1, Jntroduction

3. Outline of EIO&~ of opextion of the Spm-alm-l system
4. Shtes of aim-'

5. Detection of tile Spm element and its mode of ope::ation

6, Resnnn ses of diffe ent states of a1 m-l to the S8iYE Spm element

8, Stabi!.itg of Nutants

9. Tpes of Spn elements

10. Xodifier 02ement in t3e Spm system

11.  ~oncl%sic;ns


1

Node of *oration of a System of Controlling zlements

in i,laize,

General descriptions of controll?ng elements in maize and of their

modes of operation have been given in a number of reports aznezring in
                                                                           .

recent yezirs (for literature clitations,  see NcClintock, 19Sbb).   It is

the purpc:se of t is report to consi.der some of the e,xperi.mentsl methods

that have been emyl.oyed to discover the mode of opex>ation of the s-stem

Of controlling elements which has  been referred to fin pt'$er publications

as the Spm (Suppressor-mutator)-al m-l system.   Controlling elements may isem

defined as those unit components, carried in the chro:::oscmes,  that serve

to control gene action both wLth regard to ty-ge and degree and to the


tissues or         ,\uelF, .
       parts of a tissue m t!lfs will occur.   The differ.ent

controlling elexents are recognized by means of t'leir distinctive modes of

control of gene action, regardless of the primary type of action of the

gene substance itself.   They ex..:ibit Eendelian inher9tance pattr?- ns and

their loc3tions i-r! the chromosome complement may be determined by use of

ordinslry genetic techniques,     r;
                      ALowever,  the:? may undergo change in location

within the chrcmoso~~e comuler?ent, appearing at new locations and disappearin

from previously determined locatlcns, without  losing their distinctive

properties In the :3rocess.   This process has been termed transpos?tion and


2

the several m:t:ods that have been used to detect such transpositTon were

presented previously (McClintock, 1556b).

For man;:? years, efforts of the author were concentrated on analysis

of the system COG osed of the two controllPng elements designated AC

(Activator) and Ds (Dissociation),   This system W.S chosen for extended
                  -bA 'lx

studies because both elements cP f.'~i.. gr-&m could be identified r:adily and

regardless of the location in t2h.e chro:noso~?:e com72leme:_?t that each mignt

occupy.   mlu s , c%nge in location of these elements, and t!e effects

produced when one of them is inserted at the locus of a known gene, could be

detected and subsequently analysed,  Altogether, the operation of this

s::-stem at eight BMwn gene loci has been examined.   Also, two or more

independent insertions of one of these elements at four of t`hese eight gene

loci were detected and the co:Llsequencd of this examined in e;?c:' case,   It

was concluded from these studizs that this system should be able to operate

at a particular

m locus is not lethal.  %st imp:,rtant, however, is the realization that

the mode of control of gene actlon ma;? be predicted in advance,for it will

    6cJdtymd
follow Vhe4rules tRa.t
                           A

the operation

of the mode of operation of the AC-Ds system has been useful in guiding

of -this sy3tm.l.   Knowledge

ex-!>eriments aimed at revealing the mode of operation Of Ot3CI CO?ltPOl SFStem

'i'his aenlrics particularly to methods for identifying controlling elements


3

and for detectlns their transpositions.   It has also provided a 12odel for


recognizing and subsequently appraising the chances in state of the affcctet

gene locus.   Such chail,yes must be recognized if confusion in designing

experiments and interpreting results is to be avoided.

Some years ago, a large number of different variegates appeared in the

progeny of individual .-lants with a history of having been subjected to the

breakage-fusion-bridge c:;cle in their early development (McClintock, 1951).

2?3 the time t:-iese variegates appeared,  it WRS realized thst it would not be

possible to examine each. of them.   Therefore, only a few amon,c tFe many

that ap;>eared were selectdd for subsequent study.   The AC-Ds s::stem was

discovezXed among one of t-lese selected cases; and as the mode of ope:>ation

of t .is system became aa73ar-cnt,
                              _.     attention was focused mainly upon it.

twqty of

The other selected cases either wass discontinued or it was sharply curtaiih6

until adeauate time could be found for * detailed examination.   The system

responsible for control of gene action in one of these latter cases  is now

                                                           the
sufficientl;y unde-Tstaod to allow conclusions to be d-nawn regarding/t-es of

controlling elements that are involved in it and t1lei.r modes of open-ation,

This is the s::stem

at both al m-l

and a m-l
   2 ' mentl.cned in previous reports.   In this repoTat, attention will

be given mtiY?.y to a1 m-1
                                o


irigin of a7 m-l

   The history of origin of al mW1 from a modification tl??t occured at

a standard Al locus is as important for an an _L preciaticn of the controlling


el t:ment s involved in this s;;-stem and their modes of opc:,zticn as were the


histories of origin of modified gene action that appesred in the AC-Ds


carrying cultu;2os for an ap-r,reciation of the yreyence and Irode of ape:-ation-
                        _..y


of the elcmirants of that system,  'The modified Al locus, designated al m-l
                                                                           ,

was tne t`?lrd recognized case of change in gene action in a sequence wL;;,ich

-&mmenced with an alteration at a previously -unidentified gene locus concel?I1


with chlorophyll productl-n,   The slants kvinz this first member- of the

sequence ex'~iblted variepaticn for chlorophyll pigmentation.  This v3.17iegat

was one of t:,ose v selected among the many that appra~r-ed in the
                           .i


original cultures,  as described :>bove.  in the e2rlg stages of examination


of td.s variegate, a number of plants in one cYUllture were self-pollinated.

On the ear prodwed by one of t.kse plants, some kzmels appeared that

exhibited variegation for anthocyanin pigmentation,   Spots of deep

pigmentation appcnred in a colorless background.   The plants derived from

them also exklibited variegation for anthocpanin pigmentation,   Subse;:uent

tes-:rs of these slants and thsir progeny indicated that an alteration hcd

cccurred at the standard A2 locus in one cilrorloscme 5' of the palent giant,

'This clte:?ed locus W:JS given the designation a2m'l,   Tests w,c:re th.en

under-taken to examine the changes in e,xpression of gene action at a2 m-1
                                                            and tc


det;:ymine t-5: facto;_-s i .\volved in ccJl?tzYol of t?is.   In t-:e course  0 6
t ,.is 9 tudy ,  a numb e r of plants in a culture in Wicb the s::stem responsible ,
for control of gene action at a,;-\ was -:~i~~esent, were used as pistillate pare
in crosses with plants t:i:nt were homozygol;s for the standrrd recessive,  a 1'

in chrorosq=-e 3, and for the stz.ndard A2 locus in c'?ro,~losome 5.   On one of

t`rze ears t:;is cross produced, a single kernel was found that ax`>-`bited
                          _

       P igment
spots of ant;:ocqnin in a non-pignented background.   A plant WCS zrown

from t is kernel and t is T:lant, in turn, exhibited variegation for alltho-
                    ilLduo p&d
cyanin pigmentation.  APW+@&& Tests crosses tlt.i?;d:
                                                                 A

e&m? J- +-.G-&W indl.cated the presence in it of a?#
                 .,.

Al locus, and-

&a@@f-               m-l
  was thereupon designated a      .   It was evident tnat
w            1

         the standard Al locus in one chrom, some 3 in the L&$!$i
      -!.tu&&&            +h
parent plant,a& la-:, e in development of one of t?e ears 6P t is t3lant_, for
                                                     4

only one kernel on tq..is ear ex?ibited altered ~41 action.   Studies aixred at

determining t3e com;Jonents o. f -t&e sgs.tem responsible for control of gene

action at both a m-l and a2 m-l
              1          were contined but initially only on a

limited scale. .
           Only :-ecently :?,a~ tiz:e been found t0 exa?rine t.:is  more

cominletely.  it is now knob tizat the same system of control of gene actior

operates at both al m-l  and a m-l
                       ? o   Very likely t5is salve system also

opernted to control action of the gene that was associated with c.3oroptiyll

prodv.ction, --t:-re initial varie?a-l;e i.: the ser'uence,   This ?ostu%%te is

based

upon the patterns of varicration that were ex..ibited and upon

. lnllcritznce bchzvior for study of
                          I          t.;,is case was discontinued so;"8 years ago:


and tlsereforo direct tests to d::ter;.qine identity of control s?.7ste-s cannot

now be mzde,   3 , 0.kui-t

In order to learn of the system that is responsible for cort~.ol of gene

action at   m-1
            a 1  and a2m'1, a large number of difft:rent tyTes of tests were


conducted.  The results obtained from each are consistent witn one another

on the basis of t-e eventually determined modes of operation of the ccmponen

of this system.  In order to corn ly with space requirenents, only a selecte


set of tests 1 -kei?t   did
          be g&24-n here,  These are chosen In order to illustrate t:

salient features of t..e mode of ope:ration of tkis s:;stem ani- th.e;,r will be

confined to studios conducted with al m-1
                                                     .  Those conducted ~4th a2 m-l

will be given in a separa-tc report.

Zk-+om examinaticn of tke AC-& systctm, it was learned that  inse%-tion

of the Ds element at the locus of a gene initiated the zrimary modification

that brou&@ thi& gene under the control of tb.e Ds-A h
                  A                           system.   In many
                                                                   _,

cases, the action of the gene was noticably altered by this event and
                                                              A

detection of the          of Ds at  w-
                    the locus could be mnr:>de s?:ortl;i after
UaognP$o>ubseqt,Uent
it v.         d
                            1
  h                    the locus rnsultfi from the effects AC

exerts on the Ds element.  The consecue:`ce of tltis is eit?er remzaval of Ds

from t!ze locus of the gene or a modification at the locus, induced by 1&,

that effects a cliarqe in its organization ,--a cllan2:e in state cf t:!e locus.


7

Both t:;pes of events can give rise to recognizable c&n,?es in action of the

gene subsLz.nce,   %'ith regard to  a m-l
                             1   , insertion of a particular

Wi'ltrOlling element at the standard Acclocus is consid::red to have occurred
                              1

an& to have been responsible for the initial chnnye in gene action.   Like

Ds, it is t?;is element that directly controls We t;.pe of gene acti, n that

will occur at the A locus and tl?e
       2           4   t:!Tcs of c!?anpe in t. 5-s a- L7:tt may

occur subsequently.  1-t aDpears to be the sa=le element t?at is present at
                                  .

???
?? o  This conclt;sion is based on the r=spcnse';bBth eQ al m-l      m-1

                                                                ,I        and a2

m-1   m-l /.iMc~\clklsd @,@+-hd
al   or a2   that allow$ the Rene substance to become active e

    .
IT -    el  or in w descendant t .
    A                    The particular tyoe of e,Tressicn of the
                                                                           ._
         4

gene that th~~se modifications effect is tnereaft?r mai:-itai?:ed eittiyr in l57e
               Qh m&t kiu!&@, dt&A,-%J

presence or tl-!e absence of t31.e Spm element.

m-l

element is co:-:-elementary to the ele-scent located at            m-l
                            a3   and a2 .

It scrvcs to activate it and as a consequence of t is, stable mu-t.tions

are produced,  In t is respect, it resembles AC in tile Ds-AC system.
   trze genicmybstancem 1
i-iowevl:r,/at bot2 al   and a2 -  map be active to some extent when Spm


8

is not nresent in the nucleus.
         _                idhen it is removed, either t:?rough meiotic

segre?aticn or by menns of somatic trnnsqosltlon, antkocyanin pig:ent mag

distribution is uniform,   In otii,er words,  t47ere is no variegation,   T-he

t:?,e and intensi-iz or __
             pi67mentation is an exp ession of tl>e 9articulnr state

of &+%s tile alm'l  4J-Q m-l           a. O.&u& ciwz AdJAw
                     or a
            A 2  locus thf?t is presentA   These ska-bes and

tneir orig?ns will be co:Tsidered si:lortly.   Vhen Spm is  I' eturncd to the

nuclei by apzropriate crosses, e gene action at  a m-1       m-l
                                           1    and a 2    is sun'13res
                                                                           - ._

ed except in tose ce!.ls s&ore ~~u.~ati.on-producing" events occur.   Thus,

m-l       m-l
a1   and a2    and t::c eby conditlor&g stable mu-t?.tlo:?s z:t thsse  two

loci, but 2-t also must act upon tI:is element in ;ret another manner for its

presence results in sqozression of known :?otentials .tor eene action
                                                              J  exce7Tt
                                                                           .."

in t ose cel!.s wh`>rc lrzutatrion-lor0ducri.c~ events occur.                        * .
                                       i                 The se two SBBpLLpeks
                                                                           >

dif?orent aslleets of' the ::-.cde of control oi' the Bpm element on the element
               .

rcsidinz at al m-l       m-l
         and a2  &         ,. dd
                    respcnsible 3or Tts w designnt &
                                                                           1

Suppres sor-mutator.  kiowevor, this seemihdy dual action ma;7 be the
                                                      e

express5.l.n of' onl;~ one process rather than of two unrelated Drocesses, as wil

be indicated later.   qJ..Qsl~ if& 41"-'

All examinations of the effect of a controllTng element at a gene k


9

must be c:>nducted wit;) t5e affected locus,   Since,  in addition to stable

mutrtiozs, t:i?e controlling eler:wnt Epag also initiate structural 0~ organi-

zational  modifications o? the locus tL7.t al" OCR its subse~(uent e,xpression,-.

that is, chnn~-ne tLe state of tke locus, --it is necessary to cor?_sider

states of the aZ'?ectad locus and Weir origi?e!s before detaFled results of

experiments Q-iik%e                   m-l
             pres-nted.  With al   , a c.l:an,ged state 2s readily

recognized b7 the appearance of an .iw?dividual ke:xnel or plant t_?at

exhibits an altered response of t!le locus to the iIrcE:er?ce and abserce of

Spm.  I?- khen the controlling element first eqe~_:" lbd the locus of
                                                                           P

A1s it effected a particular t:,Te of modificatir;n in t%e structu-e or

organization,of tlhe locus,  In the absence of Spm, some gene actdon
  oxtiw*
occurred
    4  Kernels were lightly but unifo rz?Jy pLgmented as we:>e the

plants derived from them,  In the presence of Spm, however, all gene

action was suppressed except in sozie cells where mutation-producing

events occurred at thelocus of al m-l that allowed the gene substtince to

be active in the descendants of t-is cell.   aeQ
                               iki&kf& such event ikit not

&a the same degree or t;:pe of gene action but many of them restor-ed

tnc full or near full A1 t:$y)e activity.  Irany of these events occurred
                  '( __. -           :-

relatively early in plant and ke-Tnel development. c` .
           4x-        1
in dose Of Spm haL'n0 effect on  alt aring the time of occu-rence Q? t:rrese
                                                      t


10

                      aliAvd
????? o  wxu-tation-@noducing events om in germinal cells

and t&is made it possible to examine the nature of the mutation in the
                      _.

                                        m-l   u&n&% bw
next generation,  Another t;:pe of change a2 al    also occurred in a
                                             4

few of the .~erm:inal cells.   These resulted in altered expressions of the

locus both in the presence and absence of Spm,   Some of them were

detected initially in individual kernels in the proFeng Of t3e origcnal

rg a=1
  1    carr?rinr i:la.nt .   When t:lis ;Tl;lint was crossed to ulants ti?at were

L-          "    II

..hOXOZJ~~OUs for the standard recessive, aI, w%c1-1 does not reqond

but res3onds, instead to Dt, the v :x..xa~n

to Spm

m-l
al

JXBZ.X  locus in the majority of kernels that received it

was unmodified.   Among t3.e kernels receiving the unmodified al m-1  locus ani

also Spm, the variegation pattern was much t2e same.   The 72 .. we e many

    pigmented areas>
large yn indicating early occurring mutation-producing

Qna@J nox4 Al/w+ak~rnaEeeo* J
events.
  .A  .&any of these e,xhibited t'ne full A
         d&Q                 1 type of pigmentation.

ilowever,  an occasional ke-32el ap73c::
                                         -i  red that exhiblted a quite dirferent

pattern of pigmented ar:->as.  c,Ewo kernelA were found,bmong several /
                                           -.                                                        i
thousand&hat h: -7~': only small pigmented areas.
(J@J&cceQewl~~
distributed &ahe intensity of pigment
&u+     4
oyvery dark.

pigmsnted a:Ti:as as J:ell as some small ai?ezs but the intensity; of the


11

pigment in all of them was

It ranged from vcr~                                        .
              faint in soze arc:: s to 8?--
                                                        ,'t        in ot-81's.

exceptional

Plants wc;:e grown from the three described/k?:nels.  Tests conducted

with them and ti?eir progeny indicated -t;Tnt the pottcrn of varieCgation

exhibited in the presence of Sprn was,heritable.   The alte:>ed a m-l
                                                         1   locus,--

the changed s-tizte 0:; t;le locus, --in each case responded in its own

particular WEI:- to the nr:::;ence of Spm and also to its absence.  In the

absence of Spm, the state of a 1 m-1 -JFfmFsb in the first described l:n;;nel

produces !hzqzqxm deeply pigTented kernels and plants.   RBJC in the

second described kc.!-snel @.,-es rrise to very lig!?tly p?-gy?znted kcrnel.s  but

r-,t::er darkly pigmented plants,   The state of 3 m-1
                                          1    present in the t-ird

described kernel produces no pigment in either plant or kernel in the         .
                                                                           .
                                                                           r'

absence of Spm,   Subsequently,  other states of a            f
                                            1 m-1 h&%&n isolated. $
                                                     a

.&cl?- is distinguishable from fine otlrlt:r by the t:,Tes of mutr.tion and the   4
            ad *'$eeyL uJtu webvJr
                                                                           1
time and frequency of their occurrence in t++ yCB    j
                                     as r,-el1 as a
                                         A                                                 ,-

     the tyne of e,xpression of the gene su-zstctzce t.net FtikEc
pN\~;pcmy&QMa$;nc~ QQ@.wmtlnpp~~p~Sp*y. M@fhtw t?@Q cl@i[~~l~~
     Spm. 4 IJo relationship wzs noted among the diff'e-:ent states betk;cen the
         w& @qputQ
     control of` time 0:' occur
                         A      ence of mutatrion ii- the presence of Spm and that

of ty-e of gene action th::t occws in its absence.  2"igure 1 illustrates


12

%e ints9rbty of 8 state of al I?.-1 is maintained in heteroz:?gcus

plCC?t S  and t i s n-fxliss to plants t-at carry a dii'fcrent sta-Le in eat?: of
                      

their chrome so33 s 3.   In such plants, each state resp,nds to Spm in its

o+m peedictable way an: thLe variegation patterns  each 3roduces will be


                                       F *
expreaUU
    ~~~~d in the '?lant or kernel tissues.   qure 2 illustrates this.   CLe

kernels in the pDic&o&raphs carry the state shown in - of Piipre 1 in one

chromcsoi-.e 3 and the stake s?own in 6 of figure 1 in the hcmologue.   The
      CLua~m~qpo,

pattern of mutation produced by each state f3.r readily recognized in these
                    A

kernels.  In t?e plants, normal segregation of the two states occurs at

meiosis and each map,: be recovered in the expected proportion from the

gametes that these 73lants produce,

States of alm'l are maintained unchanged in the absence of Spm.   In

its presence, hop:ever, new states ma;7 arise and the frequenc:r of cccu-:roncem

of t)Tis in germinal cells is related to the time of occur-en?e of muteticn-

producing events th,zt 8 particul:~ r state exhibits.  If the sta:,e :zrodnces

some earl;: CCCV.iT!.ng mutatinns, then new sta:.:os of   m-l
                                                               a 1   may appear in the

gem ce?.ls, and the frecuency of' t..is is propc+cicnal to the frec.llency of

CC~U?I't?YlCC? of ti-lose :zarly mutations.  If, on the other hand, the state is

one in w ich mutaticns occur only late in development of t-he plant e
                                                                           t

ice tl-ieh few or no altered states may be recovered in the gametes of


13

these slants.  The mutation-producing events at al M-l
                                                                        , regardless of state,

give rise to stabilit;; of expressicn of tl>e locus.   TIce particular tT~T;e of

gene action the mutational event produces continues to be e,qrezsed in

subsecuont gcneraticns both in the presence and in the absence of Spm.

Tnis suggests that the change resncnsible for these mutations may Erave

removed the cc'ntrclling element frccl the locus or it may kw33 result& in

its inactivation with regard to Spm,   Since the chan,yes in state arise

at tAe very same developmental p ricd as the mutation-inducing events OCCUrl

it is conclxded tat the y represent a modification at the locus induced by

tne controlling element residing there &at did not resu1.t either in its

.I

removal or inactivation,   In cthcr words,
             A                      the element responded to the

presence of Spm at the appointed time and in the apFcinted cell$ but the

cons nuence of this was not the usual one,--ikek, ti!e mutation-producing

event.  Instmd, t?e responding element itself was eith z: modified or it

induced some roorganizaticn at t3e locus that modified its capacity to

mutati ns it c:ln induce W tlr:e 'c-T-pe of gene action that can occur
                          I

in tke absence of Spm,  Jt is as evident in this  c 3 9 e  as it is with 9s

that a chan,c:c in sta-i;e is one of the ccnseq.uences of the response of the


of the controlling element residing at a gene locus to the independently

located element of the sys-;em,--Spm L-
                         'n t+is case and AC in the case of 3s.

                                         ,j q
                        m-1         :J,QL "<
In the early excminaticn of al   , no evi.dence of the presence of the
                                                  4

Spm element,l= &&i~&&.  This was because the original a m-l
                                                     1   carrying

plant had a number of Spm elements located at various Y:csitTcns in the

            (17. (.k&* ~A+fJ$pQ
.+&romosome ccmplenent,  Al140f tile garnet&s it produced had Spm elements
-.. .,,
-..,
,_
GE&-them.  Since the dose of Spm hss no a-preciable e:I'r'ect on tl-le pa-ltern
1~1.

of mutation produced by al m-1 , differences in number of Spm elements in a

kernel or plant is not made directly evident by t:?is means as it is with AC,

It was only after several gener2tTons of cr0ssri.q of al

tester stocks which did not have Spm, that
uniformly p&Q.@

kl&&$ colored kernels appezl.red on the test cross ears.  These ratios

indic a-l;ed the presence in the   m-1
                                   a 1   carrying plants of an inde?-.endently

located element that is associated with control of almol expression.

They also indicated that the number and the loc2ticn of this element WRS not

the same in all tested plants,  In the meantime,  several different states

of al m-l had been isolated on t?ie basis or the alte;ed va:?iegztion patterns

that ay>:?er.red in individual kernels on several of ttic WAY& testcross

8,2rs,  as described e:.:rlier.  In successive ge- erations of cross?ng of


plants carrying these di.?f'er::nt s ;ztes of a m-1

                                       1   to the al teste;: stocks,

the same kinds of ratios of variegated to non-variegated kernels also

began to aJ-,pc' r.   It was thena-ssumed tilat the unifor5L:T pigmented

(non-vr!-:iegntcd) ke?:nels and plrnts carried al m-l
                                  but not t,:e inde::>endertly

located element.   On the otler hand, this element was assumed to be

present in trlose kernels and plants that srlowed 12lgmented areas in a non-

pigmented bnck;ercund.  $i t2.s interpretation,  tie independently located

element was exsrting a suppressor-mutator t;rrle of control of gene action

at a m-l
  1 o   Since the dose of t Is element obviousl;: did not affect the

pattern of mutational events,  control of t :Is must reside at the a m-1

                                                             1

locus Itself, the tppe de-,end$lng u77on g5.e state of the locus.   It was

eviCent that the phenot pes of t'ie nonvarieqatei kc:-.:nels and plants also

.

reflected 'c-:e st::te of the a El-1

                       1   locus that was nrescnt in t?lem.

The 'hypothesis stated above was subject to test.  If: i4.2 were correct,

evidence in su?2rjort of the PollowTng eour statements  s" ruld be obta<.ned;

(1) All variegztod kcrne1s and ?lan>s carry at Icast one Spm eleiment.

(2) igo Spm ele-- ,,lent is pres;:nt in the non-variegated class of kernels
                                                 o
               ????o?????
and I:&ants.( Germinal mutations, &WXK&A earlier,  are excluded from t:?i.s

class.)

(3) The al m-1 locus in t;:e non-variegated kr+;neBs and nlants is capable


16

of res c:ndTng to Spm if t:->is elel-.lent Zs subsequentl;.F introduced into a

nucleus.

(4) The tr,pe of rospcnse bB Spm and the p.~enotpes paoduced in its

absence is a function of t.h:: s tse of the a m-1
                                     1    locus.

A large bodp of evidence in support 02 t5cse statements is now available

and it has been obtained by various t Des of test, onlp a few of w-ich
                      I--

"'need by ou.t?.rincd hzre.

   In order to facilitate identification of t3c pry-sence or absence of

Spm in a particular plant, so-call!:d Spm tester stocks were developed.

These sto+ck have eith:.:r one or the ot:!er of the two states of   m-l
                                                                           a 1    S?lOkJ
(23; :r  I ,-&iJ
in & and -
           .I  of figure 1 (QrFiese states were selected for the following
              1


              --
reasons.    1 n the first :3lace, wl;en Sprn is present, very few germinal

mutati:ns or cn.anges in state occur.   Therefore, nearly all of the
                                                 
                           <(-j:il", .<y.&
gametes produced by plants carr;ing these states of al m-l and also Spm
                                          4

have an unmodified a XL-1
                  1    locus in them.   Seddnrlly, the pattern of variega-

tion esch r;roduces In the presence of Spm is distinctive and non-obseuring.
                               : : 1\3 q-\)
'2;7irdly, in tl:e absence of Spm, one of t?!ese states,- fi,gu:,e 1, gives rise
                                                                      1

to darkly pigmented kernels and t:is is a useful character in some tests.

'2`h.e tester stocks were made homozygous for one or t?e other of these two

states*  Tney  were also made homoz;rgous for  either Sh2 or shz, located


17

very close of a1 m-1 ( about one-quart237 of a poment crossi?? eve? occurs

bet::cen them).

An examq1.e of one serii:s of tests will illustrnte soxe or" the mct~lods

emnloyed to determine Spm constitutions of indkfidual plants.   The ~4. lks of

two ears of a variegated slant carrying al m-1


and al  and sh 2 in the :homologne and also Y in

and Sh in one chro->?osorre 3
     2

one chro-iosome 6 and y in its

homologue, recieved pollen from a -:lant that was i?omoz;rgous for al, sh2 and

                m-L                                  <gjqk-i>

Y.  The state of a1   in the pistillate glnnt was that shown in .- of
                                                                           e

figure 1.   From t.2is cross, the two ears produced a total of 745 kernels.

There were 181 Sh2 kernels in w~~!ch the aleuzone lay:,r WLIS uniformQ.y and

rather darkly nifj;mented; 69 of ti,ese were Y and 112 we::e y.   The aleurone

layer in anoC:r 188 Sh2 kernels exhibited a number of spots of the full A1

t:g2e pigment 5-I:: a colorless background and 117 of these we:'e Y and 71 were

Y.  The aleurone la::er in the remalr:lrq Sh2 kernels was coy>letel:r colorles

and the stai:-&. _i_n its endosperm was y.  Among the 375 sh2 kernels on tl:ese

two ears, the alcurone la:r,r in 373 of them was totall:: colorless; 186 of

these kernels were Y and 187 were y.  The remaTn1.ng 2 sh2 kernels ex'Ti.bited

spots of full Al type -!;igment in a colorless background.   'i'he pnenot:Te of

tile starch in one of them was Y and that in the otiler was y.   Since


18

al mm1 is c,olsel_: 1-j-i;ked to Seh2, naar1.y all of the Sh2 class Of kernels On

tiy.ese two esrs should carrp an alal locus and naarly all of the sh2

kernels should be homozpgous for the standard recessive, al.   The close


linkage of al mw1 to Sh2 is obvious for only 1 Sh2 kernel In the total of


370 was comp?_etel;: colorless and on?y 2 sh2 kernels in a total of 375 had

pigment 9x3 the aleurone layer.

   On the basis of the interp-etation given above, the uniformly

-.

pigmented kernels s?ould have no Spm in them whereas t-ose exll_fbiting spots

of the full A2 type pignent in a colorless background should hove tis

element.   From the ratio of these two classes among the Sh2 kernels

(181 to 187) it could be concluded that the variegated pistillate parent

plant had one Spm,   The ratio of 'Y to y in each of these two classes

indicated that t5i.s Spm element was carried in t??e Y bear.';rq c'nro:-lose-:e;L

It w:; s then necessary to detcrmrl_ne w;ether Or not t?ese conclusT0n.s were

valid.  For t is gurpose, 104 p\` ants were grown from various t:;pes of

kernels on ti3ese two ears and tests we:-;e conducted with them.   The


phenot:;-:-,es of the  selected k-rnels were as follows:  11 uniformly pig-xnted

Sh2 W, 13 uniformly pigmented, Sh2 yj  17 variegated Sh2 Y, 8 variegahed

Sh2 y, 1 variega'l;ed sh2 Y, 3 0 colorless sh2 Y>and 24 colorless sh2 y.

All 2!!- planiTs dzri!3ed from the uniforml:F .- qigmented kernels were themsleves


uniformly ?Tigmented.  All e 26 plants derived from the variegated

kernels showed small streaks of the Al ty-pec$i~ent in a non-pigmented


background.   And, as expected, all sic plants derived from the colorless


Sh2 class of kernels lacked anthocyanin pigment.   Each :-lant wzs then


tested for presence or absence of Spm by crossing it wit!? a plant in an


Spm tester stock.

    To illustrate how the tester stocks can serve to reveal the nr-Jsence
--,

81" absence of Spm, those tests conducted with the 511 plants derived from

the colorless, sh2 class of ksrnels will be considered first.   This is a


completely objective test since the presence or absence&n any one of them

                                             me silks of

of Spm could not be assumed on the basis of 9,Clenotr-pit eq3ressions. / 8ne

or more ears of oat? of these plants received nollen from nlants that were
                    ' 1 .I \-('I  ; ,l,rr&- :':
homcz:-gous for either state - or - of figure 1, for Sh2 and for y; l&em
                                      ,-

pollen parents we-"e uniforml~~ p igmented indicatine the ab:,ence of Spm in
                                  I             .,

them according to the stated hmothesis.  If the plant being tested has no

Spm, then all of t?Le ke:nels on the resulting ear will be u.niforKLy colored.

I f , ilowever, the JLllant being tested carries Spm,, tllen it should be present

in some of its gametes.  Following introductition of the al m-l locus from


the male pa::;ent, the presence of Spm in those kl?rnels that received it from

the female ::a-!>::nt should be revealed by the an?-ar&nce in them of small,


20

                              -)Q *&+JJf +4.&i 4 q&-f1 $
deeply eigmented spots in a colorless background due to a&z&&&w=&&

&) almml locus,h$LL.&~ 0   In t`pose kernels that did not receive

Spm, the aleurone laver should be uniformly pigmented,   Among the

30 plants derived from the colorless, sh2, Y class of kenels, it could

be det ,rmined on this basis that 15 had a single Spm element and 15 had no

Spm.  In 13 of the 15 plants that had Spm, linkage of it with Y was

evident (A, table 2) but in the 2 remaiz;ng _nlants, no linka:.e of Spm with

Y was evident (B, table 2).                    linkage of
              (me reason for the absence of/Spm i.aithe Y

                       Q&m- .
in these 2 plants will be considered LFI m .   It need only be

mentioned here that this is not une,xpected,)  Among the 24 Dlants derived

from the colorless, sh2, y class of kernels, 6 had a single Spm element

(c, table 2),and 18 had no Spm,

Each of the 24 plants derived from the uniforml:T pigmented k:rnels

                    .                i,
11-e
            ----4 . was tes.ued for presence or absen-ce of Spm

in the described manner and in none of them was Spm found to be present.

All of t:.e .:)lan!Ts derived I'rom tLhe variegated kernels s?owed str,;aks of the

Al t:nse nigment in a non-pigmented background and the test crosses indica,:ed

tine presence of Spm in ;:aci of them.  In 16 of the 17 plan.:ls derived

from the variegated Sh2 Y class or kernels, one Spm element was present and

in 15 of these plants, it was linked with Y (D, table 2).   In one N


21

plant, however, fro evidence of linkage of the single Spm element h6th Y

was noted (E, table 2).  In the remai3Tng plc-nt bn this:.&oup, 2 Spm

elerlonts we;^o present, neither of which was linked with Y (Fg? table 2).

X07-e than one test cx~oss ear wti.s obtained from11 of these 1'7 plants and

the number and 1ocr:tion of the Spm element was the same in the cells

producing all 9~~s except for one :>lnnt.   In tl=is plant, one Spm element
                              ._

SRI3 pPcSent in the cells producing rae main ear and it, Was lir;%ed Mth Y

T&3., table 2).   In the cel.ls that D?oduced tk!e ti.llZr ear, however, a

single Spm element was present but it showed no &inkage with Y (G-2, table .Z

cr ' In all 8 ;?lants derived from the variegated Sh2 y class of kexels, one Spm-
  i

was 73roscnt (I-I, table 2),  One Spm element was also p- esent in the plant

derived from the vzyieg:?ted sh2 Y kel>nel.  'his plant was uned as a pollen

                                0

parent in crosses with plants he.ving different constitutions: homoz:rgous
            -&so for I
          and y i"      0
                                3
for al and sh,/and h~u:ing no Spm, homozggous for several dis'f-rent s-bates of

al mm1        (31 4
   but xving no Spm,  and -t;4                                                S
                                 4
                  m-l
            e al    Sh 2/ a1 sh2, y/yT no Spm.  In this third group,

                             m-l
plants witA different states of al   were represented.   All tests indicste

"cne presence in thepcllen parent o:    I

      w4  gg&&i.~
and of Spm in the 'iI' carrying  chroxoso~re


The tests described above were conducted with the proffeny of a &ngle

plant in a culture.   T-here we:-e 19 variegated plants in this culture.

.&.ch vJ.S derived from- a variegated ke?nel that apnsa-ed On an ear of a

varieg :-ted plant that was a1 mm1 Sh2/ al 8h2, Y/y, wx/wx (chromosome 91,

pr/pr (chromosome 5') in constitution w??en pollen of a nlant homoz:Tgous for

a2' sh2, y, Pr, and Wx and having no Spm had been placed on the silks of

this ear.  All gepnels appearing on t is ear :-ere Sh2 and the distribution

of phenotypes among them we-ye as follows: 28 uniformly dark @Le Y, 7

uniforniiy dark pale y, 5'5 variegated (spots of deep pigmentation in a

colorless background) Y, 86 variegated y, 75 colorless Y, and 103 colorless

Y.  Among t"le kernels showing anthocyanin pigment, tne ratio of unifo~4.g

dark pale colored kernels to variegated kernels indicated t_ie Tresence of

at leas-b 2 and nossiblg 3 Spm elelrents in t5e pistillate pa--en-t rlant and

on,: of these aT>;x:r-ed to be linked with Y,   The silks of e2rs of 9 plants

de!:>ived from tine variegated Y class of kernsls on tis ear and of 10 plants

derived from the variegated y class received pollen from plants that were

hoxozy,~ous for al, sh2, and yg and h,?d no Spm,   The ratio of kernel t--pes
                               d.~ 1 ! si1-t 4. IJJ-VC 4Al L-I FpA jfi&
ap.p~~xeing on the resulting ears Droduced b:  each of thz4e 9 plants is
                                                                      A

enter-ed in table 3*   In this table, the 9 plants are -laced in four groups :

A to D, according to the assumed constition of Spm in e<-ch that the ratio of


23

kernel types suggested.   Y%e 6 plants in A of t ,is table s:e-e assumed to

have a single Spm element located i-r! the Y bearing ch-omoscze,
             .3                                    Progeny

from 11. of thex 6 planks were grown and again tested for Spm, Those

derived from plant 6629A-1, line 1 of 4 of table 3, we1:e considered sepa:y;ate

and theti        .
        v
      n .'- -.   are entered in the last lir_e of A 0: table 3.   The s e

tests verified the pres<ence of Spm in the va?$egated kc:-l?els and :Tlants and
".

Z&s Absence in the unif'orcrly pigmented ke-nels and ntants.  '52 CT;  al s 0

verified -th& assumed Spm constitution and 1oc::tion in the pistillate
       h !.Nb
parent l>lEnt,
                -2 @nly those tests conducted w%th the 116 ?lznts derived

f'rom the cbk&mrless sh2 class
                     of kexels will be swnmn??zed 1qs:r.e.   Among

the 56 ~,lan.i;s dexived from the al sh2 Y class of kelynels, 32 cazied SDrn

and 2.!4 hr,d no Spm.  In 30 of the 32 plants having Spm, linkage of it with

   Y was expressed, I, table 2.    In two -3lent s          0-d
                                 the Snm ele&ent bf%s not ',E~E\
                             4c
+;] k l:I.nked ~~~iii;h k', ,$A L4.M        '?\\,
       B ment.i:ned earlier, (5, table 3;).   Axoq t5e 60 plants
                                                     -~. - -.-
  de:i-ived from the al sh2 y class of ker:>r,els, 17 hzd a single Spm elerwnt

(J, ta.:lo 2) and 43 had no Spm,

  In ordcl* to verify the                                in the 2

plan$s ent:sred in i3 of  ' -cable 2, tests of some of the u;<ogeny of 530th or them

I-it?:-'e conduc-Led,  An ear of one of these clan-L-s i:ad been self-pollinated


and anoth-r  ear of this slant h,a:d been used in tile cross wr'i.th an a 1 m-1 Sn
                                                                2

y, no Spm tester ~?lant.   BXE progeny from both of t'xese ears were ar?ain

tested for Spm con.stitutTon and I-occl.tion,   The silks of ears of 8 plants

dorived from al sh2 kernels in the Y class on t?e self-pollinated ear

recetved ;?ollen from the a m-1 Sh2 y, no Spm tester nlants.   From the
                     1 J
                                                     a-   *aQ
kernel t:;pes on the resultcng ears it couI.d be concluded that &PIF of 6
                                                               4

plants acre Y/Y in co::stitution and that one of them had no Spm wIhe:eas the

ot!ler had 1 spm (155 pa~le colored kex-nels : 169 variegated kernels on the

test cross ear),  The remaining 6 plants wc:`e Y/y.  Cine sprl was p:-'cr,ent

in 4 of them but it W:IS not linked wj.th Y (EC, table 2). An Spm element

homologous

app: nred to be carried at allelic lzosit1ons in a pair of/ch'qc~?osomes in the

     .
rema. ing 2 nl.ants 'L, table 2).     Seventeen nlants derived Prom the
                    4Q

vaziegated Sh2 Y class of kernels++,on testcross ear of t!lFs same pal-ent
      ( ~$~w;: 6, ri , S&J 2)-        4

plant were crossed by plants in the &IQ tester stocks,  In 16 of' these

17 plants, one Spm was nresent and on none of the ears produced by 15 of

them was their any evidence of li::kag@ of Spm with Y (PI, table 2).

iiowever, the ratTo of kernel t;jpos appearing on the test cross ear of one

of .&em suggested suc'h linkage (I?, table 2).  The remai-j.ng plant had

2 Spm elerxnts, neither of wl:ich w.as linked with Y (0, table 2).


Tests of Spm constitution in 9 plants de:?ived f'rom the variegztsd Sh2

Y 1;e;nels on the ear produced by the tes-t cross with the otiler plant

ento;aed in B of table 2, suggested tilrt the Spm element in t1i.s :?lant had

been cax~ied in the Y bearing chrozosoxe but at a new location that was

farthor removed f~ m Y,   Seven of the 9 progeny planks had a sir&e Spm

                 ti+
element and in all OY? the test cross ears,            spw
                             loose linkage of W with Y was
                                                     4

expressed (P, tc.ble 2).  One plant ol" the 9 had 2 Spm elements (Q, ta':~le 2)

and jacz. the remaii?ing plant il;7td 3 Spm elements (3, table 2).

Further examples of the progeny test method of deter--lining Spm

constitution and location will not be given here.  It should be mentioned,

however, 'cl=.-t such t ests wSre conducted with the indicated progeny of

the ?,lants entered in B, C, and D of table 3, and also with the progeny of

the 10 o%tBer plants of this same culture.   Also _ a?!k es'-s
                                                                           ii

~ ~"kQa"c      pJJ&bmQ~~~
   were conducted with th
                     4     8 -0roEeny of plants having other states of  a w-1
                                      __                                      1

or coxbinntions of states, i.e., carrying di:t'fcrent stctes of a m-l
                                                     1    in

e'&h chrornosorie 3.  apT;tslpRp
           'Tke tests clearly sunport ia

h                          the 4 statements given on

page , and particularly statements (1) and (2). ikre difect support for

statements (3) and (4) was obtained by other tests to be described s;-:ortly.

Various different locaticns of Spm, eit.,:er in different chromosomes of' the

complement or in different locztions xi-thin the same chromosome,  were


26

discovered in tileze tests and each dettrxlined i-,osTti.on of it was

subseauentlF verified by me?&ns of progeny tests.   riowev r,  it is c1ec.r

from the tests so f:-T described thnt Spm dozs not ::elzain at one locztion

:,tithin the chromosome comD?-event but disa-years from a 1z~own 1oczti.m and

appears at a new location zn:i tAs till be cclns?.d:red il? a seDarate

               a-4
section.   before this is dead&~&, it is necessary- to s3ow that the

Spm element, reg?.rdless of I.oc?tion, is canable of acting upon any one of I

.I
the states of aim-',
.-. .


27

Responses of different states of aim-' to the same Spm element
                                                            --"
              &'

dvidence for statemento (31, page   , appec:;ed in all tests in :$:ich
                                 4

either one of tine two states of a m-l
                            1   , present in the teste-;? stocks, had

been used in crosses with plants carrying Spm,   dach state res>onded to

Spm in its characteristic manner.   In order to determine w~:ether tne

Spm ekemcnt present in a particular nlant would be capable of activating

ot'ncr states of  a m-l
              1   , several additional Q-ees of test were performed.

One of them utilized different ears produced by a single plant,   In one
        WiQ &&A &$I

such test, f&&w? plants were homozygol?s for al,.  Spm could be elthcr'
                    4

present or absc ncB in any one of them.   Pollen from a plant of -tskeL

                                 I-l is          j?\qR-'
tester stock carrying either state - or state - was l,laced on the silks of

an ear of one such plant.            a al-Gjv&
                 The sl lks of wwdhx=-  ear o:i " the same slant

received pollen from m plant that was homozygous for a different

stnte of al m-1 and in w.>ich no Spm wss present.   If the pistillate plant

had no Spm, then all kernels on both ears were uniforrly pigmented, and the

intensity of this reflected the state of a m-l
                                    1   introduced bp t::e pollen

                     yl,ltQ

parant (excluding state -, figure 1, $'i5.ch gi:Yes colorl~ess kernels in tlhe

absence of Spm),  If, hot-revsr, tne pistillate parent carried Spm, then

both variegated and non-variegated ki_rnels apneared on 'rwtk enr$ but the

                                 a&L&Q
phenotypes of the two classes of kernels on each earrreflected t?e state of
                                                             A


  28
d

m-l
al   introduced by tne pollen parent.         %P+
                         Again, if the D element

"'showed Liuka~c ~iti~. a genetic fnctop ms on one ear, 6.p

U,YCIZ&~ 'i&.&s same linkage on the other earJand an examp1.e of t;:is
                                       4

is given in S of table 2,   In t iis test, the main er.r of an al/a 1 , Y/y
           &
plant received polLen from on? Y- the Spm tester stocks.   The kernel

types on t.kie ear t;-is cross produced, $1, table 2, indicatir-id the precence

&%I the pistillate parent of an Spm element carried in the y chro30 sor:e.

'3%~ silks of a tiller ear 05' ti-:Is same plant received pollen from a plant
.".
                      _: J;$;"y '/
that was homo~ygous for state ':, figure 1, and also for y.  TTne pollen

parent was uniformly pigmented indicating the absence of Spm in it.   The

kernel types on the ear this cross produced are entered ins-2 of table 2.

The, ratio of variegated to non-variegated kernels in the L' and y classes

Was muw. the same on both ears,   Tllus, it could be concluded that the Spm

element, carried in the Y chromosome of the nistilla'se plant, was capable

of activatln,? ei%hr:r sta';e of al m-1
                                              .    Pollen from the same collection

Q?. I` .`; T,F?B used in the latter cross was also placed on tile silks of a plant

homr_ CST .go-:s for al and for y, but in which Spm. was known to be absent.   All

of the 294 kernels on tk$ ear this cross produced were uniformly li?ghtly

pigmented and all were y.  This test confirmed the absence of Spm in the

pollen paryent.


29

Another t::pe of test ti:?t was emplmyed to indicate the response of

                 m-l
different states of al   to the same Spm element, utiJ.ized tote pollen of

plants that were homoz~~gous for a1 and in w'iich  Spm element was present.

The t::Tes of test conducted with two such plants, number 6861-l and 6861-7,

atie illustrated in table 4.  Both of these plants wire homozygous for

          al'--`
    .      :7 <q -
-=F                     CI CL -w
                                      .             The sil!rs of an ear of each

plant received pollen from the   mc1
                  al   tester stock that carries state 5718.
       +w*

The Q,-ues of kernels on the resulting ear are entered in A of table 4 .

There ratios indicate the presence of  Spm in e?ch plant',  50th plants

were used as pollen parents in crosses with plants that were homoz:.Tgous for

state 571SrA-1 but in which no Spm was present.  The types of kernels

on the ears resulting from these crosses are entered in B of table 4 .

Again, a 1 : 1 ratio of variegated (Spm) to non-variegated (no Spm) kc:rnels

appesxx4 on these ears, indicating the presence of 1 Spm in each of' the two

pollen Parents,  These same two plani;s were also used as !lollen parents in

crosses with slants that were   mm1
                    a1  Sh2/ al sh2  in constitution and had no

                             31
Spm.  The state of alm'l     tAP-
               in these plants was that w,ich gives no anthocyani
                                            A

pigmentation in the kernel and plant in the absence of Spm (state 5720, -

figure 1) but M many mutations to the lower alleles of Al in its prescnc


30

Othe r ea.;?s of these same plants received pollen from slants 'Cat were

homoz-::-gous for al and sh2 but i-lad no Spm,  The tTTpes of kc,:nels a2pcarfng

on the ears resulting from e::.ch of ti-xse two types of cross 83-8 ente:,ed in

C of table 4 .  Again, it is evident that plants 6861-l and -7 w.ch have

one 3pm element th::t is canable of acting on this state of  a m-l
                                  _.                  1.  The


resul-ts obtained from the described tests a-,'8 t:.!o ne to be exnected if the

Spm element in giants 6861-l and -7 is c??3able of acting on different
-.-

States of al m-1
                   .  Tne same type of test as that ,ju>;t described was

conducted with al &z/al sh2 plants having more than one Spm element and

the ratio of kernel types on the test cross ea;>s was that expected if each

of the Spin elements present in the a 1 sh2/a1 Sh2 plant was capable of

acting on each of tie states of al m-l
                                                 .

Still oti?.er ty.es of test were conduct-,d to determine the cavacity of a

Spm element to act on different states of a mW1.
                                     1     One of them utilized the

pollen of a plant that was al m-1 sh /a sh2
                       21    in which a single Spm element

was present at a knobm 10~~~ "Aon in the chromosome comlexent.   1.$..en sue? a

plant was used as a pollen na:r:ent in crosses to plants that were  a 1 m-1 Sh2/

al sh2 and having no Spm but among w1;ich different states of  a m-l
                                                        1      we--e


repxsented, the tCL- es of kernels on the resulting ears clearly indicated thf

capacity of the Spm element in WC mclo pa::ent to act not on!-y up:.n the statt


31

of tine a m-l
       1   locus delivered by the z;nale parent, but also upon the state of

the aim-1 locus delivered b--- t2le female Parent.   Again, when -slants

ea. rying Spm that were a 1 m"1 Sh,/ a3 sh2, among which di;'f'er?ent states of

al m-l were represenhed, were used as udstillate narents in crosses with a

plant that w:'xs homoz;:gous for one state of a m-1 and also for
                                        1                sh 2 but in

Which no Spmwas present, activation of the   m-l
                                                     a 1   state -elivezYed by the

G&e pa:>ent  by the Spm e?.e:,::cnt delivered by tne female __ ns.:lent was indicated

ZJIlZTAll tests of this type,

Detai].ed coinsidepat<on of tile various types of test ment?cned above

c.?.nnot be given here,   ;-rOW,sVC?Z? , all of them cle:?r>l:~ es-kab'ished t;Zle

s_imi.larit:; of t27.e Spm element darried in t-lc many di:X'e-.ent tested plants,

and rc,@rdless of rits number or its location in the  ch ,ro:;?o so;-?e coy123 e-:-+.ent

of a given plant,  fi3hey also establisjled the a%ilitT- of thjZ Spm ele--lent

to act upon any of the selected states of a1 m-l and they indicated that

control of t:$-ne of gene action in the absence of Spm and control 01:' the

     as well as its ty-oe      in &
time and fre:uency of occurrence of muta

sta-Ye of a m-1
        1'