[ 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 dciiion<tr:ttc<t in cniulsiii prep:ir:itions by ICulin ('23), W~IO c:III~LI
it nielibilsc. Little Jircct \vork on this cnzymc 11.1s :IS yet LCSII done.

Dcc.iusc of the rcl.icivc iniport:ince of gnlnctosc iii niainiiinliaii physiology, .a
consiciernllc amount of work hns bccri done on tlic I nature of the crizynintic
ciinngc wliicli pcrniits n pst cell to ferment this ticxosc.  Efnrdctt nnci Norris
('IO) found tlut 3 fcrincntilig niisLtrrc of yenst-juice (from an ad;iptcil yenst)
3nd gnllactosc rcnctcd \vi& nclticd pliosphntc in n iiinnn~r siniil.ir to ordiiinry yenst
juice arid gtucose, alt1ioii~;li n loi~gcr iniiuctiori period W~IS neccss.iry.  Thc r.itc of
C0,t fornmioii was nscclcratcil, nn cxtrn ;iiiiount of CO? quivnlcnt to  the
phosphnte added wns esol~ctl, nnd thc rnte then again Ixcniiie norinnl.  'Tlic
~!10~;1li:iti~ I\,.IS ci)ii\Tcttcd into nn orb;.iiiic foriti iioc prcciliic;ib!c 12,. Jx2~~~l~S~ll~ll
citrate niihturc.  Eulcr nnJ Jmssoii ('27) showccl ~1i:it u  11 dried ntl;iptcd yeasts
;ire w.islied, they fnii to fcriiieiit galxtosc.  I-Iowcver, such prcp:ir.itios!s cw be
i~ncti~nted to gnhetosc by ntliling the co-ciizyinc prepared from uiintlaptctl ycnst.
They were thus nblc to csclucle tile possibility thnc nJsptntion is concerned with
rhc niodificntion of the csistcnc cozymnsc or the foriiintion of n new one.  Nils&
('43) ~i'ns nblc to isolate from tfic products of the fcrmrntxtion of gnlnctoosc by
3 saxnplc of dried adJptctk yenst, n ~~~p~ios~)l~ot.~c ester wliich, iii its cleiiientnry
snnlysis 2nd specific rocntion, closcly rcscniblccl the 1ic;oscdipliosplinte formed
during tIic fcrrncntntion of ~IUCOSC, fructose nnd mnnnosc.

In o&r EO csplnin the fortrx~tioii of the snnic diphosphate from gnlnctose 3s
froni tlic norninlly fcrIiiciit.ible sugm, NiIsson ('4 3 ) sugscsts that the fcrnicnta-
tion of 3 11cxosc involvctl thc splitting of thc nionophosphntc cster into n triose
2nd 3 triose phosphntc ester.  This ~ould dcstroy t!ic spntinl specificity of +e
fourth carbon :1t01n in die SxI:ictosc molcculc miti rhc rcsultirig pliosjphorylntcd
triose codd be built up into fructose' ciipliosphnte.  If tliiq were truc, it would be
.necessary to postuln te the formition' of only one enzyme in the ntlaptntioii to
plnct~se fcmicntntion.

[\'"I.. 32

14s ASNAI,S 01: TliE hIISSOURI I!O'l'ANICAT, C.'ll<fl1IN

It is ti-uc that Aicycrliof :ind I-oliiiiniin ('34) linvc showli the csistcncc of nn
cnzymc (zyrnohcxasc) in yc.ist and niusclc prcp.ir:itions cnpnblc of converting
triosci,ionoplio\phoric chtcr into fructose dipIiosph.itc.  Ncvcrtlielcss, csperimcnt.il
sL1ppot.t for Xilsson's schcriic of fcrtiicnt.ition is rcl.itivcly ~cnk.  I IC nssunics tlint
thc forination of iruciosc-~li-l)liosph.itc is :in nrtifnct .inJ th.it the fcrmcnt.ltion
proccc:ds from his pobtul.ltcd split ol ~lic hcsosc morioj,liosplintc.  I IC b.ices this on
tlic obscrvntion thnt fructose dipliospli.itc is fcrniciircd niuc1i iiiorc slo\\~ly tlinn
fructose.  I-Iowcvcr, tliis cnn be csplaiiicd in tcr~iis of the clnsricnl AIcycrIiof-
P.irn.is fcrnicntntivc scliciiic on the bnsis of the un,ivnil;iL)ility of phospli~t~ .IC-
ccpiors.  'l-hus, hfeycrhof ;ind Lolim.inn ('27) found n c<>c prodtlcLioii of only
25 per ccnt of the cn1cul;itcd values when maceration cstrxt fc'riiiciitcd I<obi!,on
cstcrs. \V:irhiir,q :ind C1iristi;in ('33) 1i:ivc slioa~n thnt thc pnrti.11 ~lc~~li~~~~~li~oryln-
tioii of 1 :3 -dipliocj,lio:;lyceric acid to 3-plio,~7hy:;lyccric ncid by tAc h~soltinasc-
nclcnosinc di~~hos~~hntc-lie~osc system tiuy bccomc lin1itiiis in  thc fcriiicnt.itivc

pLO'css.

Chttaiico ('33) hns obtninctl nclclitioiinl cvidcncc pointing to tile convcrg<ticc
of 111c fcrn1c.rit:ition pnths of g:ilnccosc nnJ glucose by isol.iting phnsplioglyccric
icid from thc phosphorylatcd products foriid durilig the fcrmcnt.ition ut' g~1.i~-
tosc by ;>rcpnrntions 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 I<obison ester in its propcr~ics.  Attcinpts to detect the prcscticc of
s:i1:ictosc-i~liosplintc by the mcthplplicnylliydrazinc test fnilctl.  Pcr1i;ips of cvcn
gimtcr weislit ~vns tlic fnct that Iic shoucci thit n prcpnrntion froin n'dnptcd ycnrt,
which \\rould fernleiit plnctosc, f.iilccl to ferment syntl~ctic:~lly prcp.ircci s,ilnctosc-
6-phosphnt-e. On the basis of this cvidciicc, hc concludes th.it ~n~;1ctosc-6-pliosphncc
is not nn intcrnicdintc in the fcrmcntation of gnhctosc by ndJptci1 ycnst cells.  Of
fiirtlicr interest is the evidence hc presents thnt thc livitis yc.ist cell continiics to
build up tlic s.iiiic polys.icchnriJcs when Sn1;ictorc is the sole cirbohydrntc iiictnho-
lizccl ;is when the cnrbohytlratc is glucose.  I-Iydrolysis of thcsc polgsncchnritlcs
iridic:itcd thnt they nre polymcridcs cliicfly of glucosc, and to n lcsscr~cstcrit of
In;lllllosc nn'l frllctosc.

Iiostcrlitz ('43) found that g~ilactosc-~-phos~iIintc is fcrniciitcd by cxtrncts
of S.iIncto;c-nd.iptcd psts.  I-e found furtlici thnt, nltliougli sucli cstr.ict$ fcr-
mcntctl gli~cosc nt Ilislicr rnt.cs tlim gnlnctosc, the fcrmcntation rntcs of g.il.isiosc.-
1 -phosphate ;ind ~IuCos~-I-plios~li;itc \\'ere iclcnticnl.  It milst be noted. Iiowcvcr,
tht the fcrnicatitioii r:itc of tlic mono-cstcrs wns lower thnn tlic corrcsponding

. .I

Rttcrnprs to obtain nd.iptntioi1 wit-h iion-vinlik ccIh or wirh cells wliosc physiolo~y
lns been seriously intcrfcrrctf with by various rcngcnts h.ivc uniiilrnilg met witli
failure. Tlic sole csccption was the report by AbilcrIi.ildcii ('25) that hc hncf
obt.iined xhptnttion with dricd dcnd yctst cells.  This csperinicnt has iicvcr bccn
succcssfully rcpc.iccd, :~nd his fiilurc to check the possibility tlint the ~d;ipt;ition
niny have bccn due to tlic growth of R fcw survivins ctils throws sonic doubt on
the validicy of his conclusions. Von Eulcr and Nilssoii ('25) clainicd that nd.iptn-
tion wili not occur when the cctls :ire suspciiiicd in ordiii.iry pliosphatc concnining
gnl.ictosc sncl [!icy mnintaincd that the addition of ``2'' factor WRS iicccssary.  ?'he
c3rlicr cxpeciiiients of Dicncrt ('00) contrndictctl this, sincc this author did dl
his cspcrinicnts with washed cclls in ordinary solutions of phosphntc.

                    AII our daptntions ivcrc
pcrformcd \vir11 thorougllly wnahctl cclls suspcnticd in pliosphatc sotittioris of
tw~cc-rccr~sc~lli~c~~ gn1l;ictosc.  According to tlicsc rcsulcs, rlic enzyme c.w be

Our o\sn espricncc ngrccs wiih tIint of 1)icncrt.

I60}

120-

N

Qco, -

80.


40.

Strain tii2G12

/Q--r-------~-r

2 4 6 8

-?

HOURS

1:;):. 2.

     h tcst of tlx XbiIity of ccils IO incrc*Isc tlrcir pl.actozyma<e acci\*ity
sr:tscqucnt IO aerobic induction pcricdr in COIICIC~ with g.rlactnrc.  Tl~c qxn circles
arc thc r.itucr .irriscd ac for a iulctirc hiving coi~t;iiui~~s ICCCSS to o>yi;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;<ygcn. ' Tn thc case of the strnins unnblc to ndnpt :irincrobicnlly, it was of SOIIIS
intci.cst to dcrcrmiric wlictlicr ciizyiiic formntion could occur :it .111 unclcr .in.lcrobic
cor,ditionc.  This \\.as done by following thc activity nnncrobicnlly subsequent to
brief pcriods of ncrobic incuhntion wiLIi plnctosc.  The results obt.iiiiccl arc given
in fig. 2.  Tlic opcii circles arc vnlucs ;ittninctl during continlious ncrol~ic incubn-
tioii urith.4 per cciit ga1:ictosc.  Tlic nnacrobic te1inv:our of tlic cnzynic nctivity,
following vciriouc pcriocls of ncrobic contact:, is rcprcscntcd by tlic solid triangles
2nd full and hnlf-shadeJ circles.

IC is sccii that 31: the cnti of the first hour of acrobic contnct, tlic Q;I:o,
~nluc is nbout 2,s.  Under nnncrobic contnct tlicrc is :in initinl slight rite above
thir valuc nnJ n subsqucnt -!ow but consistcrit drop.  At tlic end of two hours
of ncrobic cont;lct, the Q;(,? ntt.iins n v.11~~ of I 5, and snbscqucnt nnacrobiosis
does not prevent its inc[-casc, :~ltliou~b tlic rntc of incrc;isc is slo~vcr than thlt
obtnincd in thc continunl presence of oxygen.

It is clear from tlicsc cspcrimcnts r~i:it, \vIiiIc nJnpcntiori cnitiot bc initi.itcd
anaerobically in thcrc strains, it can procccd uiitlcr tlicsc conditions providing an
adcqiintc cnzymc activity has been built up. The condition to bc met hcrc nppcars
to be that enough cnzymr be formed acrobicnlly to utilize the cncrgy content of
the galnctosc n;olcctilc ~vlicri nnncrobiosis is cstntlishcd nt n ratc ;idcquatc: for
further syntlicsis.  K'c linve here the intcrcsting pliysiologicnl situ:ition xvhcrc thc
substr.itc not only stin>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<I nspcct of ad.iptntion is tlic
iiictcnsc in ciizyiiutic activity otscrv~rt in non-clivitfiii:; cells pl:iccd in axitact
with substrate. . Tlic usual description of gcric action assuiiics that the gene

    k
1' 1:
   I<'

t

>-

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 : ,<din with niclibiosc nntl WCIX
tcstcd :it wcekly intervals.  At the cnd of tlircc nionths tlicy could all fcrnicnt
niclibiosc nt r.im qual 10, or grc.itcr than, thc original rntc.  ?'his pcrioJ is
cquivi!c;ir to ovcr 2,000 cell gcncratioris. It is cvitlcnt that tlic enzypc c111 not
only ninint.iin itself in the prcscncc of n~clibiosc but it can also iucrcnsc in nbsolutc
arnoiin t.

*rm~E IV

VI\ 1.1; ES .I 1,-1 1; I:

A I< I( 0 1 I IC: IN C1 j II ,\TI() N l\'I '1 1 I 11 I<T,I li I I ) I; 1: 0 L: S'r I<;\ I S S

"()'

\VIIlC11 I~VLSl'U,\i,l~Y J.OST TIIEII~ XIIII.11'Y '1'0 Al)Al'T

ID

ID

2h

2 1%


IC


ID

6C

61)

0

5.1

2.4


10.1

6.3

5.0


4.2

1 .li

4.8

12

40

26


33

46

69


23


34

42

24

96

109

SG

73

161)


71


84

121

48

123

114


136

I (1 1


170


I34

14 1


1JO

The siinplcst csplariition wliich c;in tc offered at prcscnt for the nbovc rcsults
is tlic s.imc one advanced for the S-shnpcd curve, i. e., thc cilcct of substratc on n
sclf-diiplic~tin~ cnzynic.  Wc may thus cxplnin the cifects of nic1il:iosc on thc
inhcritancc of nielibiozymnsc 3s follows: b>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. RCV.
7:139-173.

Graxlr, G. h. (1935). ?`lie nictabolism of sal.ictosc. 1. Phosl.!ror).l.itioii duriti,; gnl.ictosc
  fcrnlctit.itioll and its rclncion to the inrcrconvcrsion of tIw hexoses. Biocfisril. Joiir.
   29 : I 66 1 - 1 676.

Guillicrlliond, A. (1903). Rcchcrchcs sur 11 i:crmination dcs sporcs danr IC Sttcchro??tyccr
lx:fri+i.qii (I farlscn). Bull. Soc. hiyc. tic Frmcc 19:17-3 1.

f-Iardsti, tt., ~nd Norris, R. V, (1910). Tllc fcrnmcnt.ition of galactosc by yeast and yeast-
X:Irstnirn, PI. ( 1935). l'nzyni~tischc Adaptation tci Mikrmrganismcn. Ergcb. Ellzyn1-
.filuyver, it. J. (I9 1J). Dioclicmischc suikcrbcpilingc11, procfsclirifc Delft, p. 7 1. (Cited

liosterlitz, PI. {I 94 >). Tlic ferincritntion of galactose -and g.~lactosc-I -phospIu~c.  ISo-

liuhn, R. (131.3). Gter Spczifitst der Enzyme. Zcirschr. f. f'llysiof. Chcm. I25:25-92.

Lcwis, I. XI. (1934). Basrerid variation with soiix special rcfcrcncc to behavior of some
  niut.il>lc strains of colon b.ictcri,i in synthetic media. Jour. llact. 2S:619-638.
tinrlc?:rcn. 6. C., arid I-iiiticgren, G. { 1943a). Selecting, inbrccding. . rccoiiibining 2nd

hylriiiizin!: coninicrcixl ycists. I&[. 40:405-419.
      , (1333b). A new method of liybridizing yeasts. Proc. Nat. itcad.
Sci. 29:306-308.
       , (1 933~). Scgrcgxtion, intitarion and copulation in Surchuronrjecs
rcret:ih=. Ann. hio. Bot. Gird. 30:453:464.
  , Spicficliiixn, S.. and Lindcgren, G. (1944). Xlcnciclinn inheritance of an
~&ptivc cnzymc. Proc. N.it. Acid. Sci. 30:346-35?.

I.inJ~~r~tro~~r-L~ii~, I<. (1940). Enzyni.ititche A(f.iptztion. Nord-\Victfciill~~c~~, I-I.~ndbuch
  Jc`r t-iizyiiio!tr$c. ~~~a~lenlis~lle \`rrlngs,:ftc!lscfinfc, ncckcr 11. Erlcr, LcipLic.
3lcycc!;q>f, O., rind I.ohmniin, K. ( 1927); ij1,c.r die cnzym~tischc SlilchF~urcbilifunfi im

  ~Iu~Lclcs:raLc. ~~~oc!icn~. Xcitscttr. 1&.5:1 I 3-16.i.
-__ ._IL- , ( 1934). ijbcr die cnzyni.itisc!ic C;Isiilil:c:a.icli:rrc.ll;rion zwisc!icn
  IIeso~cciiplio~~~lio~~~irc uiiti I>iosy~c~to~ipi~o~plio:~~urc.  IM. 27I:t19-1 10.
   , (1935). Utcr die Wirkiingsweisc der lcxokinxsc. S.itur\r.~ssciisciinftcn
  23: S 5 0-S5 1.
Nifsscm, it. (17.I)), Uber Jic Organisierung Jcr Lioclicniischcn Wirkstoffc in dcr Zellc.

  NnEiirwisscnsclixftcIi 3,1:25-3 5.
Qtiascc1, J. I-. ( 1937). Enzymc formation in hacteri.i. 1~rrzyn~ff~~'~in 2:37-42.
1<.1h, 0. (1938). On thc nniurc of adnptivc cnzynics. Growth 2:363-367.
Schultx, A. S., Atkin, L., and Frcy, C. N. (1940). Influcnce of osypn on thc fcimenta-

tion Of nnltosc and gilactow. Jour. Amcr. Chcm. Soc. 6?:227 1-2272.

Slator, A. ( 1906). Studics in fcrmcntition. 11. Thc mcchanisin of alcoholic fermcntltion.

Sohngcn, N. L., and Coollinas, C. (1925). Thc fcrrncntation of galactose by SorchJro-

Spicgclmnn, S. (1945.1). Tlrc cbcct of anncrobiosis on adaptivc cnzynie formation. Jour.

1:. E. (1943).

Factors influencing thc cnzpnic activitiss of tactcria.

juice. I'roc. Roy. Sac. Lord. (B) 82:6+!5-649+

forsclt. 7: 3,S0->76.

.by Sohn!;cn and Coolha.is, 172.1).

clism. Jour. 37: 3?2-)26.

JhI`t!. 93: 2 17-23 5.

nryrt.s cerc.t+i;ir*. Jour. Bact. 9:l 31-141.

Cell. & Comp. Physiol. (In press).
  , (1~5b). ~ii~u~lisllcd espcrimcnts.
  , and LinJc!:rcn, C. C. (19.1.)).

                 A comparison of the kinctics of ccizyrnitic
xcfapratiozi in pictically Jiomo~cneous and hctcroscncous ycasr populations.  Ann.
110. Bot. Gard. 31:219-233.

      , (1345). The rcfxtion of srorul.ttion inti rlic range of vnriition of
[he h.lp!opliJsi: to populational adaptation. Jour. 1l;ic t. (In prcss) ~
       , and Ilindcjircrr, G. (1945). i\faintcn:incc xiid iricrc.asc of gcnctic
chmcrer in tfic abscncc of the specific gene. Proc. Nat. Acari. Sei. 31:95-102.

--+----. '

13451

S I' IEG 111, hi A N -EN %Y &l n'r'IC  AL)APTh?'IO N

163

        , 2nd l-fcdpxck, L. ( 13.14). Xfeclianisn;$ of cnzyniitic ntl~ptitions
ill t;enctIc.~liy ControllcJ yc.ist populalionc.
-------, anif XO%.IW~, ?A. (19.15). 00 tlic inability of intact ywtc cells to fcrmcnr
  tllcir c;lrhhyJrstc reserves. Arch. 13iochcrn. (fn prcrs) .
S:c!lin:;-Dekker, N. ?,l. (1 93 1). Dic 1 Icfcs.inimlurig des "Ccntraal Curciu vwc Sclrimntcl-

  cultures." l`cii I. Die Sporitpmcii IIsfcn.
Scephcnsori, hf. (I 937). Bnctcrirlf hfctibofiim. Lniid011.
-, and G.iIc, Ii. 1:. (1937). 'I'hc adAptibilicy of glucozymasc and ~~lsctozyn1asc

-, and SticklnnJ, L. If. (1933). tIytlrogenlyases. 111. Furtllcr cxpcririicnts on

-------, and Yudkin, J. (-1936). Ci1:ictozymasc considcrcd 3s 3n ad.xptivc cnzymc.

Sricr, 1'. J. I).. aiiJ St.iniiarc1, J. N. (1735). A kinetic aridysis of the endogenous rcspira-

Nzt. Acnii. Sci, I'roc. 30: 13-23,

ill 13. roli. 1iiodicz:i. Jour. 31:131 1-1317.

rhc forniation of formic hydrogenlynsc by 1%. coli. Ibiif. ?7:1525-1534.

lbi(/. 3fk506-514.

tion of bnkcr's ycist. Jour. Ccn. I'liysiol. 13:461-477.

li)-dr.itc rcscrvcs in Ix~kcr's ycnst, lid. 479-+t94.


y-eisr. Jour. Cell. fc Comp. l'hysiol. f0:79-7 I.

I

, (133 5.1). The rnct:tbolic systcn~s. involver1 in dis~in~ilariori of carbo-

, (1927). On LIIC ~necl~aiiism of carbohydrate dissimilation in biker's

I

`&`:rburi:. O., 2nd ClIriTtian, V. ( 1337). Iso!ic.ruiig uric1 Krist~llisation des Proteins Jcs
. oay&crciiden Girungsfcrnicnts.  Uioc!icin. %citsc!lr. 303:40-6S.

%?age, O., nnJ Lausrwn, 0. (1337). On two typcs of spore i;crrnination, and on. pxictic
scgrcgatinnr in Sicchuoinyccs, dcmon~tmtcd through single-syorc cultures. Cornpt.
rend du hb. Cnrl\!xrg, SCr. y!iysiol. ?2:79-116.

'

I , (1938). ilrtifichf species hybridizntion in yc'JSt. Ibic/. 235-244.
? , (1939.1). On ld ncw yeast typcs, proJuccd by Iiybridizition.

lbi,f* 337-353.

__I -- , ( 1939b). SJcchromycrs Lvrfuisii Iianscn. A b31anccd hctcrozy-

FOIL`. 1L;d. 357-370.

\,-- u.tn~~nn, J. ( 1 S82). Untcrsuchungen iibcr ~.IS dizst.itische Ferment der Biktcricn.

Zcitsclir. f. pliysio!. Chcm. 6:2~7-329.

Yudl\in, J. (133 S): I~nzynic v.iri,ition in micro-orC:inicitis. IJiol. licv. (Cnmb.) 13:95- 106.