[ VOI.. 32 140 ANNALS 01: THE ?ViISSOUlu[ 1:OTAKICAL GARDEN traction was done in the abwncc of substrate. They found that growth in the prcscncc of urcn stnbilizcd L~IC. urcasc content of cells. While exceptions may be found by future study, thcrc docs cxist a relatively large group of \vcll-tlcfincd cnzymc systciiis which respond positi\rcly to thcir specific substrntcs. In the c.isc of thc "ndnptivc" enzynics, the response is rnnrked nnci thc cnzyiiics in11 to zcro or near zcro Icvcls in tlic abscncc of siibstratc. Thcsc cnzymcs sccm to differ from the constitutivc oncs solcly in thcir rclntivcly grcntcr instability in tlis abbcncc of substrate. It scciims qucstiorinblc, from this point of view, wlic~licr classification into "adnptivc" and "constitutive", implying IS these tcrnis do a diflcrcncc in origin and function, is fruitful or cvcn valid. This same point of view implies thnt enzymatic "adaptations" arc but qu.intit.itivcly cxag- gcratcd instances of a more general phcnomcnon resulting from the cflccts of substratcs 011 the synthesis and stability of tlicir enzymes. \While we shnil in ~lic prcscnt diwxssion usc thc term "adnptivc" in corincctiori with cnzyrnc formition, it `should bc cniphasixc? Llint this is not mcnnt to iiiiply tlic tlc iioz'o induction by sulsrrutc of, the enzyme coiiccrncd. The tcrni is used licrc` to describe tlic situ.ition in which an cnzynic responds by increasing in the prcscncc of its substritc and decreasing in its aUscncc. From thc stnndpoint of genetics, cn%ymntic ndaptation has scvcral interesting postibilities. Thus far, attacks on thc ikoblcm of thc nnturc of gcnc action has Iind to dcimid, for the most prt, on tlic stuJy of the final cnd products of cnzynintic rictivity. It has gcricr.illy hen assumed that gcncs dctcrminc phcno- type by irirtuc of tlicir control of cnzynintic constitution. If this be true, tlic pr occsx of .i (1 x p t n t io n 11 rcscn t s 3 n mi n iqcc o p po r t u 11 i t y for c xn in i ni n g ccr t .i i n dct :i il s of scnc ac~ion. In p,irliculnr, it is rc:icori.ible to lwlx that such studies coulJ. ticliiicntc tlic nnturc of tlic control cscrcisell by sciics over cnzymc activiLy. It is thc purpose of this ppcr to prcscnt sonic data bcnring on this problem. and thcir varintions in yeast cclls. We sh!I confiiic our attcntion EO galactozyniasc 2nd niclibiozpiiiasc activities BIOI.OCICAL hfECl IANISMS OF l'Ol'ULA`I?ONAL ADAPTATIONS Large numbcrs of individunls arc .ilwnys involved in adnptation cxpcrimciits, and it is incvit.xblc that attcnipts to clucidntc furthcr the biological mturc of thcsc modifications encounter a basic problem common to nll studies of physiological chan~cs in largc populntions. A conipnrativc biochcmical study of large popula- tions alw.iys involves ovcr-all populational charactcristics. This nccess.mrily intro- duces difficulties into the intcrprct.itioiis of any obwvccl chan~cs in physiological propcrtics. The nicchnnisms nvnilaLlc to an individual ccll for adapting itself to an cnvironmcntJ changc arc limited by its gcnomc and thc pliysiologic.il flcsibility pcrniittcd by its pnrticular dcgrcc of spccialiintion. \Vhcn, howcvcr, tlic adapta- tion of R popiilation of cclls is being considcrcd, tlicrc must bc added to tlic p!iysio!oSicnl pliability of its nicmbcrs the gcnctic plasticity of the group in tcrnms of the numbcrs and kinds of varimts it is CJ~LIC of proclucing. $cause of :his composite natwe of poput.itionat adaptability, it is chr that in any $vcn case thc s:>.me end resulc can bi. obtaincd by any one of tlic following nicchznisnis: (I) The iiaturnl selection of existent vnri:iiits with tlie desired cha;actcrirtics from a gcnot~picnlly Iietcrogcncous population; (2) induction of 3 ~iciv (.IS far 3s niensurcilieilts ;ire concerned) cnzynic by tlic substrate in all thc nlcniters of :I lionio~yncous popul;ition; 2nd (3) R combination of natural selection 2nd rhs action of iiicchai~isni (2) oil those sclcctcd. It WIS difficult to resolve these questions with b~cterin sincc genetic control over their popuI.itions is not .attainable. In two ixistnnccs, Irowcver, a decision on the SioIogicnt mccIinnism involved wns possible. Lewis ('31) slioweJ tli'1t. the ability of so-cnifcd "rrnincd strains" of U. coli to fcrnicnt lactose oriSinated rhroug!i thc natur;il selection of :I spontaneous vnri.int which \\\'as nlwnys present in the original cillturc in thc r.itio of about I:I s 10'. StcpIiCnson nnd Sticltlnrict, ('33) were`ablc to hioiistratc tlic forniatiori of Iiydro!;cnly:isc in tlic presence of formate in non-dividing CU~~LI~CS of B. coli. It is clear chat a consic1er:iblc advantngc would be g2incd if it were possible to study this phcnoinenoii x*it!i microorganismic poput.itiyns whoso .genetics could bc con:roIlcd. Asidc from the .obvious possibility of cx:iminini the genetics of rhe process, the study of its physiology could be cnorniously' siniplificd. Itc- pducibility of the nicasureincnts would thus be assured anti thc complications of nacur.iI selcctiori, which nrc nlwiys present wficn tfcaling with gcnctically hetcrogcrxous materi.il, couItl be nvoirlcd. The opportunity of using genctically coiittoflablc niateriai \viis providc.ti by tlnc funJ:imcnt.\I \vork of \Vingc nntl L3ustSen ('57, '58, '>?:I), in Ilcnninrt;, anit ttic Lintkgrcns ('432, b? c), iii this cosi1:ry, on tiic pnctics iiad Iifc cjvclc of the ye:ists. ?%E GESE*IIC CON7'110L OF GAIACTOZYMASE FOllhlhlION IN YEAS'I- POPULATIONS Diencrt ('00) \vas one `of the first to describe a \vcIl-dcfincd csnmplc of pop- &tionat adnpcntion in the yeasts. He showcti that suspensions of ycnst cells &Id ncquirc the enzymatic npp:imtus ncccssary EO ferment galactose whcn placed LZ contact with it. Since its discovery by Dicncrt tliis p.irticular problcni has bccn investigated by nunicrous workers. Armstrong ('05) confirmed Diciicrt's f;;.Jin;s xiti furc:icr fount1 that sonic yeasts were incniublc of acquiring this p5ysioiogicnt property, no mntter how long they were cultured it1 the presence of SddCtOsc. Slator ('OS) showed that those yeasts cap.ib1c of fcrmcnting salactosc psscss this nbitity only aftcr they had bccn acclini.itizcd by culture in its prcscncc, RO yenst lie invcstig.itcd WRS nblc to fcrmcnt this hexose inimediatlcy upon being incroducccl to 3 medium containing it. Tlicrc wns dways an iriductiori ycriod OE ~riable Icngth connccted wit11 thc ncquisition of this property. Scvcr;tl attempts \sere ma& to decide whether natural scIcction or a direct inicraction butwccn tlic g.~Iactox mri die cytoplnsni wns iiivolvcd in tIic aplwilr- accc of plnctozyrnasc. Sohngcn and Coolliaas ('24) grew their yeast cultures at 30" C, and measured cn7yniatic activity at 38' C. to avoid cell division during the n:c;Isurcnicnt of CO? cvolution. Thcy concluded, from tlicir crpcrimcnts, tliit tlie production of gatactoi.ymasc par.illcls rhc formation of ncw cclls. In aciJiLion, thcy confirnicd Kluyvcr's ('14) fitidings thtt at 38' C., at which tcm- pcraturc ccll division is complcrcly inhibited, 110 atlaptntion gake5 pl.lcc. Othcr invcstigntors also trid to obtnin adaptation in thc alxencc of cell division, sincc this \~;oulJ clearly cxsludc the opcration of natural sclcction as n cauwl agent in cflecting the clinngc. Eulcr and Niltson ('25) and law Eulcr and Jmsson ('27), in .I morc thorough invcstigation, tried, without SUCCCFS, to adJpt yeast in the prewncc of 0.5 per ccnt phenol to inhibit cell division. Thc fsilurc of the nbovc- n1Cncioned autliors to find atl.ipt.ition in thc complctc .~bxricc of cell tliviGon cannot bc tnlicn ns conclusive cvidcncc tti.it no such phcnomcnon could cxist. Kspeci.lIly is this truc in those cases wlicrc supprcs\ion WAS olir.iincJ bp such agents ac. Iicat or cellul.ir poisons. It is iiot unliLcly dint in culcurcs whcrc this "ideal" h.id bccn renchcd, rhc pliysiologicnl stntc of thc cells wns such dint their ability to syrithcsixc new cn-rymcs had been lost nlong with thcir ability to divide. Stcplicnson and Yudkin ('36) conclutfed fioni their cxpcrimcntr that thc pro- duction of gntactozyinasc in ycst cultures need not involve the fornution of ncw cclls. This conclusion was bascct on thc observation that the ability to cvolvc CO? anncrobic.illy, from a nicdium cont,iining g.iI.~cto~e, w35 acquired in a pcrid wheii the total and viablc count remained constnnt. Thcsc findings were ap- pnrently in tIircct contr.idiction with those of previous worltcrs 2nd in particulx of Sohngen nnti Cwlhans ('2.1). Beiorc uriJkst t.il,ing aiiy dcteilzct study of tltc physiology of this ndaprarion, it wns clcarIy ncccssuy to rcsolvc this tiiiiiculty. It is cvidciit from tlx naturc of ~hc problem of popti1;ttional atl3ptnbility that one of thc crucinl problcnis nt issuc is thc ptirnotypic hornopncity or hctcrogcncity of tlic starting population. The possibility of attacking thc problcm from this point o€ view was provided by the usc of known Iiaploid arid tliploid strains, thus pcrinitting gcnctic control ovcr the populntiohs bcing studied. Two strains of Succbrlro~~zyccs ccrcz&itic, Db2313 and LK2G12, bot11 of ivhich could acquire the abiiity to ferment gdactosc when grown in its prcscncc, wcrc sclc.cttd for study. Strain Db2313, which WRS known to be a haploid arid there- fore genetically unstabk, was shown (Spicgclninn, Lindegrcn and Hetlgccock, '44) to be plicnotyyicnlly hcterogcncous with respect to gxlnctose fcrrncntntion. Sonic individuals in populitions tlcrivcd from this strain could not adapt to fcrmcz~t galnctose, wlierens otlicrs could. Strain LK2G12, 011 rhc other hand, Tvliicli urns known to be dipl~id, was uniformly homogcncous in that all of its iridivicluals w'trc ablc to acquirc thc capacity for thc fcrnicnt.itivc utilization of plactocc on stnntfing in contfict with the sugar. The adaptive licliavior of tlicse two strains followcd what ~oultf Le cxpcctcd from the tJJt.1 obtaincd 011 thcir phenotypic 143 c!lzrJst,-ristics. Pol~u~3tions of Db2JB, stirring with a low pcrcerit:igc of the icrnlcnting typc, could incrcasc tlicic cnzyniiitic .Ictivity only through the IIl&misi;i of cc!1 divisioii nnd the subscqucrlc sc!cctipn in favor of. 111~` galactose fcrncntcrs. It was sliown (Spicgclmnn and Lindcgrcii, '44) chat the kinetics of &ptarlon by Db23B populations wcrc in :igrccniwt ivith thc niturn1 selection b.yp t licsis. Using t Iic n p propria t c s t rxins, cx Lx" i nieii ts were perf 6rrncrI \v Vhid t sought to Juplicatc the findings rcported by Sohiigci1 and Coolliaas and those rc- ported by Srcplicnson and YuJkin. The results arc sumtnarizcd it1 fig. I. 194SI S P ITXELSI AN 7 EN %Y Xi A?7C A 1) 12. P'I`ATIO N `OUi 0-Strain Ob238 a-Sfrain 012 I I 260 W -4~;i~~~'~ cv $20 Q 5 u O-@, 15 17 19 2'1 23 25 27 29 31 No. of Cells'per Square Pi,;. 1. A comparison of dic vnriAtion of crizyinc activity (cxprcsrd 2s r312 of znarrobis CO:! ixoduction) 2nd the r.um!>cr of ccllr in adnpting cul- turcs of h~pluiJ (L>b?ili) jnil diploid ($12) Stiafns. $20 5 u 15 No. of Cells'per Square Pi,;. 1. A comparison of dic vnriAtion of crizyinc activity (cxprcsrd 2s r312 of znarrobis CO:! ixoduction) 2nd the r.um!>cr of ccllr in adnpting cul- turcs of h~pluiJ (L>b?ili) jnil diploid ($12) Stiafns. It is strikiiigfy app.irunt that populations of tlic haploid striiin, Db23B, in-. cicsse their cnzynic activity by virtue of the ncw culls arising during the cspcri- cicnai period, nntf ngrce n7ith Solingen :ind Coollinas. on the othcS lianti, the r;;c3rrtrc.d activity of thc diploid s 12 populntion w;is, in the pcriod csnmiocd, ;-irtmily indcpcnJcnt of cell nurnt)cr. This strain w.15 ablc to incrc.isc its activity i:om zcro :o an activity lcvcf of 70, while maintninins its population at thc saxnc dtnsiry. The rcsults wish this strniIi thiis confirm Stcphcnson and Yudkin.. From thcsc results it is clcnr rhnt tIic contradiction noted is only an appnrcnt one and is p:ob:ibly tluc to die dificrcnccs in tIic genetic bnckgrouiiti :inti phciio- typic constitution of the strains employed. The conclusion riiny also Le t1rna;n :kt ir is fudc to ;ittempt to dccidc, 3s sonic previous authors haw rricd to do, hmcn the "naturil sclcction" hypothesis anit the one of "dircct cytoplnsmic intcrxtionl', as tk cspl~iintion for the production of sonic one ndaptivc emymc. Th! prticuitr bioiosiCa1 n~cchnr~isxn invo!vcd in rhc protluction of n givcn ciuynie or cnzynic system in a popu~ation of cc!k is n cluractcristic of thc strain bcing csafiiincd, r.~~hcr than of thc ciizync~ system itself. Such questions ca~itiot be *nswc:cd Tvithout rcfcrring to thc genetic bnckground and stsbility of the populi- [Vor. 32 144 nNNALS 01; THE MISSOURI UOTANICAL GARDEN tion being stuclicd. It is also evident dint such strains as Id1(2C12 and 812 possess tlircc important characrcristics of inimcnsc valuc for investigations into thc pIiysiology of cnzynic synthesis, nmicly, ( I) a gcnome which permits tlic syntlicsis of the enzymc Lcing stuclicd; (2) the gcnctic stibility to insure re- producibility of the pliysiofogici1 charactcristics of tlic populations, and (3) the ability to ad3pt without ccll division. THE GENETIC BASIS OF IXABILITY TO AIIAI'Y TO GALAC'I'OSE FEllhlENTATION From die earliest investigstions by kinstrong ('05) and KIuyvcr ('14), thc cxistcncc of unadaprablc yeasts was noted. Subsequent investigations have un- covcrcd many more. ,?:s niay be seen from a perusal of Stellins-Dcltkcr's ('31) ri~onogq)li on the sporogenous yc~sts, cxampIcs of noii-fcrmcntcrs of gsiactosc exist in all thc icncr3. It did not seem improbable, in view of our cspcricncc with DL23B, that tlic fiiilurc to ndnpt ccrt.lin of thc strains might bc due to inconiplctc utilization OF thcir mutation;iI potentialicies. Thris, o I~o~~-s~orul.it~it~ population of diploids or one in which SOIIX other zi~cclitnism csistcd for tlic suppression of tlic haplo- phasc would bc unlikcly LO gain a ncw charactcr or Iosc in old onc by mutation, This suggcstcd the possibility of attenipting to adapt strains, wIiic1.r hnd bcen prcviousfy labeled as non-fcrrneiitcrs of galnctosc, by encouraging the production of hiploicis and thus disturbing the genetic stability of the population. From tIic point of view of the Iife cycle of tlie yeast, this coulJ usunlly Le accomplishcd by inducing Iicnyy sporulation and allowing gcrmiiiation ,to occur, thus refcasing hnp!oid cclls into the ppu1:ition. such cspc.timcnts w!re pcrfornicd (Spicgclnian ant! Lindzgrcn, '4s) with tIircc ye;ist types, Scbizos:iccbaroiiL),ccs Ponrbr, Scbi,-osircc~~roii~~cc.s ocfostwrris, aid &ccb,zromycotfcs LdriG,yi;. All tlirce wcrc investigated by itrmstrong ('Os), who concluded t11.7~ they wcre incnpa1,le of adaptition to g~il~ctosc fcrnicn tition. In addition LO tlic fact that they liavc been studicd niorc thoroughly than otlicr non-fcrnicntcrs of galactose,' they were selected for onothcr advtritagc, which is of sonic iixiportancc from a coniparativc point of vicw. Sch. 1'owbc can, without my diiliculty, esist in thc hapiopliasc. This is not true of Scb, octosjorzis and stili less so for ~~icc~~r~)~~~co~~~~ bdwigii. Tlic nicchanis~ns of supyrcssion of thc haploplissc in thc lattcr two strains differ: \While Sch. octosjiorris sporulatcs with case, thc haploids tvliich result frotn thc gcrniin;ltion fusc rapidly to proilucc diploid cclls. When sporcs from four- and eight-spored asci from the stock culture wrc plantcd, they a11 grcw and cvcry single-spore culture thus dtaincd sporulated on thc agzr in less tlian -18 hours. TJic further spore analysis of this strain indicstcs that thc diploid stock culcurc vas complctcly homozygous and that, uiilike S. cere-vishc, the production of vifitlc sporcs 3pparcntly cfocs not dcpcrrd on tIic prCesisiencc of 3 hctcrozygous nucleus. The' sporulation of single-sprc culcurcs was never otscrvcd in tlic S. crrrvisitrc strains used in the adiptatiori studics. The fact that it docs occur in SrJ]. ar/os!ictrirs is 311 indic.ition of licnvy cliploidization which is confirmcd by dircct niicroscoi,ic obscrvntion. 1~0latcd non-fusing linploitl cells arc r.ircly seen in suspciisions of sinSIe-sporc cultures of Sc.6. ac/osl)orirs. 'I'liis proccss of immc- tliatc fusion cricctivc1)- stipprcsscs tlic h.iplopliasc, nnd in thcsc cultures jpictic variitions coliic ni.iinly from rcconibinations. This source of v:1ri:ition woulcl obviously not be cffcccivc in popu1ations \vhicli arc Iiomozygous for thc rcccssivc. The suppression mcclianisiii is cvcn :tiore highly dcvclopcd in Socc/~'lrot/s~cod~~s Liduiigii. Cuillicrniond ('03 1 rcportcd that this yeast usually forms four spo~.cs without p-cvious conjug:;tion. 011 gcrminntion, liowevcr, thc sporcs conjuS.1tc within tlic motlicr ccll, two by two, so that only t\vo vcgctativc cells cmcrgc from cnch four-sporcd ascus. \Vingc and Lnustserl ('33b) coiifirincd these ob- servations ant1 dcscribcd the successful isolation of thc haplophisc by micro- nianipulntiye disscccion of tlic ascospre and scpnration of tllc four sporcs bcforc geriiiiri;ition. AS n result of their csnmination of the haplophasc cultures they Co Ilc 1 udcd t h? t S.ICC~J~O //I~'co~IL~s Lird~Uis ii \\'AS 2 ba I liccti hC t C rozygo Le. The JlC f result of the scriiiinntion mcchnnism is thc almost coinplctc suppression of thc haploplinsc under norm.11 conditions. \When IicaviIy sporu1:icing (20 per cent and above) culturcs of Srbizos~ccli~rro- myccs Po1tiLc \VCK secdcd into 6 pix cent gnl:ictosc, 2' per cent glucosc nicdia, adaptnblc populxtions Tvcrc recovered. Tlic results arc sunimnrizcd in Tablc I. Exactly sitiiiI.ir cs;~criniciits \vitli Sc~i!lj,,sJccb,lraIti~'rc,s oc/os)orrrs and S4iczhrrro- .xL)-ro,lrs Li;JiLti%qii f.lilcd. Tlicse failures nrc iindcrst:iiidnble in terms of the in;ilility of the 13::cr scr:iin> to czprcss thc iiitltarioll.11 ptcntinlitlcs of tlicir 112~)10- p1insi.s. IC n-,ig!it also bc. notd cli:it .itl.ipt.iblc culrui.cs \vcre iicvcr rccovcrcd irorii Sch. Por~rbc in rlic nbscncc of ticavy sporulntion no iiinttcr how long contiict with gn In c t osc was 111 a in t n i n cd. It is clear from these cspcriiiicnts t1i.it inn!)ility to atlnpt is in sonic cases duc to the gcmtic stability confcrrcd by diploidy. It may, liowever, bc doubted wlicther all tlint is rcquirctl for populntional aJnptation is thc brcakJo\yn of the Scnetic stability of thc unnd.iptablc strlin. It is concLivablc tlint tlic hnplopliasc of 3 particular strain might not contain within its niutationnl potcntinlitics the sbility to mutate in rhc direction of, for csnniplc, galnctosc fcrmcnt.ition. Tlint such indccd could be tlic cnsc wns shown by tlic isolation of tIir~'c haploid strains (Spicgclman 2nd Linc!cgrc.ii, '45) \\.hicli could not Iiiucntc towards ga1.ictosc fermcntntion altiiouSli ltcpt in contact with the sugar over n four-IiiontIi pcriod. During this snmc pcrioJ they wcrc, however, throwing off nuineroiis physiological and morphological niutants of vnrious kinds. AS \vas nxcd by Lix!cgrcn ('45) , prcliminnry c.spcrinientr 011 hybritfs Lctwccn S. D~piiis and S. ccrcr.isic:e clcxly indic2tc n typicnl gcnctic control of adaptn- bility and non-ndaptnlility to snlnctosc fcrrncntntion. -. [Vo:.. 32 BIOCI-lI31ICAL ASPECTS OF ADAPTATION kfore underraking an analysis of thc genetic implications of the phenomenon, thcrc arc certain questions it: \vo;ld bc`dcsirablc to nnswcr as comp!ecely IS the data al1o.r~. `i'licse qucstiuns would involve, ariiong other things, the csistcnt cvidcricc for cnzymc focni,ition, possibbfe biochiical functions Of tlic induced crizynics, nnit connectioii of the adnptztiori with the ovcr-all mctnlolic activity of tl1c ccil. Implicit in tlic discussion prescnd ficrc, ns well ns in the entire Iirernture of tlic so-called "ad;iptive" enzymes, is the nssuniption that \vIicn a cell is phccd in coiitxt with sonic substrate and accluircs, during the course of tinre, tltc ability to 1iict;iLolize tlic added substrntc, a new enzyme must have made its apparmcc. This ~ss~iii.iption stems, of course, from the innumcrnblc observations that every nictnbolic protcss rcquircs .in enzyme. Ilirect proof that an enzyme is synthcsizcd woufd involvc its isolation from tltc adiiptcd cells and is not avail&lc in any kiiown cnsc of adaptation. Such proof, in any case, would liavc co bc prccectcd by 3 dctcrmination of the niimber and functioris of the cnzynics induccd by tIic substmtc. At present tIic bcst thx can be offsred is evicicricc of cnzyrnc activity in cell cxtr.icts after adaptation. In the cast of both galactozpiazc and suelibiozy- mire, sutficierit \vorli Iins bcen Jonc to rewove any rcison&lc doutt on the qucstion of cnzymc ilAVO1Vcnl~Iit iii the adnptation. A delayed penetration into the cell CRII ccrtairily not be invokcd as the csplnnation of tlie induction period in gal.~ctosc ftrmcn'tacion. It ' ?vas sIiown (Spicgclman, '4~1) that i;aIactosc actunlly enters the ccll imnicdiztcly and is nicrabolized by a purely aerobic nicchanisnl in thc preadaptive period &fore thc fcrnwiitativc cnzyincs makc tlieir nppc.irance, Fur- tIicr, it Ius been found earlier (I-Inrdcl1 nntl Norris, 'IO) th;it yc.ist juice ai~d maccr~tion estrxt prepnrect from ndnptcd yeasts grown 011 g.ilactosc \x*cre able to icrincrit !;.iI.tcto.sc. Similnr prcpnrn tions from glucore-j;rown ~ulturcs wcrc in- $,-rive. TIIL'SC cspcriil:ents wc:.c repented and confirrnctl with our own strains usirig toluol cytctlys.itcs. It is clc.ir from these cspcrinlcnts that sc>nicthing, yos- scssiiig ~nI.i~to~~-f~~-~ils~iti~ig cnpncity, cnn be cxtr.ictcd froiii ccllc after atl:iptnttion which \,,ras not thcrc brforc. I:spcriiiicnts of the sniiie typo on cell extracts were pcrformcrl in ndnptntioiis to nielibiosc fernwnt:ition. I. icrc ngniil nctivity cotild bc Jcxi1onstratc:tt in the cyrotysatc only niter n&pt:1tiorl wns i.stnblishcti in the inrnct cells. Ir niny be noted here that a11 sucli CXKr3CtS wcrc nxic!c in the presciicc of subsrr:ite. Ccrtninly n question of prime importance is KhC .inture of the cnzyiiintic chmgcs xicccssxry for the newly ncquircd mctabo'lic propcrty. Is x wIioIc new set of crtzynxs rcquir&I? Or, is only one or two foriiicd ~liicli woiiltl transform the supr into onc utiliz,iblc by the Slucozyniasc systcJil? In the case of nictibiozymnsc, it swills most probable that n single clizyliic only is formed which splits n1cl;biox into slucose 2nd gzlactose. tiit enzyriie of this kind 11.1s been dciiionc prodtlcLioii of only 25 per ccnt of the cn1cul;itcd values when maceration cstrxt fc'riiiciitcd Ircpnrntions of ncl;iptcd yc:ist in thc prcscncc of ndtlcd pIiosph;itc, ncctnl- dchytlc nnd sodiunl fluoride. Gr.int ('3 5) rcinvcstigntcd this pro1)leni and confirmed the work of previous invcsiigntors on tlic role of Iiliosl)lior)'lncioris in tlic . mctabolisni of g.il.ictosc L), nci~ptccl yenst. I IC \vns nLlc to cstablith, with sonic ccrt.iinty, tht tlic phosphorylntcd products \vliich :iccuniul,itcd during tlic fcr:ii~.ntaiion of ~nI.ictosc ni'c not thc cstcrs of this supr but of ~ILICOW nnd fi-uc[o~<*. i itc hcso.;~'ilij~I~os~~l~~i~~~ C~LC'~ coiisciLutccI tllc Innjot. portioii oi tlic cstcrificd phosplintc. Froin tlic monoi)lio~phntc frnction lie u-ns able to isol.ite trcl1.i tosciiionophosplintc and smnil ;iiiioiiiits of n monopliosplintc tht closcly re- scmblccl the Iyi;cn; the tri.in!:lcs, full-shsdcd arid half-shadcd circIcr rcprcswt the subscqucrtt Lcha\.ior during snierobiosis. forriicd in the abscncc of 311 tstcriinl source of nitrogcn. One must concluck, then, dint tlicrc exists in tlic ccil a source of proteins OII which t1:c cell cnii tirnw for enzyme formation. It Inusc bc notcd, Iiowcvcr, thnt tlic attninnblc activity led is About hnlf tlint nrrivcci ;it if an cxtcrnd SOLILXC of nitrogcn, in the form of nn~moni~trit snlrs or nmino acids, is pro"itIcd (Spicgclmnn, '45). Using non-div: i:~g diploid populntions, 311 nttc;iipt wns nindc to begin tllc study of tlic conncitioii between tlic synthesis of thcsc cnzynics and the over-all mctnbolisni of the ccII. Exanihtion of the effect of oxygen (SpicSclninn, '457) rcvcnlcd dint thc atfapt;itioii tv.is cs:rcnicly scnsitive to oxy!;c~i (cf. Steplicnsori ant1 Yudlcin, '36; Schultz, Atltin nntl Frcy, '40). Some strairis W~I-C iound tlut wcrc coinplctcly unnblc to form g.iInctozymssc if Khcy cspcricnccd only anncrobic 19451 Collc3Ct v,titl1 g;tI.ictow, wIiiIc otlicrs could form t!ie cnzymc anncrobic..illy. I~ow- ever, the rntc of nnnerobic ndaptntion wns ni>proxiniatcly l/.tOth of thnt nttniticd in o;ul.itcs thc forni3:ion of :in enzyme, bur, in ndtlition, :icts nr the only YOUI'CC of c:ic:gr;y for its syiit1:csis. Ti1.i~ tlic cncrgy s~ipply is critic.ii sxms clcx from ttvo facts. It h:is been sliown (Spicgc11ii.in niid Noz.i~vn, '~15) thnt for these str.iins, in coninion with others (see Sticr and St.inii;irJ, '353, b), thc cndogcnous rcscrvcs nrc not fcrnicritablc. Further, supplying cstcrnal fcr- nicntiblc substr.itc (c. g., fructose and, under ccrt:iin conditions, glucose) permits (Spicgelnim, '45b) the ndnpt.ition to tnkc place arincrobicnlly in diosc strains in which it ordinarily docs not occur. These rcsults suggcstcd th.it tlic aerobic ntlnptntion occurrcd ~CC.IUSC, uridcr these conditions, the ccll could drnw 011 thc cncrgy coniinS from thc 0sid.Ition of tlie ~IJC~O~~~~OUS rcscrvcs for synthetic activity. Esixrimcnts WCTC tlicrcforc pcr- formed to csnminc ndaptation tiiiics (timc to rcnch n QFO2 valuc of 100) whcn tlic scllsctose wns added at difTcrcnt lcvcls of the endogenous rcspirntiori. Thc results 011 one strnin,nre given in fig. 3, in which, for purposes of oriciita- tion, the cntlogcnous rcspiration curve is nlso dingranimcd. It is c1c:ir from this figure tliat up to tlic zero-rate portion of tlic cndogcnous curve Iittlc iIi5crcncc in idaptation times is cncountcrcd. I-Ionwet-, the importnnt point to notc is that, nlthougli daptation . tinics iricr.carc ;IS the galactose is atIJcd further out along .the zero-rate portion, ncvcrchclcss adnpt.ition occurs. Thcsc csperimcnts 1v0uld seem to indicnrc that atfnptntion can talrc pl.icc after nll tlic osictizablc rcscrvcs Iinw been cshnustcd 2nd that thcrc is no :ipp.ircnt SOUKC of ci-rcrgy. `rhis Sfrorn 012 situ;itiun wn~, liowcvct, clnrifcd by the iliiding (Spicgclnian, '.iSb) that the piactox itsclf is osiJizcd by sonic cnzymc systcin other rlian tlic fcriiientative one, which forms Lttcr wcicr its stimtilntion. It is clcnc from thcsc cspcrimcncs cliat rlic adaptation is intimately connected with the nictnbolic activity of the cells. Both ncrobic 2nd atiaciobic proccsscs are cqudly capaLIc of supplying the cncrgy for spnthcsis. Furtlicr cspcrimciits, which will Le detailed. clsc\vl~crc, iiidicate that agents wliicii interferc witti nitrogen assiridation (e. g., azide) comylctcty suppress adaptation. GEXETIC iiri:EiwxcEs FROM TI IE iaiwrra OF m,t PI-AXI~X~ It w3s pQinCCd @lit ill thc inf r0dl:cCioll tltnt, Silicc Cnzymntic nj.ipCations in- vol\wl erizyinc formation, R cnrciiil t~udy of such proccsscs coultl providc a clue as to tlic iixurc of the controls cscr:is+.d by thc gezw ovcc the ciizyioe constiru- tion of cciis. The tiiyst obvious :uid c;isily ~iic.;surc- t- > t- V 6 - - TIME- Vig. 4. Activity-time ciirvcs prcdictccl by (a) Clirccr pri- miry gcnc control of cnzynic \yntliccic 2nd (t) self-duplication of cnzynic molcculcr. coursc of ex.imining tlic synthesis of tlic glucoqmnsc and mclibiot.yni.isc systems, over 400 nd~ptation curves have bwn obtained. In no case docs the activity-timc curvc resemble the coursc prcdictcd by rhc above analysis. In all instanccs (see e. g. fig. 2, open circles) tlic initial part of the curvc is characccrid by a rising r.itc of cnzymc formation. This is thcn followed by 3 declining r.itc portion, wlicn prcsumably tlic indi@crcnt subscratc becomes limiting and fiii~lly cxliaustcd. Tlic increasing ntc of cnzynie synthesis, with increasing a~nouiit of cnzynic, sug~c5~cd an obvious modification of thc nicchnnism dctailcd iii diagram (1). Retaining all thc properties ascribcd to die first mcchanism, we atid tlic additional oiic that the enzyme OIICC formed cnn duplicntc itxlf witliout furthcr nccd for genic intervention. With this self-duplication liypothcsis, instcad of rcactioii dinSrain (I), wc ha~e, 13451 \.J k 155 (4) k' u;licrc thc s!+mbols Iinve tlic sm.c nicJiiing. As before, n.c suppose tlint chc gene c'in tr;iiisforiti P into active enzyme E, which is unsrnblc in the absence of sub- strate. TI:? Arrow going from E to A symbolixcs the self-tluplicntion of tllc enxynic, Ivvlticfi \vortld csprcss itsclf in terms of chnnging vclocity constant so that its vntu~ nt any p~r'ticulnr niomcnt woi3lcI ticpelit1 on tho nniounc of I:' present. 111 the picseiicc of substnte, the r3tc of fornixtion Qf tho enzyme bC'COIlXS, uii&r tlicsc asstiinptions, 2 quirfrntic function of the nmowic of enzyme present and takes the foinl: whcrc E apin reprstcrits tlic niiiounc of P transforriictl into E in unit tiinc and P is tiic initi31 nniouiit of precursor present. 1ntcgr.iting cquntion (5) n-c Obt3iIl - wlicrc (1 is nn iiitcgia~ion coiistnnt determinet1 by iniii.11 conditions. According to cqiution (5) then, the assumption of self-ctuplica~ion predicts the s-shnpcd curve given in fig. ;b AS tlic activity-timc curve Juring ndnptntion. Therc is no doubt that rhc chtn lend support to tlic sclf-duplic.ition liypotlicsis InJ rule out the simple genic riicchnnitm undcrlying rcnctioli dingrnni (I). More risorous mnLhcinaticn1 .~nd cspcrinicnt:il tcsts haw bccn mndc mid will be dctnilcd clsewhcre. An ini;imrtnnc 2nd critical prediction stemniing from the sclf-duplica- tion ni~'chanis~ii is tht, oiicc the process is st.irtcd, it cnn proccc'l in tlic dxcncc Of die sene which initi.itcd it. Attenips to tcst this prediction wcrc nindc with data on thc inheritnncc of nielibio~yn~~sc. Thc XiznJclinn nicciinnicni undcc- tying the inliciitnricc of the ability to form n:cIibioryn~.isc w.is nnniyrcd with tlic aid of two striins differing i.1 this ch~r3cter. TIE GESETICS OF THE ABILITY 1'0 FOR XI XIEI.II\IOZYhl.ASE Tfybrids Lctwecn. niclibiosc-fcrmc~iters snti non-icrniciiters find aIrcnJy been AI1 hybrids of such crosscs were fcr- examined by Wngc 3nd Lnustscn ('393). nlcntcrs, nnd their rcsults led Wingc :ind I,nustscn to st;itc tli.it `Itlie presence of n spccii`ic cnzymc is dominant to its absence in all tlic instnnccr studied." Thcy riindc m.itings. by pl;icing two spores in coiitnct with cach otlicr. This nicthocl hns tiic dis.itIvniit;ige thnt one c.lrinot clinrnctcrizc tlrc 1i:iplophasc pircnts, sincc bot11 of the originnl spores nrc coiisumccl in the m~ting. This, 2nd thc failurc to csnn-iinc. tlic plicnotypcs of tlic scgrcgnnts from thc hybrids, prcvcntcd an annlysis of thc gciictic mcchnnism. I Iiis annlysis w3s unticrt.il;cii by Lindcgrcn, Spicgclninii arid 1.indcgrcn ('~t-i). usin!: S. carlslzrgc~isis, wliicli coi,iii n&pt to fcriiiciit mclibiosc, ind S. ciwi~isiLrc*, which coulti not. Iii this invcstigntioii hybrids ~crc produced by mixing haplo- phase sulturcs. Since only p;irt of the culture is necilcd for the n1nting, the rc- m.iindcr couIJ bc used to detcrnlinc the clinractcristics of tlic [xirent strain, ns wcll ;is for bnc1;-crossing or ninting to otlicr clones of interest. All hybritls foriiicd wcrc nllo\vcit to sporulntc and the xci dissected to permit es.iminntion of tlic plicnotypcs of tlic haploid scsrcg.ints. The dntn obtnincti from 175 progcnics of the intcrspccilic nnd of rcl.itcd hybrids ~vcrc consistciit with tlic view that S. c-~rls!~crgcrisis contnins tn`o pirs of Joniin.int gcncs (nicI-{-), cithcr one . of which permitted thc protluction of nieIibiozymnsc. Sincc 311 of tIic IinpIoid segregants of S. ccrkitilic f;iiIcd to pro- duce the ciizyme, it wns clcnr that it .w3s homozygous for the rcccssivc alleles: I. SI~Ll'-I~UPI~ICA`l`lO~ OF XIELlDIO%YXI~\SB 1N TI 11: ABSENCE 01; ITS GEKE Wtli tl:c gcnctics of thc cnp.icity LO form mclibiozym.isc known, it bccnmc possiblc to clcvisc espcrimciitc which would tcst the sclf-~liiplicatiiiS hypothesis suggcs~~c! l>y tlic S-sl~:ii~~d ad.ipt:ition ctirvcs. I11 p.xticular, it \V.IS crscntinl to prol.ic!c: .ins\vcrs to the following questions: (I) If syntlicsis has bccn iiiitintcil nnd the gcnc's nllclc substitutcii by scs~-e- gition, can tlic siibstratc-cytolil.isiiiic interaction mnintain tlic cnzynic intlcfinitcly in tlic cytopl.ism in the aLseiicc of the specific gciic? (2) If sonic cnzynic is prcscnt, cnii synthesis of adJitionn1 cnzynic occur in the nbscncc of the specific scnc iicccssnry to iiiiti.itc its syntlicris? Use \v.is mndc of progenies of known gcnctic coniposition ironi tlic S. crrcr,isinc s S. c-:r,-l.sbc~~~~o~sis pccligrcc cmploycd in the study of Llic hIcndclinn nicclianism of ~iiclil~io~;~~~ii:~sc iiilicritniicc. 111 tlic cspcrinicn:s tlcscribcd in the previous scc- tion, the cells cniiic into coiit~ct witli mcliliosc for the first tiiiic in the test for nAptnbility after scgrcgntion had ;ilrc.idy tnltcti pl.icc. To niiswcr ths questions posed nlovc, cspcriments wcrc pcrformcd (Spicgclm;ui, Liridcgrcn nnd IAdcgrcri, '4S), in \vhicli the mntings ns n-cll ts the scsrcgntioiis wcrc c;irricd out in thc prcscricc of mclibinsc. The results wcrc compnrcd \viLIi matingc from tlic s:iiiic cross in which iiiclihiosc wns oiiiittctl until testing rlic pliciiotypc of the li,ii>loiJ SC~I'C:;~II~S. I o simplify tlie genetics of the sitii;itioii, R Ii.ipIop1i.i~~ cloii~ cnrryiiig t single tiicl-{- gcr~c controlling ad;ipt.ition WJS used. This wnq mntcd to R haplo- phnsc clone of S. cercuisirrc wliich cnrricd only the reccssivc nllclcs. The Iictcrozy- ,.. h 'C 11s so. -1- + 4- + -i- 4- + + -;- 4- -t- -i- -.- : + 4- -!- I i- + + A\crrr No. x 3 10 11 12 13 14 15 I6 17 s pars s o A 11 C 1) [Vot. 32 158 d\NhTALS OF 'I'i-iE hfISSOU1tl BOTANICAL GARDEN adaptatioii to fcrmentarion. Despite this, all four spores froni thew tetrads prodticed 12nplopliase cultures wliich fcrmcritcd melibiose. Since all steps were carried out in thc prcscncc of melibiose, selection of aJnptiblc niutnnts from haptoids oriijnally unable to fcrmcnc mclibiosr mislit hsvc occurretl. ( 1) Iluring tlic tcsting of many 1i;iphid scgrcgmts from S. CCTL'ViSiiJC, all of which arc ricgati~~, no mutation to 311 adnptnblc type lins cvcr bccn observed whether inclibiosc was prcscnt or not; (2) thc sanic is true of negative haploills iron1 hctcrozygous hybrids. No mutncion to adapt:iblcs in thcsc Iuvc been seen no mnitcr how often they Ii;lvc ken through mcIibiosc nicdiJ; (3). asci s arid 9, whosc segregants wcrc plnntcd on mclibiosc, yielded thc stand.ird I :I r.itio. l'rcsuniably, tlic cultures from the two spores of each ,tetrad from die first six asci werc able to fcrnicnt nielibiosc. only due to tlic prcsencc of the enzyme in tlic cycopl.tsm. On this bssis it was KO be cspcctcd that rciiiov;il of the niclibiosc would lead not only to thc disappcarmcc of fermentability in 311 c;ises, but to an cvcntunl loss of rca;ln~tability in two of cvcry'four cultures arising from cacli of the frrst six asci. To cxciudc the complication of mutation away from adapt- ability, non-dividing cultures, suspcntkd in M/15 KIH2POB, wcrc used. Portions of df 29 adapted haplophasc cufturcs originating from thc first 6 asci were dis- siniilntcd in t1ic absence of su+rarc until they h;id lost dl rnelibiozymasc activity. S.iinplcs were then removed and incubated with melibiose to test for rc.itlaptabiIity. h'ot all Ii:iplopI~nsc cultures survivcti tliis reiativcly vigorous trctitniciit whicii in sc1111c caws i.isccJ 20 tlnys. T.iblc 111 ~~inirnnr~iz~'s tlie rcsrilcs obt.iinc2 with tiiosc xci, nil four of \vIiosc ssgrcgnnts sroocl the trut nicnt. 'The rci~iov;il oi the iiicli- biosc ;ind its st:ibilizing iiitlucncc IcncIs to tlic dis.ippcsrance of thc cnzymc in thc cytoplasm ant1 tlic rcappc.mncc of the cspcctcd Mcndclian ratios, Dlta collccrcd nt rlic saiiic timc indicate tlxit syntlicsis of additional enzyme can occur in the absence of tlic mcl+ gcnc. After allowing a11 susgcnsioiis to Several spccific facts, Iiowcvcr, rule out this possibility: 19451 SPIEGELhl.~N-~h'%YhlA'I'IC ADAPTATION 153 fall to low Q:,,, ~nlucs (bct\vccn 1.8 and 10.1) in thc nbscncc of niclibiosc, portions wcrc rcniovcd rind incubated with niclibiosc nnd rcSencraLion of activity folloncd nt intervals by mc;isuring G&,,. The rcsults of thosc Iinploid sesrcgants \vhich subsequently lost tho ability to 3d:lpt arc rccordcd in Tnl~lc Il'. It is scen that in nll cascs ni;iiked incrcnscs in Activity wcrc o!)tnincd. I:urtIicrniorc, all thc str.iins listed in Tablc: I\' \VCI'C cnrrictl in staiidnrd : ,v performing thc mncing in tho prcscncc of melibiose, tlic cytoplnsm of rhc hnploid cnrryins thc mcl+ gcne is pnckcd with tlic melibiose-ferrncritiiis cnzymc. Sincc both copiil.iting haploids contributc cytoplasm cqunlly to thc zygotc it stnrts out with sonic cnzynic and builds up niorc sincc it 113s thc gene nlso. Sincc sporulntion 'occ~irs in thc prcscncc of mcli- tiosc and sincc tlic sporulation period is charncccrizcd by growth :ind considerallc storngc, the cnzymc niolcculcs nrc stntilizcd 2nd possibly insrcnsctl in nmount. Each of the four haploid sc!;re!;.ints tlcrives its cytoplnsm from thc diploid hybrid, and it follows that cxh will liavc cnzynic niolccdcs in irs cytoplxm no ninttcr fVVL 3.2 162 ,\`hlKI\LS 017 TIIIi XIISSOUR1 1~OTAN.ICAI.. GI\KI>EN --, un~ Nilsaon, It. (1925). Ubcr dcr Cil.~l;tocavcrglrurifi Jursh IIcfc nach \'orl~clia~i~llun~ ntit ciicscr Zuckerart. ?bid. 143:89-107. Bact. 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