumes of / dilute buffer solution causes a redistrihtitioh of protein - between the,cell sap and various particulate components . of the cell# ■ This may also be complicated by leakage ' ■
■ ■, ' 4 IXl, “w '
of Gomponénts put of #ie mioleus due to a, change /Im thé environment of the; mioléar membrane ,m%d any contribution from broken nuclei or<nuclear components of ■ qel3.s imdergoing. mitosis; liiclear- contamination of the oytoplaam by ouch effects is likely to be small « > lfaYort1;ie3.ese . experimentB Oil the location of engymes Involved in incorporating ribomicleotides into polyribo- nùclèotiâee are at the best.a description of thel± location in cell homogenateo.
2» Oom'pariaon of the MioroBomal Eng^vme with Other In^mea. of ' Rolyhuoleotlde BiosyntheèiB #
; . The- - micfp'Bomal ; system appears to require xrlbo--» nucleoside 51’trx?iphOBphatea ae aubatratea like all other eyateitB aynthealeing polyribonueleotideB axoept polynucleotide phoBphorylaae which requires ribonucleoaide 5 diphoaphatee # 'Breaumably phoaphodleâter bond ;formation prooeede by nuoleophilio attack of.the f 3 Wiydrozy].,of a .nuoleoaide ■ mohOphoaphate residue ; at = - > the growing end of a newly ByntheeiBOd chain on the .phosphate- group of an Ihooming; fibômîclèoside 5*-tripho8' phate with the elimination of a pyfophoaphate group#Buoh a, réaction mêohahiem has been noçtulatèd for > ■- % phOBphodleater bond formation- oatalysdd b y .the BÈâ
4 pplymerase. ) (Kornbef g/iSSQ ) f dr/which there /is. çvldemoé ' /required* - - : % analogy,
■■ / phosphpdieeter/boM^ fprmatipa Patalyeèâ^'by DEA.^primed ' '' R W À Wmogoly^ /
■ ; vrlbonucleotide ;’forming/Byetema may proceed by a-, similar .£/; meGhanlsm* though/■there;/ie -'-no. .direct-^PTMenee/for thls t ;■;.V . /.'.-'V The :..ffiiefo'spmal ëns;ÿme = ehdws iap%é' Bifilllarltiedito,./- '. pplyribohuoleotide-biosyntlieela by D M dependent jRM ,
p o l y m e r a e e e o r / d e p e n d e n t ; W A p 6 l y m e r d ^ % T h e ; / /,, former have bOen IdWtlfled in mammalla% tieëUeei (llo and./; Büeôhÿ 1964; /Raepn a a i d ' / 1965)/%m^^ p)urlfled ' from /'.;bact63?lal ■ _ Bpùyepé; (Kfatow/.;aad- Ophoa; 1963 ; ;} Makamoto fox , ' %d':'■WëiB8.ÿ: \1964;):ÿ\'i#ile,-the/l /partially - ' ,
. {purified.from: Pella /iWfepted: %vith'ÀfÆ.'VlruBèa.# , ( E a r u n a - ., .#\Êl, '3-0.62; ; : WelAsmanm:^ 1964) . ,' V CMe . inoosporatioa .:
’ .. of the : four riboùuôléôeldq 5 k-tribhPephates by ithèeè r : :. . é à ^ y m e a / r e q u l ^ ^ . i o h ê I n
. ^ ':Éle..•reàotion /mlxturé#,. '/ÜBuallÿ>£fmixture, o f ; M g ^ t and , . o r ' : i m \ / ' I o n a a l o n e a r e - u â e d f o r / a s s a y , o f t h e D E A d e p e n d e n t
' ■/. JÎIA. poXyméraBa// thpiigh/8omè-;'MA./ûàp©ndeBt''fîIAi polymeraaea , .are ■=iuhibiteâ/bÿ"îlh .t/ànd;,Bpéç ,fiëally- redRire/-Mg?' /lüns'v, ■;
■-.V;. (011me/ Easoh aiid 'Sïïieille,‘/;1963i;' ■'IMltlraore-ahd/franklln,, 1963) # :In g e n e r a l ,/ t h e / p H o p t i m a .are ; variable, p r o b a b l y .
; being influenced by thè degree .of contamination'-by -- , ../_
interfering enzjmeB W t are usually about-pH 7#5 for v ' / . purified eiisymea# The DMA dependent HITA polymerase ' is . . inhibited by DMaae, actinomyain Î) and Blase, while the . viral HIA dependent ÎIÎIÂ polyraoraBeB are Inhibited, by . EMase and not by Mase or acitinomyoih D# ’ Polyamines, . 'particularly spermine or spermidine stimulate the incorporation of the four rlbon'aeleoaide 5*—briphosphates into HIA with both types of engymê # ■
Mofmally the DMA dependent E'iA polymerase shows a requirement for double stranded DMA which is a better . primer than heated DMA* Polyribonuoleotides will also act as primers for the enaymes purified from M» Ivsodeikticueand A# vinelandil, the complementary base or bases to theprimer being incorporated# (lakamoto and Weiss, 1962 §Krakow and Ochoa, 1963)# ■ There- is no evidence;that the ' viral EiA dependent ElA polymerases can utilise,DMA as ; a template# Single stranded EMA is first converted to a double stranded form with'the purified .SMA dependent EIA polymerases which then acts aa a template for the .production of single stranded EIA* (Weiesmanxi et ai* ‘ "1964) # Simultaneous Incorporation of the four riboiiuoleo- side 5 -^briphoaphates is required for heteropolyrlbonucleo- tide synthesis and omission of any one of these reducesthe Incorporation to vi3?tually àero# (Murth et al, 1962§ %
114Haruna et al,; ,1963) * . lAllcaliae îxydrolysis ofvtheV prodmot', :BIA: s h o w o / from ( ribonuoleoside .5 triphosphate . :to hé/distributed roughly,/ëquàlXy .àmonget; the riboBùeleo** side 2* (3’ )Ariioîiophospliates reflecting thé 'bâsè composition of the DMA dr EIÂ iiBecI' ae a template* (HimYlts ^ al, ' :19G2à)* In those'respects the.DMA dependent BHÂ /polymerases differ markedly/:from the. microsomal ensyme ‘ ..wiixioh, although it inGofporates; the four rihonueleoside :5 -^triphosphates, only appears to catalyee the. aynthesis . .of limited liOEiopolprXhoniieleotido sequences and not the net synthesis of RMA* , Henoe the microsomal enzyme is ' -better compared,wdth other homopolyribonueleoticle forming: Systems# .v . ■-
A: system from hqmogenâtes of chick embryos incorporates ATP into poly A# (Qhimg, Mahler-and, Snrlone,, ■ / I960)# An enzyme having similar/aotivlty has been partially purif led. from a particle free preparation : of chdrid^ ' :allantoic membranes of efcven clay o311oh;ick/embryos* . :X^enkataraman:/and-Mahler?''X963)t / Poly A séquenoèé . ,f " '8 10 residues long Exre produced by this enzyme. An / ^enayme synthesising poly A has been purified from extracts ' .of calf thymus nuclei and appears spebifically to require à poly A primer* (Edmonds and Abrams ? I960, 1962).The incorporation of OT? into poly 0 sequences has-been.
.sho.mi: ' to 1)è\T.oàtaIÿSQ{X by;: a/sèparâte; -omâyBiG/ obtained '. ; /'<)4:/ from • the • Bamé.■ Bouroo #•; V./f Abrams,. Etooaids/èhü;’BiBwaB'v •.///- ', ■1962)?#/;-A’ more highly, purified. 'cmsyme -f.(mMlng'-: p61y A/"::'/-, BOquenecB lias bemi obtained from" E*- ••boll# ; CAUguat,■■ • ■ r" OrtiW andr-HuOTltis'jj 1962) $ / ■/in/thlB/Oage/eéparate/:./- ohalne. of poly A containing 100 r.;200: .#D?_yrOelduoB/ ■ ■ •. ' / ; appehr- totb'e - eymtheelBéd'* / ; ' - i n p o z ^ p o r a t l o h / o f - .:/a- .- :fôsidûës'.intoozlsting■•■•polyrlhohueXeotlâê.-r.ohalno• has'-..' % Vbeeh" do te é tod lA,: the nuolel :of,:'B£md-Bo3mtB7aooi:to0; tumour'.' . ;cell0 (Smeilie , • 1962) . ahd -mtolear • riboBomés-‘'frbm--: the- okme sguro'0'_;oat'aly8e 7 thë7\inèôrpOra ' of % A#X/and' '
'latp'v.rlba'egim ^ ^ ';77(33hrd%; /■/■/■ 7 / f / / 7 ^ ' " v - .7 ...'/ ''làoorpo'ratlïïg;,# .iBtq7poly--tî-/7,:- v/-,/
•Be'qnèneéé. iiavo been ■ found in ; the ^mlçroewial., fraction of/pigeoh'; llYar;#7/-::/.’( ' \:£%f •.•' " ensyme; fraotioh/hae 'beeiT.partially 4purified from- m - pn5"7:77--; prpoipitate 4àf.-avBupornhtaHt7fràatiüh7obt'ained' ■by/;//-/ ■/7>> 'f / eexitrlfugatloh of ; rat liver --hômogesiàteè at 105,000 x g/ 7 '
't’or/ohe; .hour #/';:■/Tho- : partially7puri'fled 'enëymè/iîieôrpôrates’ .WlffrgsiclueB , into, .poly-II' in the ' pré a one o ; o f iXg7'^. iono / ,m%d7AMPy3:eqi&uo'k7int0'\poly- Mn " 7.;ioiië«(IClemperej?, : 1963à, b) 7. ::: The 105?0007x g Bupornataxit:;,
f ractlOHs/'UBed/'byiKlemperer IgOgEi) and by "’Surdon 7/ ■■:7 ' /,: 7. and Bmelilé.; (1962) from ;.Xi.andschuts ; aB'citoe timour, cells /
on tho -iÿ0QtgO3?a'é|,OîV' polyreiboiwplcotxclori nuy l)ot'lx. oontàia eonolâoralilo aNOimt:; • of Hiieronoainl material. Sho fna«t:L5a/feOii asicites
...... tpBMJur-pol-lB gavo on Inoofpqnatiqn:mg proteixi i.GOiiipmzablo %o -tlieV^pgesght 4%;'-4
mlproBbma]. onpgme nfpa I b f ? : # ! ' :-A%. r gon-..-/■ - G Z ’ a t i n g B j f f l t O i i U ■ Possibly P e n t r i f u g s / t i b i l ;at 105/000'
.for. one hour remoTod the ’bixlk, of the .àfl aoG and plioa-> 4:;::4#oai;es$eru8e n o t j .Tlty' : .
" / ; : f ' / / ' : / . ' 4 4 4 ; 4 . i : / - / . 4 4 .
/ : 4 : , y ' 4 4 / 4 ' K f o r % i o 4 ' l h B o r p q r 8 t l d h ; ' # - r i h o m o l e b h i d e . . 5 ' ^ - - t r i « - ' : ' - 4 4 : , : . : ' " 4 4
: 4 : / : , 4 4 4 ; / pHpsphates'. i a t b ; exist i S g i 4 E M P h a l n y . în;,;gënéi?al / : 4 ; . ■ ; /
4 ' 4 . 4 4 . ' . : 4 . . 4 4 l o n g Q ' p l i b i % B o l ÿ 3 4 1 h o m % i Q l p appéàr t o - h e '
.4 4 : 4 4 4 4 . . 4 b y h i W â ÿ î à s " t h e : ' W a y m q # : ] y u . ? ? l f l o d / ’ , i f / : / : - ; 4 4 / 4
' 4 ; 4 " ; 4 ' : . - ' 4 4% ’ 0 , f / t H e ’ 4 b p e q t f l a ' 4 ' 4
■ 4 ' ; 4 / 4 H 4 4 4 , : 4 ' r l h à r i i i o l è ô b l 5 ' f f - t r i # i o 8 p h a t G 4 4 1 a 4 : . t h e 4 ' ' p r e 8 b à G ' e 4 4 b f ' 4
; 4 4 ' 4 4 4 4 / : / : ' 4 t h . e / 4 o % # ® P ^ t # y r i h o n u e l ç 0 q i d e t r l p î i q s p î i a i è ® > - ; / 4 / 4 4 ' / 4
/ ■ r . i 4 ' 4 4 4 4 ' i # g ^ % i 4 l 6 3 ï B . Y a r # 4 O Q m G à a s e s ' 4 s ® b s ? 5 i 4 d l n o . ' 4 4 :
; - , 4 - : 4 - 4 : 4 4 4 \ : : # x i l i # l . t , 8 : 4 b h e " l n o b Z ' ' p ô ÿ à t l q 3 i . / 4 . / '
44 4:.v4444f::4i95U;»;/iv'r |s*a Is littlÔ;4s9ooiflûiiÿf‘‘fp4r4'the4"P 44:4.:•; thqngh.4 tho' ®2cte.u:c of incorporation oftcî» TO'elaa ... ■ ■ ’
(îojîsiûorp.hly with sys'ithotio pQlyfihqxipolGQ'bldtiB /Shd:'' froE!.;di i'«‘ofoat ' 8ourq.Qo/.:\;'4011goriWmïoloôtidGq4,:witK.4é4q0^4 :4'
444444i .4 : 44 4';'4T3,l?)iydro%yl..grq'UP844o'#4%Gt4.#8 ^ ^
.of-AWiP raoidueB 1)y a rat liyer-: eraîsïïie.i _ ami fetra- oiigqmuQleqtfdoa Eire the mo at e f f é o five - primer a V . - , (Kleïàpoferÿ 1964) • f M a iè ' in Vconfràst to the speciflo foHitxirament ' for bEIA hy ' mi pnrified from thé, oelX■ sap / ail'd ' oatalys'iaag • the-' formation of the pOpÔpA terminal sequence of, theae' molèoules * (îïeoht ^ameonik, Stephenson a W BoottŸ 1958; X Prélss, Dieolmann and Berg?' 1961),
■ . $ho microsomal system differs from systems of the• ahoye." typo: in that the four rihanucleoslda: '5 '-trlphosphateB are independently incorporated into Itomopolyrlhomicleoticle;/ ■produots? though of % short chain length® It is possible, that the. oligoieihomicleoticle , chains which are formed are separate from the - primer or template i " . Other■crude systemB may also incorporate the four ribonuoleoeide 5 ’ tflphosphatoB bxxt such ,an activity may he, lost with partially purified enÈÿmeÇ which show a specific substrate requirement for a single ribohucleoside 5 —trIphosphate# filé' EIA requirement of the microsomal system differentiates it from système 'synthee 1 sing'' homopolyribonüoléotide requiring DMA' aa a primer. / lllïe highly purified. baoteriaX ;d1A /dependent -EIA polymerases catalyse ' the formation of separate .homopolÿrihonucleotidé chains at a higher ,substrate ooncehtratiph than required-for replicative syhtllesiB'of a DMA or EE A template. 'ihiis reaction is
118
prlined by,, RFA -and heated. M A le better primer than native M A . i(in . lone,, are reQUlrèd and the; réaction is inhibited by the other complomentary rlbgniicXeoside 5 L.. ,triphosphates and/spermidine# (P0% and WeIsa, 1964? : StoYonB f 1964) « Mammalian: BÿBtems - cataiyBirtg the-MA ■dependent Incorporation of AMi'and. IMP. residues into , homopqlyribonuoleptides have been deeoxlbed. by Oh#iboh: ' et al : ( 1963) hen ' livexl and by .Burden (1963b) iii nucleifromjhandâohùtA^asoitee tumour cells «. Ohaine of about100 'residues- are formed with both systems which' are probably separate from the primer DMA# Heated DMA is à more effective primer than native, ÊMA /for the incorporâtion of /and this reaction"is also Inhibited by the preBehce rOf complementary ribonucleoside 5 . triphosphates ® (Burdon? i963b)* All four ribonucleoslde 3’-^triphosphateB 'are' ixaeorpprated onto the ends of BIA chains by an enzyme described;by Krakow et al (1961)#" ' ' . ' ^ - « M M H J M «ÉipaWart ^ ^
One x\ibomiolebBide; residue‘is added to the: ends, of 33IA , ^ohainsv.
' 3, Heaotlona Interferinvb with the MicrosoBial îneoxxpox’aition of llibohuoleoeide 5 *W-^riphosphates.’ Enzymes present. lii the micz'osomal fxviçtion that
aotild interfere :V/ith the inporporatibii of, ribonucleoBide
■ g^^rlphpsphatee are of two;-typea? (a) other DBA or ilA . , A , V polymoraseB?'■(b),, enzyïïieB'aotlBg on the substrateB or ..■ -. produots of : thè-yreaetlon-è -.-In- the former group BHA '_• polymerase is %ow a to ho a splûhle enzy#ë which ooqürs
' in the cytoplasm (ICelr, 8mGille and Blehort, 1962) and /It eould oondeiyably he absorbed to tho-miarosomal.fraction*_ The êpeèifiolty of substrate and template
•■■• requirements for this ensyme ’ indicate,. it is not - affecting . the ihobrporatio# of ■rlbonuoie.oaide ■h’wtriphosphatée by \
, the microsomal system* She DMA dependent RNÀ polymerase is probably not present as this enzyme is more likely tobe entirely located in the imcleua * laok of priming ,r ;;v;x
; by BHA and lack: of inhibition by jDlaee and aotinomycln B indicate that it is not oontributing to the mioroeomal. '
■ incorporation * '.An I#A dependent RWA polymerase of the viral type can be eliminated as this enzyme requires ' the presence of four ribomioleoaicle 5'!—triphosphates..-to %. replicate ah ENA template whereas, the\miprosomal enzyme . synthesises homopblyrlbohuoleotide sequences * Eui'ther- more? a eompariaon.of ribonucleotide incorporation in uninfected and infdoted cells showG the ENA dependent
v-^EIA pd3-ymerase , activity'.•appear preddmlnantly in a, large paftiolc fraction Of cells infected.with 'an BIA 'virusë (Eason, Qline mid Smelliej 1963; .Baltimore' andi-i
Pranklin # 1963)/, Bolynupleotlde phosphbrylasG liasnever been oonciuslvGly demphstrated In mammalian oells, • In; any oase stimulation of riboxmoleoBide, 5^M^riphosphate % incorporation by an AÏB- regenerating ay stem, laek of inhibition by ihoÿgahiç orthophosphate and a slight inhibition by inorganio pyroplipephate indicate rlbomtcleo- >:lde. 5 triphosphates and not diphosphates are the true /substrates for thO mioroebmal' syàtom# ^ome oontaBiination of the microsOmal prephnatipn with the bE M terminal ■ 'Bequeuea qsynthe.BlBing-onzyme is indicated' by stimulation of the, inaorporation of ' H. by sEIà compared with the . ' lack o:h stimulation of the incorporation of % UTE, Presumably sEMA would also stimulate the;,; incorporation of '-;rH',A plby\ the mieroBOBial eystèBi* $he presence of such ' an êhzymè. activity. aBBoolated with the microsomal system , should not seriously affect the results of the inoorpor» at ion of % A®P or % OBI as only traCe ' amounts of bEB'A ■are; present in : the micro somal preparations and the primer -, E K used in routine assays. ■
. ,\ Amongst ; the enzymeb =, acting ■ on the subs trates or products of the re act ion ABPaee (10. 3*6*1.3) is certainly preBont in the naiorpsomal fractidh$ (c>2%) W P présent'in/', the reae/cion mikture -was ■ shown to ■ be ''oonsiderably . : ;.degraded.during,incubation•unless■an ASP ; regenerating ' .
system was, Included in the rèactlom mixture. /Enzymea may also be^present.which degrade ribonuoleaaldeh . - residues hut these preeiimahly. act; oh- the rihomioleoBide ; " or: the rlhomeleoside b. VmonopliOBphate as-.would exizyaiee - catalysing opnveraiohB:hetween each of the . purine or pyrimidine nuoleosiclee. In the presenoe of- an ATP / ■ -regenerating, system the effects of suph;enzymes would he expected to he megllgihle* . Ezaiiiination. of the - ' release -of ...acid soluble mxdleotides in the absence of ; added substrates shows eonsiderable hydrolysis of riboBOBml EIA and added primes BBA# : lack of degradationof. ribosomal REA OKtraotecl from ■ reaction mixtures incubated, for one hour and analysed by sucrose density centrifugation Buggeste the presence of aix-e^conuoleaee in the microsomal system* Direct assay with p**nltrophenyl thymidine 3*- /phosphate and with p^nltrophenyl tliyaiidine 5 — phosphate as subetrates confirms:the presenoe of both 3 and 5* . phosphodiesterases » Such an activity may seriously, limit the Incorporâtion.of ribonucleotides observed, account for the short .chain lengths in thé product and tliO lack of labelling Of ribosomal E M aeouBlng incorporation of . ribonueleotldee into existing 'EM. chains as3has airead been discussed in section lâ*.
■■ - Y ; - ' V . - - 1 2 2 - - , . . . . ,
- '• ■'■4.3, mMlfio8mee^of_#G:':#p™80mal , I i; „ ' :, / ‘ of Eibonueléotlcle# into Hosiqpp&ribpmielo.otidesv, : , . ;
,, ■ V ■ ' .Xf ;homo.polyribonu0leotidn,:-., eeqix0no0B'-.‘'axi?e synthosised. in vivo, thoy àre likely 'to bo of a / limited nature. / She % .OS^tBCSe . » W *Niii**WsW V , .■ ' ; y . - ■ ,, , ,
experiment involving-ftlAse.' trëatEient';:pf ,-;îfc ÈNA: prepared ' . in vivo ;in land s chût z , as cite 8 tuAour ; GGlla exoludaa the . popBibility. .that; - large ; sequences of poly. A : exist v in vivo * - ;. ; ; Such, an experiment does not exclude limitedshomopQlyriho-i. ' . nucleotide aeqUenpe formation;either of new chains or ;. involving exptension of exiating'',EM3;chain8.. : \. -.■■•.;■■;■■ V
The microsomal i location ; of the .synthetic activity . suggeets an -involvement ; in protein éÿnthesis, Slightdifferences ■.•between 'the Be.dimehtatlon coefficients of . newly formed rihoeomes.ahd = mature ribosomes.have been ' detected* (Kohdi .Oteka 'ànd-’Osawa, 1964) * Such 'differences^ could perhaps be accounted for : by limited synthesis of ; ' ;3 hpAOpolyribonucleqtide. BequeneéB.: onto 3 the vends of ^ 'exietlng a?lbosoBial ,BM ohaina by .the micrpaornal onzyjuie • ; : detected in vitro » -Such Bequemoes may be necessary ; to ... prompte' adequate binding; of ;-.meaBenger-,BM .or-bEIA; ' moleoüleê qto. the rîboèomea making them functional in . :.y protein- eynthealB# . '= - : y; - } ■ ■ _ -' .it- is imomi that the^ secondary structure of poly-*ribonudleptides detemilnea its/affinity; for the ribosomea
(Oteafflota and Salcaiaora,' l’965a,. b) -whloh in in vitro ■ / ; ■ ' :BjBtémB affects the ability of a given polyribohuqleotideto code for various''asiIno: aoids* ^'XBlngerA/tfoneà^^à 3;. : Mlrémbèrg, ;l965)-*33'‘ADimlt6d-:-hoffiopolyrlbonuelGOti^o ' ' :y;Bÿnthoois onto thé ends pf matural messenger ÈMA . . .3",:moleouleB may be nepéssary to reduce •.tlio'helioal. oonteni; 3 3 of the ends of : 3thè ehaln ahd:3 thu interaction ’of the meoaenger REA with a ribosome# Buoh terminal ■^eequenoee may lnte]efere with the oodingIpropertieo of ■ ' 33the Aeseenger- EIA and the.,pequeno©'would have to he . - "3\3 .0UCÎ1-' that 3it ■ does'. not oode/for any " amino aei’d. ■ • However, poly H, poly A and poly G epde for phenylalanine, lyoino and prpline reèpèetlvèly # y Only; poly à appears not to -' 3/3- :pprrespoBd to a ebding triplet yand this on it a own'has ; -3%' ^ooneiderahXe helioal sodôndany etruoture "though ■ liBiited'" 3 33- sequeneeB' at the ends of meBaenger3BHAymoIedulee may , - ,3;■ ; not # There la ho 3evidehee ■ itmx -in: vitro ' Byatems that 3.: : ; :any .incorporâtion, of. rihpnuoleoside : triphoBphatea la , ■. 3 -':3^required fbr..-proteih', eyntheaiB :exdept in crude Bystêma. ; ■•3-; GTE? .a,B;;far'’aë‘ is at prWeht know appears to he involved in the formation- of peptide hohda oh the rihoeomal surface ahd3npt ih any -''limited-: poly G synthesis': at the 3 33' .endry-of meesenge'r-EMA, moleculea* ; The above^ euggé'ations' - also tend to3he excluded-ae no lahdlling of rihpsomal
3 3-
■ or "primer E M ôùtild be ydeteeted ; by Bucroee density ■ 'gradient . oentrlfùgat ioû : in. E M êgctraote.d fhorn reaotioh ; .3;/y., mixtures following inoorporation of rïbonuqleotidé 5 L. ■'triphosphat08* , -. '■■';3y3‘-333 y'-;3K:\3.... . , " \3;3 . .-■ - . : /. ;■3" . If limited hombpblyrlbonuoieotide; eynthesie of
' -, .:/ . ;3mew ehains ooburs in vivo,. rather than being involved - •. KWH I III» '
in protein eyhtheelê these Aay be à product of the enzymic aqtivity of newly eysitheeiaed subunlta of a M A dependent : 3E1’A polymerase éàph qr is reèpêneible for" catalysingthe formation of3phosphodieeter■bonds-’between* specific ■■ ribbnuolaôside S^^trtriphosphates and thé 3*-hydro:cyl o*f::/3% .A the growing point of the M A chain* ■ Bvepy though the
■ subunit nature-/of;3B» .’coXi M À dependWt M A polymerase . ■ ■■- 3 has .been demonstrated XEuehB- al, -1964) the individual3 enzymic aotlvitiea of the eubunits would be3expected to , . - be 3primed3by;.-3MA’*;■ ■ ■ ' - not3'the -oafâ'e with the ascites . tumour cell 'miiG3roaomal3. - An alterhative possib*^llitj is that the activity .10. ,due to 3 subunits of a* viral BMA/dépeiàdënt^ïdlÂ' polyAe.i*use.- ''3'3'Thla■’GO.iO.d'bo-due to ■' . _latent virus infeqt^oh or partially inhibited viral, replication: .or,' as a result of the induction of the -■ ascites tumour by a-viruA whioh/ia :lnéorpdràted .into wldie cell 3ge3iome/ahd'3repllcat',ed With it * y Partial expression Of 3
3 the- viral- ge iome ; may-^prhduce:^ l'#A- dependent ERA' polymerase
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thtm they ahx±mira‘;adtly Ydf ■ tllèyfràdtioîi' inobnitoratiagY aY%:m:YY: .Yy j?ihonuûlebs±Ü0:.,5*”-ttlpho6phGtës yiht.GY homopY/Y yyyY:/y Y ;Y ; ;Y Y w ./y / /Y /y /% :y:yy^yyY-yYy\yy:; ':■ -Y' :tMë.'ë'X'!;\':;%O:y#bs,tratëX':Gpnbèht ati0m-3y3'\ -y/z-y'; y'yy:y;y;:; ÿyyy:iy;3:myY3m/"3yyr yY':y.yyY:y:'y YY:. i, qq#'arabIe'';'y$pprpzmatelv\;batùratihgi^ooâô0 \ :ÿyt, : of.üh8tràt«àppeai'tohavbWenu«odformo^^^
■the incorporation o:C rihcmnelootidoo'(sog klguro 16);:y;'y:'yy\:::Y but 3 twenty y time's the e^mount '
‘Y.YY-Y
yYY::y:/YwY:m:âyy#3M
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tîayaiidino S’ -phosphate: wao iieed for meaBinzoment of 5*. phosphodiesterafae activity* , -
, Reversal of pliospliodieoterase activity would accoimt for.symthealB of new oligonucleotide chains as, is Buggested by analysis of HMA extro.cteci from the reaction mixture by aucro8e deno1ty: gradient centrifugation and determination of the chain lengths of the products *•If it XB asBumed that ribonucleoside 5i-monophosphatea from the hyclrolysio of EMA by the 5 h xHiosphodiesteraee can initiate the reverse reaction this would explain ptimulation of xâbOBucleoside 5 — triphosphate incor%)or- at ion by RWA and appearance of label af tex"ÏÏSE incorporation amongst the Complementary'ribonucleoside 2»(3^)-monophosphate8 obtained on. alkaline hydrolysis of EIA productB in the reactiom mxxtures. It is then . neoe.Bsary to postulate that the revei?8e reaction only syarbheèiBG’B phosphodioBtor* bonds at a significant rate bet?7oen the same ribbnucleoside 3 — triphoephates as substrates* RIA hydrolysis can howevGr occur between all sixteen, poseible diniicleotide pairs; though individual : rates of hydrolysis of siieh pairs, may. vary eonsldera.bly* furthermore@ the inoorx)pration of TMJ? residues into poly T occurs, to. lesB tlia.h one , twentieth the extent of the incorporation of UMf reeidues into poly ÏÏ even though
thymidine 5 l^hopghate la -readily hyd^zqlysea : by the miGrosomal fraotloh* .Buoh côaeldérAtlôns .. . 3indloate a reversai of S^ phosphodleeteizaee activity Is , not l3:ivolvGd In the Gyathotlo réaction* This Is also -indlGated by a J:?equlrememt ,fdr3rlbonuol#08lde / 'phatoB for the lnoorpoj^atlon.:;rôaptloa\.,whereas rlhonuolooside 5 monophoepbatea would be required for reversal of the hydrolytio reaetlon, Thorny Im. e V M that snake ; voDom plioephodieeteraBO ahowB AT3? pyrophoepliataee 3activity hydrolysing ATE : to AMP and pyrophosphate but 3 .\
not hydrolysing - ABEê (Bomaii, , 1959 ; Bazzell and JChorana,' 1959) # In this way euffloient rlbonuoleosidë mpnpphos^ 'phatès may be provlded.ln proximity;to the active 8ite_ of the phoaphodldsterase; to/pause rGVere'al. :Of the \ . :)iydrolytlo, 'reaction#. OOnoelvably the. èÿhthetlo reaction .meiy not be a true rëversal^of .t'hè hydrqlÿ'Uloj reavôtioi and may require rlbonucleoelde 51— triphoqphatee ae siibstratee, Thé' elimination - of pyrophosphate by nuoleO"phillc attack of the 3^4ïÿdrèî%yl3,of on the cxiphosphate group of another rlbbhuplepolda 5i—trlphoephàte may. oçour.morè/readily than.the ollmlhatlon of water between two molebulee of flboimelapeldo 51— monophosphate* . . 3.. ./ .
. .There bib definite byidenoe t M the_ Synthetic
• . • - 1 2 9 - '
réaction la ' catalysed by the pliospliodiesteraBe though euch a pOBaihllity cannot,, he entirely ruled out* The eiizyiae oa-talyaing the ineorporatiou or rihonucleoside'5^—t3?ixihospliate£}‘ into homopolyrihonùeleptides is therefore separate fro2â the 5’ phoephodiestoraBe, > though the latter enzyme fnay he expected to reduce the chain lengths of the homopolyx'lhonucleotideB synthesised and poseihly account for the lack of lahelling of rihosomal or primer E M * 3 ' '
' 5# Odnclusidns# ■■■lpew*friNw«*»i"i»NNfa»jwij*î*erta;*toTT»w»es*
‘ Attempts made with in vitro systems to showJ.. piiPM m p u iii iiMi iii i M .» V
RIA BynthGBiB? 1 *e * tranBox^iptioh of the hase sequences in a template RIA molecule involTing simultaneous incorporation , of the four rihonubleoside 5 --triphosphates catalyGod hy cytoplasmic fractions proved negative.’. This, is in agreement' with te vivo experiments3 - - -3 ■ ■on H M synthesis in which evidence from several types •of experiments-^ the kinetics of nucleotide incorporation?followed autoraciiographically or by cell fractionation?'pulae. /chaseexperimonts in the presence. and absence ofaotinomycin .10? nucloar tazansplantation end removalexperiments both surgically? and as observed.naturally .in crythrohlast. maturation ? a3.1 indicate lack of
nytopXaBmic (BMK /syntlieeiB *,/> \flme 31t Would appear that - / all aelliiXar. E14? mesaehger? rlhoaomal and eEIA ,ie ' ,eynthesieecl rlh thé nucleus on / & ; template* : Hencethe rihoBOÈiea would niot appear, to he 3self iTeplleating part1cles 0oourring In the eytoplaam and the pbaeihlllty ; ,±e elimiriatecl - that tranedr 1 p11 oh bf M A ■ templa,tee^ producea only a limited numher ■ o f/: copi# - of HWA ?' 'which are then further replloated in the . oytoplaam to give an -adequate :; rate of protein ayntheaia'* - A: poaaihle exoeptlon to this scheme ie that, mitochondria may he organelles which , can self'^replicate independently of nuclear dlvlaioh in : the cytoplaam# There is eyidehce that mitoohondria contain MA; which could ;'Bpeeifyl;sU'fficient meeaenger EIA for,the aynthesis of all mitochondrial proteins,' 3 ‘ Hov/evex j : the results: of ■the present' expérimente: ,and, other investigatore do show that aytoplasiiiic fractions are cap.ahle of limited homopolyiWHohualeotidti synthbsie in vitro Hesidea catalysing the aynthosia of terminal ■sEMA; sequencea* There is: ho ohvioûs requirement!# • for such a system to function in vivo nor have homopOly?- : , rihonucleotides existing eitheriin the free;state or . attached to existing EÉA chains been detected in vivo#The pDSBihllity la diaoussed that the inôprporàtion of rihonuoleotides, in vitro ie due to reversal of phosphod"*# * * « = * imurn'Wirrt 4# -
- 151 -
iestsrase. activity whicîiiaiay: be preoluâed >in vivo' bydifferent. Ionic- conditions and. aiibetrate eonoentrations.
s tî:l H A îi t.
1 3 2 -
1* , Expérimenta were performed in vitro to detect enzymes ceitalyaing the. net ayntheaia of HEâ in the cytoplasiB of. Iiandachutz ascites; tumour, oella# Enzyme : fractions were obtained by differential centrifugation of disrupted cell homogenatea# , The incorporation of .T). 3!P into acid insoluble material ,in a system containing
? bentonite ATE? and OTP was found to.occur mainly in the microsomal fraction. An acetone powder of the micro some B was prepared and its properties further Investigatedo '2, AT’P, GTP and ÔTP .were shovm to stimulate (m incorporation but this effect was abolished by an ATPregenerating : system# ‘ . . .
' 32 \ -3. The incor%)oration of ( oc P) HTf showed a pH optimumof 8,5? requi3?ed BÏÏA but not M A as a primer? %ms inhibited by Blase but not Mase or aotinomycin D and showecl an absolute requirement for Mg' ions,4# Alkaline hydrolysis of the reaction products after
32 \(cx P) HTP incorporation and determination of the distribution of radioactivity amongst the ribonucleoside 2*(3*)-monophoBphates showed HMP to be mainly labelled,This wae so whether ( ÏÏTP? ( HTP plus OTP or( o<^^p) DTP plus ATP? OTP and OTP were included in the
. ' :y. - - - -
réaction mixtures* / Hence hornGpalyribonucleotide :synthesis was indicated* ■
5# : Time curves were obtained for the incorpora,%io5a of( <s<52p) tJÏÎ> alone- aad ( 0^5%) tJS]? plus AffP. 6ÏP and OÏP Y; .in the presence and absence of an ATP regenerating system, The stimulation of the incorporation by the,ATP regenerating system could be related to the extent of (oC^^P) trap degradation.*6* Yereene and 2-m6rcaptoethanol v/ere'found to stimulà
\ " ' '"-32' ' ' " ' - - -the , incorporation of ( 3?) HTP# These reagents were,henceforth routinely isloludèû in the reaction mixture *
’ 0,4.. .Mn" lone? epermine ancl putresoine inhibited the incorporation of ( <sc? p) tJfp and KOI slightly inhibited the , , incorporation *7* ÏÏoïaopolyxrabonuolebtide synthesis was. further . : ■
■ ' ' , ' ■ z ' ■ " ' .investigated using H fibonueleoeide 5^—triphosphates* .% ATP? %r crap and % OTP were incorporated under essentially the same conditions as (oC' P) iraP*
- • -A' ■: 3 ' ’ ■ ■ ■ ;8# The incorporation of H ATP at increasing substrateooncentrationB was inhibited by equal concentrations of crap? OTP and UTP over the range of concentrations tested* 9* Dotermination of the .average chain lengths of'the .homopolyiraboBuoleotides showed the main products were oligoribonucleotidfôB of short chain length,
/-’y id//A / AttemptBywerë 'm W é ? 16 : show. tho. rqïe ; :o:P-;priiper .EMAy;:;/ te ' Éztrao tion.t ô f /
' ;' / f r o m : y { m l x t i i i r W Y - y \ ; t/;/'/;A::gràd;iént''0031 '/labelled'' ma;t érlaî : oiily ' ’y:y: -fyY, ; ; between, thé mehi sous'/and ; /AË' ■. règlôA/H f y:;tHe ; gradient V-and ;■ \- no la/foéiliûg of;.: the mibpa «: ' y yy///.: A : '.y/ÿ//':' y= . y : ;
y:/"\ll;/'/ '■ q o m p a r A ' s p A / f o ^ f '% "Ÿ'-ti'domm amd;--$éplté^ ::''t%ou;b-\'VAp èéll Hltàly ;/ /-Y:"':0ho%véd'Y'..tiié'latté'r :two;'-tp-Âbè'/éqüàï%y::etfé6t: . thé : teea%éÿàtion " è f : ; no t /ay'primer'*,y;;y;,0BIA?; hoîToVor
///fteotiom/lé'.- spécifié for -Yfi'boBUClëosiàè 5 triphosphatéé#:y::/x.:V4./:Yy/YY:y;Y^gly
V vMdATP;%and/("'ç ,,..p), TTP.yVfere.'-'tecorporatod tô/l e a s A"; ; 6 n ë % t w ë n t i e ^ . .-yf-
ynuciëqéidéy3 *Y::Y.t%\lp,|ioëphat Â,^reguibement/yfor .’■.;/Ay ’y/.:/"" /y .tripÜdéph^
■■''■y ,/.pf'/ibéy :teoo ;0 f H T E ; bÿ iB0t%^td’;pyro|Hoo-; .4-yYphate/yiôhe /Md. 'riot; by; orïkoprioripWtéyipriË;^:./;.' '.of.'trie ;;teeOriporat'i ;éf riljomuçïeosidé 5 *--triph08phateri - -f bÿ %H;ÂTÿ''.:'régene -'-'éÿstémi. y . , .. -y’ /V; .//yy : y/,\y Y-:^
y /'■ i 5 /y : ': ' Thé - mlor p Bomal, riat ur e pf-trié, incorporating enzyme.," ■■■; waa yreinvestigated,;" -y Brizÿmo. : f raù tlori è y p b t alrip d , by y//" idifferéntialy:eentritegàtion.-rif/ .dirirüpted;céli Wmagèriates.
• - 1 3 5 : - y ■
were.assayed under the reaction eonditiens used to detect hoBiopolyrlbomioleotlde : synthesis , . The microsomal fra.qtionwas again, shown to he the Biost active, Oentrifugation of the mioroeomeB on sucrose deaisity gradients and repeated washing hy streptomyoin.precipitation demonstrated the particulate nature of the incorporating enzyme,14/ Attem%)ts were ma.de to demonstrate hoHiopolyriho- nuoleotide formation in vivo * ^^0 HMA prepared fromascites tumour cells ? treated with,HHase and analysed on ¥ikK columns showed no Mase resistant sequences with sedimentation coefficients of 48 or,greater,15* 3 and ,5 phosphodidsterases were detected in themicrosomal, fraction, These enzymes are likely to affect the results and interpretation of the incorporation reaction, , ' ■16, It is'concluded that no.HIA primed: HHA syn thesis occurs in the eytojilaBm though an enzyme catalysing the synthesis of homopolyrihonucleotides- appears to he present. The significance of such a reaction is discussed
.. r
.1- '..y -Y
/yy yry .-..vyy
///y;/://.V.y,yY,::;y:
/ # / : y
'.y y;
;Y,
.... Y /.yy/. ', ' T, Y ■'
B I,'?!-'0 if vK. A V hyÏ-Y. Y ■ .
'■y-y y'
/'•
' . -y'_ 156 -
;- y.;..
■Ateams? ■ Ht ?; Edmomls? 1* â Biswas?’B,B, (1962), . 'Colloques Internationaux du Centre latioiial cle la ■ Seoharclie Boieatifiquot lo* 106? Aoides Hiboiiuoleiqueè et Pplyphoephatea; Structure ? Synthèse et Eonetions? Strasbourg? p# 323, Haris g Centre National de la Seoherche, Soientifiqùe.*
Acs? 0* 5 Eéieh? 1, & Valamju? B, (1963), Bioohim# : biophjev Aeta? %6?y .68.* - ; - '
Idler? ’!.? Weiesman? B, Ji Cutraan? à.B, (1938), J, biol, Ohem., 230, 717.
Afflano, Mi,.;g5 ïifâblonfl, O.P* (i960). Bjcp. Gsll Ees» ,- . 250. Y : ■' . y ■ ; . : :
Apgar, .J«, Holley, R.W. & Merrill, 8.H, (1962). Y «T. biol.Ohera.,:237, 796, .
August, OY, j Ortis, P.J. & Hurv;lta, «T. (1962) . ?T. biol.ObeiB. , 232, 5786, Y
Avery, O.T., Mcleod, 0.1. ê leOarty, M., (1944). J, exp.; -Med.:, 2â, 137,
Baltimore, B., 38ggers, H.J#, Prankllu, ïamm, I.(1963), Broc, nat. Aoacl, Soi,, Wash., 49, 843.
Baltimore, B,. à Frmikllu, R.M, (1963). J. Mol; Oîiem» ,^ , 3395, . ' ' . ■ :
Bather, H, & Bùrdlé-Pèpper, -(I96I). Gan. J, Biochem.PhyMol./59r l625.Y:': ; , ' V'/y /" , , 'Y .
Bautg, B.IC.P. & Hall, B,ii.. (1962),. Proc. iiat, Aeacl. Scx>, ■Wash., 48, 400. , ■ Y
. , - <*irjBSSSM ' ' • '
Benclich? A#? Hoeenltranz? H>S* à D'ltt ? S* (1961) *: Deoxyriboîiuoleie . aclcU . Structure? Synthesis and.
Himctioiv® , Prooeed,ingB of the 11th .annual reunion of the ïSocieté"; de Ohimie Phyeiqué » : ' Pergamon Press,
Berno ?. If ® I #;; <% ThoBias ? C*A* (I96I). I* molee. Biol * ? i ; , y : --'ÿ : ,y;; ;:,.yi \ . . y -i:Beesm&mj - J * ? Behmah? I.H* ?;,Simms? 3ÎÎ.B., .& Kornherg? A/: (1958)® J* biol, Ohem*?. 233? 171#BirnS;tiel',J:MYIi,-, OliipchaseM.I.H. & Hyde, YB.B. (1963) .
Biociiira. liiophys, Acta, 76, 454.Bock, R.M. Y& Bing,' I. ,'(1954). , Analy.t. Ghem.,, 26, 1943. ,,Bpdgahova, E .3., ,f avsilova, B.P., Bvorkin, G .A*Eisselev,
■ E.A. & SpiMn, A.8.:, (I962) . Blokhimlya,, 2;;, 387. Boilura, 'B.J, (1963). : Cold Spr. Harh. Syrap,. quant. Biol., „,Y ,y .,28,':21., ,, ' ■ ■ ' Y -,-: Y, ' ' ' , : ■ ' Y-Bollum? P/#J* 9 Croeniger? È* f'j Tonecla? M*: (1954) • Proc*
nat* Aoad* Sol* ? Wash* ? 53,? 853•Bollvim, I'.J. è Potter, ?.H. (1957). J.'Araer. ohem. Boc,,' ■ '79,Y3603. /. , ' '■ :
; ,; Y'"Bp/#y#.G.Y'(1959y. ' Ami.Y E.y. Acad. Uo i 4800./ /Y/:;:Y/Y YY/ o /E . Ygoc;- m o i r parlp .YrA3;YY88:;'/YYY/Y^Y':
Y;,4} :YŸ Y :./apïë^/yyYyi ;■ Xrohy Bipl;;;:%," :2d#% ''//YY-Y'/'Y/YY: Y :Y{,: " , ; ; / ÏCù8hèv?:/%Vr#'-\#_ lÔë'aTitskil?/;/;;,Y,^;y;rY/:;,7YYy.^/y;q96
r:/ 'Yy ; Y®^9Wn'V f ,«IM Y:( l963.) ', .Y '1%1' YYf y'Y:Y;y/;/y Ip/ y:;/y p;:/2^ , Y'Y,YY" ■; ;Y\YYY%W .Tork;, ,'Y,AëadPmie:;Pre8f Y Ind;. ;Y Y/YYYY- %, Y ; Y;/:.,, :'YYYYYY"YY’'Y/Y:
Y’: YYYYYYl&c/na/;:f >t>:Y(l960)v/Y-/tïY‘ShéYH/l'eiuMe ?, Y.Y>Y.’YIY;'; : YiV'Y/ 4>: ./:" YYY'P;- 3 /Bd» ' by .Ohàrgaï'fi YB. &; DavidsonJ .1;, : Y.;; ;.y - Y;,YYYYYYY;YY:YYY#:MYY/m»'Y/Wm:YYYY;^
' Buîriioïi /'E*H* Ul9&3a)/'- Hio,choÉ-*y.biopHys-HéB*yOommuri*ë""/; / - ; Y Y Y / : i i Y Y Y / ^//;■■ . Bürdon g :: R*H'. : ( l96gb ) * ' ' '%,qohem*\Y5Ï'ogliy84#yYEè8
...y:"Bürdon>; R*Ë* ./&/SBiell-ie? É'*;M*8V:./ (1961 );* Blooliim*' HiopMÿY4®'Y’;'/.-
■ y;ylv-Bùrdori?' E #ÎI i'- ,/„-( 1962 ).,* Bioohim # blophyB
,Y YyYY-YYyy Y-:Y.o& Y É y w Yyy:yYy ;; ' , 4: Burma 5 ' B .B., ' Krogçr, > H », ■ Oohoag.YS Wariier y,:YB .0 i ' à ,‘Weiiï'Y Y/ . .. ., Y" Y :,: S>33.-, (igëÙ VYvYpypp . . natY.Y A/d.'Y ,YYWash. ,Y 749 Y ' ; Y ■‘.'Y'./-, YYoa/ns,:: J, Y(i963)Y/-:ï*YY/pe/iBiQ/vYi9'- - y /YYyYy.'/4 ■ Y;. Y . , Oaiàweli Y Y :?'YYyY:Hin/eiwàp/Y,XY;YYq95d7YY YBoë;. y YY;,, :Y: './■'
■./ri/;'y;'lBtërnritiongdxxv:àtiy'Oè dë';/ia ' Rèphérohe :_ ';
y//■:■ y;BçSentlfiqiiaj';/lo fyl06y:yÂeideè: Ei>omolâ.qri0Byet y ■ ' ’■ yy.. y-: /',;y:; , ÿ'/Bÿn^thepG; - é t 'Poùctioiis:?; ry"-/
/ y / ' - ;y;0t3?aqb0iirig ? ’ ':'p y / 361;/: B a r ié # / ;yqémtr e ;y là t lom àl de ' l a ’ "
y4 /yy& éh ërùh e /S o ÿen^^^ .y /f: /y/' /ÿy;!
Y e a r tp rY Y f /tY ^ W S y ) Y/Y:yy'Araér.Y;elaem:..Y^ Y-";:
Qaepërson Y $ *Y4'('.19 . Bop'*;, 50; ' 8.: ■;. 4- — ;„, ; ;
Ô a e p / a p y ^ y Y Y ; ( ) . ; Y y 8 % # > y : # y / : ;SKP. ; . / p l Y j / / , ;.127, , Y ■■ Y-4-,
;dhatebpriîïî YY te .Y^Bj^Qd/YnatY,; » ,;'
y:/%#yy:/yy:y:y://OharaisQrliffY # /y4 B # g y# Y Y Y (# 0 7 yY Y ’y , .Y /lpa.Y B ip l . yY ; Y ' 'YY- Y; '
GtiamW IxhY.'YiyYYY • ( 19'54 ) ;' ;Y";J:jY 'iholecVY3ioiY.,;., Y; • ’ ' /Y 4 Y
;,Qiimbpy,\Y#Y''^YY:Y,^0l%;YYyv^
'Oliawpq,. :-B , B i '{.éyB&is0v, S . {1962 )Y-.'YvYYPrdO;v-'. M : S o l .Y, ■ ■■ ,■
¥ Ê m & m , YiY/YYYYYYYYY:;::Yg:YY:YYHhèrig ÿ, $ eY*y,y%//3u0qka:y. H * ( 19,62 ) y : ; i; *, ymôXeo;* ; yB161/ ? :,_/y: y ■ ,Yry,. "y
yOÎxipoK^é'p’yll *T *11 * à[ 'Blteétiéï'py Y*Y;YyÉrpG//nat:#yy:y y •;■
- A o a a * , b c . ? Wash*, 1101 .
I 40, ^
:\;;y Gîiemvwr goa.. 19..
;;■;. :'X-' % : /32g'.:: j , Ê ^ ^ '& /Dà?;â.fisbïi>;#il>' »; ;>,; 'x
' ■■ :■ V';;,: " ;î)£iviabbri> ïd><fc E v M i i , S i V ; ' ; K e J ; 3 ? ; ;
;> 'P'.Cji;;.; ;®OTdâEjqîi'ÿ;''S #;%iÿmqüWjl:0
■'"; .: ■- :'’;(I9611’.V ':aai{.: :Èb#a;!::Sqi;.,;;>.taBh47V :a::x4. - ,
141 -
. de '?IDuye, V-0 ( 1959')-... -T!!sWjôeliula}? ' ioles’IjV- gâ'ÿ 128...• lilcl. 'by -Ilayashl','; %# # i . B e w Æ h e 'Eonal’d' ;teess *;v- ' , ; ’•
- ])oiÿ llji.■\ ' .' Wash..). .52, ' ; \ : : ' -, Doty p i ? *; ( 1961 ). ; : Proo \5rbh\ l a t \ O q n g : c . CGlooliem . ■ '- .
/, ' \Mo8oaw. IWpt#' 8,t .1 \ - %'/,%'. '
jMlbeopo.g R. (1962) 0 . Xn .#Bâ8iô ‘Problem'^,;iü':;%ëpplà8tie'.• • -\ ' . MBeaëe", :P * : 95 # ■ ■ IM .' .by Sallhorn p ■ A é ; ; Eireohberg E,; -
. Ooltmibia lJni¥erBity ' PresB.,. . V - . : 4.,. ■ \ Dwani B.B# (1959) # Blochimo biophyB.. Acta^lxM,. 286. .:Dmmÿ PéBi h(l961)4 - BioehiBV» bXQpîïya* Acta^ 45{, 198#
■■■-Bunn ' P.B# SMtii'V.:p ’Spaîir'p P.P. (I960) raolêe.
EaBcm^ :R. g'Oline^ M. Jv & Bmellie^ R.M.8. (1963) biol.- : Ohém;i 238, -3978#%::/ \ ' i ' ri '
' ^Éâepnv E# aTrBraellie, E.M.8# (1965)#/ Bioèhem. Avti 84» 7P# : 'B#ioz.id8g .M# u: Abrams, E#' (i960). i J#: ,biol$ Oheïn., £2âî 1142#: Edmond s, -M. ;& Abrâma# ' • E è ;•.. (1962),#' .\Ji>;b'ipl # Ohom # -'E3Xf ^636#Edmondsp-M. à Abjramsg /R , (1963). A . 3)iol. Ohem,, 258, lU , y
y BG1I86. ;■. . V y:' - : :"1'-4 v : .EdstrBüi-ÿ J è'Eè ('1963) # ' J * biophys # bloohemL ' Oytpl. . •; ,. .:;
- . V ' \ v v/.y:. .Pamvlok, M#l# (.1964) * ‘ ■ BlcohlBi. , biopbjs « Acta,. .87 » 388. '
i'.;Hdpp'e--Seyi»' :■ '.
,: ■ ;lis^ers Hâs?ï*±çii H. ; (l962)i / ProcVvaQy.. 8oq..à, ^:
: Plelgqabri ..Bqrek,, 1B 1962) Pÿoq:;/:m@t k , Sel*, ;' ' . V , y . . - ,;Ws!3tej,t ,:.;;il99.., . ' y■ ;,'1?ô3sy: G /ÿoblnbop, .Raqeikorhy/ Ri- Weiss^. ;S üB» ;." v, ■ ■ ;
' '- / ' ÿ l ' \ l : -pq% ;yd #(sl88 y: S . B * 1 1 9 6 4 ) -.f.iblol.. Ohem.R2£, 176',":
;. I ? x à n k 3 . i 3 j (1963).*; :', Bl0G.hlm.y.\blQphy8 :555 *;:: ■•' . gucli ;, E*:,',.Zil.lig, .W...yiIof SGbheld.dy yYB *11,. Proias-,' A . - . , ..
C1964):/- ' . ' V: .- Pnrth;. .Ji j,y::Ilucwltâ, 'd ! Â h â e r B M i (1962) « / J , biol.
: y . 'Qh8m^y.g2Z,:;26:%»yj/: . ;y y -, ., '; :' Eurth, H'urwits'j., , dj.:fâoldman', II. (1961a). ' .BioelieaiV ,■i ■ : blORhy.s.xRsg. Goàmim., A, 362* / y';"'
;Riiï’th.j / J.*y', , Éüç#.t0; J & .Gold%ag; : (1961b) ,.;',Bio'cHenf*:.; : \ \bl#hy8. :Ree!,::'Oqm >■
: ,' Q-ëiâtise.l'iqk,s \ Ev'J?.* . .lalçamêto.f ■ Æ * è.-,|(0'X&‘6, S:.B',. ,■ (1961)\. .Iroo*., m.at * u&oaa.. 601. :* Wash, «l .,47,.1405'; ■
1: : GeiduBOhek, R .$« 'i, -, $0Gchliii-Talentinl, G.P. .&-, Sarfiat , M,T»'/ ?roG $/ nat # Aoad. 8oi,k, Wash* 486 . ; .
";..aebrgley-; G*^ Mantieyà^, y : (1963 ). / Bioohëmiqtrÿy /' ;A , '805..:':;: ' : ' --% -
r ■ : : ■ I-;-; :: ' - 143. X'.-
GeqrgleT^ #,$, ;& ; Bamarlha^, ,0 (l96l). Biokhlmlya; , '"y : / . ^ - 4 5 4 ^ ^ / ;\ '. ■::: Ç6id, ■ M; ::&. He.llelàei?-, .O .W, ((1964) » '■.Blocliim. „'biophfs,. ,, ■ -, :
' Ac%, 80.,:(193:..(:.':\ .;(( .( (\4,.. ; ^eoldi imrwM t.*X/(l964a):,.(r .d. ibiol.' Oheaiiv 239, 3858.'..'■ I::.-',!! ■(■ (\ - —Gold, M.;: & iluz-witE:, : J,. (196:4b),. . J. biol. Ghem. . 239. 3866. Goldberg, : ( H (('1961 ):,.(- Blq.bhim., bio.phys. i'Acta, ;3r, 201.
. Goldberg H « I, - & Eablnov/ii;s,..:M». ;(1962).Sqlenoe,, 136, 315 « GoiaBiQin:, 1.( & 3?l:mt j .W. (:(1955 ). Prooi: ,kai. 'Aoad. :8oi., ,, V ; . .Wà#. ,\41. 874. ; . ' % ; ( . ,(//:/.- % , .. ■,-Sqo ûiaan >,..1, .M. ( % i111 cb y : A « ■ • (19 62 ). . > :px>o c .1 nsit.; ' ibèd, \ S oi., ;
;.Wasb. ,.:4.8y.2lGl.^.■ ' : X ' ( ■(; -Gràhàràv. A.Bi. ; Bl'miabvitoh, (Ii. -(1957) : ' Bipcliim, ..biopliÿe . '
, iC : l Aota^ ;427:. - 1 ' , . ' / / / ( . i:('' ( ' ' :CrPunberg^iaHago, .IS.' ( 1 9 6 3 ) In - %.ro gr eb 8» la. ( Miiol e ie Aoid
Bèséarcli'.’,;'Yol., 1 p. 93« : : 33cl.( bjr (3)atid.s,on, J.H., &' ' (' ( G o b b , ( ' 1 7 , E . . /-AùèAëmie: lue . , ( ■ " ( , '
, GrunbergrManago', : K. , \ Ortlg, I?.J , &. Ochoa. 8. ( 1 9 5 6 ) .. , .’.Bipchiia.;- biopliyb. Acta,.20, 269.4. " / .,, I;.', ,, G-uilfl,, % ,É. ' & Robison., , M. , (,1963).. Rroc ... hat. . Acad,. Sol.,: :':( ^W£ihh. y ;^,4;106. : ' :7:(( : ( v ; ;., . :y(;: : ' ; " , ■' 'Eall, : E.H. (1963) « ' Bioohem, .’biophys. Reb. . Commun.,
. / . . :4y4..y/ .i\/ 4 .. 'y"'( , . .
’ ■ . ■ ■ 1.44 A';-. ' ' -
Hall^ . & ;8pl0gel%ani) 8. C M » ?roo\ nab * Aosd * 80!#;:. Wa8h.:,:42,y,#V:-':^ '. .Hallinaiiv T* & Mmiro, H*M*. (1965)* Quart, J. exp. IPIiyslol#4
'%%# I.,- 93:#:;....':,,Hamilton ». LJ)# j.lktller-, ,%V.. & Reich $ B# (1963) # . Nature,
liondon, . . . ,
Harberog E* A .Muller,.-W* (1962’.) #- 13iochem. hlophjB* Res.Bommim., %» 10?. .
Harris, g/ (l959)y ,Bioohem. J# y 362. :Harris 9 " . H # - c t . Watts*, J . V f * ( 1 9 6 2 ) , Proc* R o y * 8 0 0 # B . , - ■ '
H a r u n a , : I t y N o . z u , O h t a k a , : Y * è . B p l e g e l r a a a i ^ 8 . ( 1 9 6 5 ) #
' ?rOo. mat# .AcàÆé -Sei*y-'fashVy:^^yv:;905-y ' r . ,Haselkorh, R*. (1965) # tlold* Bpr# Harb#,' 8ymp#. quant. Biol,, :
Hayashiy’:iy, Hayashl, HI* 8piegelman4.,^ (1963):# ..ProOiTv: "■■■■ : : : nat # Acad, 80I# y>feBh;.y,.:gQ.;. .;664-."l ■ '- ".yys.v: . ' ’ : y:Ijiayashi, , xHaŸâshi, M JI#:: & % Spl'ègèïman, -, 8 *'( 1964), ' Hrocv 4
' . I n à t # . 8oi,,' WashvL’: 51'y"35i'. ''' I ' k 1 - : ' ' - - - ’ ' ;
Hayashi, M. & Spiegelmanv ;S, (1961). :l%6o.4aa*(;, AoadSci»',■ ' ImBH*, (4%, ^564. -y : ■ / C-.‘ : : ■ 4 ' ,:;
l î e o h t ' , î i ' i l . , . K a m B G i i i k y . . R i G * , . S t e p h e n s o a , l l c l i . & S c o t t , J « P . .
(l958) .''A j . 'bi61:..0hemiÿ2.^3 954. ',-., : ,^ ^ f KMMtiÆ a fcaM ^ '
Helde.lber-ger, 0 4, Harbors^' • V Betbman, ' K.O* @ ■ Wakagl,-.Ty'iY& rpott.ers, ■..T41V , ■(•1956) #■ ■ Bioehim# blophys# Aeta, 4.45#
Heppel,, : , -Whitfeld, 3#R# & Markham g ;R# (1955) * . •■•' • '• ■ * v .Biooliem# J. g 60. 8* .
Hiatt, H,E. (1962)# J, moleo# Biol* , 217*Hogebooiiig §#H* § Bolnnelderg W*0# & Balade g (lyE* :(X947),
Broo# .Boo# exp*,Biol, & Mëd.. 65, 520* .Hogeboom, 6..H,.Sclmeldery ,f .0, as Palade,. G.E. (1948).
J. blol. Oliem., 172; G19. .Huang y H.O* & Bomiery (1952)* fed* .f roc.*21, 384*Huaiig, R*0*, IfeîteBîwariÿ. if* & Bonner, J. (i960) * Bioohem*.
biophÿB* lleB# 0ommuh*v j g 689* ' :''Huang g 8 *Ii * y & Oavalieri, B*f #i (I964) # \ Broc* nat* Acad* ; ’ .- '- • 8oi*g'.#a8h*q 51, '1002*% r /.'Hurvfit^g JVj Dreseler, 'A*Ê* /& Biringer, 'llv (i960) * Biochem*' . biophyo* Rea* Oommwi&gl^g' 15* ;' l ,r - ,Hurwit g g ,J * g : F urth ÿ J*JV v:Andèr#, :MV. & Evane, 'A*il* (1962a) .
" JmVbioi* Ohern*, 237g';3752#9 ^
’HUtwit^g -J*, :furth, vy Éalaëÿy M* ,& Alexander, M* (1962).■' PrQc*- lïàt * Acad* Sol*, Waeli* y .48, 1222* - ' ■. :
^ ^ ïiïÆîcraP! f
liurwit^^ 1*, CtoM, ' 1* â AhderBg Fu ( 1964a) * J* blol* Ohem*,
Hurwitm,-J* g Gold, M * Anders, M* (1964b)« J * blol* Ohem# » ^ g 3474o
;,f Qssê I ; ,■ ; (19,61) *:,' ::Ph8m.:y0,x#y
;/y v : - : ■■':7,; ■ ; ;: ',; Kmif gG#h8i# m . ;: &, Gr&y y. ny ' (1951) .;•' j .''eeiiy; :.,, 'y,y,::emp:..»y8 ' 8ûgpi.;;iyi71.: 'i , . ' _ ‘ "
leir', lî.l*, :'Stèlliey ailia. à Slebeh:!;, 'G. ■ (196g),,. ■ ’ îîaturë,,
,:,;,.'ic@'ir$,,::É;#. & m.: (iggg)hr''aaïo.ohim., b iop%8Aota, iVy.'
Kiâfâqhj :0.* ■& Kl$'byyCK«8« (1963) •, ' Biocfiiia'.,,, biqphÿey Â'o,’fe',;.
■ 'ÉÎr-byyiC.8. ,(l956):y':'mÿ6mm.,'Vy:y;,M»':V',; 'îCirby, îC'.'p»;,'.(IgôS):.Slochlmy,'; Aota. ',.61,;: 50,6., &:';-,;'y;:
, " gîJ?by,,,;i.i.,y:,Ëasi»gsV'4V*S*:',Vt’èhlllvaaV, ' ' Sloohim,.:^MQpfeygi. -Acia.,v61. 978. //r; ■■7.i.;7;- -.1',
;,, glhaelev, :-,:,î|>V>,:y : GraYx’lïôva i,; 1, yp, • # aplrin, ;&, ,S.;',:( 19"61')j.,: ,
y .7(i;9'6o)Viàreto iBlWiya, wv;7;': ■,ïvli j'-,:.B. ',(;1Ô62):*,'■,■ Eÿbwe^ ïiüjiâbn1-93., 274 :V -■■ ■;',,-
y ;Ei,eim8oïmiÀt,,:;Àyi{y A. Dwimebaoke, ' T,My- (I964),y,i ^
V'/'CLeîapërar,g\ HyG. V’(‘X963a):v^:;.■BioàMfâ#vi)ibpHya;V âotBV-•; . Klemperer -, - 'Hi * @ 1 ' 9 6 gb ) o;;' ' Bioohim » blopHy a V 'Aotay "72 y ' 4K*-
ï.'i'
: , , : : - y :: : x ; - v y .-• IClômp'erox'i Æ «Ct# (1964)# Mooh^mv.-blophy8# .-.Ees#-- O ommn» # ; , ■ :' \ i: ' v'" */• "v yy y\,; / ■ :-vr:hr<--» V:., , > ;:■ ■;••■“■■'■■' V\ . ;v..■ ■' ' ■ : ' ■ ■ / F - ' X : ;
ICoerher, J ,P. & Sl^iehqlmez', R .I,. ( 1957) iT* biol. /Ohem., /
, Kono, H. g Otalm, E*. & Ooawa,. (1964) • ' ■ Dioohlm* biophysÀ
Eorn.'begg. A’« (1959) » Rov. Bnodem Phys.. 31. 200.Kognbei'g» A»> Relmah,, I.îl. ■&. Sisiiras,' B.B. (1956), , ■fôd. 'Proe.,,
■"\^'V;"3^,:'29iy; - :;y y, ICï»akov7, çT.S. ,’ vOoiigeogeorgopoiîloE, Ci & Oanellakis.'^B.S. :. : V-' ' F'-"'/ '' : l'Vy ■ ■ --i 'v-: yv,,. ■ : (1961)-. ■ Bioehlm, bldphyB:* . Acte., y§2» 659. . v :■ , - '.y : . y : . y-; ; y; y \ V / , y . . .... , ■
'.ICrakow, J*B>, vKsAien, H.O# : & Oanellalrls, E*B* . (1961) ♦y : ^ , y y .. y'::y 4
Erakov/, J.S. ' & yOphoa. 'S* (1965). Pi oe, Bat, .icacl. Soi.,: Y/ash,, :49,. 88. : . . ' ' .y'/'/l - y:A'. '.■: 'V.. h;,.'; ' -.y:':yY'.y yyry-' ■:. './"y y '-.y-y y v-' y ' y''
. Kuby,„;S*A., Bpêa, ly & Iiardjr-s H.A, ,(1954);,,. . J;, Mol,. Oîigei. >.
, Kuoiii ,ïùH.. (1962). y Bio.oh3Æv,y;biopî^8 .y'; y-: .y;" y y :laBgridge, H.,, ïïilleteg, .îl.Ay, .Borot, P., .BmMon» S.H*;- . . :
y g) WoleemaBB,; 8, y(1964),,; hat. Aoaa.' Soi.y . ■ -,y ." m8h,',x^,y' ii4:,y/'' ' y.' ■ :y' yyVr\y:, .;,:y, /.k'y .. ".Tiangridgey E.y ,& .fâomatfts,, P.,tT,. (1965) » SoleBOe.' 141. 694. . 'Iiehamh, yi.R.y: BesomaB», .M.JBlmmo, l.S, & Itornberg».. A.. ■ (1958) . J. biol, Ghenu’,. 163.: X •■ ". ■■ , y' " . ■: ■ , ..; . "-yy ",yy^ y ",:-.y ■ y .-. ■. '. '
; KeucîiteBbergsg.;, 0.»,,' bouqMeab꣒geE,, E., Tendroy,, Ci fis .. ,yy ,'y. % 0Bdgey,yÉ, ( w :%ÿy o,Gii: 840. yy .. '“yyyiy
Rowry y' il.0,: 'IWsebrqugh:,. Jil *6 yy B a r g B y :'& '-Ràhdallâ'/ R '%»' :• Vy j;"V(i95i) V V c , ; V i ô i » y;03iem4
H&gàé^ikv''Bir;;:(ïOSB)%y .!iB.y#i#y#0 y :-,'ÿ:;yp!;:,y373;,yyBd(^
., /;:y ;y :.HGW:?ork; Adadèmioy'By&SG'lho, :y':.:\ '' y; \Mahs^y >ë; B q y è l l l ' ( BXqGhimi, y 'b l 'ô p h y # 'A o ta ,
.M aàtsaYiBÔ sy yR. : | l9 6 4 > i. ; Y J y 'M d l . ômem, ,:. 2 3 9 . 3451> ■;y;'V 'y y-
:MàrBmr> j * #: iGréèiiè^n §VO*M v ':Pé3.eçe}t r".p»:3.>Kàhan>V f,#1 , V-: • ■■', : /Vh#iBe)yW: âyïî^âelV; i v y ( Ï 9 6 3 ) ^ y y 'ô d ia y s p h y - ^ ■) y:/ y ,yyy#mp.;,#0 t..;# '■'■ ■ .Yyy-y y, Xy.McO.onkéÿ» :E iHv;;Ê: S b p lM h ih €i)^.î ; ( l9 6 4 ; ) y
; y>'.yy8Gi'^Vwà8h-% y • ;, v-iyV'y::yyy#.îîssèlsoh.,' '|i;. ,& StàJil-j-vp ,Wv, ,(195.8) vPîîoc. 'rmt i. Aeèdyyse’i-,,.):
..y:::?_y:rÿ;#y:,Miaglèy$yj-#.M, ,:(,l9ë2):« ::.:Bioéh i ..ActSs ' 6lÿ‘515'.-
R i h ' li.yXig^ . j,.{: gén, ;my#iQi.y^ 45iy,:MoTleX* :'-'.b ' :.B ded tk . ' OqilQCÎiiëB, _IhtegàÉ,tloma%
du 'Oéàtre yRatldBS'l:'de? layKéqhehGhe: 8 '' '?:.:'AqXd0dyÈibdhuolelgyidG:' et': '
y; y y SÿiiXV^ e t; I ' 'o a o t io â j ysb râ sb o ù ï'g y ■ p>.;.;99 Viy’B à g is s ; ; ‘ - ‘ .
; S': ; ■; ■ ' O eâtÿe; î îa M o h a l- . dè, ' : lé . i Îîè oîaehehe y S è i é n t i f iq ,li® »- " : ; y; ' : ;
Morrison,. .E• .O"?Biillivan;,.-J.# & tOgefori, G* (1961),.BloqhiDV*ybiophÿu#. 82* . . •. ■ ,
eamateu,‘\M.., Hodnett, ?J$I,# /& Buschv H* ..(1964)# Bidchim, biantes*; Acta.*:: 93:*. 592# :
SB, 8.* & mBB.,: M.E* (1963a.):* .J# cell- Biol..-,.. 593#NaoB, 8*. (% .masB:, m*E:* (1963b)* J* cell Blol*., 1^, 613* Hakamoto, T.,- Fox, 8.33*/ (1964)* J*. biol*. - ;
■' Chem., .239, 167# -\ . . . ' ; . '■H'akamotO;,/ (R,: Weiss., (1962) ... Proc# mat* Acad* Bei. ,.
.Waoli.48* 880# . /. •.. ■, ... • ,Ochoa, B* V D.H*., Kroger, H*. & W.eill, Jÿ.D* (1961). ^
. Bro.o'#. .mat Acad.. ,_Bcl *,/, W a s h ,670..: . ., . 'Okamoto, T.akhnaml,, M*, (1963a)Bioohik. blophys. Acta, ;':. '■ : %6., 266. ■' \ , ; - \ i-' ; ,Okamoto,: JA. (%' .'Bakanami,, M . , (igGgb) v 1 mol'ec. Blol., %, 323. Oika^akl1., & Komiberg,. A*.: .(1964 ).. J.' biol. Ghem., - ,
Balad'e,.GJ3., (I956a),# tT. biophyo. bloohemi. Gytdl., 1, 59. lalade,, G # B ( 1956b). J. blophyB. bloohem. O^^tol., 85*Balade-, ‘ Blekeviti^, B. (1956a). 1:. blbphys# blocliem.' ; : O#0l., 2, 171. . . ' ...: A3?alade, G.B. cK SielceTxtSi B, (,1956b),. 1. biophys. bioehem.
C j t o l . , 2 , 671* . ‘ F . : : , .
Berry, R . B (3,962) Broo*. mat.. Acad# Bol.WàBh*-,v;. :j:8,-2179.'. - \ % ' \. ' .. ' : '
Berry, .R,1?fBrrera, M*, Hell, à. &. Hurwald, II.. (1961) *. , ;J, 'Mophys*, bloohem.V Oytol., ; 11, I*
Berry, R*B,, Hell,. A* &.Brrera,. M, ■(1961a)*; Biqohlm# i blophys* îâ# 47v'l'i, ' /; u.- : -
3?etermann,; M.Ij*-'â-HamlltpBv/ll*#:* (196I) * IH.-‘Brotein. ■;■ Biosynthesis^*, ;p* 233* M * , by Harris, E. J.O# :New. York: ■ildademio .Bress'I'no* ; %/- .. , ; = • _ ' / ■ ;
iinlieiro,/ft, lobloM, OHP* è Bros, 3# (1963) * Exp. Oell517.} - \:'iv B
Bogo, Â*0 *, j p û g o , . - 7#§*, ' .IlirB'ky,. A.E. & Hamilton# (1962) * Bioehim* biophys#
.^Aqtay '849r ' -, -"'i' " '- , \Bollister,, A.W# (1955) # Ha(l*kBiol., 2# 205*%; : . ' - :Breioa;,,. 1*,. Hieekmsnm,, M*, & 33e%,/B# '(1963-)*; -u * Biol#;.'-
Ohem*;, .236* 1748*. , ■ . ' -Prescott, /B.l# : (1962) . 1 V HiatoeWm* Oytoehem#10,145*. Preeoott,, HfM*, (I964) * . In ipi’ogress: in luoleie Acid Eeeeareh
. arid ' Molecular ...Biology ** *. /7ol # " 3:,; p’•. 33 * Id by ......Havldéçm,. J #W * : & .Oolm, W*E* . New.: York; Academic ^
' •: Press Inq* 'RaBssel, W*E, (1961a) * J# biol. GhemV, 236* 3028,m s 00l, W*E, (1961b). J, biol. Ô3ièm*, 236. 3031, y -
*■ 1 5 3.
Raszol, ;W.B. =l£horanaV- H.G* / (1959) • • J-« ' hlol. Ohemi,,. 234, 2105. :■ , . • . , , .
Eàs^el, & Khorana, .EéG.*. (1963 ) J#. biol,. Ohèm*,*4MN»--u=«tiU ' .
Reich, E •, -franklin, R ,, Bhatkin,. A « <T, 8: {Datum, B • L,, (1962) ,' i?roe.,: xiat* Acad. Boiv, Wash,, 48. 1238.
ReVessî, 1,, forasberg, A,. 61 Klein, G. (3-955) * ' J.^nat.Oanoer, I n s t ; 1%, 3?*^ ■
Eho, J.E, & 'Bonner, J , (1961). Broc#, mat* Acad. Sci#, Wash,:,.;.£7.,- 1611. : 1 ’ ' . - "
Rich,.. A..& Watson, - J.'i). (1954a) . . Nature,, london, 3.75. 995 Rich, A,. VifatGon, J*D. (1954b) * = Broc# - mat, Acad; : Sol.,
Wash., ; 759, 1 A .. ; . , ,RichardsonC,G., Inman, I.B. & Kornhorg, A. (1954)*' 1;:- molec, Biol;,, 46 , ' ■ ' • , — , . - ■
Eiolmrdson, 0 * 0 ,, Schildkraut0 .L., Apoàhian, V ,. & F Kornbcrg,. A, ; (1964) . J. Mol. Oheiin, 259. 222.
Ro, 1,8. . a BuBchi E.: (1964)* Oaneer ,Reai, ^,1630. . I / EoMnBon,'W.S,, Hsu, W.A,, Box, 0,B. & WeisB, (1964).
J b i o l , Ghom.. : 239 * 2944 * ' : • -Balsman, ‘ H.3?., Bagle, II. &? Behring, E*l). (1956). J; hiol.
Chem.,:230, 1001.Bal^man, N.3?#, ê Behring,. B*B*/ (1959) * Arch. Bioehem*
ÎBiophyB., 84, 145* - ■ * - , ; :
■ - -152 - . : ■
Sato-Asemo, ?C* à Bgaml, ’]?♦ (1958) # Biocliim* hiophye*Aota, 29# .655*
Belier:eer,..K# d3 Darnell, J*E, (1962) # Bioehem. hiophys# /. Eea# Oommmi., %, 486# . . . '
Bolierrer,-IC., latham, H, & Darnell, l.B. (1963) « Broo.nat* Acad* Bci#, Wash.* 24Q. , .
Bchildkraixt# O.Iu, HiehardsoB, 0*0. & ICornberg, A# (1964) *J* mole.Of Biol*, 24* ■ -
Boott, J.P* & Taft, E.B* (1958) * Biocliim* Mopliys* Aeta,28, 48*
. m m v u t yBcott, J.f*, Taft, B*B* & letoiirmeau, N*W* (1.962) * Bioohlm
hiopliya* Acta, 61^ 62»Bernal, J*, BpenGer, D*, ICim, %.T * &,WHdmann, B»Q» (1964).
Bioehim*. biophya* Aeta, vgl, 205* - , .BiMtani, A*,-de ÎCloet, B *R #, Allf rey, . G * & Miraky, A.31*
(1962)$ Proc* mat* Aead* Bei..'Wash., 48* 471* Bibitaai, A*, Yamana, IC*, Eimura, 1(* & Okagakl, H* (1959) #
Ilioohlm* biophya* .Acta, 390*Bibl t ani, A *, ïamana, . K ♦, ICiiuura, K * è ‘ lakahaBhi, T.
( I960 ) *. Mâture, Iiondon, 186. 215 *BiminOTitch, L* & Graham, A*P* (1956)* A* Histochem* •
Oytochem*, Af 508*Binger, M.P*,, Jones, O.W. & Mirenberg, M*W*. (1963) *• Proc*
mat* Acad* Bei*, Wash., 49,* 392*
';:% i;;%yy;?y y%:Y 3 9 3 . , r m y ^ ^ io w i V a f f , - e :B&vids6n;, - y - ■ -;' ■ '
%;f - y / A f y A y y y J;Mv^:::L$Gw/.Ÿork:4 ' :AoaâemicyPx^eés" I m o ' * ^ ■; -- ■ y ' -■' .;
V. '(ï'962 )/.:^[XfOllQ>qii0B In to rn â tïd m s iid r,:dU-%;"■ ;■, .i-fy '
':'#:gr:%%-:'yT''"''%y}y::6emtro H & tio h à ly d & v ia - IWoherGhe .H o lem tlficm e ,, V- ;
;%:v ::\;y ÿ : ihlboxmoleiçmes e t P o lyphosphate ; %''. y ' .
y'A,' y--y ‘'V '-y,'; % ÿ B ÿ a t h p s p f o W t l q i i ^ . R t r à p b # =::--543*. . ;
%; y y :.i::yV%pàMG§.:.;^Hèntro 'N a tio n a l-■: de; iàynG cïie rcke 'S o iex 'itifiQ .ud# -
: y y. .B m ellle -, .B *fi*8 *;y^(1963)■.-'y>ln;''^ProgresB in ■■Hticleio. Acid;-' ■'■■ '■
- ';y-ÿy-%y- ";,' yy'--' /& ' Oohn, . ‘ NeWvYgrk’i {Académio -TrcsÈ In o #' - ■ v . - ' ''
;:ï % '
;y ;y v :Y y% i;:;;p i,o cM m yy iyp îîys . : -;y
' îiWi'Wr,^ . J ., Aisieri 'ohom. S oe ,,:' ';-
' . ; ;y : ;y : : ?
:' : ( M , 'loU?-;"';vv;;• ;;■;;%.- ' ■■ ■ ■ ■ ; ■■Y > ; ; ; : 7 r ' - Y / y - y / y ; ■ ;-:;--:y ;:y - 7 - 7 - ;; \ 77 —7 - S p l r la t ;'4; (:1:96I) . 7 W k h im ly a ^ : __g6 7 5117. 7 7 ^ % 7 .,,
77''7: ''' 7:7:77piy%^777&* 8 « 7'.(:1963 Iiyy'l'#ë^G6% :.% n 7lK l ,
;77%,7777:?-77;7'-v"foi7 l i l ÿ w 73017 . 7:Bd:» 7 # :m v ia : j ' & '0ohïT, .w. B. . '-..
7; 7 7 '77C:777 .l7&GW-:Tdi&i;'7- Aoadem ibLlresÊ/ i n G ',} 7. 7. " '7777-7,7.77 - 77.::7 . ■ ■. ■ .
... 7..7y- 7 ' '':. ..SK'lnlYaëcüiy . i ,R7y ’'yi'»',. . s.: . 'f ir re la p ■'■ '
77 '.7 -'7 7:; ... 7:7- ' :-II« 7 i l% 3 .) 77 72 ; 209 ; :'■ 77'' 7
7 ; ; y y : m 6 % w m y y ; y # # y y : ) ^
- • — X54 *** ’ % -
BteTene, A* (1964)# J* blol# Ohem*, 204# ,Btevena,, A. = & Henry,. J* (l964')V- J*yblol*7 Chemr#, 239, X91* StrauB, 3).B*. c%.GoXdwaas©r, E, (1961 ) # «I*-biol, Ohom,,' ' 236, 849:*::"/: : . ' - , ' . /i - ''Bueoka, N# ê Oheiig,, T*Y* {1962a)# I'Proo.ynat# - Acad#- SoiV W;;..;
Waoh*, 48, 1851* : " .. -y : ,Biieoka, M* d?, Oheng, T*T* (1962b) * J* moled * Biol *, :161,Sue oka, N* : & Oheng,.. S.Y. (1963), Soienrie141. ' 1194#Sue oka, ID #1C# /ê. Bplegelman, S.- (1962)# '3?roo*-. nat*< Aead*
Bei..,. Waah*-#';^V 194.2# ■- . V . . ^Tada, M* .& Tada, m* (1962). J, Bioehem,, T o k y o , 92* Tener,. a*M* (1961) * Amor*: ohem* Boo. , 83* 159,Too ehlnl-V àlent iài, G * P *, Stoâoleky,.M*, Aurialoehio, A,,; -
ÙraBiosi.,, f *V 8arnat, .Mè,7 Weias,. ■Geidueohek, ' 1■ ■ '. B*P* (1963)# Proo# nat. Aead* Soi*, Wash*, 50, 935#
V enka tar aman, P*R*:& Mahler, Hèll*. (1963) # J.#; biol# Ohem#,
\ 238* 1059*. . : - : 4- : 1 -Wake, R#0# & Saiavhi,%/R (1962)*; ^, 201#Watson,, J*!0* .drOriçk, P*H*0* (1953) * Nature, loùdon, • ■% ■ ./ m , 737::1\'\ : yC > ^-y' "" " y^:Watts, J*W* (l9G4â) # Bioohem* J* V 293# ■' .Wâttê, J*W, (1964b)Bioehem* d*, .306*Watte, d$W* # Harr#, E$ (1959) # Hioehem* jv, 147#
■ ■ , .. . - 155 - ■ : y . ' " ■ 7:
Vfeiss-i 8 7(I960)?.' \Proe*. mat;. Aoad*. Soi,., ■ Wash,, J;6, 1020. Welss,-S.B.,';,;ê7 01âds1;oîiê, .Îj, (1959). J, eîiem, Soo. ,
. 81, 4118,' ! : .7 V. . ,. , . ... . .. .w * w i * e "
Weie'eraanh#: .0* g .FBo'rst, : P#, Surdon#. R»H*, Billot or, 1*A* ■' &,Oôhoa, 8* (1964) * PPoG*. mat, Aoàd# Bel*, * -Wash* v y
Rn y.AiniltfeM,,##!!*:',: H:eppe3. - & Màr3cham, .Rr; (1955) • .
Sloohem* H » # ; 60 #' 15* ■ . : ë' y; ■ , , F FW i l k l n ë . i (1956) Oold B])r.yHarb# Bÿmp;, quant# Biol*,
Wyatt, G#R* (1950) * Nature, londmi# : 166v 237* ,/•Wyatt». G#R#Oohen,; 8*8#: (1952).,.-.Nature,;. Ijonclon-, 1?0, 1072 Yamama, If# & Bibltsal, A# (i960) * ' BioGhlmv blùphys* > '
Aota# 41', 304# ' : y 7 7 ' . \ . , . ' : - . - . . ' 1.TankofBkÿ, El VA# y& Bpiegelman, 8* (1962) *y Proe#,nat* Aoad,
box#., ;Wash#', 3- 466# „ . , "7 3 ■,■ ■ < < .r- - 7fâîîkofsky# ' S#A*y'Sî Bpiogelràati-, 8# (1963). J?roo# mat* Acacl#./
, Bol*, Wash# $ lÉâ,. 538* . gubay,rG*=&.Vfilkiâ8,.M#I^^^^ J. moleo# Biol*,- 2#/io5V:\;. ' .y: . :\,i-
