MUTABLE LOCI IN MAIZE

Barpara McCrintock

For the past few years, investigations perimental procedures and results have
have been focused on the origin and be- been described in previous reports. In Year
havior of mutable loci in maize. The ex- Book No. 48 an interpretation of the

14
158

mechanism associated with the origin and
behavior of the Ac-controlled mutable loci
was presented. During the past year, em-
phasis has been placed on expanding this
evidence, in order to determine whether
or not the premises of the interpretation
would continue to be supported by further
tests. The results of the year’s experiments
have substantiated the main conclusions
given in the previous report, have pro-
vided more information with regard to
the mechanisms involved, and have sug-
gested the probable nature of the chromo-
some materials concerned.

Variegation has been described in a
wide range of organisms. Some cases of it
are known to be associated with the irregu-
lar mitotic behavior of ring-shaped chro-
mosomes, or with chromatin deletion pro-
duced by the breakage-fusion-bridge cycle.
Another class of somatically expressed
variegation is known to be related to irre-
versible changes in self-reproducing cyto-
plasmic elements, such as the plastids. A
third class of somatic variegation is asso-
ciated with detectable changes in genic
action. The terms mosaicism, mutable
genes, somatic variegation, mutable loci,
and position effect have been used to desig-
nate this class. It is on this third type of
variegation that the investigations in maize
have been focused. It has been described
in many organisms. Because the general
nature of the phenotypically expressed
instability is so similar in these unrelated
organisms, it is difficult to avoid the con-
clusion that a common principle of nuclear
and chromosomal organization and func-
tioning is concerned. The evidence ob-
tained during the past year has made it
possible to formulate a working hypothesis
that incorporates such a principle. The
hypothesis is an expansion of the inter-
pretation of the origin and behavior of
mutable loci presented in Year Book No.

CARNEGIE INSTITUTION OF WASHINGTON

48. Its salient features may be reviewed
here:

A normal, wild-type locus may be
totally or partially inhibited in action by
the insertion of a foreign piece of chro-
matin adjacent to it. Total or partial re-
lease from inhibition will occur when this
foreign chromatin is removed or altered
in organization. The insertion, removal,
or change in organization of the foreign
chromatin occurs because this chromatin
becomes adhesive in certain somatic cells .
at very precise times in the development
of a tissue. The adhesiveness causes a
rupturing of the chromosome at the ad-
hered positions during the subsequent
mitotic cycle, which results in removal,
transposition, or alteration in  constitu-
tion of the chromatin materials involved.
The chromatin primarily concerned in
these events is heterochromatin. Its be-
havior as revealed in this study of the
origin and expression of mutable loci may
reflect one aspect of its normal behavior
in the development of an organism.

Mopbe oF Derecrion oF TRANSPOSITIONS
or Ds

The first direct evidence with regard
to the mode of origin and the operation
of mutable loci was obtained from study
of the Ac-controlled mutable c”* locus.
The origin of this mutable locus by a
transposition of Ds has been reviewed in
previous reports. Because transposition
of minute bits of chromatin from one loca-
tion to another in the chromosome com-
plement is basic to the concept of the origin
and behavior of mutable loci, extensive
investigations of this phenomenon have
been undertaken during the past year.
Twenty cases of transposition of Ds from
its standard location in the short arm of
chromosome g to another position within
this arm have been studied. Several cases
DEPARTMENT OF GENETICS

of transposition from the new position
to still another position have also been
investigated. Transpositions of Ds from
one location to another within the short
arm of chromosome 9 were selected for
study because the design of the experi-
ments makes it possible to detect such
transpositions shortly after they occur, and
also because it is possible to locate, readily
and accurately, the new positions of Ds
activity.

An example may be given of the
methods used in detecting and locating
new positions of Ds. Pollen of plants that
have one or more Ae loci, and carry I, SA,
Bz, Wx, and Ds-standard in one chromo-
some 9, is placed on silks of tester plants
carrying C sh bz wx but having no Ds or
Ac \oci. The state of the Ds locus in these
plants is selected for a high frequency of
dicentric-chromatid formation as a con-
sequence of events occurring at Ds. On
the resulting ear, kernels that receive an
Ac locus and a chromosome g carrying the
markers I, Sh, Bz, Wx, and Ds-standard
should show sectors of tissue with the
C sh bz wx phenotype as a consequence
of dicentric- and acentric-chromatid-form-
ing events occurring at the Ds-standard
location, which result in elimination from
the nuclei of the acentric segment of chro-
matin carrying the markers J, Sh, Bz, and
Wx (for details, see previous reports).
With very few exceptions, the expected
phenotypic characters are present in the
sectors of the kernels carrying the stated
markers. The few exceptional kernels
that have sectors showing unexpected
phenotypic characters are important, for
from them are derived the strains having
new positions of Ds activity.

The new position is often immediately
revealed in an exceptional kernel. As an
illustration, we may describe a kernel
having Ds transposed from its standard

159

location to a position between J and SA.
During the development of such a kernel,
a dicentric-forming event, involving sister
chromatids at this position, will produce
an acentric fragment that carries the two
I loci and a dicentric chromatid that in-
cludes the two sister proximal segments
of the short arm, each carrying SA, Bz,
and Wx. In the dicentric chromatid, the
Sh and Bz loci are physically close to the
position of sister-chromatid union. A
bridge configuration will be formed by
the dicentric chromatid in the subsequent
anaphase. Breakage of this bridge can
occur at a position anywhere between the
two centromeres. The break is most often
nonmedian. Consequently, the broken
chromatid that usually enters one telo-
phase nucleus carries two Sh and Bz loci
and one Wx locus. The sister nucleus
receives a broken chromatid carrying only
the Wx locus. From each nucleus a sector
of tissue will subsequently be produced.
When the kernel is mature, twin sectors
arising from these two sister nuclei will
be evident. One sector will show a C sh bz
phenotype, within which subsectors of
Wx and wx phenotypes will appear. It
will be derived from the nucleus that lost
the Sh and Bz loci as a consequence of
the nonmedian position of breakage in the
first-anaphase bridge configuration. Varie-
gation for Wx and wx within this sector
is brought about by the subsequent break-
age-fusion-bridge cycle that eliminates the
Wx locus from some nuclei. The sister
sector will show a C Sh Bz Wx pheno-
type; but within this sector subsectors will
be present. Their constitutions will be
C sh Bz Wx, C sh bz Wx, or C sh bz wx.
These subsectors arise from losses of Sh,
Bz, and Wx loci from some of the cells as
a consequence of the breakage-fusion-
bridge cycle initiated by the original Ds
event. The variegation pattern appearing
160

in the kernels with Ds located between I
and SA is thus in striking contrast with
that produced in kernels having Ds at its
standard location. Other locations of Ds
activity in the short arm of chromosome 9
are likewise readily detected by the type
of phenotypic characters expressed in the
sectors of the exceptional kernels. These
exceptional kernels, therefore, make it
possible to identify immediately those
kernels that carry newly arising trans-
posed Ds loci. From such exceptional
kernels have been derived the strains with
changed locations of Ds activity. A study
of these strains has provided the informa-
tion needed to interpret the mechanism
responsible for transposition.

Many of the exceptional kernels appear-
ing in the cross described above are defec-
tive in growth of either the endosperm or
the embryo. They do not germinate. Other
exceptional kernels appear to be morpho-
logically normal in both endosperm and
embryo, but many of them also do not
germinate. Less than half the selected
exceptional kernels have given rise to
viable plants. The plants obtained from
the viable fraction, however, have been
subjected to extensive cytological and
genetical tests to determine the exact loca-
tion of the new positions of Ds activity
in each case. These tests have revealed
that Ds may be inserted at various posi-
tions within the short arm of chromo-
some g. Its insertion adjacent to the C
locus, which gave rise to the mutable c”“,
. has been described previously. Recently,
a mutable bz locus appeared in an excep-
tional kernel; and a plant was obtained
from this kernel. Preliminary tests sug-
gest that the behavior of this mutable bz
locus may be similar to that observed for
e™*, It is now being investigated to deter-
mine whether or not it arose by insertion
of Ds adjacent to the Bz locus.

CARNEGIE INSTITUTION OF WASHINGTON

Events Occurrinc at THE Ds Locus

Considerable progress has been made
during the year in understanding the
nature of the events that occur at the Ds
locus in somatic cells, It is believed that
only one primary type of event occurs at
this locus, and that it has several different
types of consequences. These consequences
resemble those produced by treating cells
with X-rays, ultraviolet rays, chemicals,
and so forth, in that they involve some
mechanism leading to chromosome break-
age and fusion. The several types of chro-
mosomal alteration that are repeatedly
observed when Ds is present can be readily
understood if it is assumed that the pri-
mary event produces a physical change of
the material composing Ds: that this
material becomes sticky or adhesive in
certain cells at predetermined times in the
development of a tissue. The known con-
sequences of events occurring at Ds will
illustrate the reasons for the assumption.
They may be enumerated: (1) Dicentric
chromatid formation, with fusion of sister
chromatids at the position of Ds. This is
accompanied by formation of an acentric
fragment composed of the two sister-chro-
matid segments, from Ds to the end of
the arm. (2) Deletion of chromatid seg-
ments of various lengths adjacent to Ds,
usually with concomitant loss of Ds
activity but occasionally without loss of
this activity. (3) Loss of detectable Ds
activity without visible alteration of the
chromosome. (4) Reciprocal translocations
involving chromosome 9, in which one
breakage point is at Ds. (5) Duplication
of segments of chromosome 9, with one
break marked by the known position of
the Ds locus. (6) Transposition of Ds
activity from one position to another in
the chromosome complement, with or
without an associated gross chromosomal
rearrangement. (7) Composition changes
DEPARTMENT OF GENETICS

at the Ds locus that result in precise
changes in the relative frequencies of oc-
currence in future cell generations of the
various events enumerated above.

In previous reports, this last occurrence
has been termed “change in state” of Ds.
A state giving a high frequency of event 1
and relatively high frequencies of events
2, 4, and 5 sometimes changes, after a
somatically occurring event at Ds, to a
state giving low frequencies of these
events and a high frequency of event 3.
A return from this latter state to the
former state requires a progressive series
of events at Ds. This was determined in
the following manner. The frequency of
the various types of events at Ds may be
deduced by observing the type and pattern
of variegation on the Fi ear in crosses
of Ds-carrying plants to tester plants. If
the Ds locus gives a high frequency of
event 1, the majority of the kernels on the
test ears will express this frequency in a
similar manner. There may be a few ex-
ceptional kernels, however, that show a
greatly reduced frequency of event 1. The
latter kernels are selected and plants are
grown from them. These plants also are
crossed to tester plants, to observe the action
of Ds in the kernels on the resulting ears.
With very few exceptions, the action of
Ds in these kernels is quite similar to its
action in the kernel that gave rise to the
plant carrying the modified Ds. Occa-
sionally, however, a kernel is observed
that shows a slightly higher rate of event
1. This kernel is selected from the ear,
a plant is grown from it, and a similar
test is made for the action of this selected
Ds locus. Again, the action of Ds in the
majority of the kernels on the test ear is
similar to that in the kernel that gave rise
to the plant. A few kernels, however, may
show an increased rate of event 1. These
are in turn selected, plants are grown from

161

them, and the test of Ds action is con-
tinued. In the experiments so far con-
ducted, it has required three or four gen-
erations of such progressive selection to
obtain plants carrying a Ds locus giving
a frequency of event 1 that resembles the
frequency given by the original Ds from
which it was derived.

Tue MECHANISM OF TRANSPOSITION oF Ds

An interpretation of the mechanism of
transposition of Ds was considered in Year
Book No. 48. It was based on analysis of
those transpositions of Ds that were ac-
companied by gross chromosomal rear-
rangements. During the past year, two
such cases have been extensively analyzed.
The nature of the alteration, in each
case, with respect to chromosomal con-
stitution and genic arrangement was
determined with considerable accuracy.
In each case, a duplication of a segment
of the short arm of chromosome g accom-
panied the appearance of a new location

cof Ds activity. Each case arose from a

different male parent, and was originally
identified because of an exceptional varie-
gation pattern appearing in a single kernel
on an ear produced by crossing a C sh
bz wx tester plant by an Ac-carrying plant
that had two morphologically normal
chromosomes g—one with the markers
1, Sh, Bz, Wx, and Ds-standard, and the
homologue with the markers C, sh, bz, and
wx, and no Ds locus. In each case, the
duplication arose from breaks at compa-
rable positions in sister chromatids of the
I Sh Bz Wx Ds-standard chromosome.
Likewise, in each case, one break occurred
at the Ds-standard position, the second
break at the position marked by the new
location of Ds, that is, just to the right of
the J locus in one case, and a short distance
to the left of the J locus in the other. The
duplicated segment in the first case was
162

in the inverted order, in the second case
in tandem order. Diagrams illustrating
the chromosome composition and genic
order in the two cases are given in figure 1.

As the diagrams show, two Ds loci are
present in each chromosome. Their posi-
tions in each case mark the breakage points
in the sister chromatids of the 1 Sh Bz Wx
Ds-standard chromosomes that gave rise
to the duplication. In the case illustrated
in figure 14, no Ds locus is present at the
standard position in the distal segment,

 

 

CARNEGIE INSTITUTION OF WASHINGTON

breakage points, genic organization, and
altered positions of Ds activity, can be
understood. According to this assump-
tion, the Ds material in the 1 Sh Bz Wx
Ds-standard chromosome adhered to an-
other position in the short arm of chromo-
some g—immediately to the right of J in
the first case, and to the left of J in the
second case. Stresses produced by chromo-
some movement during the mitotic cycle
that followed resulted in rupture of the
sister chromatids at the adhered regions.

I Ds Sh Bz Wx Bz Sh Ds
\UZ | nee
Ds I Sh Bz Wx DsI Sh Bz Wx
(1 Vi | _
b

Fic. 1. Diagrams a and 4 illustrate two different duplications of segments of the short arm of
chromosome 9. The wide line indicates the duplicated segment in each case; the open circle repre-

sents the position of the centromere. The arrows

tied by each of these chromosomes.

although a break in the chromatid oc-
curred at.this location. This position marks
the proximal breakage point of the in-
verted duplicated segment. In the case
illustrated in 4, no Ds locus is present at
the standard location in the proximal
duplicated segment, even though it is
obvious that a break occurred at this posi-
tion to give rise to the chromosome with
this particular duplication. If the primary
event occurring at a Ds locus is related to
a physical change causing adhesiveness in
the material composing Ds, then the origin
of these duplications, with the described

point to the locations of the genetic markers car-

Subsequent fusions of broken ends pro-
duced the duplications with the altered
positions of Ds.

Various types of chromosomal rearrange-
ment, with or without accompanying
transpositions of Ds, could result from the
above-described mechanism. Figure r illus-
trates only two such cases. It is readily
apparent that in these cases the fusions
could have given rise to morphologically
normal chromosomes 9, -each carrying a
transposed Ds locus but showing no other
alteration in genic organization. The types
of fusion that followed the breakages
DEPARTMENT OF GENETICS

would determine the consequences. There-
fore various other kinds of gross chromo-
somal rearrangements, with one breakage
point at the known location of Ds, could
be anticipated. Just such rearrangements
have frequently been observed in the
sporocytes of plants carrying Ds at known
positions in chromosome 9. The majority
of viable exceptional kernels carrying
newly arisen transpositions of Ds, however,
have not produced plants with such gross
chromosomal rearrangements. Most often,
a chromosome g with a transposed Ds
locus is morphologically normal. This was
to be expected, for the kernels having
newly arisen transpositions of Ds were
selected from ears produced from crosses
in which the Ds locus was brought in by
the pollen parent. The pollen grains with
unbalanced chromosomal complements do
not compete favorably with those having
a normal chromosome complement. Con-
sequently, many of the pollen grains carry-
ing transpositions of Ds associated with
gross chromosomal rearrangements may
be expected not to function. It is reason-
able to believe, therefore, that the above-
described cases having a transposition of
Ds associated with a gross chromosomal
rearrangement are representative examples,
illustrating the mode of transposition of
Ds.

As stated earlier, the transposition mech-
anism introduces Ds at various positions
in the short arm of chromosome 9. Ap-
parently, it may enter any position within
this arm. The analysis of c™? indicated
that the insertion of Ds adjacent to the C
locus brought about an inhibition of the C
action, which was withdrawn when Ds
was subsequently removed from this posi-

tion. If Ds inhibits the action of adjacent —

genes, then newly arising Ac-controlled
~ mutable loci should continue to appear as
the result of continued transpositions of
Ds to new locations. Detection of such

163

mutability depends on experimental pro-
cedures. Crosses of plants carrying known
recessive markers in chromosome 9 by
plants having the dominant alleles and
also Ds can reveal the presence of muta-
bility of one of the marked loci directly
on the F; ears. Sectors within a kernel
that show newly arising mutability for the
marked loci are not uncommon after such
crosses. In order that a strain having such
a newly arising mutable locus may readily
be obtained, it is necessary that the causa-
tive event occur before the two sperm
nuclei are formed, so that both the endo-
sperm and the embryo will carry the
mutable locus. Since Ds events may occur
in only a few cells, late in the develop-
ment of the sporogenous tissues, relatively
few kernels can be anticipated that will
show new germinal origins of mutability
for the marked loci. The few exceptional
kernels that do show such mutability may
be selected from the ear for continued
study.

Newly arising mutability for previously
unmarked loci may be detected in the
progenies derived from self-pollinations.
Ease of detection requires a sharp delinea-
tion of contrasting phenotypic characters
capable of being expressed in small sectors.
Morphological determiners, or physio-
logical determiners requiring special tech-
niques for detection, are not suitable for
such a study, even though they may con-
stitute the largest group of mutable loci.
In the culture under investigation, many
new recessive mutations effecting marked
changes in morphological characters have
appeared in the progeny of self-pollinated
plants. It is not practicable to make a
precise study to determine whether or not
mutability is being expressed at each of
these recessive loci, even though the
mutable behavior of a few of them has
been obvious.
164

TRANSPOSITION AND CHANGE IN AcTION
oF Ac

The transposition of the Ac locus from
one position in the chromosome comple-
ment to. another, frequently from one
chromosome to another, was described in
Year Book No. 48. The relation between
loss and transposition of Ac was likewise
described, along with the methods used
to detect the kernels that show loss or
transposition. During the past year, this
study was greatly expanded. All together,
69 independent cases of loss, transposition,
or change in action of Ac were investi-
gated. For 58 of these cases, the method
of analysis was similar to that described
in Year Book No. 48, and therefore only
the results obtained need be considered
here.

In 42 cases, kernels were selected be-
cause they showed no Ds activity in the
cross of de Ac (allelic positions) by Ds ac
tester plants. To determine why Ds activity
was absent, plants were grown from these
kernels and tests conducted for the pres-
ence of Ac. In 19 plants, no Ac locus was
present. In 17 plants, two independently
inherited Ac loci were present, indicating
that a transposition of Ac had occurred
in a cell of the female parent of these
plants. In 6 plants, there was present an
Ac locus that was inherited as a single
unit. Its action, however, resembled that
of two doses of the Ac locus from which
it was derived. The reasons why Ds
activity did not appear in these 42 selected
kernels have been presented previously.
They are related in 19 of the cases to the
absence of dc, and in the other 23 cases
to the high dose of Ac present in the endo-
sperms, which so delayed the occurrence
of Ds events that none was observed.

Eight other kernels were selected from
the ears of the mentioned cross because
they showed a very obvious delay in the

CARNEGIE INSTITUTION OF WASHINGTON

time of occurrence of Ds events. Only a
few very late-occurring Ds events were
evident in these kernels. Analysis of the
Ac constitution of the plants derived from
them showed that in 5 cases two non-
linked Ac loci were present. In 3 cases, an
Ac locus inherited as a single unit but
giving a double-dose type of action was
responsible for the delay in the timing of
Ds events.

In 8 more aberrant kernels selected from
the ears of this cross, the dosage-type
action of Ac appeared to have increased
moderately, although not to an extent
resembling the double-dose action in the
above-mentioned cases. The analysis of Ac
action in 7 of the 8 plants derived from
these kernels indicated that the cause for
the altered timing of Ds events was re-
lated to an alteration of Ac that had
occurred in a cell of the parent plant late
in its development. In the eighth case,
the Ac action resembled that given by the
Ac loci in the parent plant; no obvious
change in action was detected.

Tests of Ac action in 21 plants derived
from kernels that showed no alteration
in Ac action in the above-mentioned cross
served as a control. In all ar cases it was
obvious that no detectable change in Ac
action had occurred in the ancestor cells
of the Ae Ac plant that gave rise to these
kernels. With respect to events that occur
at the Ac loci in some of the Ac Ac (allelic
positions) plants, resulting in transposi-
tion, change in action, or loss of Ac from
the gametic nuclei, the above findings are
similar to those previously described.

Seven independent cases of transposition
of de from an unknown location in the
chromosome complement to chromosome
9 have been detected. In 4 of these cases,
Ac was transposed to the long arm of
chromosome g; in 2 cases, to the short
arm of chromosome g. In 1 case, the exact
DEPARTMENT OF GENETICS

location of Ac remains to be determined.
The presence of an Ac locus in the short
arm of chromosome g is most strategic
for determining the events that occur at
Ac, because of the excellent group of
genetic markers carried by this arm. One
such case has received sufficient study to
permit some definite conclusions regard-
ing events that occur at Ac. Plants were
obtained that carried Ac in one chromo-
some 9 and no Ac in the homologous chro-
mosome 9. The pachytene configurations
in some of these plants were examined.
It was possible to detect in some cells an
alteration in one chromosome g at a posi-
tion slightly proximal to the middle of
the arm. At this position, one chromosome
showed a deep-staining chromomere not
present in the homologue. Sometimes a
tiny loop was present at this position in
one chromosome. Linkage experiments,
utilizing markers in the short arm of
chromosome 9, place this Ac locus between
Bz and Wx, at a position approximately
four crossover units distal to the Wx locus.

This position accords well with the posi-

tion of the observed alteration in chromo-
some 9. Although there is not yet enough
evidence to state definitely that the de-
scribed alteration in chromosome 9g is at
the site of the Ac locus, it may be antic-
ipated that a correlation will be found.
Transposition of this Ac locus to another
chromosome of the complement, and
absence of this locus in a few gametes pro-
duced by plants that are homozygous for
it, have been observed.

It has been emphasized that Ds and Ac
are alike in that both undergo the trans-
position phenomenon. Breakage events
will occur at Ds only when Ac is present.
It cannot yet be stated that the reciprocal
is true—that is, that Ds must be present
for Ac to act. This may be difficult to
determine with the required degree of

105

accuracy in the material now available.
Events occurring at Ds produce chromo-
some breaks, as previously described. That
the transposition of Ds is a reflection of
this type of event has been emphasized.
Is the transposition of Ac brought about
by a similar mechanism? In general, are
the events occurring at dc similar to those
occurring at Ds? Preliminary study of
chromosome events at the location of Ac
in the short arm of chromosome g has
shown that breaks occur at this locus in
somatic cells. In this respect the events
at Ac very much resemble those at Ds.

CONSIDERATION OF THE CHROMOSOME MatE-
ras RESPONSIBLE FOR THE ORIGIN
anp Benavior of Murase Loct

Because the same types of mutability
as those observed in maize have been
described for a wide variety of organisms,
it is probable that the same events, in-
volving the same chromosome materials,
may occur in all organisms. Heterochro-
matin is suspected to be the nuclear mate-
rial primarily responsible for the origin
and behavior of mutable loci. It has been
observed in many organisms; it appears
to be present in all those examined, and
occupies rather precise positions in the
chromosome and nucleus. Its apparently
universal presence indicates that it must
have a special function in the nucleus.
Since it is suspected that one phase of the
function of heterochromatin may be re-
vealed by the behavior of mutable loci,
it is necessary to explain why heterochro-
matin is believed to be associated with
mutability. Only the more pertinent evi-
dence pointing to this relationship will be
considered here.

In the present study, the original burst
of newly arising mutable loci appeared in
plants whose chromosome-9 short arm had
166

been subjected to drastic structural altera-
tions as the consequence of a number of
successive breakage-fusion-bridge cycles
occurring in early developmental periods.
A cytological study was made of the chro-
mosome constitution in a number of these
plants, in order to determine what types
of structural change had occurred. In
addition to the expected types of altera-
tion of the chromosome-g short arm, a
number of quite unexpected types of chro-
mosomal aberration appeared in many of
the plants. With few exceptions, the
heterochromatic materials—that is, the
centromeres, the knobs, and the nucleolus
organizer—had been involved in events
leading to the unexpected chromosomal
aberrations. In most cases the structural
alteration arose from fusion of the knob or
the centromere of chromosome g with
other knobs or centromeres, or with
the nucleolus organizer. Unquestionably,
the somatically occurring breakage-fusion-
bridge cycles involving chromosome 9
were responsible for the origin of these
unusual types of structural alteration in
the chromosome complement.

To assume from the above observations
alone that alterations in heterochromatin
are somehow responsible for the origin of
new mutable loci would not be defensible.
When considered with reference to other
evidence pointing in the same direction,
however, the correlation becomes more
apparent. Some of this additional evidence
will be considered. A case in maize
analogous to the Ac-c™” relationship has
been investigated by Dr. M. M. Rhoades.
The activator in this case is designated Dt
(Dotted), and in the strains used was
found to be located in chromosome g. It
causes a recessive locus in chromosome 3,
a; (colorless aleurone), to mutate to domi-
nant alleles of the A: phenotype. Just as
e™* does not mutate unless Ac is present
in the nucleus, neither will a: mutate

CARNEGIE INSTITUTION OF WASHINGTON

unless Dt is present. Tests have shown
that Ac does not substitute for the known
Dt locus; the two appear to be different
controllers of mutable events. The simi-
larities between the two cases are striking,
and a common type of causative factor
undoubtedly exists. That a heterochro-
matic element is involved has been sug-
gested by the genetic studies of Rhoades,
which have placed the examined Dr locus
in the heterochromatic knob terminating
the short arm of chromosome 9. The
following questions arise: Is D¢ action a
reflection of some specific alteration in
this heterochromatic material? Could this
alteration be produced anew? It was
thought that since the breakage-fusion-
bridge cycle gives rise to various altera-
tions in heterochromatic elements, it might
be possible, by subjecting tissue to this
cycle, to re-create the Dr effect.

The experimental procedure aimed at
re-creating the Dt effect has been as fol-
lows. The plants used as the female par-
ents had normal chromosome constitu-
tions, were homozygous for ai, and carried
no Dt locus. Extensive tests by several
investigators have shown that no muta-
tions of a: to A: will occur when such
plants are crossed by plants of similar
chromosomal and genic constitutions. In
this experiment, however, these plants
were crossed by plants that were homo-
zygous for a. and carried no Dt locus,
but whose chromosome constitution was
not normal. In these, one chromosome 9
was deficient for a long terminal segment
of the short arm; the homologous chromo-
some g carried an inverted duplication
of the short arm. More than half of
the functioning pollen grains of such
plants carry a chromosome g with a newly
broken end; and breakage-fusion-bridge
cycles occur in the endosperms of kernels
receiving such broken chromosomes. If
DEPARTMENT OF GENETICS

the Dr effect is due to a specific structural
alteration in the heterochromatin, then this
particular alteration might be re-created
in some of the kernels as a consequence of
this cycle. Should this occur, the newly
arising Dt-type effect would be evident in
the appearance of spots of the A: pheno-
type in some of the kernels resulting from
this specifically constructed cross. The
kernels on 95 ears resulting from this cross
were examined. Most of them were color-
less, but a total of 15, coming from 14
different ears, had typical 41 spots. Nine
of these kernels had a single A: spot,
three had 2 A: spots, two had 4 A: spots,
and one had 5 A: spots. The 4: spotting
on these kernels was similar to that pro-
duced by the known Dr locus. These
preliminary results are in accordance with
expectation. The experiment must be con-
tinued, however, to determine whether
or not the mutations arise from altera-
tions in heterochromatic elements, as re-
quired by the basic assumptions.

It is in Drosophila melanogaster that.

the greatest body of evidence bearing on
the relation of heterochromatin to genic
instability has been accumulated. The
“position effect” variegations in this or-
ganism have been extensively investi-
gated. Heterochromatin components are
known to control the type and time of
somatically occurring changes in genic
action. Comparisons of the behavior of
the various types of heterochromatically
controlled “position effect” variegations in
Drosophila with the behavior of various
types of mutable loci in maize have shown
a marked similarity in the kinds of varie-
gation expression in these two organisms.
Even the dosage action of the heterochro-
matic Y chromosome in Drosophila and
that of Ac in maize have resemblances
that may not be coincidental. These simi-
larities make it necessary to consider a
common causative mechanism to account

167

for the occurrence of the same types of
phenotypic expression in the two organ-
isms, especially since the evidence in maize
likewise points toward heterochromatic
control. The converging evidence from
the two organisms may provide the sup-
plemental information necessary for a
better understanding of the normal func-
tion and behavior of heterochromatin.
That an interrelation exists between the
heterochromatic elements of the chromo-
somes and the gene-carrying euchromatic
elements can hardly be questioned. The
apparently universal presence of hetero-
chromatic components of the chromosome
complement, the particular spatial posi-
tions that they occupy in these chromo-

‘somes and in the working nucleus, the

phenotypic effects produced by altering
the quantity, organization, or position of
heterochromatic elements with respect to
euchromatic elements, and the absence or
infrequent occurrence of phenotypically
recognized Mendelizing units within the
heterochromatin, all suggest a functional
property of heterochromatin quite different
from that expressed by the euchromatic
elements of the chromosome complement.
These interrelations and differences make
it necessary to intensify efforts to clarify
the functional aspects of heterochromatin
in the working nucleus. This must be
done before it will be possible to interpret
adequately the mode of operation of the
euchromatic elements that carry the Men-
delizing units. If further evidence sub-
stantiates the present indication that
changes in heterochromatic elements are
responsible for the origin and behavior
of mutable loci, then all the observations
made concerning the behavior of these loci
will be relevant in unraveling the func-
tional aspects of heterochromatin, and
some of the details of the mode of opera-
tion of heterochromatic elements will be
made evident.