1.

Ze

36

9°

10.

ll.

ea

MODE Ci OP[RATICN OF A S¥SULM OF CONYROLLING Locusts Th MAT

Introduction

. m=)
Crigin of a,

m1

Outline of motte of operation of the Spina, system

States of a

Detection of the Spm element and its mode of operation
Responses of diffe ent states of a, mt to the same Spm element
Transpositicn of Spm

Stability of Mutants

types of Spm elements

Modifier efement in the Spm system

Conclusions
 

Mode 6f 6peration of a System of Controlling “lements

in liaize,.

General descriptions of controlling elements in maivse anc of their /
modes of operation have been given in a number of reports annearing in
recent yesrs (for literature citations, see MNeClintock, 1956b). It is
the purpcese of t is report to consider some of the experimental methods
that nave been ermloyed to discover the mode of operation of the system
of controlling elements which has been referred to hn otier publications

as the Spm (Suppressor-mutator)-a, "1

system, Controlling elements may be-
defined as those unit components, carried in the chromosomes, that serve

to control gene action both with regard to type and degree and to the

. . i
uw

tissues or parts of a tissue térewe this will OCCUr, The different
controlling elements are recognized by means of their distinctive modes of
control of gene action, regardless of the primary type of action of the

gene substance itself, They exnibit Mendelian inheritance patte-ns and
their locations in the chromosome complement may be determined by use of
ordinary genetic techniques, lowever, they may undergo cnange in location
witnin the chromcsome complenent, appearing at new locations and disappearin
from previously determined locations, without losing their distinctive

properties in the vrocess, This process has been termed transposition and
the several m-tnods thet have been used to detect such transposition were
presented previously (McClintock, 1956b).

For many years, efforts of the author were concentrated on analysis
of the system com-:osed of the two controlling elements designated Ac

7

(Activator) and Ds (Dissociation), This system wes chosen for extended

w w&
studies because both elements @&-teis—-seseem could be identified readily ane
regardless of the location in tne chromosome comolement that each might
OCCUDYe Thus, change in location of these elements, and the effects

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

detected and subsequently analvsed, Altogether, the operation of this

system at eight Bh6wn gene loci has been examined, Also, two or more
independent insertions of one of these elements at four of these eight gene

loci were detected and the consequencd of this examined in each case, it

was concluded from these studies that this system should be able to operate -
at any gene locus provided tiat the effects of i+s-operation at a particular
sees locus is not letnal, Most important, however, is the realization that
the mode of control of gene action may be predicted in advance ,for it will
Aotoyentd My T
follow the rules that eee see the operation of this system, Knowledge
mn

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

experiments aimed at revealing the mode of operation of other control svstem

his applics particularly to methods for identifying controlling elements
and for detecting their transpositions, It has also provided a model for
recognizing and subsequently appraising the chanres in state of the affcetec
gene locus, Such changes 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 cycle in their early development (McClintock, 1951).
‘At the time tuese variegates appeared, it was realized thet it would not be
possible to examine cacn of them. Therefore, only a few amone the many
that appeared were selectdd for subsequent study. The Ac-Ds system was
discovered among one of tiese selected cases; and as the mode of operation -
of t.is system became apparent, attention was focused mainly upon it.

Stugy of
the other selected cascs either wase discontinued or it was sharply curtaihe
» (3 Wot 2 » s
until adequate time could be found for @ detailed examination, The system
responsible for control of gene action in one of these latter cases is now
the

sufficiently unde»steod to allow conclusicns to be draw regarding/tynes of

controlling elements that are involved in it and their modes of operation,

 

at both a,™1

This is the system 1

mu . . : a ; : ;
and a, » mentioned in previous reports. In this report, attention will

sa m1
be given mani y to ay °
 

Cat pe Ti
‘rigin of ay i
a ss Ties spe . :
The history of origin of ay i from a modification tnat occured at
a standard A, locus is as important for an appreciaticn of the controlling
elements involved in this system and their modes of opcvaticon as were the

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

carrying cultures for an appreciation of the presence and mode of operation.

m=

of the elements of that system, The modified A, locus, designated a 3

was the tiird recognized case of change in gene action in a sequence which
commenced with an alteration ata previously unidentified gene locus concern

with chlorophyll producticn, The plants having this first member of the

Fs)

sequence exnibited variegation for chlorophyll pigmentation. this varierat
was one or toose ceteimaisdy selected among the many that appcared in the
Original cultures, as described above, In the early stages of examination
of tois variegate, a number of plants in one culture were self-pollinated,.
On the ear produced by one of these plants, some kernels appeared that
exnibited variegation for anthocyanin pigmentation, Spots of deep
pigmentation appeared in a colorless background, The plants derived from
them also exhibited variegation for anthocyanin pigmentation, Subsecuent
tests of these vlants and their progeny indicated that an alteration had
eccurred at the standard Ao locus in one chromoscme 5 of the parent vlant,
This altered locus wis given the designation aml, Tests were then

m1
undertaken to examine the changes in expression of gene action at ap and te
 

»

detormine ths factors i-veolved in control of tris, In the course of

touis study, 2 number of plant s in a culture in weich the system responsible |
for control of gene action at ae’ was vresent, were used as pistillate pare

in crosses with plants that were homozygous for tne stand: rd recessive, ay»
in chromosove 3, and for the standard Aj locus in chrovosome 5, On one of

the ears this cross produced, a single kernel was found that ex’ bited

igment
spots of antnocyanin/in a non-pigmented background. A plant was frown

from tois kernel and t is slant, in turn, exhibited variegation for antno-

wan oe

cyanin pigmentation, As~epocted, fests crosses | i tI sm dq:

 

egret bats indicated the presence in it of op det AL locus, and.

+, Ula Loewe m1 AOWL cove, chang

was thereupon designated a) . It was evident that

. wll
Aad CLvuvilo, ot the standard Ay locus in one chrom some 3 in the pals
Run Teor plore peerghuud

parent plant, ae late in development of one of tre ears GF t is pliant, for
A

 

only one kernel on tis ear exhibited altered ay action, Studies aimed at
determining the components of the system responsible for control of gene

action at both a,™t

1 and ase were contined but initially only on a

limited scale, Only recently has time been found to examine tis more
completely. It is now knogh that the same system of control of gene actior
operates at both a mt and ast Very likely this same system also
opercted to control action of tne gene thet was associated with crbkoropryll
production,--tne initial variegate in the seruence, This nostuhate is
based cs on the patterns of varioration that were ex .ibited and upon

vance behavior for study of this case was discontinued some years ago-
and therefore direct tests to determine identity of control systems cannot

now be made, No Cul Lng ' tho anade) Oporaligy of hp Oy 0 Set
* caipemieernee . — 9 —

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

 

 

 

 

Me» | m=

action at a, and Bo » 2 large number of different types of tests were

conducted, The résults obtained from each are consistent witn one another

on the basis of the eventually determined modes of operation of the componen
of tnis system, In order to com ly with space requirements, only a selecte
set of tests ee be gien here, These are chosen in order to illustrate t
Salient features of toe mode of operation of this svsten and they will be -
: as ; : Mme) 4 4 eas m=]

confined to studics conducted with ay . Those conducted with a.,

a
will be given in a separate report.

From examination of the Ac-Ds system, it was learned that inse=tion

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

tho

wy
that brouggt this “gene under the control of the Ds-Ab system, In many
A L
wusiol

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

detection of the Potues, of Ds at the locus could be mede shortly after
A

 

Wor dn rrntod tha - recluse
it eecurred, Subsequent chat the locus resultfs from the effects Ac
exerts on the Ds element, The consequence of this is either renaval of Ds

from the locus of the gene or a modification at the locus, induced by Ds,

that effects a chnanre in its organization,--a chanre in state of the locus,

‘
Both types of events can give rise to recognizable chanrses in action of the

oe re m=1 . :
gene substance, With regard to a,” ~, insertion of a particular

*

ecntrolling element at the standard Asclocus is considered to have occurred
ang to have been responsible for the initial chanse in gene action, Like
Ds, it is this element that directly controls the tyne of gene action that

smo eoet gine

will occur at the Ay locus and the times of change in tris aetsen that may
A

A
occur subsequently. It appears to be the sane element thet is present at
m1 * m= 1 m=
ao . This conclusion is based on the response bBth ef ay and a.

at

to the presence of an ind&@vendently located element designated Suppressor-
Wenn Spam ve pat wo dustin © das wy Mock ean

mutator (Spm). All action of the gene se ee tb ethan end -es

Rovul or plo -2yeopt ia Utd Ulbs,

is_suppressed when Spm is omesont in the—nuctet ofr Srartb—excert—=n

 

 
 

 
 

Oo the element r siding at eiticr

m1 Ludivol te wtdlcatoy aye lo

a,™t or ao that allowg the gene substance to become active ebteee—in-
Geb wy, be ozuens Cnty
gs 2 in Ue descondanty, fhe particular type of expressicn of the-
7 A

gene that these modifications effect is thereaftor maintained eithor in the

wes we tonslglin thon, Uat

the Spm

 

presence or the absence of the Spm element,

m= 1
. , , , m=1
element is cormlementary to the elerent located at ay and a5 .

It serves to activate it and as a consequence of t is, stable mutations

-are produced, In t is resvect, it resombles Ac in the Ds-Ac system.

tne genic meyostance

However,/at bota ay and ap ot

may be active to some extent when Spm
 

is not present in the nucleus, whe it is removed, either through meiotic

Ssegreraticn or by means of somatic transposition, anthocyanin pigment may

h mo
- ert ot Apre Med Mea ]
appear in the kernels and plants nevine either a or a@ and Lts
i iP a,

y
oa: yaa &

2

ee

1 2

Fas

distribution is uniform, In other words, there is no variecation, The

tyme and intensity of pigmentation is an exp ession of the particular state

jw me wea DULY, oR Adon
a . m= = . . ea 4
of ettaexr the ay 1 or a. locus tnat is present, these states and
“A

their origins will be considered snortly. When Spm is returned to the
: . . m-1 m=-1 .
nuclei by ancropriate Crosses, wl fene action at ay and a, is suppres
en 5 c
ed except in tiose cells where mucation-producine events occur, thus,

Spm serves not only to activate the complementary elenent residing at

m= aad

ay and a5 and the eby conditionsag stable mutations ct these two
loci, but it also must act upon this element in yet another manner for its
presence results in suppression of lmown potentials for rene action, excent
in t ose cells whore mutation=producing events occur, these two S-O-Otiiapy

different aspeets of the mode of control of the Spm element on the elenent

m~1 m=1 - dual :
residing at ay and Bo APLesponsidle for its besee designa cody ,

eo

Suppressoremutator, nowever, this seemingly dual action may be the

€
expressicn of only one process rather than of two unrelated processes, as wil
be indicated later,

Uo sive, ot Que

All examinations of the effect of a controlling element at a gene ptwn
must be conducted witr the affected locus, Since, in addition to stable

mutations, the controlling clement may also initiate structural or organi-+
3 gz

zational modifications of the locus thot alter its subse uent expression,-
that is, chanre the state of the locus,--it is necessary to consider

States of the affected Locus and their origins before detailed results of
—

An Mea]
experiments ean—be presented, Wita ay » 4 changed state is readily

recognized by the appearance of an individual kernel or plant that
exhibits an altered response of the locus to the oresence and absence of
Spm, Fomexeuipie, When the controlling element first oneted the locus of
Ais it effected a particular tyve of modification in $*9 structure ov

organization ,of the locus, In the absence of Spm, some gene action
occurred, Kernels were lightly but uniformly pigmented as were the

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

action was suppressed except in some cells where mutation-producing

m=1
events occurred at thelocus of ay that allowed the gene substance to

aud

be active in the descendants of t-is cell. Eeeh such event did not

OE an the same degree or type of gene action but many of them restored

ne full or near full Ay type activity. Many of these events occurred

yng . bo
relatively early in plant and kernel development, -Umidke Ac, vwarli¢ats
A. . ,

Tron,

* a = wf. ay . 7 .
in dose of opm hat.*no effect on altering the time of cecurrence. ofthese
)

Ou wa Tr sdageet WOU Sey nibfor» Yon that 0) he.
LO

alan ocevied)

CVEGRES« Ammer of \rutation-pnoducing events oeewmpmed-in germinal cells

and this made it possible to examine the nature of the mutation in the

ues wittel T Love

next generation, Another tyroe of change af ay also occurred ina
A

few of tne serminal cells. These resulted in altered expressions of the
locus both in the presence and absence of Spm, Some of tnem were
detected initially in individual kernels in the progeny of the original
—_- Me]

“ay 0si«@eaarpyiing, plant, When this plant was crossed to vlants trat were

-homozygous for the standard recessive, ass wnich does not regs ond to Spm

, .. meL
but responds, instead to Dt, the maguxkkyxwRxkermekx - EEEXEXXEXAN a,
Humeikkindxayxxxxkeems locus in the majority of kernels that received it

oe «os ; oe m~J .
was unmodified. Among the kernels receiving the unmodified a locus anc

1

also Spm, the variegation pattern was much tre same, Thers we e many

pigmented areas,

     
 

Sy oa Oe Le eS ctx R me]

large 3 isumm indicating early occurring mutation-producing
OnB.oe Aove Rania pacaniTH Ota a
eventS. any of these exhibited the full A
A A
A Onter

However, an occasional kernel appeared that exhibited a quite different

1 type of vigmentation,

r
pattern of pigmented arsas, Pwo kernels) were found bnong several |

‘
thousand that had only small pigmented areas. ree were uniformly .

Nove oldoune Loup dnt OMAR us awed, ann Secu ;

distributed pad the intensity of pigment in quem was either quite, light

nue “the otha de ally thp puceutay Ott: ibe, db bee ili

or very dark, Anotoar kernel appeared that had a numbe? of large

b+

ovigmented arsas as well as some small arens but the intensity of the
11

 

pigment in all of them was

b
qe ,

It ranged from very faint in some arcxzs to excrecciiin | i in ot erse

excepticnal
Plants were gpown from the three described/k>rnels, Tests conducted

with them and their progeny indicated tirat the pettern of variegation

ar : 1 ; “ m=
exhibited in the presence of Spm was, heritable, The altered ay locus,--

the changed svite of tne locus,--in each case responded in its own

particular way to the presence of Spm and also to its absence, In the

mo 1 ose=aee in the first described koarnel

absence of Spm, the state of ay
. ‘ m . .

produces tumpxmkemenk deeply pigmented kernels and clants. +e t in the

second described kernel gives rise to very lightly pismented kernels but
4 * my a a m=] a 2 iy

ratner darkly pigmented plants, The state of 5 present in the tvird

described kernel produces no pigment in either plant or kernel in the

bsen e § < ayy + m1 , Whe . a:

absence of Spm, subsequently, other states of a, heve=bean isolated,
ach is distinguishable from the other by the tyoes of mutetion and the

Jind repder wild Spam io \rewet
time and frequency of their cecurrence im the=presemrencéutipm as well as
A

Wades, L2.r TMA Zee

ope’ y
the tyne of expression of the gene suvstance thet ss in the absence of
Sr ath Comets, q2ue Oebion iy OOF proarcad wa The Prteornp {Spa nergy wa Tapp ex0la (ord Heber Le eendasdt) aula”

Spies No relationship was noted anong the different states betwoen the

out {rb uth

control of time o* occur ence of mutation in the presence of Spm and that
A

of tyre of gene action thet occurs in its absence, ‘igure 1 illustrates

the distinctiveness of several of the states of amy due ahle flere
U
He range dpruutd Bhar ii eriysercet Oey, Thy pursue, aettd Vokes of a,
Le

m-1 , . . :
The interréty of a state of ay is maintained in heterozygous

plants and t is apolics to plants touat carry a different state in each of
their chromesonmes 3.6 In such plants, each state resp:nds to Spm in its

own predictable way anc the variegation patterns each vroduces will be

way . — 7 ml,
expressed in the plant or kernel tissues. “igure 2 illustrates this. tne
kernels in the phosographs carry the state shown in — of figure 1 in one
chromosome 3 and the state shown in « of figure 1 in the homologue, Lhe -

dna The Aiutaliqn ty po

pattern of mutation produced by each state OY readily recognized in these
kernels, In the plants, normal segregation of the two states occurs at
meiosis and each may be recovered in the expected proportion from the
gametes that these plants produce,

states of at are maintained unchansed in the absence of Spm, In
its presence, however, new states may arise and the frequency of occurronce-
of this in germinal cells is related to the time of occurrence of mutation-
producing events that a particulsr state exnibits, If the staze vroduces
some early occurring mutations, then new statcs of By may acpear in the
germ cells, and the frequency of tis is proportional to the freaveney of
occurrence of tnese carly mutations. If, on the other hand, the state is

one in wich mutaticns occur only late in development of the plant om
~ a

kepned, tnen few or no altered states may be recovered in the gametes of
13
these olants. The mutation-producins events at ay » regardless of state,
give prise to stability of expression of tre locus. tne particular teve of
gene action the mutational event produceg continues to be expressed in

subsecuent generations both in the presence and in the absence of Spm.
This suggests that the change resnonsible for these mutations may ewe
removed the controlling element from the locus or it may ewe resulted in
its inactivation with regard to Spm, Since the chanrses in state arise
at tre very same developmental p riod as the mutation-inducing events occur,
it is conelvded teat they represent a modification at the locus induced by
tne controlling element residing there dhat did not result either in its
wh
removal or inactivation, In other words, the element responded to the
presence of Spm at the appointed time and in the appointed cell¢ but the
cons quence of this was not the usual one,-mifef, the mutation-producing
event. Instead, the responding element itself was eith x modified or it
induced some reorganizaticn at the locus that modified its capacity to

+ four d oremmune 7p “mutation On Welhao
respond to Spm with regard to vime Witten tits rdeleecocun, the trpes of

dink alno wid Aare
mutations it can induce  e=tedet—as the trpe of gene action that can occur

in tne absence of Spm, Te is as evident in this case as it is with Ds

that a chanre in state is one of the consequences of the response of the
of the controlling element residing at a gene locus to the independently

located clement of the sysvtem,--Spm in this case and Ac in the case of Ds,

Qe toe Dery faad Sun Lhe dvd) Uo smydt

aT o ates pomeeny

  

 

 

 

 

 

Pk z per td

In the early examination of ay » no evidence of the presence of the
A

Y=.

“ . m .
Spm element wee detected, This was because the original a, carrying

plant had a number of Spm elements located at various »nositions in the

OL Oyrtorute Warde all
fhromosonie complenent. All, of tne gametes it produced nad Spm elements
2n- them, Since the dose of Spm has no anpreciable e*veect on the pattern
Ma

of mutation produced by ay » differences in number of Spm elements ina

kernel or plant is not made directly evident by tris means as it is with Ac,

Basu by

ma
It was only after several generations of crossing of ay 4 Pees tO ay

le ut wean To

tester stocks wnich did not have Spm, thet e,ratos of varierated to

uniformly pale

léeht colored kernels appesred on the test cross ears, These ratios

re . MeL . .
indicaved the presence in the a, carrying plants of an inderendently

located element that is associated with control of at expression,

They also indicated that the number and the location of this element was not

the same in all tested nlants,. In the meantime, several different states

mel . _: : og . .
of ay had been isolated on tre basis of the altered variegation patterns -

that appeared in individual kernels on several of tne Gkest testeross

ears, as described cxcrlier, In successive geverations of crossing of
m-1 , ;
plants carrying these different svates of a to the a, tester stocks,

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

began to anne r, It was then assumed that the uniformly vignented

m= ; .
(non-variegated) kernels and plents carried a5 but not tne indevendently
located elenent, On the oteer hand, this element was assumed to be

present in tnose kernels and plants that snowed pigmented areas in a non-
pigmented backegrcund, On this interpretation, tie indenendently located

element was exerting a suppressor-mutator tyve of control of gene action

at aw, Since the dose of t is element obviously did not affect the

; . 7 : : m=1
pattern of mutational events, cortrol of tois must reside at the a

locus itself, the type de -end¢ing uron gne state of the locus, It was

evicent that the phenot pes of the nonvarieratei ke:nels anc plants also

m= 1
reflected tre state of the a5 locus that was present in trem,
The hypothesis stated above was subject to test. le it were correct,

evidence in supvort of the following four statements s>culd be obtained;
(1) All variegated kernels and vlants carry at least one Spm element.
(2) No Spm element is present in the non-variegated class of kernels
. | mud outel . -
and whants.( Germinal mutations, deseribed earlier, are excluded fron this

class.)

(3) The ay™1l locus in tee non-variegated kerneks and plants is capable -
16

nucleus,

(.) The type of response b6 Spm and the prenot»pes ppoduced in its

. . 4 Lo, , Mee]
absence is a function of the state of the a Locus.

A large body of evidence in support of these statements is now availavle

and it has been obtained by various types of test, only a few of wich
; y3 ’

*

“need be outlined here,
In order to facilitate identification of the pr=sence or absence of

Spm in a particular plant, so-callscd Spm tester stocks were developed,
= 7 § :

me=1
These stofck nave either one or the otner of the two states of a, snown
cate gah)

in = and - of figure 1) dul Prese states were selected for the following
reasons, In the first place, wnen Spm is present, very few germinal

mutaticns or cnanges in state occur. Therefore, nearly all of the
cer ve ck

mM
gametes produced by plants carrving these states of ay and also Spm
A

, as m1 ae aa .

nave an unmodified a5 locus in them, eedondly, the pattern of variega-

tion each produces in the presence of Spm is distinctive and non-obseuring.
: etV2B-V)

Loirdly, in the absence of Spm, one of these states,- figure 1, gives rise

a

to darkly pigmented kernels and tis is a useful character in some tests,

The tester stocks were made homozygous for one or the other of these two

of resvonding to Spm if this element is subsequently intrceduced into a
tn he . , . . .
statese 4ney were also made homozygous for eithsr Sho or sho, located
L7

Yi .
very close of a7 ( about one-quarter of a percent crossing over occurs

between them).

An example of one serics of tests will illustrate some of the metiods

employed to detcrmine Spm constitutions of indi¥idual plants, The di lks of

a : : M~ :
two ears of a variegated nlant carrying a and Sh in one chromosome 3

1 2

and a, and sh, in the homologue and also Y¥ in one chromosome 6 and y in its

homologue, recieved pollen from a «lant that was homozygous for a, sh, and
‘ eessah-vy
Mo J "| . < t 5 7 > ,
Ye The state of ay in tne pistillate plant was that snown in ~ of
nn

figure 1. From tais cross, the two ears vroduced a total of 7lL5 kernels.
There were 181 Sho kernels in wnich the aleurone layor was unifornly and

rather darkly pigmented; 69 of these were ¥ and 112 were y. the aleurone
layer in anothsr 1388 Sh, kernels exhibited a number of spots of the full A,

tyoe pigment in a colorless background and 117 of these weve ¥ and Yl were

Ve The aleurone layer in the remaining Sh. kernels was comeletely colorles

2
and the stascn in its endosperm was y. Among the 375 sh. kernels on tnese-

two cars, the aleurone lay-r in 373 of them was totally colorless; 186 of
ae o mm s . 4 1, s
these kernels were Y¥ and 107 were y. “he remaining 2 sno kernels ex ibited

spots of full Ay type vigment in a colorless background, The pnenotzpe of

the starch in one of them was Y¥ and that in the otner was y. Since
 

1é

aml as colsely linked to Sho, neariy all of tne Sh, class of kernels on

1

Te a — a 44
these two ears ghould carry an ay 1 locus and nsarly all of the sh,

kernels should be homozygous for the standard recessive, Bs The close

Linkage of a,

to Sho is obvious for only 1 Sh, kernel in the total of
370 was completely colorless and only 2 sho kernels in a total of 375 had
pigment in the aleurone layer,

On the basis of the interpretation given above, the uniformly
pigmented kernels should have no Spm in them whereas those exnibiting spots—
of the full A, type pigment in a colorless background should have tis
element. From the ratio of tnese two classes among the sho kernels
(181 to 187) it could be concluded that the variegated pistillate parent
plant had one Spm. The ratio of Y¥ to y in cach of these two classes
indicated that this Spm element was carried in the ¥© bearing chromosome)
ti was then necessary to determine wnether or net these conclusions were
valid, For t is purpose, 10h plants were grown from various tyves of
kernels on these two ears and tests were conducted with them, The
phenotyvoes of the selected kernels were as follows: 11 uniformly pigmented
Sho Y, 13 uniformly pigmented, Shp y ‘17 variegated Sh, Y, 8 variegated
Sho y, 1 variegated shy Y, 30 colorless shop Y and 2h. colorless sho ye

All 2h. plants derived from the uniformly vigmented kernels were themsleves
Ye
sO

unifornly vigmented,. All ef=the 26 plants derived from the variegated
kernels showed small streaks of the Ay typecbigment in a non-pigmented
background. And, as expected, all 5) plants derived from the colorless
shy class of kernels lacked anthocyanin pigment, Hach »lant was tnen
tested for presence or absence of Spm by crossing it with a plant in an
Spm tester stock,

To illustrate how the tester stocks can serve to reveal the oresence
er absence of Spm, those tests conducted with the 5l, plants derived from
the colorless, sho class of kernels will be considered first, this is a
completely objective test since the presence or absence,in any one of tnem

fhe silks of
of Spm could not be assumed on the basis of »henotypic expressions, / 6ne

1

or more ears of cac oF these plants received pollen from plants that were

eu) ema)
horicargous for either state - or - of figure 1, for Sh and for y; these
pollen parents weve uniformly pigmented indicating the abcence of Spm in
them according to the stated hypothesis, If the plant being tested has no
Spm, then all of the kernels on the resulting ear will be uniformly colored,
Lp, nowever, tne plant being tested carries Spm, then it should be present

: * . . : : 1 m=
in some of its gametes, Following introducintion of the a 1 locus from

1

the male parent, the presence of Spm in those kernels that received it from

the female waront should be revealed by the anvn-argnee in them of small,
20

Tha unt Sam suet
deeply vigmented spots in a colorless background due to activation-ef-—the

att locus be-tire—Spm—etement, In those kernels that did not receive
Spm, the aleurone layer should be uniforrily pigmented, Among the

30 plants derived from the colorless, sho, Y class of kernels, 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
evicent (A, table 2) but in the 2 remaining plants, no linkace of Spm with

Linkage of
¥ was evident (B, table 2), (The reason for the absence of/Spm iwithe Y

Latin,
in these 2 plants will be considered im ton. It need only be
mentioned here that this is not unexpected, ) Among the 2h vlants derived
from the colorless, sho, y class of kernels, 6 had a single Spm element

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

Hach of the 2h plants derived from the uniformly pigmented k: rnels

 

was tesved for presence or absence of Spm

in the described menner and in none of them was Spm found to be present,

All of tne plants derived from the variegated kornels stowed stroaks of the

A, t:we pigment in a non-pigmented background and the test crosses indicaced
1 2 PLE

the oresence of Spm in cach of them, In 16 of the 17 plants derived

from the variegated Sho Y class of kernels, one Spm element was present and

& 2 3 t

in 15 of these plants, it was linked with Y (D, table 2). In one ef—sRese
el

plant, however, ho evidence of linkage of the single Spm element with Y

was noted (E, table 2). In the remaining plent tn this’ group, 2 Spm

elenents wero present, neither of which was linked with ¥ (Fy table 2),

More than one test cross ear wis obtained from 11 of these 17 plants and

the number and locetion of the Spm element was the same in the cells

producing all sars except for one plant. In tnis plant, one Spm element

Was present in the cells producing the main ear and it was linked with ¥

(G1, table 2). In the cells that produced the tiller ear, however, a

sincle Spm element was present but it showed no Binkage with Y (G-2, table <
(xin all 8 plants derived from the variegated sh, y class of kernels, one Spm.

was present (H, table 2). One Spm element was also p-esent in the plant

derived from the variegated shy Y kernel. “his plant was used as a nollen

\)

parent in crosses with plants having different constitutions: homozygous

Lso for:
ana/y (01
for a, and sho/and having no Spm, homozygous for aeveral diiferent states of

A
Tee 1 * “ (3)
ay but asving no Spm, and be pdbeutertoeteeeeeescre
A

 
 

=p Te]

Ca ewewe a) Sh/a, sho; ¥/3s no Spm, - In this third group,

. . m= ye
plants with different states of aj were represented, All tests indicate
' s
We
in one sho corrying chromosome
A

4 : Tee
the presence in thepcllen parent of a4 i

or lewind uy a
’ 3
and of _oP™, in the Y carrying chromoso: "ey Orel rect hin Spa DEMME

nate ‘ink vil, he Meise hdfoup tales A Ql
 

The tests described above were conducted with the progeny of a ai ngle
plant in a culture. There were 19 variegated plants in tnis culture,
Hach was derived from a variegated kernel that appsared on an ear of a
varieg:ted plant that was amt Sho/ a, Sho, V/y, wx/wx (chromosome 9),
pr/pr (chromosome 5) in constitution when vollen of a plant homozygous for
ay» sho, y, Pr, and Wx and having no Spm had been vlaced on tne silks of
this ear, All Yernels appearing on t is ear vere Sho and the distribution
of phenotypes among them were as follows: 28 uniformly dark pale Y, 7
uniformly dark pale y, 55 variegated (spots of deep pigmentation in a
colorless background) Y, 86 variegated y, 75 colorless Y, and 103 colorless
Ve Among the kernels showing anthocyanin pigment, the ratio of uniformly
dark pale colored kernels to variegated kernels indicated tne vresence of
at leasce 2 and vossibly 3 Spm elements in the pistillate pavent nlant and
ons of these anpeared to be linked with Y, The silks of ears of 9 plants
derived from the variegated ¥ class of kernels on t.is ear and of 10 vlants
derived from the variegated y class received pollen from vlants that were

honozysous for ass sho; and y3 and hod no Spm, The ratio of kernel tvpes

if .
Oona ak ee “Hat ‘| Keeraty
appearing on the resulting ears produced b> sach of these 9 plants is
A
entered in table 3, In this table, the 9 plants are rlaced in four groups,

A to D, according to the assumed constition of Spm in esch that the ratio of
_
“A Ut

 

23

kernel types suggested, The 6 plants in A of t:is table vere assumed to
have a single Spm element located in the Y bearing chromosome, Progeny
from l. of these 6 plants were grown and again tested for Spm, Those
derived from plant 6629A-1, line 1 of A of table 3, were considered separate

or
above, The total number of progeny,plants in this group that were tested

i a A
prada ote epg SUM | jh Wg Oryre
and theiw omteins are entered in the last line of A of table 3, These
A

tests verified the presence of Spm in the varkegated kernels and rlants and
2ts absence in the uniformly pigmented kernels and plants. ‘They also
verified thes assumed Spm constitution and location in the pistillate

Yowlter
parent oleant. Only those tests conducted with the 116 plants derived

A
“pom the cbkbirless Sho class of kernels will be summarized nore, Among

the 56 vlanits derived from the aj sho Y class of kernels, 32 carried Som

and alt ned no Spm, In 30 of the 32 plants having Spm, linkage of it with

Y was expressed, I, table 2. In two PTANGE yp Ee Som elethhent wes not appear
- , \
vhosts Usbe TT \
linked with Y, ae menticned earlier, (B, table 3)\, Among the 60 plants

Single Spm element

9

devived from the ay sh, y class of kernels, 17 hed

(J, tacle 2) and 43 had no Spm

Whotlee , Ruse 1 DI voc
: location in the 2

    

In order to verify the

plants entered in B of table 2, tests of some of the progeny of both of them

wore conducted, An ear of one of these plants had been self-pollineted
2h

fal)

a eae ’ Me
and another ear of this olant hed been used in tne cross with an By 1 Sh,
¥Y, no Spm tester vlant, Kee Progeny from botn of these ears were arain
tested for Spm constitution and location, The silks of ears of 8 vlants

dsrived from ay sho kernels in the ¥ class on the self-pollinated ear

Mel gh

5 Ys no Spm tester plants, From the

D.orxe

Q
oh
O

received vollen from the a,
kernel types on the resulting ears it could be concluded that +we a
plants wore Y/Y in constitution and that one of them had no Spm whereas the
other had 1 Spm (199 pale colored kernels : 169 variegated kernels on the
test cross ear), the remaining 6 plants were Y/y. One Spm was present
in l of them but it was not linked with Y (K, table 2), An Spm element
homologous
app: ared to be carried at allelic positions in a p&ir of/chromosones in the
renaioing 2 plants (Ly table 2), seventeen vlants derived from the

Ae

variegated Sho Y class of kernelsgon testcross ear of touis same parent
. a A
. ( dyn Bk yO ze
plant weve crossed by plants in the Spm tester stocks. In 16 of these
17 plants, one Spm was present and on none of the esrs produced by 15 of
them was their any evidence of linkage of Spm with ¥ (M, table 2),
iowever, the ratio of kernel types appearing on the test cross ear of one

of them suggested such linkage (N, table 2), The remaiving plant had

2 Spm elements, neither of wich was linked with Y (O, table 2),
25
Tests of Spm constitution in 9 plants derived from the variepated Sho
Y kernels on the ear produced by tne test cross with the other plant
entcred in B of table 2, suggested that the Spm elenent in this plant nad
been carried in the Y bearing chromosome but at a new location that was
farther removed fem ¥, seven of the 9 progeny plants had a single Spm

thoy peodead sp

element and in all of the test cross ears, loose linkage of +6 with Y was

A
expressed (P, table 2), One plant of the 9 had 2 Spm elements (@, tarle 2)
and xa the remaining plant had 3 Spm elements (R, table 2).

Further examoles of the progeny test method of determining Spm
constitution and location will not be given here, It should be mentioned,
however, that such tests were conducted with the indicated progeny of
the plants entered in B, C, and D of table 3, and also with the progeny of

avery, bdel oun)

tne 10 olber plants of this same culture, Also, @ummaer=-e sueht esis

<Weeet cud morue 7Hheur Udse
P." were conducted with the progeny of plants having other states of a, et
A
ats ‘ 2 gtek . : ae ; a m=1 .

or combinations of states, i.e., carrving different states of ay in
a 1 we ob 2

ech chromosome 3, tre tests clearly supvort the l, statements given on
aA

page » and particularly statements (1) and (2). Wore difeet support for -
statements (3) and (lL) was obtained by other tests to be described shortly.
Various different locations of Spm, eitver in different chromoscmes of the

complement or in different locetions within the sane chromosome, were
 

26

discovered in tnese tests and each determined ~osition of it was
subseaucently verified by means of progeny tests, howev r, it is clecr
from the tests so frr described that Spm does not remain at one location
within the chromosome complement but disarrcars from a known location and
appears at a new location and this will

ALrCurroel

section, Before this is desemibed, it is necessary to snow thet the

be considsred in a separate

Spm element, regardless of location, is capable of acting upon anv one of

the states of amt,
27

Resoonses of different states of a, to the same Spm element

a
svidence for statement) (3), page » appeared in all tests in woich
A

re * Tile * 3 dee oye } 1
eitner one of bhe two states of ay 1, presont in the tester stocks, had

been used in crosses with plants carrying Spm, tach state ressonded to
Spm in its charactcristic manner. In order to determine wiether the

Spm ekement present in a particular vlant would be capable of activating

other states of ats several additional types of test were performed,
One of them utilized different ears produced by a single plant, In one
wee acicebeel taph
7 oe _ ~ \
such test, these plants were homozvgous for Bip Spm could be elther
A

present or absenc&’ in any one of them, Pollen from a plant of thea

ou Sua}
tester stock carrving either state = or state - was placed on the silks of

2 4 ryt . Oo acord e -
an ear of one such plant, the silks of gnether car of the same plant

CL.
received pollen from anether plant that was homozygous for a different

a Me . _s sats
state of ay i and in weich no Spm was present, If the pistillate plant

had no Spm, then all kernels on both cars weve uniformly pigmented, and the

intensity of this reflected the state of amt introduced by the pollen

6120
parmt (excluding state ~, figure 1, wiiech gives colorless kernels in the

absence of Spm), If, however, the pistillate parent carried Spm, then

both variegated and non-variegated kernels appeared on beth earg but the
a MnP
«<

phenotypes of the two classes of kernels on each earsreflected the state of
A
28

tu!

me 1 a > S 4
ay introduced by the pollen parent. Again, if the amhtyebiegs element

“showed linkage with a genetic factor ameng-bee-ke-ne}s on one ear, The
Urn We wash RA tected
usuatiy shened thee same linkage on the other ear and an example of tris

A J
is given in S$ of table 2, In tris test, the main ecr of an a,/8,» ¥/y
plant received pollen from on? 8S the Spm tester stocks. The kernel
types on the ear tris cross produced, §-1, table 2, indicated the presence

im the pistillate parent of an Spm element carried in the * chromosome,

The silks of a tiller ear of this same plant received vollen from a plant

ioe ree
“ Wbo oS

that was homozygous for state -, figure 1, and also for y. The pollen
parent was uniformly pigmented indicating the absence of Spm in it, The
kernel types on the ear this cross produced are entered in §-2 or table 2,
The. ratio of variegated to non-variegated kernels in the Y and y classes
was mucn the same on both ears, Thus, it could be coneluded that the Spm
element, carried in the Y chromosome of the vistillate plant, was capable
me]
of activating either state of ay e Pollen from the same collection
tesS was used in the latter cross was also placed on the silks of a plant
homozygous for a, and for y, but in which Spm was known to be absent. All

of the 29) kernels on tht ear this cross produced were uniformly lightly

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

pollen parents
29
Another tyne of test tnat was emplbyed to indicate the response of
different states of at to the same Spm element, utilized tne pollen of
plants that were homozygous for a, and in wich Spm element was present.
The troes of test conducted with two such plants, number 6861-1 and 6861-7,

abe illustrated in table lL. Both of these plants wore homozygous for

 
 

a, and sh, i , —e Om C1 3—BPeSOR >i. pe ;

7 Rekexmkankx The silks of an ear of each

plant received pollen from the a, 7+ tester stock that carries state 5718.

Wekop prowl

the tyoes of kernels on the resulting ear are entered in A of table ho,

Athao To

These ratios indicate the presence of "Spm in exch plant, Both plants

A
were used as pollen parents in crosses with plants that were homozygous for
state 5719A-1 but in which no Spm was present, The types of kernels
on the ears resulting from these crosses are entered in Bof table lt,
Again, al: 1 ratio of variegated (Spm) to non-variegated (no Spm) kernels

appearmd on these ears, indicating the presence of 1 Spm in eacn of tne two

pollen parents, These same two plants were also used as pollen parents in

+ 1. I~
crosses with plants that were a, 1 Sho/ ay sh in constitution and had no
_ m an Ti2\
Spm. the state of ay -l in these plants was that wich gives no antnocyani
A

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

Bd

figure 1) but mixes many mutations to the lower alleles of AL in its prescne
Other ears cf these same plants received pollen from plants that were

na - . ~ m p :
honmogygous for ay and sh., but had no Spm, +he trvpes of kernsls appearing

a

on the ears resulting from esch of these two trpes of cross ave entered in

Cof table h . Again, it is evident that plants 6861-1 and -7 cach have

+ m

one Spm element that is capable of acting on this state of qa ™1, he
1

results obtained from the described tests are tnrose to be expected if the

Spm element in vlants 6861-1 and -7 is canable of acting on different

Me)

states of ay : The same type of test as that juct described was

conducted with az sip/a, sh, plants having more than one Spm element and
the ratio of kernel types on the test cross ears was that expected if each

of the Spm elements present in the ay sh,/a, sh. plant was capable of

; nh one ; m=]
acting on each of tre states of ay °

Still other ty-es of test were conductsd to determine the cavacity of a
Spm element to act on different states of amt, One of them utilized the

pollen of a plant that was amt sho/a, sh, in which a single Spm element

2

was vresent at a known location in the chromosome com-lement, When such a

plant was used as a pollen varent in crosses to plants that were amt Sh/

ay sho and having no Spm but among which different states of amt were

represented, the t.-es of kernels on the resulting ears clearly indicated thi

capacity of the Spm element in tne male parent to act not onivy upon the stat:
31

of the amt locus delivered by the male parent, but also upon the stete of

i

the at locus delivered by the female parent, Again, when »lants

. m=] : es
ca ryving Spm that were ay Sh,/ ay Shoy among which different states of
Mod : 1. . ea.
ay were represented, were used as vistillate parents in crosses with a

1 , TM 7 .
plant that was homozygous for one state of a, 1 and also for sh, but in

waich no Spmwas present, activation of the a state celivered by the

mabe parent by the Spm element delivered by the female parent was indicateé
Imall tests of this tyne,

Detailed consideration of the various troes of test mentioned above
Cannot be given here, ilowever, all of them cleerly established the
Similarity of the Spm element durried in the many diffe vent tested plants,
and regardless of its number or its location in the chromosome complement

of a given plant, They also established the atbility of th#& 5pm elenent

m=1 ; er + 2:
to act upon any of the selected states of ay and they indicated that

control of type of gene action in the absence of Spm and control o* the

as well as its type in the presence of Spm is solely
time and frecuency of occurrence of mutation Amex eeKxR ake a function of the

m=]
stace of ay e