FURTHER STUDIES OF GENE-CONTROL SYSTEMS IN MAIZE
Barbara McClintock

Continued study of the Spm system of
control of gene action in maize has pro-
duced additional evidence of the system’s
versatility. A control system of this type
is capable of regulating the action of
many genes during development. In view
of the large number of genes present in
the nuclei of higher organisms, and the
need for coordination of their action
during development, it is reasonable to
assume that evolutionary processes would
have initiated superregulatory mecha-
nisms to accomplish that end. The Spm
system serves as & model of the mode of
operation of one type of superregulatory
mechanism. Such a system can activate
or inactivate particular genes in some
cells early in development, and activate
or inactivate other genes later in develop-
ment. It can turn on the action of some
genes at the same time that it turns off
the action of others. It can adjust the
level of activity of a particular gene in
different parts of an organism. Some
evidence to support these. statements has
been presented in previous Year Books
and other publications, and additional
evidence will be reported here.

At the time they were first detected,
controlling elements were distinguishable
from genes because, unlike genes, they
could be transposed from one location to
another in the chromosome complement.
Thus the operator element of a regulatory

system may be inserted at the locus of a
gene, whose action is then subject to
control by the system to which the
operator element belongs. Because the
detected controlling elements of a system
may reside at different locations in the
chromosome complement, they have been
likened to episomes in bacteria. This
comparison is justified, since it is known
that the activity of bacterial genes may
be subject to control by episomes. In
maize, however, the operator element of
either the Ac (Activator) or the Spm
(Suppressor-mutator) system may be-
come integrated into a chromosome in
such a way that it no longer undergoes
transposition. In its fixed position it
continues to respond to signals from the
regulator, and its responses are made
evident by changes in action of the gene
with which the integrated operator ele-
ment is associated. At present, there is no
direct evidence for or against episomal
origin of the components of known gene-
control systems in maize. It is conceiv-
able, nevertheless, that some superregu-
latory mechanisms in higher organisms
may have originated through incorpora-
tion and adaptation of the gene-control
components of episomes.

The Ac and Spm gene-control systems
are not considered to play a vital role in
coordinating gene action during develop-
ment of the maize plant, at least as far
GENETICS RESEARCH UNIT

as the known states of the component
elements are concerned. If they did play
such a role, it is doubtful that they could
have been analyzed so readily; for then
the selected mutants of the regulator
elements, which have provided much
evidence about the modes of operation of
the systems, would have so altered coordi-
nation as to affect adversely the growth
of plant or kernel. Adverse effects have
not been noted. Thus, the controlling
elements of the examined systems may
represent foreign, nonessential, episome-
like components that have been inte-
grated into the maize genome; or, on the
other hand, they may be true chromo-
somal components of present-day maize,
whatever their evolutionary origins and
histories may have been. Some evidence
supporting the second consideration will
be reviewed briefly.

My study of gene-control systems in
maize was initiated with an experiment
conducted some years ago, which uti-
lized the chromosome type of breakage-
fusion-bridge cycle. The unexpected re-
sults of this experiment, reported at the
time, should again be emphasized here.
In the self-pollinated progeny of a number
of plants that had commenced develop-
ment with a chromosome pair undergoing
the breakage-fusion-bridge cycle, it was
startling to observe that many unrelated
genes, whose action had previously been
normal with respect to expression in plant
tissues, were now exhibiting marked
differences in activity in different parts
of plants. The patterns of change in the
somatic tissues, regardless of the cellu-
lar function with which a particular modi-
fied gene was concerned (that is, plastid
development, chlorophyll synthesis, an-
thocyanin synthesis, or capacity of the
cell for growth or division), reflected some
mechanism of control of action of these
genes during development.

Studies were then begun to examine
these gene-control mechanisms. Later,
when the components of a control system
were recognized, it was possible to recon-
sider the basic question posed by the
results of the initial experiment: Why

487

were control systems so suddenly revealed
and at so many different gene loci? It was
concluded that regulatory elements, dis-
tinct from the genes, must have been
present in the nuclei of the maize plants
before the breakage-fusion-bridge-cycle
experiment was conducted, and that this
cycle, in some yet unknown manner, had
induced modifications in the pre-existing
elements. The evidence was so compelling
that it was later decided to test the
conclusion. If it was correct, the breakage-
fusion-bridge cycle should be able to
induce a known type of regulatory ele-
ment from some unknown element in a
nucleus whose ancestor nuclei, in their
past history, through many plant gener-
ations, had never given evidence of its
presence. The experiment, reported in
Year Book 50, was successful. There was
no doubt that Dé-type regulation could be
induced, independently, in a number of
different nuclei where the cycle was in
effect. It was also evident that each
induction was the consequence of a single
event involving some component in an
individual cell. After induction, the regu-
latory activity of the induced element was
registered in the descendent cells.
Additional evidence for considering
that the examined types of controlling
elements are derived from components
normally present in maize chromosomes
comes from studies of induction of change
in action of a presumed ‘‘wild-type” gene
by particular alleles of the gene. This
induction effect is termed ‘‘paramuta-
tion” by R. A. Brink in studies of alleles
of the R gene, located in chromosome 10,
and is termed ‘‘conversion” by E. H. Coe
in studies of alleles of the B gene, located
in chromosome 2. With regard to either
R or B, the initially recognized allele that
induced change in action of a presumed
normal allele would have been placed in
the “mutable gene’ category had its
behavior been analyzed in earlier years.
Indeed, genes under the control of the Ac,
Spm, and Dt systems were at first
referred to as mutable genes, since they
exhibited phenotypes to which this term
had been applied in many early genetic
488

investigations. It seems most likely that
induction of change in gene action of
‘““wild-type”’ alleles, and the subsequent
capacity of some of the changed alleles to
induce similar changes, resides in con-
trolling elements normally associated
with the genes.

The resemblance between effects
brought about by the inducing alleles,
described above, and those produced by
elements of known control systems is
further illustrated in studies conducted by
Dr. Brink and his students with the R
alleles. The initially recognized inducing
allele, Rt, gives rise to a distinctive
pattern of pigmented areas in a nonpig-
mented background in the aleurone layer
of the kernel. The frequency of occurrence
of change from inactive to active expres-
sion of the R gene in the cells of the
endosperm during its development may
be determined readily by examining the
numbers of such pigmented areas in the
kernel. There is a dominant modifier that
markedly increases the frequency of
occurrence of these events without alter-
ing the time during development when
they take place. Initially, this modifier
was placed approximately six crossover
units distal to the locus of R*'. Its effect
resembles in detail that of a dominant
modifier element in the Spm system,
reported in Year Book 67, and like that
element it undergoes transposition. Thus,
accumulating evidence obtained in studies
of the R and B alleles now makes it
reasonable to consider that ‘‘paramuta-
tion” and “conversion”’ reflect alterations
induced by and in components of gene-
control systems normally present in
“maize. It would be instructive to deter-
mine, therefore, whether or not the
individual components of these systems
could be identified through transposition
and insertion elsewhere in the chromo-
some complement.

Modified States of a”?

In Year Book 61 the mode of control by
the Spm system of gene action at a"?
was reviewed. As was stated there, the

CARNEGIE INSTITUTION OF WASHINGTON

regulator element, Spm, resides close to
the locus of the Ai gene. When this
element is in an active phase, the A; gene
is active, but during an inactive phase of
the element gene action is inhibited unless
an active Spm element is present else-
where in the chromosome complement.
When an active Spm is present, release of
gene action from the control of the Spm
system occurs in some somatic cells, and
is accompanied by the production of
mutants of the A; locus, the classes of
which were described last year. When the
Spm element associated with the a"?
locus is inactive, and no additional active
Spm is present in the nuclei of plant or
kernel, such mutants are not produced.

In this study of a,"-%, an occasional
kernel on a testcross ear exhibited an
atypical phenotype with respect to ex-
pression of the A; gene. Plants were grown
from some of these kernels, and each was
subjected to testcrosses in order to
explore the cause of the modified gene
expression. It could be determined by
this means that the atypical phenotypes
arose through modification of either the
regulator element, Spm, or the operator
component residing at the A, locus.

Modified states of Spm at the a,"—* locus.
There are several readily recognized types
of modification of the Spm element. One
type changes its phase of activity;
another type alters the time of its trans-
position during development; a third
markedly alters its capacity to effect
responses of the operator element that
result in release of gene action from the
control of the Spm system. The third type
of modification, discovered initially in
studies of a,"-! and a;"—!, was designated
Spm” and was described in Year Book 56.
With a,”—! and the class I states of a2”"1,
the inhibitory effect of Spm’ on gene
action at either of these loci is pronounced,
but its capacity to induce responses of
their operator elements that release the
genes from Spm control is very much
weakened. Few mutants of this type are
produced.

The type of control of Ai gene action
GENETICS RESEARCH UNIT

when an Spm” is present at the a,"~?
locus is that expected. The gene is active,
since the inhibitory effect of Spm on gene
action at a,;”-? is the reverse of that on
gene action at a,"—! and a2", In the
kernels, pigment of medium intensity is
distributed over the aleurone layer. The
weakened capacity of Spm” to induce
modification of gene action is expressed
in the kernel either by an absence of
mutant areas or by the presence of one or
several very small areas showing a mutant
phenotype. Thus, Spm” differs from
standard Spm (which in this account will
be designated Spm*) in capacity to effect
mutation-inducing responses of the oper-
ator element of the system.

Two isolates of a,"-? in which the
associated Spm? element had mutated to
Spm” were examined this past year. The
Spm” mutants differ in frequency of
reversion to Spm, one reverting frequent-
ly and the other rarely. Previous studies
of Spm” mutants, conducted with plants
having either a,""! or a2”"!, had shown
that, when Spm’ and Spm* are both
present in the nuclei of a plant or kernel,
Spm! exerts a dominant effect. Spm” is
not altered by the association, however,
but can be recovered quite unchanged: in
the progeny of plants having both the
Spm elements. Each of the isolates of
a,"-? with Spm” located close to it
responds in this same manner to Spm
located elsewhere in the chromosome
complement, and the response is similar
to that seen when Spm: resides at the
locus of a,"~?. Moreover, tests conducted
with one of these isolates have indicated
that Spm” is recovered unaltered in the
progeny of plants in which an indepen-
dently located Spm element is also
present.

Altered states of the operator element at
a,”—*, Kernels exhibiting atypical pheno-
types were selected with the intent of
finding among them examples in which
the regulator element, Spm, but not the
operator element had been removed from
the locus of a,"-?. Tests were initiated
with plants derived from 7 selected

489

kernels, and continued with their progeny.
It was learned that the modified pheno-
type in 4 of these kernels could be
ascribed neither to an event that had
removed Spm from the locus of a,"-? nor
to change in Spm action. These 4 modified
states of a," differ from the original
state mainly in the frequency of occur-
rence of each of the types of A, gene
expression that arise in conjunction with
release of the gene from control of the
Spm system. The remaining 3 altered”
states show no evidence of the presence
of Spm at the modified a,”-? locus. Each,
however, responds in its own way to an
active Spm element located elsewhere in
the chromosome complement. Two of
these states promise to provide new
evidence about kinds of control of gene
expression during development.

One of them illustrates the manner in
which anthocyanin pigment of different
intensities may be produced in individual
cells of a tissue. Such differences may be
due to “cross-feeding” between adjacent
cells that, individually, either cannot
synthesize pigment or can synthesize very
little. The phenomenon is suggested by
the distribution of pigmented cells in
kernels having one of the two mentioned
states of a;”~? and an active Spm element.
These kernels have pigmented areas in a
colorless or nearly colorless background.
Some areas are not solidly pigmented, but
are bounded by a band of pigmented cells.
The intensity of pigment in the cells
composing these ring-shaped bands is not
uniform. Many of the bands have an
inner row of deeply pigmented cells. The
cells on both sides of this inner row
exhibit a decreasing gradient of intensity
of pigment, fading gradually into colorless
or nearly colorless cells. The origin of the
ring-shaped bands, in which some cells
exhibit pigment intensities approaching
that produced by the normal A, gene,
may be explained by cross-feeding be-
tween adjacent cells that differ geno-
typically. The shapes of the pigmented
bands indicate that the cells enclosed by
each band are descendants of one in
490

which the genotype was modified, and the
modifications appear to have been con-
trolled by the Spm system.

The second of these two modified states
of a,"-2 was mentioned in Year Book 61
during a discussion of a”~? that con-
sidered potential isolates having an
operator element of the Spm system but
no Spm regulator element associated with
the locus. This state was detected,
initially, in a kernel that exhibited an
atypical phenotype characterized by some
lightly pigmented areas in a colorless
background. The intensity of anthocyanin
pigmentation differed among the cells of
an area: some were more intensely
pigmented than others, and some ap-
peared to have no pigment, but none was
deeply pigmented. As was reported last
year, there was no evidence of the
presence of Spm in the plant derived from
this kernel.

The results obtained in initial tests
conducted with this plant and in more
extensive tests of its progeny will be
summarized here. It was learned that the
phenotype described above appears in
kernels in subsequent generations as long
as the altered state of a,:-? has not passed
through a plant generation in which an
active Spm element is also present in the
nuclei. When an active Spm is introduced
by appropriate crosses, the phenotype of
the kernels that have received the altered
a,"~? locus from one parent and an active
Spm from the other parent, and also the
phenotype of the plants derived from
these kernels, is similar to that produced
by the original state of a:"~?. When test-
grosses were conducted, however, with
the plants that had received a newly
introduced Spm, some of the kernels on
the resulting ears exhibited. a decidedly
modified phenotype, which appeared only
among kernels that had received the
altered state of a:"-? but no Spm. These
kernels had mottled areas, within which
many of the cells were intensely pig-
mented. The presence of intense pigmen-
tation in the mottled areas allowed
recognition of confluence of these areas in

CARNEGIE INSTITUTION OF WASHINGTON

a manner that gave rise to a clearly
defined pattern of pigment distribution
over the aleurone layer. Microscopic
examination of the kernels showed that
the distribution and intensity of pigment
in individual cells within an area very
much resembled those produced by the
kind of cross-feeding between adjacent
cells described above.

Among the kernels on these ears that
had a,"-? but no Spm, a wide range in
expression of maximum pigment intensi-
ties was noted, although within an
individual kernel all the mottled areas
showed the same range of intensity of cell
pigmentation. Among the different ker-
nels the maximum intensity ranged from
very dark to rather faint. Kernels with
strikingly enhanced pigmentation in the
mottled areas appeared on ears produced
by crosses conducted with each of 11
tested plants that commenced develop-
ment with a newly introduced Spm. They
did not appear on ears produced by
similar crosses conducted with 26 sister
plants that had not received Spm. There-
fore it is suspected that Spm induced, in
many cells of the germ line, a particular
type of modification of the operator
element of the altered state of ai". This
change enhanced the capacity of the Ai
gene to contribute to pigment formation
in cells of a mottled area after Spm had
been removed from the nucleus in the
meiotic process. In some respects this
phenomenon resembles the paramuta-
genic effect described earlier.

Study of ai™-? has added to our
appreciation of gene-control mechanisms
in that it suggests cooperation between
two or more regulatory mechanisms. With
the original state, activation and inac-
tivation of the Ai gene are controlled by
Spm. Another regulatory mechanism
must operate, however, when the gene is
active. This fact was revealed early in the
study of a,"-?, by the restricted distri-
bution of anthocyanin in plant tissues
described in Year Book 61. It is also
strikingly revealed by the very distinctive
pattern of arrangement of mottled areas
GENETICS RESEARCH UNIT

in the kernels just described. It is again
manifested by the distribution of pigment
in the cob: large areas of deep pigmen-
tation often appear in cobs of plants
carrying the original state of a,"~? and an
active Spm element. They are not due to
a heritable modification of a,”~?, as is
shown by the results of tests and is also
indicated by the fact that kernels above
these areas have the same phenotype as
other kernels on the ear. A heritable
change would be reflected both in the cob
areas and in the kernels overlying them,
since the a,"~? locus in the cells of all is
descended from one that was present in a
common ancestor cell.

Extension of Spm Control of Gene Action

These studies, over a period of years,
have uncovered a number of examples of
gene control by foreign systems that have
not yet been reported because of inade-
quate identification. Whether or not the
system controlling a particular gene was
Ac could be determined rapidly, because
precise methods of testing were available.
Early methods for testing whether or not
a system might be Spm were much less
direct and, as applied to some genes, were
considered too indirect to be efficient.
Therefore some of the isolates, after they
had been found not to represent Ac
control, were temporarily dropped from
study. When it was determined that
wa™—§ was under the control of the Spm
system, it became possible to construct
tester stocks that could directly reveal
Spm control of gene action at other loci.
Five suspected examples of control of
gene action by this system could now be
tested with wz™-§. Two, isolated some
years ago, involve the C, locus in chromo-
some 9 and are designated "75 and
c”-*. Two others, recently isolated, in-
volve the C, locus in chromosome 4 and
are designated c2”~! and co™~*. The last
involves the Pr locus in chromosome 5.

This past year, the required combina-
tions of gene markers for such tests were
available only in plants having co”.

491

Plants that were o> Shi wz/a shy
wa"-= in constitution were crossed with
plants that were homozygous for c, sh,
and wax and carried no Spm. Other plants,
a> wa/e, wx in constitution, were
crossed with plants that were co. wr”—®/e
wx and had no Spm. Among the kernels
on the ears produced by these crosses,
response of wx™—* to Spm was correlated
with the presence of c,”~5 in the kernel.
These initial tests placed Spm close to the
locus of c™~> in the examined plants.
They also indicated that the origin of
some of the stable mutants produced by
o> was associated with transposition of
Spm to a new location in the chromosome
complement.

Further confirmation of Spm control of
gene action at a> was obtained from
tests conducted with one plant in which
Spm underwent change from the inactive
to the active phase late in development.
On the ears of this plant, some kernels
that had received both o”~* and wa™8
gave no evidence of the presence of Spm
except in one sector, produced by
descendants of the cell in which Spm had
changed to the active phase. A well
defined area in the aleurone layer exhib-
ited the types of pigmented spots that are
produced by change in gene action at
a”->: and only in the underlying cells,
derived from the common ancestor cell,
did the starch in individual cells or
clusters of cells display a phenotype
produced by mutation at wx”.

It may be added here that wx™* has
also been useful in analyses of change in
phase of activity of Spm at the a7”
locus. Responses of the two loci to phase
of activity of Spm are similarly correlated.

Further Studies of Topographical Relations
of Elements of a Control System

In Year Book 61, the origin of a two-
element system of control of gene action
from an apparently single-element con-
trol system was outlined with respect to
the Ac system in its relation to bz”~? and
one of its altered states, Bz”. Initially,
492

the regulator element, Ac, was present at
or very close to the locus of the bronze
gene, which is involved in the biosyn-
thetic pathway leading to anthocyanin
formation in plant and kernel. In studies,
with bz”, of the relation between trans-
position of Ac away from the bronze locus
and initiation of Bz gene action, one
instance was found in which Bz expression
appeared in conjunction with transpo-
sition of Ac to a position in chromosome 9
between the locus of the Bz mutant and
that of Wz, approximately 10 crossover
units distal to Wz. In one type of test-
cross, pollen from a plant that was I Sh
Bz(standard) wx Ds/C sh Bz(mutant) Ac
We in constitution was placed on the
silks of ears of plants that were homozy-
gous for c, sh, Bz, and wx and had no Ac.
On one of the resulting ears, a single
nonpigmented kernel was present in
which changes in Wx gene action, from a
low level to a high level of gene expres-
sion, had occurred in some cells during
endosperm development.

In order to examine the system
responsible for control of Wx gene action
in this kernel, a plant was grown from it
in the summer of 1961. When pollen
collected from this plant was stained with
an I-KI solution, half the grains stained
a red-brown (wx) but, unexpectedly, the
other half stained a deep blue as if to
indicate the presence of a normal Wz

CARNEGIE INSTITUTION OF WASHINGTON

gene. It was then suspected that only the
endosperm of the kernel that gave rise to
the plant had received the modified Wx
gene, and that the normal Wz gene had
been delivered to the zygote. To verify
this suspicion, a few testcrosses were
conducted with the plant. It was crossed
reciprocally with an Ac-tester plant,
homozygous for C, Sh, Bz, wx, and Ds
(standard location). It was also crossed to
plants that were homozygous for C, sh,
bz, and wa, either with or without Ac in
their nuclei, and to a plant whose
constitution was ¢ Sh bz wa/c sh bz wx, no
Ac. The kernels produced by these crosses
revealed that the zygote had, in fact,
received the modified Wx gene and that
its action was under the control of the Ac
system. Moreover, Ac was found to be
located very close to this modified Wz
gene, which was then designated wz” °
because it represented the ninth detected
change in the Cold Spring Harbor
cultures whereby the action of the normal
Wz gene had become subject to a foreign
control system.

Results of these initial tests and of
others conducted during the past year
show that return to a high level of Wz
gene action is usually associated with
removal of Ac from the locus of wr™®.
The results of two types of testcross
conducted with plants carrying wx"—® are
given in tables 1 and 2. They demonstrate

TABLE 1. Phenotypes of Kernels on Ears Produced by Reciprocal Crosses of Plants Having
the Constitution ¢, wz"-8/e wx with Ac-Tester Plants That Were Homozygous for Ci, wz, and
Ds (Standard Location) and Had No Ac

g = heterozygote employed as ear parent; 7 = heterozygote employed as pollen parent.

Pigmentation of Aleurone Layer

 

 

 

 

Level of Expression Uniformly Colorless Areas in
of Waxy Gene in Pigmented Pigmented Background
Starch of Endosperm (No Ac) (Ac) Totals
Q a 9 a
High level throughout (germinal mutation) 8 1L 12 11 42
Low level with sectors of high level 0 0 1834 986 2820
Low level throughout : 5 3 0 0 8
Null level (wz allele) 1818 1003 30 64 2915
Totals 1831 1017 1876 1061 5785

 
GENETICS RESEARCH UNIT

TABLE 2.

493

Phenotypes of Kernels on Ears Produced by Reciprocal Crosses of Plants Having

the Constitution J wx™-*/c, wx with Ac-Tester Plants That Were Homozygous for C;, wa, and
Ds (Standard Location) and Had No Ac

The colorless kernels received J, whereas the pigmented kernels received the allele c, from the

heterozygous parent. ¢ = heterozygote employed as ear parent; co’ = heterozygote employed

as pollen parent.

Pigmentation of Aleurone Layer

 

Colorless Areas

 

 

Level of Expression of Waxy Colorless Piemtet in Pigmented
Gene in Starch of Endosperm (No Ac) ~~ Totals
9 a g J g a
High level throughout
(germinal mutation) 17 1 1 2 2 0 23*
Low level with sectors of high level 1212 117 0 0 418 33 1780
Low level throughout 3 0 0 1 1 0 5
Null level (wz allele) 479 51 1214 110 27 0 1881
Totals 1711 169 1215 113 448 33 3689

 

* Six plants derived from these kernels were examined; 4 had no Ac, and 2 had Ac unlinked to

markers in chromosome 9.

the location of Ac before transposition,
and the relation of transposition of Ac to
release of Wx gene action from control by
the Ac system.

Among the total of 9474 kernels
recorded in tables 1 and 2, 13 kernels with
the wx”? phenotype showed no evidence
of the presence of Ac. Two of them
appeared on ears produced by crosses
conducted with the original plant carry-
ing wz”-*. During the past year, plants
were grown from these two kernels and
each plant was tested for the presence of
Ac in its nuclei and also for the response
of the derivative of wx”-® to introduced
Ac. The tests showed that neither plant
had Ac, and that the low level of gene
expression characteristic of derivatives of
wx"-* remained unchanged as long as Ac

was absent. When Ac was introduced,
however, each responded to it. Mutations
occurred in individual cells, whose de-
scendent cells exhibited a high level of
Wz gene action. The time during develop-
ment when the mutations occurred
reflected the dose of Ac present in the
nuclei of the kernels: the higher the dose
of Ac, the later the time of occurrence of
mutations. The genic marker constitution
of the chromosome 9 carrying wz™~® in
these two plants indicated that the
removal of Ac from the immediate
vicinity of the original wx7—* locus could
not be attributed to crossing over. Thus,
wx™"—* provides another example of origin
of a two-element system of control of gene
action from an apparently one-element
system.