THE EEEEOT OE'EXCESS I-METHIOHIHE-OH THE FTIEIZJLTIQH OE ADEEIHE-8 -C1'4 BX AH ADEHIHE-BEQHIRIHC YEAST MFTAHT
Ey
Henry H. Heimey Jr.’,
‘ vj1 '.:4', A Thesis' ' A; ' ;submitted to the faculty of the :
DEEARTMEHT OE;BACTERIOEOGT ' 'in partial fulfillment of the requirements
{ 'for the degree of -: f MASTER OE .SClEiCE . - „
in' the Graduate College, .Hniy'erS11y' qf Arizona
STATEMENT BY AUTHOR
This thesis has "been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in The University Library to be made available to borrowers under rules of the Library.
Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in their judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
SIGNED:
J ImTNG YAH DateAssistant Professor of Bacteriology
ii
In sincere . appreciation to. Dr , ■■ Irving Tall for M s , guidance and ‘encouragement throughout this investigation.
ITu'cleic acids were found in pus cells in l868i . \- The disch-very • of .the ■ purine:: bases ia,-hUcleic acids was - made in 1874 in extracts of salmon sperm« • Pyrimidine constituents were discovered in 1893: in hydrolysates of / the nucleic acid.s tbf,;;ca thymus "$nd: heef' dpleen' (Davidson and Char gaff y 1955» BeMlchy 1955)» By 1930 ;a . dfefi-=; nite picture had emerged"of two. distinct:t^pes of nucleic acids» . -%ne.'o.f them, thernucleic aci;! from, yeast g':faedd" .:ed on hydrolysis adenlnet guanine, cytosine, uracil, ^phosphoric acid and a pentose sugar identified as rihose. The ; other, - the - micleic ;aeid-: from thyms;9't:yi^ adeninesi'. ' ■. guaninei •cyfpsine.; ;thymlne>: phbsphoricl/acidi and",a : decxyr. pent os e-:- sugar . identified' as deoxyribose „ - These, two nucleic .acids, were<;subsequently named ribonucleic acid (EDA)' and de oxyribonucleic acid (DDA}S\ respectively^ .See' structural \; f brm^e^ if: v. , i; j : :. ':■ 1 % Da ter it, was found; that, both types of nucleic', ;; 'acid were, present in: all otypes „of ' celis7 . i0 e . s iplanti, ' ' , animal .and microbial, (Leslie, 1955h HagaSanlky 1955) »• The main; biologleal distinction 'betwgem EDA'and DHA was, that the former was present in the cytoplasm.(Hogeboom and Schneider ; 1955)' and 'the-'.iatter in ■■ the nucieus.y (Veudreiy ;
2■' 1955) <. .Mloroorga%ii8m'B''have. bee#5p for: 1' ;:ofv ftiev'geaeral problems of .the chemistry - and :/hio“•
. :v 'x chemistry ;o:£ : the -iauclei-c- acids, he cause - they are charact- --i.;V.: : l:erized; by a high percentage dig: nucleic acid- in comparison .
i ; /-' with that/ of the cells and tissues of higher organisms' ' / :/.:; • ' ,//. (Belozersky and Spirins 1960) 0 Deoxyribonucleic acid :
/constitutes1'' the chromatin-?-!n cells s and,!ts/biological - /?: : - role is a genetlc one (Hotchkiss9: 1955)« Ghromatin is .
- • ‘: ? • ' Composed ’ of smaller genetic units';/" or genes } .which are -responsible for the control’ of specific’ biochemical re-
,/; ■ actionss, i„e0 the metabolism'of cells (Fincham$ '1959)».i? 1; • - > . /in /alteration ..of ■ the .genetic sate rial. Jf a cell often/• ' //-,/ - leads to an inheritable change in the cell , i„ e« a muta- .-:'/? ;•' 1 Vi . tion ; (Beadle and latum, !94-llatum iand Beadle, .1942; ' ...' i; ? 1 /.; .Bonner;: 1916?; iatumV 1946|’ Romanril956Br' Beadle-g i959}» . /- /. ’ Mutagenic agents /.ahd/.mutations' have also been reviewed by//: ' /■ : ./ Braun.< (1953) and Alexander; (1957) « The biological role ' .•:-,/ ( 1 of; BBA is 'principally that of ; protein synthesis;' carrying;. ;
/ /.! // out/.the ./commands imparted: by DEAi(Brachet9 /1.955 ) o ./ -' ; 1 /' '" ; ? ;/ The purine -base adenine is also involved in trans-/ /! :. : // ' 'methylatibh reaGtiohs !h-hlolbgical .systems, ; 1 -
(1251) 9 ;. using "liyer . slices/ ittdlcated/: the. f oie / played/by; a r “t condensation product of methionine and- a high energy phrihe?
,. /:.. / /compound, adenosihetriphosphate" |42P)in the ;ifansmethyl-/
FIGURE 1NUCLEIC ACID,.CONSTITUENTS - FORMULAE
H-C-OHIH-C-OHIH-C-OHICHgOH
D-ribose
H-C=0ICHoIH-C-OHIH-C-OHICHgOH
2-deoxy-D-ribose
C-N,I « ;o-h
J > - ¥ 'X / II
9H
A > .H2N-a C-^r:T H
adenine guanine
QN^ \-CH:I HD-q, C-H ^NZ
thymine
f 2y n c -hI IIHO-C. C-HH
cytosine
9HS 0-H
HO- L G-HrlN
uracil
OHl0 = P — OHIOH
phosphoric acid
FIGURE 2 FORMULAE
.0.1 HO OH H-C— 0— C— C— C — S— CH-z I H H H H2IH-C^ XC-R
I II > - H1Tv ^ c-H
h h2thiometliyladenosine
HO OH J CH5 NH2H-C— C— p— f —- C ——d—— (JJ—— C— 6— CO I H H fip Ho lip HI
1 i ' V
hh2"active methionine"
(S-adenosylmethionine)
:ation processo The "active methionine" produced could - i function as a methyl donor even in' the absence of a high energy , source {XT'S) (Oantdnl, 1952B), Thi ome thy lad eno s ine .'tras described a,ud llhked;' •f dv transmethylation reactions - by Baddiley (1951) s Smith and Schlenlc (1952i.: and 1952B ) ,■ Schlehlc and Smith (1953 ) $ and Bmith,; at al (1953) <,"• - Smith and Schlenh (1952B) reported that the compound.accumulated in yeast when excess amounts of.methionine were present in .the - medium * Oantonl (1953), chemically characterised "active methionine", as S f»adeno sylme thionihe ‘and as si gned ' a structure tbr.it (Pigufe - 2);«Schlenk and Be Palma, (1955) established that thi ome thylad eno s ine was formed' as a chemi= cal breakdown product of S-adenosylmethionine during .perchloric acid- extraction of yeast.'-
• The. amino;: a c i d me thi onl ne $ ; di s co ver e <3. by ■ Sue 11 er- ; in 1923-5 has . accuired. a central position' in the metabolic transformations that result in the syntheses and transfer of the biological labile methyl groups, i.e. transmethyl- , ation (Cantoni,'1952)» The function of methionine as a precursor :of sulfonium compoundss notably S-adenosylmeth~ iohine s rivals in importance Its utilization for .protein - synthesis and as a precursor of "other . sulfur contaihing.. . biochemical .compounds (Schlenk, 195.8) =. S-Adenosylmethion-. "ine is a key intermediate in group transfer - especially transmethylation reactionsp(Schlenk, Shapiro and’ Parks.,
' v - v : 6:• ■ Sciiiaidt, et al (1954) and Schmidt et al (1956)
reported' that 'met^lohine isas: a,{: drala: 011 the "pool" hf adenine . ghoti.ps• Of, ha$:ers treast and ' produced an accumulation of the sulfonium compound»- The yeaSt overcompehsated this "trap - ■-•ping11- of - adenine gnoups by me-thlonine by a, net increase in ': ’the biosynthesis of purines„ Reddi (1955) demonstrated that methionine caused an Increase :ln growth and' synthesis, - ‘of1 prbtein and RHA in Pseudomonas hydro phi la e - Also / the . presence"; of methlonine in growing',cultures of Po hydrophila brought about an Increase.-in the turnover: cf: SEA phosphoruse - Ehrensuard (1955);Reported the transfer - of' intact methyl '- groups from sulfur'to nitrogen through the participation" ot' methionine (as S-adenpsylmethionine) as a methyl donor for the R-methyl • group' in' Mordenine and the Hr6-methy 1 group': • - ‘of ricinine®, : • •-y." '.V'V - ' , v f -■;/ ''v; :;' 1 tit ' : ■,' - \ ' ' V
labor "et al (1957) and labor et al (1958) reported;, , the role of S-adenosylmethlpnine,;• I n ;the.: formation 0f spef- ;' midine -.from putrescine in extracts- of Escherichia coil;
:. - Alexander' and Schwenk (1957 )■ and Alexander et al: . ."(1957) reported:: the transfer of intact methyl groups from- sulfur to carbon';;in:;yeast'.by the participation, of methidn-^ - ■ine in ergosterol; synthesis„ Parks (1958) reported that S- adeno’sylmethionine -was more efficient 'as a methyl' donor In - . -- ergo sterbl synthesIs - in,: yeast ' than: was methionine alone andt .was used in preference,'to1 methionine 1 .'
v: Remy (1957) reported the formation of S-metti^l^ :vamino-6-amlndpurine from 2s,-6-rdlamlnopurlne In extraptsvof
coll. The requirements 1 noluded^' 1 methlonlne,' iifiP,:. and- magnesium ions apd it .'was sho-wn;that, the methyl group of methionine- ;was incorporated» Bemy (1958),' Isolated and • \- purified the. intermediate - in this; incorporation ’ and itttas; ■ shown to be S-adenosylmethionine = 1 Remy (1959) demonstrate . ed' S-adenosylmethlonine to- be the actual methyl donor In - this reaction replacing the requirement for ATP, magneeium ions , and 'methionine» . - / ry , '
' Mudd and Oantoni (1958) purified., the - enzyme - from ' b.afcers yeast that., catalyzed the formatioh .of' S-adenosylt . ' - methionine ..from. ATP and i-methionine0 ; ''' ; -' : ; . The role- of 8-adenosylmefhiohine and its numeroustransmethylation reactions are also reviewed by Prut on .' and'Simmonds (1958), Cantarow and Schepartz (1957) and
- Haurowltz' (1959) » . ‘I. . r ' ';:Jail (-1958), using-an adenine-requir 1 ug mutant of' .
Saccharomyces cerevisiae, found.that in the presence of: e x - . cess1-methionlne Lthere has. a propoftionate 'production. Of ' S-aienosylmethiohlue and cell .grohth as ' the adenine coh-;
teehtfation of .the. medium was increased»’ • ' v-Svihla and Schienh:,(’19.59 and i960) reported that ■
in the' presence' of methionine they yeast., Candida: utill's:,:.; produced large amounts of S’-adehosylmethionlne and stored' '' it in . its yacuol'e» ' ''They also found that this compound was.
iiot readily; ca-tabollzed and. that part of tlie material‘was ^transferred from the vacuole-of- the mother -cell Into- the , vabuolas of the "buds and daughter cells „ • ■
Recently, Tall .and Henhey- (1961) found that the ,pre'sence'' of excess l-methionine ■ in ihe-. medium''altered1 the " ' t purine, and. nucleic 'e Wild and an adenine• requiring mutant" of S6tcerevisiaee
Of fEOlEW
v; The" phrpo se :ofphis investigshioh "was:;Po' sPudy Phe .effect of ezoess h-mefhionine on Phe nPflization of radio- acf ive adehi^e ' by an. adenine. f e%niring fe spe oiad referenc.ePo r (f) the prodhb'Pibh ;of:S^adeno syl-- mePhiohine, (2).■ hhcleic acid aynPheSis and (5) inPer=■ conyersion af vpurines»; : v V ,;v -.P :f' ; r/’
' : V hbthobb \ ; " - '. : '
j- o : Media a ' : V ; ' ; : : 'y' . . / ■ : ' : ' ' " :' Saocliaromyces oerevisiae (strain 80-10-80^3-5) ,
an adenine -requiring mti'tant, was^ p:riginallyv obtained from the University of Washington. .-iThis -mtitaht was produced . . by exposing the parent s.train to: radioactive/.cobalt. It - .'Can- utilize the purines adehine or. hypoxahthine ;for growth. ; but . cannot: utilize guanine: or xanthine; ( Tall 1 9 5 . 8 5 The culture was maintained"oh yeast extract peptone (TEP)' slants of the following beroentage compositionglucose (2) s .peptone . ( 2 ) yeast extract (1.) s agar (1,5)1 (Roman . 19561)»
The synthetic, .completed, medium of Romany 1956A) oohtalned the following ingredients per liter' of soiution:
ammonium sulfate 1 gr PeClxeSHgO 50 ug '. glucose ' • i: ? ; PQugr' V ' ZnSb^.THpO- 1 70 ug. 1 L-histidine. HOI .. 10 mg :;/H%io '. 10 ug .;
. 1 -mefhionine ; ". 10 mg ' : 'OuSQA .bHgO ’ 10; ug ;,. .L^tryptophan• ;' • ' 10 mg ' , KI ., 10 ug
. .pyridoxiue HOI 400 ug ; : 0a01p„2Hp0 100 mgthlamihe; Hbl 400 ug : g \ :
. The pH was adjusted to} 5.8. g ) : ):} v /...'v ,; :/■ /; } 'Excess methionine refers to,,:597Itg. h-methionine
)Hi,fop laboratories, Detroit I7 Michigan u) -
1 '"'}:' }}}g’' ' .10 ' ' :' v /;;,:}g.
per liter (4- m liole/ml) of medium; and. normal methiozLine;' .to 10 mg 1-methlonine per liter' (0«06 u mdle/ml;)of med
Media .were prdpdred' w^ normal and with excesa methionine and dispensed In 100 ml quantities into 500 • ■'mhfBrlenmeyerflasks.^ . Ihese flasks were cotton vPlugged ; V and, sterilis.ed in ' at 1,5 IPs pressure' for 15 . iminuteso : ■. ■ : v - v ^
. Adenine~8“0-*- i'?.was sterilized by auto clave, guah- titated in a 'hee3aaah;DU Spectrophotometer{Beareh, Holidasr and’Johnson, 1955) and 317 u moles were added aseptically,.to the flasks'of media* flasks to which no adenine was .added were used as control si , Ihe radio a ctiwi t% of the : adehine-8-ot^ was determined by combustion to-carbon di- oxide, and'-plated: and read as Ba00% „ : The radioactivity of fhe:adehlhe"8^0t4 wsed = in.the experiment Was 1,472,000 :■ODM/flasko 'Since there war el377s;uif:,moles added per flash, this was a specific activity, of 398,000 OEM/ u mole, This figure.was verified and the purity of the adenine-8-0^ 'determined by column ' dhromatography, v paper chromatography ' and ultraviolet absorption spectrum. . :
\ / All,materials used'were of the highest purity - available . : ■'.-b'yl ’ , • /■ , -y i. ' v' -. -
,B . Growth of Organ!sm. • '7 :;■ . • The inoculum was. prepared by-'growing the cells for: . California’Oofporatloh for Diochemioal U Dos Angeles':
1248 hours ; at 22c*0 in YEP ."broth. In 500 ml Erlermieyer flasks, on a -Brunswick; .s-]3ken';.i;::::':v$hc ■ cells were,", sedlmented by centrl- : fugatlon^ washed 3. times .with sterile distilled, water, and t resuspended In sterile distilled water6 Replicate aliquots were added to-Vthe:;normai;, f:eweess and uontrol flasks contain- ;; Ing synthetic mediae '" Sterile tubes' containing-:20 per cent ''ICOH were' .inserted Into some of the flasks in order to absorb) the Cafboh diozide formed. :lhe flasks were'incubateds wklle - shakihg', "for 48 hours at 22o0| after which time the pells were sedimented- and' washed three times by' .centrifugation. The washings and medium were combined and-Called-’ " super- nataht''. The cells -wefe ;>esns:pended in : distilled e > -The -supernatants. and cells ware frozen at -20°0 until'need- ; r edo The tubes containing 20 per- cent ICOH- were; removed and - a solution. of Ba (OS;) g and_ BaGlg :wus :;added. ih crder to pre- - ;-4 ‘ cl pi fate the absorbed Carbon dioxide as BaCO-^«. -. The radio - , . activity of the BaCO-j was determined. : .'" ; - .; '
';0.:--.%traCfionS'p;u:; ; ’ yv, v'lx,'. vl:;y - ; V;,,'/; /: vlvl-'lS-AdenosyImethiohine was extracted from the cells b
using the cold 1.5 B; perchloric acidrmethpd of S'chlenk, - . 'Dainko and Stanford- (1959). The S-adenosylmethionine was assayed by chromatography cn Dowex .50,;.:H+ resin, and by . ; . Ultraviolet spectrophotometry (Schlehk and BePalma. 195.7A- and- 1957B). The Bowex 50 column was , developed" with B,'' 2 B t, -, • ; and ;4;:B'BC1 in. vdlumes pf: 300.,’ ,300- and 200 mis respectively. 4-
; v \ \ r : \ ^ 13.The nucleic acids were extracted and' hydrolyzed
"by a modification of the method of Ogur ejb al (1952) o fhis method consisted of extraction with- 0,2 H perchlofic - 'acids acidified alcohol,■and a1coho1-ether to remove all - non-nucleic acid ultfaviolet absorbing material./ The BHi" was ' extracted by treating with'I" perchloric acid at 4°0 for ■18-24 hours' followed by 3 washings "with same in the cold«' - The DEA was extracted, and hydrolysed by treatment with M perchloric acid at 70^0; for 40 minutes „ Ribonucleic acid .-was hydrolysed by similar treatment4 'As a result, the : - hticlelc acids were split to purine bases and pyrimidine : ■nucleotides (lorlug-. 1 9 3 5 ) ’• Ihe- hydrolysates; were adjust- . ed to pH 8- with E KOH (Davidson 1952), The insoluble. EOIO4 was. 'removed, by filtration through a coarse sintered glass. f i l t e r T h e solutions were adjusted to pH 4 with H HOI and then applied to Whatman $1 chromatographic paper„ ' _ ■
V. m s a z t e i s x m . ,( ' . ..One dimensional chromafography was performed - 'using the isoamyl-dibasic sodium phosphate system of ; Garter .(1950) and the butanol •‘•water system of Marlcham and , Smith (1949) o 2he purihespyrimidines, and their, deriva^ ":: tives were detected.by using an ultraviolet lamp at 260 mu (Wyatt 1955)o The ultraviolet absorbing spots were cut ; :from the paper, eluted ■according to the method of Garter /'- (1950) and .quantitated with the. Beckman DU Speofrophbto-- " ‘ .
.mete*- (3esire3i$ ; Holiday a M Jolinson 1955)« ’ . v
E» Total Qomlustlono : . : ■' • ;Radioadtlve assays of organic' materials were per- '■
formed using the combustion methods of Van Slyke and Folch (1946) and Calvin et al (1949)0 ■ The former/method was onlyfor non-volatile compounds' and the latter for volatile or
•: : .. : : ■. ' . ■ ; , - , non-volatile compoundsThe: apparatus used was describedby Stutz and Burris' (1951)».Combustion by these methods produced a,- total conyersion of;;carbon/to cl^Og whioh was collected as BaC- Oj,''- The BaC^ Ot was then filtered through tared; Whatman #. 542 hardened .filter paper (2ol4 cms in diameter). %lh':'an -S429', filter / tower apparatus^. ■ The filter / / paper with: BaC^Gj was/, weighed5 placed on a disk and the radioaotiyity/ determined.'in a D-4? gas flow geiger counter^. The''weight of the BaC^ O^ was used to correct for selfabsorption (iCamen 1957) « . 1 \ ,-./ /
.Fo 1 lowing' he , 48 hour . exposure ; to : adenine ~8=rG * g. the ceils and. hupernataiit were oomhustea separately and theVradioactivity, 'determined by methods previously described,, The results' ■ .are rShoM'En ..Table 1 „ -' : / ‘ '
The radioactivity of.carbon dioxide collected during the exposure' was .700 OEM/flask: for excess 1-meth- ; idnlna and 1>5Q0 ■GP31C//fIasi: for normal ; B~methionihe«1
The superhatants,of the normal l^methionine and the excess 1-methionine accounted for. 1705000 and 175,000 Oi%;respectively*/: y : - : r ■ ' ■ '
i^The:cells in the presence of both excess•and normal L-m.ethionine,. contained 87 per cent of the radioactivity .detectable in the'flasks after.thev iuonbation period®
The ■supernatants of both the horiaal and excess cells, were chromatographed on paper and ion-exchange col umn' as. previously described® . The:, various fractions were combusted to determine' which compounds contained the re- sidual' radioactivity„ The radioactive,compounds were ident ifled as nucleic acid fragments or.polynucleotides. There - was no- free adenine,-8~G^t";'detected in - the supernatant;, : :;thereforey ..it was concluded that complete uptake of the,' /- .supplied, purine ty the cells had occurred®.
15
16
Dlstrlbutloii" of radioactivity following a 4-8 hour exp os- ure of yeast. to, adenlne-8- in. the. presence of normal
lr ■ / 1 ■ ' ' and exdess' -aiiouhfs h'f 'h™methionlh'e' f 1 ■ ■.
- 1: . OPM/flaslt in thousands . Total / Activity ' y Activity Detected'' ; \ . : ' . - Detected -, ( Oells ) ■Sample ’ : V Supernatant ' ' Intact' (super &' ".I
■i' Excess L~ '. - v ;'.y. .. y . ■y. Methionine;: ; 175: O-' 1:,125: 1,300 . 87
Activity :introduced_ 1,472»000; OpM/Elask
. - ' ;: Ilie S~adenosylmethlonlne extracted from the ex- >cess L-methiotiirie cells was quantitated and com'busted for radioactivity^ Therevwere 0 0 7'n moles of S^adenosylmeth-ionine vf orfeed,. vwith . a-^. Which was. 15. per cent of the radioactivity fixed’-in the .: intact cells« Extraction of the normal L-methionine cells revealed only a trace,of S-adenosylmethionine and radio- ' ’. a c t i V i t y o . i - ' ' " r- .
. . The cells were extracted, for hucleic acids and ‘ :. the various fractions com'busted for - radioactivity determine nations« • In the ‘presenoe: 'of normali.L-raethibnine> 79 per ” can of the fixed radioactivity was in the nucleic acids of the cells» This was in', oontras t to - the cells grown in the t ; pre s ence: of-excess^: l~methi onlne, -wh contained only 53 ' ' -per / cent - * of -; the fixed, fad lo a c 11 vlfy- lh:;'the nucleic acids 0 -
• In the presence.of .excess 1-methionine5 42 per cent of the fixed radioactivity- was found in the .initial acid-soluble $ - .non-nucleic acid fraction. However,' in the pfesence of‘ i ■ normal•1-methionine only 18 per cent of the fixed radio- .activity was present in this fraction« The distribution ; and per cent .activity .inv the .various fractions' are listed in Tables 2 and 3..
The 0.2 H HOIO4$:• acidlfled-alcohol and alcohol-, ether extracts, were -chnomatographed on paper and the compounds detected■were combusted and their specific actiyi- '
ch •
18
;'v.; . . : $ablb a- - :I)lstrltiu:tloA of/Radioactivity in Yeast /Following a 48 Honr Exposure to AAenine-8^ol^/in the /Presence of Excess -
X :: X- X ' ■ - : MetMdnine' / ". ' 1 'X ■ . ' ■ ' /
• ' . TABLE 3' 'Distribution of Radioactivity in Yeast following a 48 Hour Exposure to. Adenine-r8>0^^' in the Presence : of Hormal
.L-'Hethionine
fractionActivity . OPM/flask ■ in Thousands
■ ^ ofActivity; of Intact
Cells
Intact/ Cells:-, : \Cold Qy2l HCIO4 'Extract 'Cold Acidifi ed Alc0hoi; Hot Alcoholylther 'Cold -H HCI64 (REA) ' /1; 4 Hot E HCIO4 CDHA). V'Cell Debris . . ’ h
1,102 200
- 93
803
18 : 1
727
ties were deteriainedo The specific activity of the guanine was less than that of the adenine-isolated; and the speeifio aoti$ity of the -latter was: still less •than that ;■ of the original adeuine-8-0^ supplied in the mediuml. The specific,activities of the compounds isolated from the . normal,l^methionine cells,were.higher'than thbse; from the -. excess itmethionine ce11s. The specific activity of the : S-adenosylmethionine found in the 0„2 M H0104 fraction was in agreement .vrith;, that phtained hy Ion-exchange 'chrom-- atographyt, The results . aip: summarized,'in Tables 4 and 5>
/ , - ’ ’ The hydrolyzed:- nucleic -acids were chroma to graphed on paper and specific actiyities: were obtained fop the '.purine bases'isolated.®; • Both adenine and guanine were : - radioactive® The specific" activity of; the guanine, was lower than'that of the adenine' whiohyln turn was lower , ' than the;original adeninet8-d^^ added to the:media® .The specific activities of the compounds isolated from the ., cells grown; in the. presehce 'of:normal l~methionine, were , ; higher than those grown With-- excess h-me thi onine In the medium® These data are summarized in tables 6 and 7.
, - The cell; yield in the' presence ef normal L-rneth- • ionine, was about twice that of the- cells grown with ,ex~ -; cess l-methionine,,.; ,
: ' : . ■' • TABIiE 4 - :M S trl’biition of ,EAdioa6tivity ..in Tea.s't Following «, 48
.. ■ ■: - ' . v *1 - ' ..V -Hour Exposure to Ad;,enine-8-G-^ in the Presence of Excess' E-Methionine . .■ :
Extract, Eadioactivev . . , OPM/uMole: V. : ? ■ ; / : , C o m p o u n d s ; i n . ' A; : ' • : ' 4 . ; ' / ' ' / ' I ' 1, ' . ; ■ V : t h o u s a n d s . . - -
Acldifled-AlcohoA Adenine \ \ 250
Gold 0»2E HG104 S - Ad eho syIme thi oni he , 240Adenine 250Guanine 210
Alcoho1-Ether Adenine- 237
# Adenine-8-0^ Introduced - 398»000 CPM/uMole,
'22
Distribution of Eadioaotivity 'in Yeast iollo#ing a- 48 Hour Exposure to Adeline- 8 in the Ere8enoe of Hormal,
L-Methionine ' V
Extract •
Gold' 0>2B HOIO4
Isolated Badioactive ’ Compounds
Adenine Guanine:
.-Specific Activity - OPM/uMole in ' Thousands
300250
LcidiYied-Alcohol v 275
Ale ohoI-Ether ' Adenine 310
Adenine- 8 introduced; 398 ? 000': OPM/'uHole
23,
3 ' . ' ; TABLS 6 ■„ ' 'DistriTmtioii' of Radioactivity in- Teast Following a 48 Hodr Exposure to A d e n i n e ~ 8 i n : tlie Presence of Excess
■ TABLE 7' 'Distribution of Radioactivity;' in' least Following • a 48 Hour Exposure to Adenine -8-0- ' in ■ the Presence of Formal
L-Methionine
ExtractIsolated
RadioactiveCompounds
SpecificActivityCPM/uMole"■ ■ in . Thousands
Ribonucleic Acid Adenine .Guanine
340240
Deoxyribonucleic Acid Adenine 'i . /- : ; f A; 3 f Guanine’: .
' 340 270
4 A4eniue-8-C^^ introduced 398,000 CPM/uMple
DisdussicS
Tiie.. results obtained are iu agreement, with those- of Tali' (195.8) who -reported the. accumulation of S-adeno^-: . ■
' ■ sylmethi onin'e. bT an: ad enine »requir ing ..Teast mutant in the ■:. presence of . excess l-methionine. .V
The finding of nucleic acid fragmehts in the media is in agreement with Boreh et :al (1955) who- reported the ;,presehce/of, such ffhgmenfs in the media in which both ; ;; - r lysogenic and non-lysogenic strains .of 'Escherichia coli ' were growno .These could be excreted products or accumulat- ." ed dehrls from dying microo.rgahisms '
' ' : : The presence m f . free puiine. "bases In the acid- hisoluble noh-nucleic acid fraction of the cell; has been
• demonstrated previously. •Schmidt et al (195^)$ Siminovitch . .■and ■ Graham::i 1955') $tSpbmidt-. et al>';(1956), ;0owie and Bolton •' (1957;) s and Roush et al (1959) h H suggest the presence • of purine popls in. microorganisms . ; . _•. ■ \ , The ; observed presence . of S-udenosylmethlohine ) in the. 0>, 2; hi SOlOmtnon-nucleiobacid fraction' .greatly inm
. .creased the radioactivity of this fraction of the excess ’1- methionine cells over that of . the. normal L-methionine cells,
• With the limited amount1 of adenine -8-0^^ supplied to the.;. ;■ cells, tbs;shift;in'metabo.11 sm toward1 the production of = ;■
25
;S~adenosyimethioBine due to the presence' of e x c e s s h-meth- ionine could- also. explain the- decreased, radioactivity observed in the nucleic acids of these cells„ The same • r-■ amount of radioactivity.was fixed by both normal 1-meth- ' ionine andt excess h-methionine',cells but adenine utilization ; was shif ted : toward the activation of methionine rather than v nucleic, acid synthesis in the presence, of excess i-meth- .ionine0 •';:This- effect -is. demon&trated ;by tne: decreased cell -t■ yield obtained in the: presence; of excess l-methionine. ,
. ICornberg. et al (1955) isolated from yeast an en- :.■ ;;n.yme . cataiyzing,’ .the : cpnyersion,cf purine"'bases to mcleo- .; ;Vv;■ tides« An example of this reaction■ is hypoxanthine ' t . ,:; phosphoribosylpyrophosphate: inosine -5 phosphate. -I; :py r opbo splia t e» •;;; Ino sine pho sphat e' can -be cohverted ; to / '. :■ both adenylic acid and, guanylic acid „ ' The finding'of ■ - 'radioactive guanine revealed,that an adenine to guanine ::
1* conversion; tooh. placeSince 9 .with; this mutantg hypo- . . . V,Xanthine can substitute forAdenine hutIguanine cannot
• . (Tali', 19585 9 a bio eh In the transf ormation of guanine to ,, adenine ,is .indicated<,1. fixamples of., this' type, of mutation '' in.microorganisms are found repeatedly in literature:-.fierce; and horing (1945) s heurosnora; Mitchell and .Houla-, \: han: ;(1946A)-,:::45 independehtly occuring mutahts of heuro- , spora; boring and;fairley (1948), heurospora; Fairley and - •boring (1949), beurosnora; Kerr- et al (1951) Torulopsis
utilis,? HortJaan and ilorris (1951) Schizosaccharomjces :oatoSpo'nus:? ^brams -(195$) / 3, oerevibiaeT Pamper' (1952) o ' ." ■7 mutants of 8. careYislae-; Abrams (:1952) 5 -So oereyisiae ;V Hamilton, at 'al (1952)9 Aerobacter aarogenes; Weygard (1952), : lactobacillus lelchmanil: Pries (1955) $ Opliiostoma mnltl- annnlatnm; OBamlerlain and; Eainbow (1954), 8. cerevi'siae’: and 8» o ct o.sporns i Aarons on .-.(1955 ). ? 8 taplxylo co c cus -. flavo - cyaneus ; Siminoyiten and. Graliam (1955) $ B= coll; Bllner v(1956), ifibrocoecus pyogenes var, aureus ;' Oowie and Bolton(1957), 0 . 'ntilie « Pagersten'(1957 ), 7 •mutants of 8» cere- • ylsiae; Sots and ; Sollub. (1.957) ' B. -jOoli and ,■ typlilmurium;; Mc'Iutt '(1958). Heurospora; Suriano (1959) , . 'Staphylogoccus aureus; and Wood and Steers.. (1959.), 8,sssuso ; ft-i: 1 ' : ^ : - :n . V :
■' Indicate; a-'metatoilc biock prior to inosinic acid), since the organism is deficient in its ability to synthesize ' , purines from simpler constituents«/ ': llie: discovery ithat the; specific activity of the ■•
isolated :purines.ms- lo'wer than the supplied 'adenine-8-'01^; ms; vCdnsistent;' with the vf611 owing .’reports in the- liter- : ature : ■ ' I "I-'" -x ■■ v ■ : \ ; . : Eorowitz ' et val.;; (IS^il ,and : Horewitn; (1946) report . . :that a cholineiess strain of EeuroSppra was not.complete- . ■ ly deficient in the ability to- synthesize choline. An incomplete m-etabpiic; ,-b 10ok was :suggested® Mitchell and 'Houlahan (194-6B') ■ reponted a riboflayinle ss mutant; that produced riboflavin at'low temperatures.. They suggested that either the gene involved had not been-completely in-.' aotivated and 'controiled; the pnoduOtlon of 'a modified; and temperature sensitive enzyme or the gene was completely inactivated, and that the temperature sensitive riboflavin . synthe si s' /was. d.ue;, to .tne;. adaptive capacity 'Of. some'' other ' ... - ,.system in /the organism^ - Wagner (1949) reported the. in■ vitro . synthesis of pantothenic / acid by pantothenieless.. strains ;.01 Seurospora. / He Goncluded that the mutants possessed, a ■ mechahism which was active in vivo in/preventing'this enzyme system from operating in pantothenate synthesis. Wagner and Eaddox (1990 and. 1951) reported the , in-vivo:?synth&siscnf 'Pantothenic/acid by "pant0thenicless"; WeurosPOra. -. kalckar ;'-■ ■■;"
v :;:-v ■ .
(1949-5C)j Jr.eported. thd synthesis adenine /by 'an adenine-; .less '"Laoto'toaoillusl'i'cti'aeiV Bonner et al (1952 ) studied - mutant,strains of,Beurospora' genetically:Hooked in the formation of niacin and"required this .substance for growth/ .as well as- similar;: .tryptOphanless .strains, and found .. •• ,them capable of synthesizing the specific compounds re- ; ' speotlvely required for growth<, •He indicated that altera- tion of the; 'Speelficity of an enzyme ;could lead' to. a de- ' crease- in.-the- rate of the reaction;It catalyzedThe time : . at which, an .enzyme was formed could be altered or that inhibition of enzyme activity could occur<, -Relief; from such ' InhihltiOn - at "some 'stage of growth would -then restore '; normal actlyity* Abrams (1951, 1952) reported.a yeast ' mutant of St cerevlslae which would not grow unless supplied adenine cr' hypozahthine. but then proceeded ' to-, synthesize-- 'the purine s. / Balls ejr.gj, (1956) reported -an A a- aero genes -.mutant that began de novo synthesis and also - reduced .thespecific activities' of "its supplied purines, . Wood and Steers' (1959) had the same results-with.So. aureuS and Me- '
- Hutt (1958) found this occuring in a Heurospora mutant„ Morris (1959).suggested incomplete metabolic blocks in■ vitamin''B6. requiring strains '• of -Bo • col 1* Straus nhd • Min- ' . • agawa (1959) suggested that the observed methionine syn- ■ 'the.sis by a methionine-requiring mutant occurred by the y :normal, not an alternate: (pathway, I ; : ; ' ' ; /;.:v.• / ;•■.
SUMMEY
;, ; ■ An adenine^Yeq.uirYB^ ;;iimtant 'c>f 'Saccliaromyoes \ Ycerevislae (styaiii S0-10 =-80-3-5) was Inoculated, into synth- ..etic "complete" media containing aienlne-S-Ol^ as the sole ; source:>ol: punihe 1; ;.In:; one' Set .ol experiments themedium : contained the normal' amount ol L-methionine and in another set. the* medium contained excess amounts of the amino acid.0 .The cells were harvested' after' 48 hours incubation on a sliaker a t '22 0, and the. 'fete of' the* adenine-8-0^ r ' was defennined-bY; ahalytica.1- procedures:,. The following . 3 Observations whre- made : - .■/; " : '• :
(1) In the presence of excess amounts of L-meth-. : ionine, 15 per cent of the radioactivity fixed by the cells was: found, in the S■-adenosylmethi0nihefractionv : This was in distinct contrast to the cells cultivated in the pres- / ence of normal amounts of h-methionine, which.produced only a trace pf 8-adenosylmsthioniueo ' ' : I- *
. ■112) ;• In';the, presence ■ of excess amounts of L-meth-. ionine.5 53 per cent of the .radioactivity fixed by the cells , was 'detected in* the nucleic'.acid fraction, .Forty-seven * :. , per cent of the radioactivity was in', the ribonucleic acid fraction and 6 per cent in.the deoxyribonucleic acid fract- iono * vln .the presence of normal .amounts of' 1 - m e t h i o h i n e :
.79 per cent of the radioactivity fixed by the cells was detected- in. the nucleic acid fraction. Seventy-two per cent of tle- radioactivity Was in the ribonucleic acid . : ’fraction and, 7 per cent in the' deoxyribonucleic - acid fract- i ion. .'The specific activities of the' purines isolated from the cells •constituents were in all cases lower than-that of the a d e n i n e ' originally introduced« This "'indicated • . that the Cells were able, to a limited extent, to carry •out do novo synthesis of the material for which'they-were , . 'dependents \ 7 \ 7 , \
. (h). There was no. significant: difference shown,in the ^interconversion of purines due to - the presence of . ; . excess amounts of 1-methlbnine«- Radioactivity was present in the guanine fraction indicating the conversion of ad- 1 enine to guanine although the reverse.process had previously been; observed :: to be blocked; ; ' . : - 7 r / '
; - REKSEMOES \ ^ ' 'v: Aaronson, S» . • 1.955 The purine requirement of Staphylo - ' ' .coccus flavocyaneus. ; ' Q6h«. Mlcrqhiol, P -129: T?7-155«/.Abrams $ R. . 1951 Purine synthesis .In a purine •“requiring
yeast, mutant» J, Amer* Chem<, Soce $ :75> 1888-1889»' AbranlS'g; R < /ISSg - - Inbo^Pora^ /g3.ybine™l“»Q^^ and i:. • '' adenine- 8 in the purines of purine-requiring
yeasto Aroh9 Bib'chemov Biophys»„• 57o 270-275.Alexander,. G» E« $. Gold, A t- Hi, and /Schwenlc# ,Es 1957 ; / ihe methyl group of- methionine as'a source of Ogg
; in ergpsteroii- / ; it Amer, Ohem. Soc6 s 7£, 2967.o / V - AtAlexander,"G,.E« and Schwenk, Ev; 1957 • Transfer of the
; methyl group of methionine to carbon-24 ergosterol.. - Ji Amero • qhem,.: Sop t,. 455A«4555i '•Alexander, P0 .1957 Atomic :radiation - and Elfe o / •; Penguin ' .Books,. Inc, ,/ Baltimore* t ./Baddiley, A* ' 1951 Adenine-51 «deoxy-5 1 =*methyl-thio.pento- -
: ; side(adenine- thlpmethy 1"pentoside) i; A proof of. : ■" structure • and synthesis0'/ ii:9hem0 Soo«* 1951.,: 1348- :
../", .. T l■/^ .'■/•'■:' ■ • ' • ' y ■ ' / ' . ■..' A t ' :Balls,- Ho. So., Brooke, H a, 8*,- Bro.wn,: G* B», and Magasanik,.B„ f he utilisation of = purines by purine less
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S c h l e n k , ]?<> and Smith, E0 L« 1953 The meohi.nlsm of adenine', thidmethyIrlhoside- formatlono . J0 Blol6 ' Chem» s '204,* ;27-3a.: ; : 7:;.::
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