CYTOGENETIC STUDIES OF MAIZE AND NEUROSPORA

Barsara McCuintoce

THe Mourtasre Ds Locus in Marze

General considerations. In last year’s re-
port a summary account was given of
several newly arising unstable gene loci.
The instability of all but one of these loci
was phenotypically expressed by ‘the ap-
pearance in an otherwise recessive plant
of sharply defined sectors of dominant
tissue or of tissue showing an intermediate
condition between recessive and dominant.
Each of these sectors arose following a mu-
tation in the unstable locus occurring in an
individual cell during the development of
the tissue. When an unstable locus is
Present, the tissues of the plant show a
pattern of variegation which is related to
the time and frequency of mutations oc-
curring in particular cells during the de-
velopment of the tissue. Observations of
the behavior of the unstable loci have sug-
gested that a common underlying phe-
nomenon is associated with the expression
of instability in all the cases examined.
Several generalizations may be formulated
concerning this phenomenon. Two sepa-
rable factors are known to be associated
with the expression of instability. The first
factor is concerned with the particular state
of the unstable locus in the cells of a de-
veloping tissue. The state of a locus is

reflected by the time of occurrence of
phenotypically visible mutations and by
the frequency and distribution of these
mutations. The second factor is concerned
with the mutation at the unstable locus
that gives rise to the phenotypically recog-
nizable altered expression of the locus.
During the development of a tissue, the
state of a locus may remain unchanged.
This results in a tissue showing one par-
ticular and readily recognizable type of
variegation pattern. Changes in the state
of a locus may, however, occur. These
changes arise abruptly and appear to be
associated with an event that occurs during
a mitotic cycle. Following such a change,
the variegation pattern is altered in the
descendants of this cell. There may be
fewer or, conversely, more mutations in the
descendent cells than would have occurred
had the event that gives rise to a change
in state not taken place. The evidence
suggests that the change in state may be
related to the reproductive cycle of the
chromosome, for it has frequently been
observed that when a change occurs the
state of the mutable locus in each sister
chromatid may become altered. The state
of the mutable locus may be quite different
in the two chromatids, and the state in
each chromatid, in turn, different from
DEPARTMENT OF GENETICS

that which existed in the immediate
mother cell. In brief, it is the state of the
locus at a particular stage in development
that determines the time and rate of future
mutations, and this state may be altered
by an event occurring at a mitosis often
considerably in advance of the genotypic
mutation itself.

During the past year attention has been
concentrated on one of the mutable loci,
because its action and its location are par-
ticularly favorable for an analysis of the
factors associated with mutability. Further-
more, the type of action at this locus is
unique in its cytogenetic aspects and of
considerable general interest in this respect
alone. In this one case, mutability is ex-
pressed not by a visible phenotypic change
in the action of a gene, but rather by dis-
sociation of the bonds that normally would
maintain a linear cohesiveness of this locus
with an adjacent locus in the chromosome.
As an ultimate consequence of the muta-
tion, the chromosome is dissociated into
two completely detached segments. Be-
cause one of these segments is acentric, it
is not capable of directed movement in the
spindle figure and subsequently is lost to
the nuclei of descendent cells. This mu-
table locus has been designated Ds because
the most readily recognizable consequence
of its action is this dissociation. By both
cytological and genetic methods, the Ds
locus has been placed in chromosome 9g at
approximately the position that demarcates
the proximal third of the short arm. The
acentric segment that is produced as a con-
sequence of a dissociation mutation is
composed, then, of the distal two-thirds of
the short arm. This segment contains the
loci of most of the known mutants of
chromosome 9. Collectively, these mutants
affect characters of the pollen, the endo-
sperm of the kernel, the seedling, and the
mature plant. Consequently, dissociation
mutations at the Ds locus may be traced

147

by genetic analyses in all stages of the life
cycle when a plant carries dominant alleles
and Ds in one chromosome g and recessive
alleles and a normal ds locus in the homol-
ogous chromosome 9. Whenever a disso-
ciation mutation occurs in a cell during the
development of a tissue of such a plant, an
acentric segment carrying the dominant
factors is produced. This acentric segment
is subsequently lost from the nucleus dur-
ing a mitotic cycle. The result is a nucleus
having only the recessive alleles that are
present in the homologous segment of the
ds-carrying chromosome g. All the cells
arising from this cell will be recessive in
genotype and also in phenotype, if the
expression of the particular recessive factor
is cell-specific and if this phenotypic ex-
pression is not subject to changes that may
be caused by diffusible gene products from
the adjacent dominant cells. The presence
of a recessive sector in a mature tissue in-
dicates that a dissociation mutation oc-
curred in the ancestor cell that gave rise
to this sector. In plants of the given consti-
tution, therefore, the mature tissues can
be expected to show variegation for reces-
sive sectors. From the size, frequency, and
distribution of these recessive sectors in
any one tissue the state of the locus in this
particular tissue or sector of tissue can be
recognized.

Control of Ds activity by Ac. Accumu-
lating evidence indicates that the Ds locus
will undergo dissociation mutations only
when a particular dominant factor is
present. This factor is designated Ac be-
cause it activates Ds. Ae is probably lo-
cated in the long arm of chromosome 9,
but its exact position has not been deter-
mined. By the end of this growing season,
the analysis of the action of de on the Ds
locus should be well advanced. At the
present time the evidence suggests the fol-
lowing relations between Ac and Ds. If
Ac is not present, the Ds locus is com-
148

pletely normal in behavior and indis-
tinguishable from ds. If, by appropriate
crosses, however, Ac is introduced into
the primary endosperm nucleus, the Ds
locus again becomes mutable and disso-
ciations may begin to occur shortly after
the introduction of Ac. Ac will not affect
a normal ds locus, however; in the pres-
ence of Ac, ds remains stable.

Cytological aspects of the action of the
Ds locus. Genetic evidence indicated that
the dissociation mutations take place rela-
tively late in the development of the
sporophytic tissues. This was confirmed
by cytological observations of the sporo-
cytes of Ds Ac plants. In the sporogenous
cells of the anthers of the majority of
plants examined, the dissociation muta-
tions—recognized by the constitution of
the chromosome g bivalent—most_ fre-
quently occurred in a late-premeiotic ny-
cleus or sometimes in the meiotic nucleus
itself. Genetic and cytological evidence also
indicates that dissociation mutations may
be delayed throughout the period of
meiosis and only begin to take place in
the following gametophytic nuclei. In
some plants, however, dissociation muta-
tions were observed to have occurred in
relatively young premeiotic nuclei. The
relation of this variable timing of dissocia-
tion mutations to the particular state of the
Ds locus will be discussed later.

In making preparations of the sporo-
cytes with the usual aceto-carmine staining
techniques, considerable difficulty was en-
countered in obtaining an adequate num-
ber of well spread and sharply stained
meiotic prophase figures. Consequently, it
was Necessary to attempt an improvement
in the techniques. Methods that had been
developed in the fall of 1946 for the study
of meiotic prophase chromosomes of the
fungus Neurospora were tried, and found
to be likewise superior for similar stages
in maize. These methods introduce the

CARNEGIE INSTITUTION OF WASHINGTON

use of lactic acid, either in the fixing fluid
or in the staining solution. Young anthers
were fixed for 12 to 24 hours in a fresh
mixture of four parts of 95 per cent alcohol
to one part of lactic acid. The sporocytes
in the meiotic prophase states were forced
out of the anther in a drop of aceto-orcein;
a cover slip was placed over the drop, and
the slide gently heated. An unusually
sharp differential stain resulted. The cyto-
plasm was only slightly stained; the
chromosomes, in contrast, were brilliantly
stained, and the centromeres were sharply
delimited in each chromosome. Consider-
able stretching of the chromosomes some-
times occurred, however, during the flat-
tening of the sporocytes. When equal
parts of lactic acid and acetic acid were
used in the fixative in place of lactic acid
alone, the chromosomes stained sharply
with aceto-orcein but were less subject to
stretching during the flattening of the
sporocytes. A third method involved the
restaining of aceto-carmine preparations
with an orcein stain consisting of 1 per cent
orcein in a mixture of equal parts of lactic
acid, acetic acid, and water. Brilliant con-
trast in staining resulted. Initial use of
this stain on the sporocytes did not give
satisfactory results.

Some of the major aspects of chromo-
some g behavior that are associated with
the presence of the Ds locus were reviewed
in Year Book No. 45. It is now suspected
that the dissociation mutation process is
not a simple breakage of bonds at the Ds
locus, although this is usually the eventual
consequence. In making observations of
the chromosomes, it was necessary to be
able to identify accurately the Ds-carrying
chromosome in the sporocytes of a Ds ds
plant. Crosses were made, therefore, be-
tween Ds-<arrying plants with morpho-
logically normal chromosomes g and ds ds
plants having a chromosome g with a
small terminal knob at the end of the
DEPARTMENT OF GENETICS

short arm and a short duplication of
chromatin extending beyond the knob.
The heteromorphic end of the short arm
of the chromosome 9g bivalent in the
meiotic prophases of the resulting plants
allowed the Ds- and ds-carrying chromo-
somes to be readily identified. In these
plants, a number of sporocytes were ob-
served in which the Ds-carrying chromo-
some was deficient for the terminal two-
thirds of the short arm as a consequence of
a previous dissociation mutation in an
ancestor cell. In all cases, without excep-
tion, it could be determined that only the
Ds-carrying chromosome had been affected
by this action. Some sporocytes were ob-
served, however, in which the Ds-carrying
chromosome had not simply lost two-
thirds of its short arm, but had been sub-
jected instead to some other modification,
whose history is not understood at present.
Among the aberrant types, those showing
the complete loss of the Ds-carrying
chromosome in a sector of sporocytes in
the anther were the most frequent. In the
recognized cases, the losses occurred earlier
than the more frequently observed dis-
sociations at the Ds locus. In other cases, a
small sector of sporocytes was present in
which the Ds-arrying chromosome was
missing; in these cases, however, a small
ring-shaped chromosome was present in
the cells of these particular sectors. This
ring chromosome probably is composed of
a segment of the Ds-carrying chromosome
g, although positive identification could not
be made. A few other types of aberrant
configurations also were observed. In all
these relatively rare types of aberrant be-
havior, only the Ds-carrying and never the
ds-carrying chromosome was involved. It
is obvious that the presence of the Ds and
Ac loci is in some way responsible for
these aberrant types of chromosome g be-
havior. It is hoped that a more complete
analysis of these relatively rare types of

149

sporocytes will yield some insight into
the nature of the action that occurs as a
consequence of the combined presence of
‘the Ds and Ac loci. It will be necessary
to observe many thousands of sporocytes
before a sufficient number showing aber-
rant types of chromosome g behavior can
be found. With the improved techniques
recently developed, it is hoped that this
may be more readily accomplished. Until
this needed information has been obtained,
it would be premature to attempt to pro-
ject a sequence of events that result di-
rectly in a dissociation or in one of the
rarer types of alterations involving the Ds-
carrying chromosome 9.

The stability of the state of the Ds locus.
As mentioned previously, abrupt changes
may occur in the expressed pattern of dis-
sociation mutations in a tissue or sector of
tissue. The general pattern, in each case,
is the product of the time, the frequency,
and the distribution of dissociation muta-
tions that have occurred in individual cells
during development. At present it is not
known to what extent these changes in the
expression of dissociation mutations are
controlled by altered conditions at either
the Ds or the Ac locus, or at both, or by
other genetic conditions not yet identified.
Until further evidence has accumulated,
the observed patterns will be considered
a reflection of the state of the Ds locus
even though this restricted definition may
later require modification. The patterns
of dissociation mutations are visible in
tissues of plants that have a ds-carrying
chromosome 9g with recessive factors in its
short arm and a Ds-carrying chromosome
g with their dominant alleles. The re-
cessive factors wd, pyd, and yg (wd, white
leaf tissue; pyd, pale-yellow leaf tissue;
yg, yellow-green leaf tissue), located at or
close to the end of the short arm, have been
used to determine the pattern of dissocia-
tion mutations in leaf tissues of seedlings
150

or mature plants. The series of alleles
¢ (colorless aleurone), C (colored aleu-
rone), and J (inhibitor of C color)—
located approximately at the position de-
marking the distal third of the short arm—
and the recessive factors sh (shrunken
endosperm), bz (bronze, modifier of C
color), and wx (waxy starch in endosperm
and pollen)—located, in the order given,
between C and Ds—have been used to
examine dissociation mutations in the
endosperm of the kernel. The alleles Wx
and wx have been used to estimate the
number of pollen grains in individual
anthers of Wx Ds/wx ds or wx Ds/Wx ds
plants that are deficient for the terminal
two-thirds of the short arm of chromosome
9 because of previous dissociation muta-
tions in ancestral nuclei.

When silks of plants that were homo-
zygous for ae and for C, sh, bz, wx, and ds
received pollen from plants carrying Ac
and a chromosome g with the dominant
alleles I, Sh, Bz, Wx, and Ds, a number of
kernels on the resulting F. ear were
variegated because of the presence of sec-
tors of cells with the phenotypic constitu-
tion C sh bz wx. With a few exceptions,
each sector composed of multiple-recessive
cells arose following a dissociation muta-
tion that had occurred in the ancestor cell
of the sector. Subsequent elimination, dur-
ing a mitosis, of the acentric segment of the
chromosome 9 carrying I Sh Bz Wx re-
sulted in the absence of these dominant
factors from the descendent nuclei. In
crosses involving any one male parent
carrying a particular Ds and a particular
Ac locus, the majority of variegated ker-
nels fell into one main class with respect
to the type of variegation pattern they ex-
hibited. Great differences in pattern types
exist. For example, the majority of varie-
gated kernels on the F: ears may show a
speckled appearance because of the pres-
ence of a number of small patches of cells

CARNEGIE INSTITUTION OF WASHINGTON

that are C sh bz wx in constitution. The
size of a speckle here depends on the time
of occurrence of the dissociation mutation,
If it occurs very late, the colored speck
may be composed of only one, two, or a
few aleurone cells, but if it occurs some-
what earlier, the colored speck is com-
posed of more aleurone cells. Crosses in-
volving a different male parent may give
rise to Fi ears in which the majority of
variegated kernels show early dissociation
mutations. These kernels are character-
ized by large areas of recessive tissue and,
in extreme cases, by only small patches
of dominant tissue—residual areas where
dissociation mutations have not occurred.

In a number of variegated kernels, there
were relatively large, sharply defined sec-
tors in which the pattern of dissociation
mutations within the sector contrasted
greatly with the pattern exhibited by other
parts of the kernel. These sectors in-
dicated that a change of state had occurred
in the ancestor cell that gave rise to the
sector. Often these changes in state occur
at a relatively early period in the develop-
ment of the endosperm. The most in-
structive cases were exhibited by those
kernels in which a change of state could

be traced to the first or second mitotic

division in the endosperm. A change in
state in the first division may give rise to a
kernel one-half of which shows one pattern
of dissociation mutations and the other
half a contrasting pattern of dissociation
mutations. Or a kernel may be divided
into three or four sectors, each with its
own particular pattern, following changes
of state that occurred in the first and
second mitotic divisions of the endosperm.
Because of the free nuclear division that
takes place in the early development of
the endosperm, this tissue is not ideal for
a study of early cell lineages. Nevertheless,
early changes in state can be recognized
in many kernels. The prospects of de-
DEPARTMENT OF GENETICS

termining the contrasting nature of the
altered states of two sister chromatids, fol-
lowing a mitotic cycle that introduces a
change in state in both chromatids, are
considerably better in this tissue than in
the sporophytic tissues.

In the sporophytic tissues, dissociation
mutations occur late in the life of any one
tissue. This is in contrast with the endo-
sperm tissue, where dissociation mutations
may occur at any time during develop-
ment. Changes in state, however, may
occur at any time during the development
of the sporophytic tissue. In this respect,
the two tissues are comparable. Barring
heterofertilization, the Ds locus in the
first endosperm nucleus and that-in the
zygote nucleus are carried by sister chro-
matids. If no change of state had occurred
in the division that gave rise to these two
chromatids, the conditions that govern
the states of both Ds loci should be alike.
Several lines of evidence have indicated
that this is probably true. Kernels show-
ing early dissociation mutations in the
endosperm give rise, in general, to plants
having relatively early dissociation muta-
tions in the sporophytic tissues. Con-
versely, kernels with late dissociation
mutations in the endosperm tissues give
rise to plants showing relatively late
dissociation mutations in the sporophytic
tissues. Present evidence indicates that
the state of a particular Ds locus may
remain relatively unchanged in most of
the cells of a plant. Plants arising from
kernels that showed early dissociation mu-
tations gave rise in the next generation to
variegated kernels the majority of which
showed early dissociation mutations. Con-
versely, plants arising from kernels that
showed late dissociation mutations gave
rise in the following generation to varie-
gated kernels the majority of which
showed late dissociation mutations. Even
though a Ds locus may remain in one

8

151

state throughout a number of consecutive
mitoses, changes in state nevertheless are
not infrequent. At present, no critical evi-
dence is available concerning possible
genetic or environmental factors that may
influence the state of a locus or its ex-
pression as reflected in dissociation muta-
tions. Although changes in state of a locus
and the subsequent changes in the fre-
quency and distribution of dissociation
mutations are interrelated, the alteration
that is associated with a change in state
and the alteration that results in a dis-
sociation mutation are distinct and sepa-
rable.

CoNTINUATION OF STUDIES OF THE CHROMO-
SOMES OF NEUROSPORA CRASSA

Study of the mutable loci in maize was
interrupted during the fall and early winter
of 1946 in order to continue the investiga-
tions of the chromosomes of Neurospora
crassa begun several years earlier. The
earlier work was preliminary and explora-
tory, and no time was spent in obtaining
the necessary illustrations of chromosome
morphology and behavior during asco-
sporogenesis. In addition, the preliminary
study indicated a need for improvements
in techniques in order that consistently
good preparations could be obtained of
the chromosomes and nuclei of the ascus.
Efforts were concentrated, therefore, on
these two objectives. The investigations
were conducted at the California Institute
of Technology, with the collaboration of
Mr. Jesse R. Singleton. Approximately one
hundred photomicrographs were taken, il-
lustrating chromosome and nuclear be-
havior from the prefusion stages in the
crosier to the binucleated stage in the
ascospore. New or modified techniques
were devised, which greatly improved the
quality of the preparations. This applied
significantly to the meiotic prophase stages,
152

where the chromosomes are greatly ex-
tended. In the previous investigation, the
minute morphology of the extended
chromosomes could rarely be observed, but
with the present techniques this morphol-
ogy is sharply defined in many figures.
Mr. Singleton has succeeded in mapping
the chromomere organization of each of
the seven chromosomes. Each chromo-
some has an individually recognizable
chromomere organization, including deep-
staining regions which, because of their
positions in the chromosomes, probably
represent the heterochromatic regions
known to be adjacent to the centromeres.
It can now be stated with certainty that no
heteromorphic pair of chromosomes is pres-
ent in Neurospora crassa. These tech-
niques have also made it possible to ob-
serve more critically the mutual relations
of the two homologues of a bivalent during
the mid-meiotic prophase stages, that is,
before diplotene. Many bivalents were ob-
served in which the two homologous
chromosomes were lying side by side but
not in direct contact at anf point along
their length. These favorably oriented bi-

CARNEGIE INSTITUTION OF WASHINGTON

valents showed no relational coiling of the
two homologues about each other, and it
was obvious that the distance between
them was quite constant—amounting to
approximately half a micron—so that in
their spatial relation they resembled rail-
road tracks. Technical methods were de-
vised by Mr. Singleton for softening the
ascus wall in order to flatten the asci, and
also for achieving a sharp differentiation
of the spindle figures and the centriole.
From preparations kindly donated by Mr.
Singleton, photographs were taken to il-
lustrate the peripheral position of the
chromosomes in the spindle figures and the
rather bizarre organization and behavior of
the centriole in the third division in the
ascus.

Though the primary objectives of this
interim study of the chromosomes of
Neurospora were fulfilled, a supplementary
factor of possibly greater value was the
progressive realization of the possibilities
for utilizing this material in attacking a
number of cytological and cytogenetic
problems, both old and new.