THE SUPPRESSOR-MUTATOR SYSTEM OF CONTROL OF
GENE ACTION IN MAIZE

Barbara McClintock

Continued attention has been given dur-
ing the past year to the manner in which
controlling elements in maize affect the
phenotypic expression of known genes.
Controlling elements, because of their ca-
pacity for transposition, are considered as
chromosome components apart from the
genes, which appear to be stationary. Once
this distinction had been made it was pos-
sible to examine the mode of operation of
controlling elements independently of
genes and to study relations among them.
It was found that some elements interact
with others to control gene expression. In
this respect, the elements can be segre-
gated into groups whose members inter-
act with one another but not with mem-
bers of other groups. Each group exhibits
a particular type of control of gene ex-
pression. Therefore, a group of interact-
ing elements has been referred to as an
operational system. Progress has been
made in interpreting the mode of opera-
tion of several of these systems, but some
important questions about controlling ele-
ments themselves remain unanswered. We
do not know, for example, how they are
incorporated in a chromosome or by what
process transposition is accomplished. Ex-
periments aimed at answering such im-
portant questions are needed. My atten-
tion has not been turned in these direc-
tions, because I believe that we should first
expand our knowledge of different sys-
tems of controlling elements in order to

learn, if possible, whether the modes of
operation of all controlling elements are
basically alike. It is conceivable that these
elements may be composed of one com-
mon type of substance and have one com-
mon mode of operation, as is now thought
to be true of genes.

Appreciation of the way in which some
systems operate may be gained rapidly;
with other systems, analysis may be diffi-
cult and progress slow. A system may be
responsible for the appearance of an array
of quite different phenotypes, even within
a single plant. Also, in some cases, analy-
ses thay be complicated by what appears
to be an irregular form of inheritance of
components of the system. That apparent
complexities in phenotypic expression can
be resolved into ordered patterns when the
operation of a component of a system is
comprehended became apparent during the
course of studies conducted this year; the
findings will be considered in the discus-
sion that follows.

The Mode of Operation of the
Spm Element

The relation between ai", located in
chromosome 3, and a2”, located in chro-
mosome 5, was considered in Year Book
56. It was mentioned there that the action
of genic substance at each of these two loci
is under the control of elements belonging
to the Spm (Suppressor-mutator) system,
and, further, that the Spm element in a2"
416 | CARNEGIE INSTITUTION OF WASHINGTON

cultures does not behave quite like that in
a."* cultures. During the past year, dif-
ferences between these two elements have
been examined. As a consequence, some
previously puzzling aspects of the system
of control of gene expression in a2" cul-
tures have been clarified.

The difficulties encountered in earlier
attempts to analyze the system of control
of gene expression at a2""" were not ex-
perienced in the analysis of a:"-*. Whereas
in the a2” cultures detection of the Spm
element was much delayed, Spm was easily
identified in the a."* cultures. Its mode
of operation in these cultures could be
readily discerned, and its inheritance be-
havior did not seem complicated. Trans-
position of the element from one location
to another in the chromosome complement
could also be followed easily. It was
learned, however, that this Spm element
is not altogether stable. Some changes in
its action were recognized; and one of
these, effecting a reduction in its capacity
to inhibit gene expression at ai." and to
cause mutation at that locus, was discussed
in Year Book 56. This finding suggested
the possibility of a direct relation between
the degree of suppressive capacity of an
Spm element and its ability to induce
mutation.

Most of the studies of control of gene
expression at a:”* were conducted not
with the modified Spm element, just men-
tioned, but with the originally identified
Spm element, whose capacity to suppress
the expression of gene action at a2
is complete. In the presence of this Spm
element there is no visible evidence of ac-
tion of the genic substance of the a,"
locus except in those areas of a plant or
kernel that arise from cells in which muta-
tion to or toward A: has occurred. The
cells having such mutations contain an-
thocyanin pigment; and the mutants so
formed are thereafter stable. The pheno-
type attributable to a particular mutation
will be produced by the mutant either in
the presence or in the absence of Spm in
subsequent generations of plants.

It was found that another type of change
also can occur at the locus of a;”", Each
such change is reflected in an altered re.
sponse of a” to given conditions jn
subsequent cell and plant generations. The
alteration must modify some component of
the locus that is capable of being repro-
duced during each chromosome replica-
tion; in other words, the modification ef.
fects a heritable change in the state of the
locus. Control of the time of occurrence of
mutation during development of a tissue,
of the type of mutation, and of the num.
ber of cells in which mutation takes place,
was found to reside at the locus of ay")
each state expressing a particular muta-
tional pattern in the presence of § pm. The
pattern peculiar to each state is not altered
by increased doses of Spm. The same pat-
tern appears when one or more than onc
Spm element is present. In the absence of
Spm, on the other hand, some gene action
at a." is expressed as anthocyanin pig-
mentation in both plant and kernel by
all but one of the examined states, and the
degree of this action, as indicated by the
intensity of pigmentation, is also a reflec-
tion of the state of a". In the absence
of Spm, however, no mutations occur and
each state retains its integrity through suc-
cessive plant generations. In short, the ex-
pression of types of mutation and of their
pattern of distribution in plant or kernel
in the presence of Spm, and also the type
of gene action exhibited in its absence.
define a state of a:""*. No unresolved am-
biguities in this respect were encountered
during an extensive study of states of a”.

In the a2” cultures, in contrast, such
clear-cut distinctions between states werc
not evident in the early studies. It was
realized, nevertheless, that not all deriva-
tives of the original a2”! were alike. At
first, the different isolates could be segre-
gated only into two main classes. Difficul-
ties were encountered in interpreting the
mode of control of gene expression of
members within each class. A few ex-
amples will be given of the irregular pat-
terns of gene expression observed in
members of the first class.

In plants carrying isolates of a2” be-
longing to the first class, it was noted that
abruptly initiated changes of some kind,
apparently occurring within individual
cells, resulted in the appearance of well
defined areas in which the phenotypic ex-
pression of gene action at a2™" differed
from that in the surrounding tissue. Such
areas also appeared in ears, all the kernels
within an area exhibiting the modified type
of gene expression. Again, such modified
areas were often seen even within a single
kernel. Different types of modification
were sometimes observed in the same plant.
Anthocyanin pigment would be totally ab-
sent in some areas whereas most of the rest
of the plant was deeply pigmented; or the
intensity of pigment within an area might
simply be reduced. Areas were also noted
in which streaks of deep pigmentation ap-
peared in an otherwise nonpigmented
background. Both the number of these
deeply pigmented streaks and their size
seemed to be controlled in some manner,
for each such area exhibited a precise pat-
tern in these regards. The patterns differed
among the areas, however, and often over
a very wide range. Some areas had only
a few small pigmented streaks; others,
many such small streaks. In still others,
the size of the streaks was much larger,
and there might be either a few or many
within one area. In the course of study of
isolates belonging to the first class, it was
found that mutation to or toward A2 oc-
curred, but only in those cells of an area
that otherwise exhibited no anthocyanin
pigment. It was also found that the mu-
tants were thereafter stable with respect to
the phenotypes they produced. No varie-
gated phenotypes appeared in either a plant
or a kernel having one of the mutations,
when tested under conditions that would
be expected to reveal any instability of
expression had it occurred.

Often, in plants showing the above-de-
scribed irregular patterns of distribution
of anthocyanin pigment, similar irregulari-

DEPARTMENT OF GENETICS 417

ties were noted among the kernels. Some
kernels were uniformly pigmented, and in
most of them the intensity of pigmenta-
tion was low. Others were variegated, ex-
hibiting deeply pigmented spots in a color-
less background. Sometimes all the varie-
gated kernels on an ear exhibited similar
patterns with respect to size and number
of such spots, but on other ears wide dif-
ferences were observed among the varie-
gated kernels. Some had only a few small
specks of deep pigmentation, others had
many such specks; in some, a number of
medium-sized pigmented spots appeared,
and in still others there were relatively few
spots but most of them were large. Very
often, on an ear produced from a testcross,
the ratio of variegated to pale-colored ker-
nels, and also the proportions of different
kinds of variegated kernels, gave no evi-
dence of orderly meiotic segregation of
heritable elements that might be responsi-
ble for the appearance of the different
kinds of kernels. On some other ears, in
contrast, the ratio of kernel types was quite
consistent with an orderly segregation.
Frequently, the different ears produced by
one plant were not at all consistent in this
regard, even when the same pollen parent
had been used in making the cross to each
of the ears.

Confused impressions of the operation
of the system of control of gene expres-
sion at a2" also resulted from attempts to
analyze the constitution of plants derived
from the pale-colored kernels on ears
that segregated both pale-colored and varie-
gated kernels. Some of these plants had
variegated phenotypes. Others, however,
showed no evidence of variegation, the
anthocyanin pigment being uniform in
intensity and distribution throughout the
plant. Study of the progeny of these plants
led to further difficulties of interpretation.
When the ears of one such plant were
used in crosses with tester plants homo-
zygous for the stable recessive, ae, all the
pigment-containing kernels on each ear
of one plant might be uniformly pale
colored; whereas, in another plant, one ear
418 CARNEGIE INSTITUTION OF WASHINGTON

might have all uniformly colored kernels
and another ear might have also a few
variegated kernels. These variegated ker-
nels exhibited deeply pigmented spots in
a colorless background; the pattern of spots
might be the same among all, or might dif-
fer widely. On still another ear of the
same plant, a clear-cut Mendelian ratio of
pale to variegated kernels might appear.

That some system of control of gene ex-
pression was operating in the case of a2”
was clear, but evidence from the early
studies did not allow ready recognition of
its components. The above-described ex-
amples of irregular phenotypic expressions
and inheritance patterns observed in plants
carrying a class I state of a2”" may sug-
gest why this was so. Only when the be-
havior of the state of a2”* belonging to
the second class was being examined was
initial evidence obtained of the presence in
this system of a heritable element responsi-
ble for suppressing phenotypic expression
of a2”"*. In this aspect of its operation, it
resembled the Spm element previously dis-
covered in the a:”* cultures. We now
know that this resemblance is more than
coincidental. These elements are basically
alike, and it is altogether probable that
they arose from a common progenitor.
Each appears to represent a different state
of one controlling element. The following
discussions will consider the differences
between them, and will explain the con-
fusion experienced in earlier attempts to
interpret the operation of the system re-
sponsible for controlling gene expression at
a”,

After it was recognized that an Spm-type
element was involved in this system, the
Spm element extensively examined in the
a:"~* cultures was introduced into some
plants having one of the states of as”?
belonging to class I, and subsequently into
plants carrying other states of this class.
With this Spm element present, the mode
of control of gene expression was as
sharply revealed as it had previously been
in the case of ai", Moreover, some states
of a2”"* were seen to resemble some states

of a:""*. In the absence of Spm, both plants
and kernels having one of these class |
states of a2" were pigmented. The in.
tensity of anthocyanin pigmentation in
plants was rather high, whereas in kernels
it was always low; and the distribution of
pigment was uniform in both plant and
kernel. In the presence of Spm, no pig-
ment appeared except in those areas of
plant or kernel that arose from cells in
which mutation to or toward A: had oc-
curred. Such mutations occurred in some
germinal cells, and, as a consequence,
mutants could be isolated. They were
found to be stable in expression in sub-
sequent generations of plants, either in the
presence or in the absence of Spm.
Through the use of this Spm element,
it was learned that class I states of a”?
differ from one another in the manner
in which they control the time and fre-
quency of occurrence of mutation during
development of plant or kernel; and some
sharply expressed distinctions among them
were noted. These class I states of a:”"!
and the states of a"? are much alike.
The similarities include the appearance
of uniformly distributed anthocyanin pig-
ment in plant and kernel, and stability of
this phenotypic expression in successive
generations of plants, in the absence of
Spm; suppression of all visible evidence
of this kind of gene action in the presence
of Spm, and also control of mutation type
and pattern by the state when Spm is pres-
ent. The Spm element originally present
in the a2" cultures, on the other hand, did
not always effect suppression of gene action
in all parts of a plant or kernel. Mutations.
however, occurred only in those parts of
plant or kernel in which gene expression
was suppressed. The pattern of mutation
produced by any one state was not set, and
the types observed ranged from early-oc-
curring mutations, which resulted in the
appearance of large areas of mutant pheno-
type, to late-occurring mutations, which
produced only specks of deep pigmenta-
tion. Invariably the mutations were con-
fined to regions in which the background
phenotype was nonpigmented, that is, in
which the suppressive component of Spm
activity was evident.

It is now apparent that one of the major
difficulties in analyzing the control system
responsible for the many different pat-
terns of a2” gene expression was occa-
sioned by alternating phases of activity of
the Spm element in the a2" cultures. But
one other factor also contributed to the
confusion. Early in the study of a", a
new type of state appeared, whose expres-
sion was in great contrast to those observed
with other isolates of a". It was there-
fore set apart and designated as the class II
state of do”. By means of various kinds
of experiment with this state, it was first
learned that the Spm element in the a2”
cultures may undergo frequent changes in
activity during the development of a plant,
each such change affecting its capacity to
serve aS a suppressor-mutator. Clearly,
some regulatory mechanism controls the
time of occurrence of such changes, al-
though it is not yet understood. Such a
change may evidently occur within an in-
dividual cell during development, for well
defined sectors in a plant or kernel often
exhibit the result. The direction of change
may be from active to inactive, or from in-
active to active, and alternating cycles may
also be observed. The class II state of
a™+ readily reveals these changes in ac-
tion capacity of Spm, for, with this state,
the Spm element in its active phase serves
only to inhibit expression of gene action at
as™*, No evidence of mutation has yet
been obtained with this state, although
abundant evidence has been obtained with
the class I states. It is important to bear
this fact in mind in order to appreciate the
significance of the phenotypes arising from
the class II state, which will be considered
below. All the variegated patterns to be
described reflect only changes undergone
by the Spm element itself.

The class II state of a.™*. When Spm
is absent, or when an Spm element is pres-
ent in its inactive phase, the class ITI state

DEPARTMENT OF GENETICS — 419

of a2" produces a phenotype similar to
that given by standard 42. Both kernel
and plant are deeply pigmented. In a plant
that is a2" (class II)/a2 in constitution
and carries a single active Spm element
derived from the a.”* cultures, suppres-
sion of the effects of a2"? gene action is
not complete. Pigment is present, but it
is less intense than the pigment of plants
having no Spm or Spm in its inactive
phase. A change in Spm from an active
to an inactive phase during the develop-
ment of such a plant is made evident by
a well defined area of very deep pigmenta-
tion in a more lightly pigmented back-
ground. The positions of such areas in a
plant, their number, and their relative
sizes reveal both time and frequency of
occurrence of such changes in Spm activity
during development. If the Spm element
is inactive during early development, a
change from the inactive to the active
phase is represented by areas of lower pig-
ment intensity in the background of in-
tense pigmentation. In kernels, on the
other hand, this Spm element in its active
phase will suppress all expression of gene
action at @2""". Those parts of the kernel
in which it is active are totally colorless,
but other parts, where it is inactive, are
intensely pigmented. Changes in Spm ac-
tion phase may alternate, and both the
times and the types of change are revealed
in the kernel phenotypes. In kernels hav-
ing one Spm element, these alternating
changes may be observed readily. For ex-
ample, a large pigmented area may be
seen in an otherwise colorless region of a
kernel. Within this large pigmented area,
smaller colorless areas may be observed,
and within these, in turn, specks of deep
pigmentation. In this illustration, the se-
quence of changes of phase of Spm activity
during development of the kernel was
from active to inactive to active, and again
to inactive.

The dose of Spm can alter the patterns
of variegation produced by the class II
state, and this dose effect can be seen in
420 CARNEGIE INSTITUTION OF WASHINGTON

both plant and kernel. It is particularly
well expressed in the aleurone layer of the
endosperm of the kernel. The endosperm
is triploid and therefore is useful for ex-
amining the effects produced by various
doses of any chromosomal component. The
female gametophyte contributes two ge-
netically identical haploid nuclei to the pri-
mary endosperm nucleus, and the pollen
grain contributes one haploid nucleus. The
different patterns of variegation exhibited
by kernels having one, two, or three ac-
tive Spm elements provide a means of de-
termining whether a plant has no Spm
element or an Spm element in its inactive
phase. Before explaining this method of
differentiation, it is necessary to review
some of the tests that have been made with
the class II state of a27.

The first example concerns a plant that
carries this state and also carries one Spm
element, which was in its active phase in
the cells that produced a particular ear of
the plant. The endosperm of any kernel
appearing on that ear after pollination
should have received from the female par-
ent either no Spm or two active Spm ele-
ments; and the ratio of this distribution of
Spm among the kernels is expected to be
1 to 1. If the plant serving as pollen parent
for this ear is homozygous for az and has
no Spm element, half the a:”*-carrying
kernels produced by the cross should have
no Spm, and half should have two Spm
elements. Kernels of the former type
should be deeply and uniformly pig-
mented, and those of the latter type should
reveal the presence of the active Spm ele-
ments. In the many tests of this kind so
far conducted, the Spm-carrying kernels
have shown a number of rather small,
deeply pigmented spots in a colorless back-
ground, and the ratio of fully pigmented
kernels to variegated kernels has been 1
to 1. When the reciprocal cross is made, a
1-to-1 ratio of deeply pigmented to varie-
gated kernels again results. In this case,
however, the variegated kernels show a
number of large, deeply pigmented areas,
within which smaller, colorless areas often

appear. It has been learned from other
tests, one of which will be described below,
that this is the characteristic pattern of
variegation in kernels that start develop.
ment with one active Spm element in the
primary endosperm nucleus.

In a cross similar to that described above
except that Spm is absent in the ear-pro-
ducing parent, all the @.”"-carrying ker-
nels on the resulting ears should be deeply
and uniformly pigmented, for none of
them will have an Spm element. If, how-
ever, the pollen parent that is homozygous
for a2 has one active Spm element, half its
pollen grains should have no Spm and
half should have one Spm. Two types of
a:"-carrying kernels are expected to ap-
pear on an ear produced by this cross, and
they should be present in equal numbers.
Those with no Spm should be deeply and
uniformly pigmented, and those with one
Spm should be variegated. More than

‘fifty crosses of this kind were made, and

all the eats so produced had a 1-to-1 ratio
of uniformly pigmented to variegated ker-
nels. In all cases, the variegated kernels
exhibited pigmented spots in a colorless
background; many of these spots were
large. Within most of the large pigmented
areas, smaller colorless spots were present,
and some of these contained specks of
pigment.

If pollen from a plant homozygous for
a2 and carrying one active Spm element
is placed on the silks of an ear of a plant
homozygous for the class II state of a:"”
and carrying one Spm element that was
in its active phase in the cells that gave
rise to the ear, then four types of kernels
appear in equal proportions. One type 1s
fully pigmented (no Spm); another shows
many pigmented areas, many of them
large, in a colorless background (one
Spm); a third has pigmented spots, all
rather small, in a colorless background
(two Spm); and a fourth shows only
small specks of pigment in a colorless
background (three Spm). Higher doses of
Spm elements in the endosperm have been
produced by other kinds of testcrosses.
These tests have shown that kernels hav-
ing four Spm elements exhibit at the most
one or a few tiny specks of deep pigmenta-
tion; much more often they are totally
colorless. Kernels having more than four
Spm elements are all totally colorless.

The relation between pattern of varie-
gation and dose of Spm was confirmed
by tests in which the location of Spm in
the chromosome complement was known
because of its linkage with some genetic
marker. The distribution of the linked
marker among the various categories of
variegated kernels was that expected in
the event of a direct relation between pat-
tern of variegation and dose of Spm; an
illustration will be given later. Confirma-
tion was also obtained in tests of the Spm
constitution of plants derived from the
variegated kernels of each category.

As was mentioned earlier, the relation
between pattern of variegation and dose
of Spm has made it possible to determine
whether a plant whose phenotype exhibits
no evidence of Spm actually has no Spm or
carries an Spm element in an inactive
phase. This procedure will now be de-
scribed.

Among a2™*-carrying plants, those hav-
ing an inactive Spm element and those
having no Spm element may be pheno-
typically the same. Spm may be present
although no evidence of it appears in any
part of the plant. In other plants, there
is no sign of the presence of Spm in the
main stalk but one or more of the tillers
have sectors of the phenotype produced
by an active Spm element. Several kinds
of test may be employed to determine
whether or not Spm is present in a plant,
or parts of a plant, where it is not re-
vealed phenotypically. The most significant
of these was formulated after it was dis-
covered that the introduction of an active
Spm element into a nucleus carrying inac-
tive Spm elements will result in activation
of the latter. In this test, ears for crosses
are selected in plants showing no evidence
at all of Spm, or in parts of a plant that

DEPARTMENT OF GENETICS 421

show no evidence of Spm although it is
known to be present in other parts of the
plant. When possible, the first and second
ears produced by the main stalk are used.

The silks of one ear of a plant receive
pollen from a plant that is homozygous
for a2, and for other recessive markers if
these are useful for the test, but that carries
no Spm. If Spm is absent in the female
parent, or if it is present but was in its in-
active phase in the cells that gave rise to
the ear used in the cross, all the a”"*-
carrying kernels on this ear will be deeply
and uniformly pigmented. Silks of a sec-
ond ear of the same plant receive pollen
from a plant of the same constitution as
the first pollen parent except that it carries
one active Spm element. Half its pollen
grains will carry an active Spm element,
and half will have none. If Spm is absent
in the female parent, two types of a2”?-
carrying kernels will appear on the second
test ear, in equal frequencies. One type
will have deep pigmentation, uniformly
distributed over the aleurone layer (no
Spm). The other type will be variegated,
with a colorless background and many pig-
mented areas in a pattern that character-
istically appears when only one active Spm
is present. If the ear-producing parent
carries an inactive Spm element, however,
three instead of two types of kernel will
appear on the ear produced by this second
cross. Half the kernels will be deeply
and uniformly pigmented; the other half
will be variegated, and will fall into two
sharply defined subclasses with equal num-
bers of kernels in each. One subclass will
show the heavily variegated pattern char-
acteristically produced when one active
Spm element is present, and the other will
show the pattern produced when three ac-
tive Spm elements are present. All the
variegated kernels receive one active Spm
element from the pollen parent. The dif
ferences in pattern among them discrimi-
nate between those that receive no Spm
from the female parent (the one-Spm pat-
tern) and those that receive two inactive
422

Spm elements from the female parent (the
three-Spm pattern). The three-Spm pat-
tern appears because the two inactive Spm
elements derived from the female parent
are activated by the Spm derived from the
male parent. Among the fully pigmented
kernels, half receive no Spm from the fe-
male parent and half receive the two inac-
tive Spm elements. In the latter category,
the Spm elements remain inactive during
the development of the kernel, for no
active Spm element is introduced into the
primary endosperm nucleus by the male
parent to trigger them into activity.

Evidence to confirm the above analyses
of Spm constitution among kernels of dif-
ferent types was derived from tests of
a2""*-carrying plants in which one inactive
Spm element was present and was located
near wx in one of the two chromosomes 9;
the homologous chromosome 9 carried
Wx. The results of tests conducted with
one plant may be examined for illustrative
purposes. Two ears of a plant that was
ao” (class 11) Bt/as bt, Wx +/wx Spm
(inactive) were crossed by a plant homo-
zygous for a2, bt, and wx and carrying no
Spm element. (The locus of Bz, normal
endosperm, and its recessive allele, b2,
brittle endosperm, is close to the centro-
mere in the long arm of chromosome 5,
and it undergoes approximately 6 per cent
crossing over with the locus of a2”* in the
short arm of that chromosome.) None of
the a2”"*-carrying kernels on these two ears
was variegated. All the kernels were deeply
and uniformly pigmented, and the ratio
of Wx to wx among them was | to 1.

A third ear of this plant was crossed by
a plant that, again, was homozygous for
a2, bt, and wx, but had one active Spm
element. (Twenty-eight testcrosses had
been made to determine the presence and
activity of the Spm element in this pollen
parent. Among 5997 kernels produced in
these tests, 3041 had received no Spm ele-
ment from the pollen parent and 2956 had
received an active Spm element. It could
therefore be assumed that half the pollen

CARNEGIE INSTITUTION OF WASHINGTON

grains of this plant had no Spm and half
carried an active Spm element.) When
pollen of this plant was used on the third
ear of the plant under consideration, 468
kernels were produced. Among them, 245
were completely colorless—34 Br (18 Wr:
16 wx) and 211 dt (105 Wx :106 wx),
Undoubtedly, these were homozygous for
the stable recessive, a2. The remaining 223
kernels (195 Bz :28 St) had pigment and
so had received a2" from the female
parent. Among them, 114 were deeply and
uniformly pigmented (57 Wx :57 wx);
the other 109 were all variegated, with pig-
mented areas in a colorless background.
In 57 of these, the pattern of variegation
was characteristic of the presence of one
Spm; 45 of them were Wx and 12 were
wx. The other 52 variegated kernels had
only specks of pigment in a colorless back-
ground, the characteristic pattern produced
when three Spm elements are present; only
9 of these were Wx, whereas 43 were wx.

The ratio of kernel types on this third
ear, and the relation between pattern type
and the alleles of Wx among the variegated
kernels, indicated that the female parent
had some component in the wx-carrying
chromosome, and closely linked with «ax.
that was responsible for change of pattern
from a one-Spm type to a three-Spnr type.
That this was the Spm element in its
inactive phase was indicated by two kinds
of test. One was conducted with sib plants
and with related plants in which an Spm
element was obviously present and active.
These plants were either a2”"* Bt/a:”* Bt
or a2" Bt/a. bt, and all were Wx/we.
The silks of ears of these plants received
pollen from plants homozygous for a2, 2.
and wx but having no Spm; and this set
of testcrosses produced a total of 3007 a2"""-
carrying kernels. In 1538 of them, the
aleurone layer was deeply and uniformly
pigmented; 1345 of these were Wx and 193
were wx. The remaining 1469 kernels were
variegated, with pigmented areas in a color-
less background; 166 were Wx and 1305
were wx. Relatively close linkage of Sp”
with wx was demonstrated by the ratios
of kernels on each of the ears.

The second kind of test confirming the
presence of an Spm element in its inactive
phase was much more direct. It was made
with sib plants of the one considered above.
In them, the phenotype of the main stalk
showed no signs of Spm, but one of the
tillers gave obvious evidence of its presence,
in an active phase. The two kinds of cross
described above were conducted with ears
of the main stalk on such plants. The re-
sults in each case were similar to those
described: no evidence of Spm when the
pollen parent contributed no Spm, and evi-
dence of an inactive Spm, closely linked to
wx, when the pollen parent contributed
Spm. The presence in the main stalk of
an inactive Spm element, closely. linked
with wx, was inferred. Confirmation came
from tests of the constitution of tiller ears
of such plants. They were used in crosses
with a plant homozygous for az, bt, and
wx, in which no Spm was present. Segre-
gation of kernel types on these ears clearly
indicated that the Spm element in the tiller
that produced each of them was located
close to wx in one chromosome 9. There
appears to be little doubt, then, of the ef-
fectiveness of the described tests in re-
vealing whether Spm is absent in a plant
or is present in its inactive phase. The
tests have also shown that a change in
phase of Spm is not associated with a
detectable change in its location in the
chromosome complement. Evidence of
transposition of the Spm element has been
obtained; but the observed changes in loca-
tion have not altered the expression of its
cyclically occurring changes in action
phase.

Tests of Spm activity in plants having
class I states of a2"-'. The deeply pigmented
areas in a colorless background observed
in kernels having the class II state of a2”
and an active Spm element are an expres-
sion of events that affect only the Spm
element. They do not express mutations
to stable 42, as do the deeply pigmented
areas that appear in kernels having a class

DEPARTMENT OF GENETICS 423

I state of a2". Therefore, the class II
state has been particularly useful in eluci-
dating changes in phase of activity of the
Spm element, as described in the previous
section. Nevertheless, similar analyses may
be conducted with the class I states of
a2” if attention is focused on the patterns
of palely pigmented areas that may also
be present in kernels exhibiting the deeply
pigmented spots that represent mutation-
producing events at a2".

As was stated earlier, kernels carrying

class I states of a2”-' have uniformly pale
pigmentation if Spm is absent. The same
phenotype will appear if Spm is present
but in an inactive phase. Thus, the pale-
pigmented phenotype in the kernels carry-
ing the class I states of a2”™ is the counter-
part of the deeply pigmented phenotype in
kernels having the class II state. The rela-
tion of pattern of these pale-pigmented
areas to dose of active Spm elements is the
same as that of the deeply pigmented areas.
By observing these patterns in kernels hav-
ing a class I state, therefore, it has been
possible to conduct the same kinds of test
as those described above, to distinguish be-
tween absence of Spm and its presence in
an inactive phase.
‘In kernels having a class I state, then,
two distinctly different types of variegated
pattern appear: deeply pigmented areas,
arising from mutation, which are solidly
pigmented throughout; and pale areas,
which reflect inactivations of Spm. When
a pale area is large (one-Spm pattern) it
usually contains smaller colorless areas
within it, and some of these, in turn, may
exhibit small specks of either the mutant
phenotype or the pale phenotype. Mutant
spots, it has been noted, always appear in
a part of the kernel where Spm has been
active in ancestor cells; they do not ap-
pear in the pale areas. In other words,
mutations at a2”? occur only in those cells
in which Spm is in its active phase.

Examination of kernels carrying the
class I states has also shown that the size
of mutant spots is always small if activity
of the Spm element commences late in
424 CARNEGIE INSTITUTION OF WASHINGTON

development. Thus, if a change in Spm
from an inactive to an active phase is de-
layed until late in the development of a
tissue, only small spots of mutant pheno-
type will appear in the areas in which Spm
is active, even when the state that is pres-
ent is known to be one that would produce
many large areas of mutant phenotype, as
a result of early-occurring mutations, if
the Spm element were in its active phase
during early development. Therefore, the
size of a mutant spot produced by class I
states of a2™* is conditioned not only by
the particular state itself but also by the
timing of change in phase of activity of
the Spm element.

Knowledge of the activity cycles of the
Spm element in the a." cultures has
clarified many of the bewildering aspects
of a." behavior encountered early in its
study. Its seemingly disorderly patterns of
gene expression and apparently unorthodox
types of inheritance behavior can now be
interpreted and no longer give cause for
confusion. The conditions responsible for
change in activity of Spm are not yet un-
derstood, but studies are under way that
may help elucidate them.

Before leaving this discussion, it may be
useful to point out the resemblance be-
tween the patterns of pigmented spots,
arising from inactivations of Spm, that
appear with different doses of Spm and
those that are produced by different doses
of Ac. In the former case, the pattern is
produced by change in phase of activity
of Spm, whereas in the latter case it is pro-
duced by mutation-inducing events insti-
gated by Ds, the complementary compo-
nent of the Ds-Ac system. The resemblance
of the two systems with respect to pattern
type and dose effect is so great that it
suggests some basic similarity in mode of
operation.

A Modifier Element in the a:"*-Spm
System

When the standard Spm element of the

a,""* cultures is present, mutations occur

at the a." locus in plants and kernels; the
size and number of mutant areas reflect
both the time of occurrence of mutation
during development and the number of
cells in which it takes place. As described
earlier, the different states of a:”7 are
recognized by the distinctive patterns of
mutation they produce; and the expression
of a state of a1” is not altered by dose of
Spm, for the same pattern appears when
one or more standard Spm elements are
present.

During the course of study of a par-
ticular state of a:" 7, a single kernel ap-
pearing on one ear of a plant reflected a
mutation frequency much higher than that
expected of the state that was present in
the plant. The plant grown from this ker-
nel likewise exhibited a marked increase
in mutation frequency. From testcrosses
of this plant and examination of the prog-
eny it became evident that a modifier ele-
ment, independently located in the chro-
mosome complement, was responsible for
the increase in mutation frequency, and
that the action of the modifier was ex-
pressed only in kernels and plants that
also carried Spm. Further examination re-
vealed that this modifier element under-
went transposition. Preliminary findings
about the modifier element were given in
Year Book 56; and the investigation was
continued this year, as reported below.

Effects of the modifier element on the
expression of four different states of ai”
were examined. When Spm is present,
without the modifier, one of these states
gives rise to a few, relatively late-occurring
mutations; a second state produces more
mutations, most of which occur relatively
late in development; a third gives very
many mutations, all occurring late in de-
velopment; and the fourth produces many
mutations, some of which occur early in
development. It was found that the modi-
fier alters the mutation patterns of only
the first two of these four states; it does
not change the patterns associated with the
other two. In the two affected states, an
increase in frequency of mutations when
the modifier is present is expressed in a
stepwise manner.

The first-mentioncd state, which gives
relatively few mutations in the absence of
the modifier, gives many more in its pres-
ence. The mutation pattern so produced
resembles that of the second state when
the modifier is absent. In turn, the effect
of the modifier on the second state is to
increase the mutation frequency so that
the pattern of mutations resembles that of
the third state described above.

In the testcrosses made with the first
plant in which the modifier appeared, no
linkage of the modifier with genetic mark-
ers in chromosomes 3, 5, or 9 was detected.
In the progeny of this plant, however, it
was early discovered that parts of some
plants lacked the modifier and that other
plants, or parts of plants, carried different
numbers of modifier elements. Tests were
conducted with a number of these plants
in order to detect changes in location of
the modifier that might place it in one of
the three above-named chromosomes. It
was detected in chromosome 3 in one
plant, and in chromosome 9 in another.
Tests were then carried out with some of
the progeny of each of these two plants,
to determine the constancy of location of
the modifier.

One of the testcrosses made with a plant
whose constitution was a:"* Sh2/a she
suggested that a modifier element was
located in its a1 sh2-carrying chromosome 3.
The state of a1"? in this plant was the
second of the four described above. In
another test, pollen of this plant was placed
on the silks of a plant homozygous for the
second state of a:™* and for Sh2 but carry-
ing no Spm or modifier element. This
cross produced 356 kernels, classified as
follows: 1 deeply and uniformly pig-
mented (germinal mutation); 206 unt-
formly pale colored (no Spm); 65 varie-
gated, with a number of small, deeply
pigmented spots in a colorless background
(Spm, no modifier); and 84 variegated,

DEPARTMENT OF GENETICS 425

with very many more mutant spots (Spm
and modifier).

The silks of ears produced by 11 plants
derived from kernels in the last category
received pollen from plants homozygous
for the second-described state of a.™* and
for sha but having no Spm or modifier
element. Eight of these 11 plants were
a" Sh2/a: she in constitution, and the
other 3 were homozygous for a™* and
Shs. The modifier element was present in
all 8 plants having the former constitu-
tion; some plants had one modifier ele-
ment and others had two, but each carried
4 modifier element in the a: shz chromo-
some 3. In the cells that gave rise to the
tested ears of 5 of these 8 plants, a single
modifier element was present. A single
Spm element was also present in these 5
plants, and was inherited independently of
the modifier element (table 9, A). The

TABLE 9. Spm and Modifier Constitution of
Different Plants of a Culture, as Determined
by the Cross a,%1 Sho/a, shy, Spm, modi-
fier 9 Xa,"1 shp/a,"" shz, no Spm, no
modifier

 

 

* Spm No. of Ears
number . Plaot
Group A
ai™-l Sho +/a1 sha Mod 1 2 t
4 3
5 1
7 1
8 L
Group B
ai™-1 Sho +-/a1 she Mod, 1 1 1
plus independently 3 1
located modifier
Group C
Same as B 2 6 2

 

cells that produced the tested ears of 3 other
plants had two modifier elements; one was
located in the a: she-carrying chromosome,
and the location of the other was not de-
termined. The cells that gave rise to the
tested ears of two of these plants contained
a single Spm element (table 9, B), and
those that produced the two tested cars of
the third plant had two independently lo-
cated Spm elements (table 9, C). The fre-
quencies of the different types of kernels
on the testcross ears, which revealed these
426 CARNEGIE INSTITUTION OF WASHINGTON

three types of constitution, are given in
table 10. In A of table 10, the percentage
of recombination between sh» and the
modifier is 172. This value need not
represent the percentage of crossing over
between sz and the modifier, for some of
the kernels in the recombinant classes prob-
ably reflect the consequence of independent
meiotic distribution of the modifier and
sh» in some sporocytes in which the modi-
fier had been transposed to a new location
in an ancestor cell.

The modifier element located in chro-
mosome 9 was detected when an ear of
a plant of the constitution a1" Sho/ai sho,

in either chromosome 3 or chromosome 9.
A modifier was present, located in the Wx.
carrying chromosome, in at least some part
of 6 of these plants. In the 7th (plant 5, table
11), the results of the testcross, made with
the ear of the main stalk and two tiller
ears, indicated that the cells producing
each of these three ears had one modifier
element, but that it was not located ip
chromosome 9, or, at any rate, not close
enough to Wx to show evidence of link.
age with it.

A similar situation was found with te.
spect to the ear of the main stalk in plant 1,
table 11, although tests of the tiller ear of

TABLE 10. Types of Kernels Appearing on Ears Produced by Plants Whose Constitutions
Are Entered in A, B, and C of Table 9

 

Phenotype of Kernel

Spots of Deep Pigmentation

 

 

 

Deeply Uniformly in Colorless Background
Consti- Pigmented Pale (Spm Present)
tution (Germinal Colored Totals
Mutation) (No Spm) Few Spots Many Spots .
————_—. ——_——_ (No Mod) (Mod)
She shy Shy Shy —_—_—
Sh, shy Shy shy
A 15 0 531 548 479 107 92 476 2248
B 5 0 193 166 67 21 89 141 682
Cc 4 0 78 89 95 21 112 192 591

 

Wx/wx was used in a cross with a plant
homozygous for a:"7, Shs, and wx and
having no Spm or modifier element. The
cells that gave rise to this ear had one Spm
and also one modifier, the latter located
in the Wx-carrying chromosome. Kernels
from this ear that were Wx and showed
the presence of both Spm and the modifier
were used to grow 8 plants, which were
-tested for Spm number and for modifier
number and location by means of crosses
with plants homozygous for a:""", shz, and
wx and having no Spm or modifier ele-
ment. One of the 8 plants proved to have no
Spm element, and therefore the presence of
the modifier could not be detected by this
cross. In all examined parts of the other 7
plants, however, one Spm element was
present, and it was not linked with markers

the plant and also of the pollen of this
tiller indicated that the modifier was
linked with Wx in at least that part of the
plant. In a tiller ear of plant 6, no modifier
element was present; and absence of the
modifier was also observed in part of a
tiller ear of plant 2. In plant 2, the cells
that gave rise to the main ear carried two
modifier elements, one of which was
linked with Wx. The distribution of ker-
nel types derived from the testcrosses of
plants 1 to 7 is given in table 12. Linkage
of the modifier element with Wx is clearly
expressed by the data of parts A and B of
the table; but absence of such linkage 1s
evident from the results of the tests of the
three ears produced by plant 5 (part C).
The types of kernels appearing on the
tiller ear of plant 2 are recorded in part E.
DEPARTMENT OF GENETICS = 427

TABLE 11. Number and Location of Modifier Elements in the Progeny of a Plant
Having One Modifier Element Linked with Wx in Chromosome 9

 

Modifier Elements in Different Parts of Individual Plants

 

 

Plant B Cc
No. A Wx Mod/wx-+;1Mod Wx/wx;1Mod W i. .
Wx Mod/wx + Independently Not Linked No i a
Located with Wx
1 Ear of tiller Main ear
Pollen of tiller
2 Tiller ear * Main ear
3 Main ear
Tiller ear
4 Main ear
5 Main ear
Ears of 2
tillers
6 Main ear Tiller ear
Pollen of main stalk
7 Main ear
Tiller ear

 

* A sector of this ear had no modifier element. See part E, table 12.

TABLE 12. Types of Kernels Appearing on Ears Produced by Plants Whose
Constitutions Are Entered in Columns A to D, Table 11

 

 

 

Phenotype of Kernels
we Spots of Deep Color in
on Parent- Deeply Pale Colorless Background
Shownin age in (Gonna Colored (Spm Present) Totals
Se Cross Mutation) (No Spm) Few Spots Many Spots
olu a (No Mod) (Mod)
Wx wx Wx wx ee —_—
Wx wx Wx wx
A g 14 «17 498 490 150 388 364-109 2030
é 18 «#617 354 = 330 125-241 238 69 1392
B g 6 5 77 89 6 31 90 48 352
Cc 2 2 5 215 229 99 119 98 102 869
D g 0 2 77 55 56 62 0 0 252
E* g 3 0 48 63 15 34 35 23 221
Et 0 1 17 19 29 22 0 0 88

 

* Tiller ear of plant 2; modifier present.
+Sector with modifier absent.
428 CARNEGIE INSTITUTION OF WASHINGTON

In one sector of this ear the modifier was
absent. The cells producing the remainder
of the ear carried a single modifier ele-
ment, which appears to have been in the
Wx-carrying chromosome, although the
data are few and the expression of linkage
is less marked than in the results entered
in part A of the table.

The evidence outlined above, together
with that obtained in other studies of the
modifier, indicates that this element, like
the elements Spm, Ds, Ac, and so forth,
may undergo transposition from one loca-
tion to another in the chromosome com-
plement, at a time either early or late in
the development of a plant. Another find-
ing of the past year is that the expression
of the modifier is the same whether the
standard Spm or Spm-w is present. (For
differences in effect produced by these two
states of Spm, see Year Book 56.) In the
absence of the modifier, on the other hand,
the distinct pattern of mutation at a:™*
effected by each of these two states of Spm
is clearly expressed. The difference may
be observed among the kernels on ears of
plants carrying both states of Spm as well
as the modifier, when such ears are used
in the testcross described above. Those
kernels having one or the other state of
Spm but no modifier are readily distin-
guished from each other, whereas kernels
with either state of Spm and also the
modifier cannot be distinguished from one
another. Thus, the modifier element ap-
pears to be a component of the Spm system
that can modify the expression both of a
state of a."7 and of the Spm element.

Continued Investigation of Transposition
of Spm

A report was given in Year Book 56 of
two progeny tests made to determine the
time and frequency of transposition of the
standard Spm element carried in the a:.”7
cultures. The ear-producing parent in one
of these tests had one Spm, located in one
of its chromosomes 9. The numbers of

Spm elements present in different plants
of the progeny, and in different parts of
a single plant, and also the location of Spm
with reference to the marker Wx, carried
in chromosome 9, were summarized ip
table 6, page 394, Year Book 56. The tests
reported there were extended this year,
with plants derived from variegated, Wx
kernels on five of the ears recorded in
that table: the second and tiller ears of
plant 7285A-1, a tiller ear of plant A-2, a
tiller ear of plant A-7, and the main ear
of plant B-6. The cells that gave rise to
four of these ears had one Spm element,
located in the Wx-carrying chromosome 9,
The cells that gave rise to the fifth ear
(tiller ear of plant A-1) carried one Spm;
but it was not linked with Wx, even
though testcrosses of both the first and
second ears of the main stalk of this plant
showed linkage of Spm with Wx. The
plants studied last year provided a num-
ber of examples of transposition of Spm
occurring early in development. The
tests conducted this year were made for
two purposes: first, to learn whether or
not early-occurring transposition of Spm
would continue to be expressed in the
progeny plants; and second, to learn
whether or not the transposition of Spm
away from a known location in chromo-
some 9 might be followed later by transpo-
sition back-to its former location.

To carry out the first of these purposes,
Spm constitution and location were de-
termined in 37 progeny plants. Each plant
was grown from a variegated, Wx kernel
on an ear produced from cells in which
Spm had been located in the Wx-carrying
chromosome 9. Twelve plants were de-
rived from one ear, 8 plants each from
two other ears, and 9 plants from a fourth
ear. Table 13 summarizes the results of
these tests, which have been incorporated
into a single table because early-occurring
transposition of Spm was observed among
the plants of all four cultures. It may be
seen from the table that early-occurring
transposition of Spm, evident in the parent
plants, was likewise exhibited in their
progeny.

To determine whether or not sequential
transpositions of Spm may be directed in
such a way that removal of Spm from a

DEPARTMENT OF GENETICS 429

mentioned above. In this tiller, the Spm
element had been transposed away from
a location close to Wx in chromosome 9.
‘Twenty-one ears in all were obtained from
these 11 plants. Among them, there were

TABLE 13. Spm Constitution and Location in Different Plants and in Different
Parts of Individual Plants

 

 

No. of Ears Position of No. of Spm Constitution No. of Plants
Tested per E vane ° i. “0 and Linkage with Given
Plant ar in Plant Plants with Wx Constitution
1 Ist ear, main stalk 13 1 Spm, linked with Wx 9*
2 Spm, neither linked with Wx 3
No Spm 1
2 1st and 2nd ear, main stalk 1 1 Spm, linked with Wx 1
2 Ist ear, main stalk 15 1 Spm, linked with Wx, in both
Tiller ear : ears 10
1 Spm, linked with Wz, in Ist
ear; 1 Spm, not linked with
Wx, in tiller 1
1 Spm, linked with Wx, in Ist
ear; 2 Spm, one linked with
We, in tiller 1
2 Spm, one linked with Wx, in
both ears 1
1 Spm, not linked with We, in
both ears 2
3 1st and 2nd ear, main stalk 6 1 Spm, linked with Wz, in all
Tiller ear three ears 3
1 Spm, linked with Wx, in Ist
and 2nd ears, main stalk;
2 Spm, neither linked with
We, in tiller 1
2 Spm, one linked with Wx, in
Ist ear; 1 Spm, linked with
Wx, in 2nd ear; no Spm in
tiller 1
2 Spm, one linked with Wx, the
other linked with Pr, in all
three ears 1
3 1st ear, main stalk 2 1 Spm, linked with Wx, in all
Ear on each of two tillers three ears 2

 

 

* One ear with sector in which Spm was absent.

known location in chromosome 9 may be
followed by a return to that location, tests
of Spm number and location in different
plants and different parts of the same plant
were conducted with 11 plants derived
from the variegated, Wx category of ker-
nels on the tiller ear of plant 7285A-1,

six certain cases of successive transpositions
of Spm; but in none of the ears was Spm
found to be linked with Wx. Although
this test is limited, it does indicate that
a directed type of transposition of Spm,
which returns it to the location it previ-
ously occupied, does not occur regularly.
430

CARNEGIE INSTITUTION OF WASHINGTON

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Campbell, A. Effect of starvation for glucose
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Campbell, A. Synchronization of cell division.
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Campbell, A. Transduction and segregation in
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Campbell, A. The different kinds of transduc-
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Clowes, R. C. Investigation of the genetics of
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Clowes, R. C, Nutritional studies of cysteineless
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Demerec, M. Structure and arrangement of
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PERSONNEL
Year Ended August 31, 1958

Balbinder, Elias, Associate in Research

Balbinder, Evelyn (Mrs.), Research Assistant

*Banié, Stanko, Fellow of Boris Kidrich
Foundation, Yugoslavia

Bennett, James L., Maintenance Man

Buchanan, Jennie S. (Mrs.), Research ‘As-
sistant

Burtch, Barbara J. (Mrs.), Stenographer

*Campbell, Allan M., Associate in Research

Carley, Catherine, Switchboard Operator
and Computer

Catcheside, David G., Carnegie Institution
Fellow

Datta, Mihir Kumar, Research Assistant

Demerec, M., Director

Fisher, Agnes C., Secretary to Director

* Resigned during the year.

*Fredericq, Pierre, Fellow of the Belgian
American Educational Foundation, Inc.
Gay, Helen, Associate in Research
Glanville, Edward, Research Assistant
*Goldman, Irving, Research Assistant
*Gross, Julian D., Research Assistant
*Haggerty, Michael J., Maintenance Man
*Hashimoto, Kazuo, Carnegie Institution Fel-
low
Hershey, Alfred D., Microbiologist
Jones, Henry H., Photographer
Karossa, Judith, Research Assistant
Kaufmann, Berwind P., Cytogeneticist
Koch, Gebhard, Associate in Research
Lahr, Ernest L., Associate in Research
McClintock, Barbara, Cytogeneticist
McDonald, Joseph L., Janitor
McDonald, Margaret R., Chemist