~26-

to be generally true, it will explain why the crosses combining
different o-ml wx ds chromosomes with o Wx Da chromosomes showed
different coincident rates of visible mutations of the two loci.
If the observed coincident rates are relatively low, the rates of
hidden and visible mutations, in the two loci, may not be properly
balanoed to show the rates of coincident mutations that actually
occurred.

The mode of origin of mutable loci

The analysis of the c-ml case has been of considerable interest
because it points towards the mode of origin of mutable loci. It is
only necessary, it would appear, to insert a Ds locus into (or adjacent
to) a normal dominant locus. Inhibition of the dominant locus must be
associated with this insertion, if a changed phenotypic expression
is to be recognized following insertion. If the organization of the
inserted Ds locus gives the "few-late" pattern of behavior, which
usually involves loss of the Ds locus as a consequence of a mutation,
the inhibitor action will be removed and reestablishment of the normal
organization and genic action may occur. Such mutations will oceur,
of course, only when Ac is present. If this projected mode of origin
of mutable loci is the correct one, many newly arising Ac controlled
mutable loci should appear in Ac ac and Ac Ac plants that also have
Ds loci somewhere in the chromosomal complement. That such newly
arising Ae-controlled mutable loci are continually appearing in the
Ac Ac and Ac ac plants is well known already. The rate at which
such new mutable loci could arise would depend upon the frequency
of transposition of Ds loci from one position in the chromosome

complement to another position and also upon the relative number of
-27~

loci present in the complement that can give a changed phenotypic
expression following such insertions. The type of mutable phenomena
to be first observed after such insertions of Ds loci, would “epend
on the organization of the Ds locus at the time of its insertion.
There is sufficient evidence now to indicate that the rate of trans-
position of the Ds locus is rather high. This adds considerable
strength to the plausibility of this Ds-insertion concept of the
origin of new Ac controlled mutable loci.

With regard to changed locations of Ds, the c-ml case has been
particularly instructive. 1t arose in a C 3h wr Ds / Cc Sh wx Ds, Ac ac
plant. This plant had been examined cytologically and was known to
have two morphologically normal chromosomes 9 (except in those cells
that had undergone a Ds mutation). Among the 4000 tested male gametes
of this plant, one pessessed a new mutable locus--c-ml. When this
chromosome , carrying o-ml, was tested, no Ds activity could be
detected at the standard looation--the position it was known to occupy
in the two chromosomes 9 of the parent plant. Instead, typical Ds
activity could be detected at a new location: close to or at the newly
arising o-ml locus. No obvious chromosomal alterations apnear to have
accompanied this change in location of Ds. The chromosomes 9 in plant
4204, . were morphologically normal. Also, the c-ml carrying chromosome
gave no evidence of reduced transmission through the pollen.

Groga chromosomal alterations may accompany a change in location
of Ds. One such case has been analyzed (from plant 4306; for a summary
of this analysis, see "Notes to accompany annual report” included with
this memorandum). This analysis points towards the mechanism under-
lying such changes in location of Ds and also supports the conelusion

that Ds mutations are associated with some process that often results
in tearing out of the Ds locus from the chromosome. This tearing-out
process produces broken ends capable of fusing with other broken
ends, not only in the torn chromosome but also in the torn-out Ds
locus. The torn-out Ds locus, with broken ends capable of fusion with
other broken ends, may be inserted into a new location if a coinci-~
dental break cccurs elsewhere in the chromosome complement. Fusion of
unsaturated broken ends, a well established phenomenon, is all thet
is required to complete the process of change in location of Ds.
A second oase, possibly quite similar to this one, is now receiving
analysis. Other cases involving gross chromosomal alterations that
aocompany transpositions of Ds which should also add to the evidence
on the nature of Ds mutation and transposition, will certainly be
found now that I am sensitized to recognize them.

Because of the changed nature of variegation appearing in
individual kernels, numerous cases of changes in location of Ds
have recently been recognized. Such chenges in location of Ds are
readily detected in crosses of C sh bz wx 4s ac x I Sh Be x Ds, Ac.
These transpositions include insertions of Ds to the left of qT,
between I and Bz or between Bz and Wx to give Ds I Bz Wx, I DOs Be Wx
and I Bz DOs Wx constitutions, respectively. Some of these kernels
have been germinated in the greenhouse this winter to obtain lines
with these various altered locations of Ds. Unfortunately, germinae-
tion of some of them did not occur. This suggests that gross chromo-
somal abnormalities affecting germination capacities may have
accompanied the transposition of Ds in some of these cases.

Before considering a possible mechanism responsible for all of
the Ds mutation phenomena, it would be profitable to present briefly,

some observations of relatively infrequent but nevertheless very
~£9 =

important types of events associated with Ds behavior. These are

most easily detected in crosses of C sh bz wx ds ac by I Sh Bz yx Ds-

early or Ds-few-late, Ac males. A change in location of Ds, as

mentioned above, is one of these relatively infrequent events, These

infrequent events are additional clues to the fundamental mechanism

underlying the Ds mutation phenomena. In examining the variegated

kernels of the constitution C sh bz wx ds / C sh bz wx as / I Sh Be wx

Ds-early, Ac ac 4c, some very clear cases of internal chromosome

Geletion in the Ds cérrying chromosome have been observed, These

involve (1) deletions of the *x locus. The resulting sectors are TI Bz wx

Typical dicentrio Ds behavior may or may not be present in Such sectors,

(2) The deletion may include both Bz and Wx. The resulting sectors

are then I bz wx in phenotype. Such sectorg may show typical Ds

behavior within them or no Ds behavior may be registered. The relative

frequency of these deletions is low but their total frequency is

sufficiently high to have been observed numerous times. In kernels

of the above constitution but having the Ds-few-}ate instead of the

Ds-early, this aberrant tehavior is much more frequent per visible

Mutation then occurs when Ds-early is present. Tf believe that meny

of the observed visible mutations produced by the Ds-few-late state

are due to such aberrant conse uences of Ds mutations,
Q

without question, the presence of a Ds locus, whether in the Ds-

eerly or Ds-few-late state, introduces chromosomal abnormalities other

than those resulting from the more usual types of Ds action. Any hypoth~

esis of Ds behavior must consider the origin of these numerous cases

of well defined types of chromosomal abnormalities that occur when Ds
-30~

and Ac are present but do not occur with Ds ac, ds Ac,or ds ac. The
frequency of appearance of these various classes of duplication,
deficiency and transposition is too great to be neglected. They must
represent some of the consequences of the underlying mechsnism
associated with Ds mutetion phenomena.

The genetic observations of odd types of chromosomal abnormali-
ties accompanying Ds mutations have received cytologics] confirmation.
In plants that were Ds Ds Ac ac, clusters of sporocytes or individuel
sporocytes have been observed with duplications or internal deficien-
cies in the short arm of chromosome 9. A few have shown inversions
(some pericentric). In some cases, only one full chromosome 9 was
present. The other chromosome 9 was represented by a ring-shaped
chromosome, usually relatively small. In Ae ac plants having ea ds
cerrying chromosome 9 with a heterochromatic extension ef the end of the
short arm, and.a morphologically normal chromosome 9 carrying Ds, it
could be determined that thé*aberrent chromosome events involved the
Ds carrying chromosome and not the ds carrying chromosome. These
observations support the conclusion that the aberrant types of
chromosomal events are associeted with the De mutation phenomene
itself--something must occur at the Ds locus itself before the
observed types of chromosomal rearrangements will arise.

These cytological observations were made early inthe study of
Ds behavior. They had caused me much concern, even though they were
relatively infrequent. Here, again, their frequency was much too high
to be ignored. It is realized now that jast such conditions are to
be expected. It should be possible to obtain from the Ds Ao plants
@ number of strains with various chromosomal abnormalittes. Already,

T have obtained plants with duplications and Plants with deficiencies
-31-

in the short arm of chromosome 9 that arose from Ds te Plants having
morphologically normal chromosomes 9. The methods of detecting

such plants pertain meinly to abnormalities within the short arm of
chromosome 9. However, I do have a case of the involvement of the
short arm of chromosome 9 with the lone arm of chromosome 8, T have

not teen looking for such cases. More should he found.
Possible mechanism responsible for mutable phenomena

Knough information has been collected on Ds Ac behavior (1) at the
standard location, (2) when transposed to the right of I (case 4306)
and (3) when st the C locus (c-ml) to allow consideration of «4 possible
mechanism that could account for the observed types of behavior,

These various types of behavior may be enumerated.

In the presence of Ac, and only in the presense of Ac, the Ds locus
is associated with different kinds of abnormal chromosome. or gene
behavior. These varicus kinds of abnormal behavior surely must arise
ag the consequence of one fundamental, primary type of event occurring
at the Ds locus. It would be difficult to picture 4 number of
different kinds of primary events but it is relatively easy to picture
a number of different consequences of one primary type of event.

This event must account for the following behavior:

I. Ds at its standard location gives rise to:

1). Dicentric chromatids: the fusion of sister chromatids
occurring at the Ds locus. This is the usual event with the Ds-early

fet gita

organization of the Ds locus.
2) lheDe-early state can mutate to the Ds-few-late state by a
change at the locus that arises in one cell (at a mitosis®). This is

shown by the sectors of Ds-few-late appearing in Ds-early kernels.
-32-

These sectors are sharply delimited.
3). Ds-few-late is more stable in organization than Ns-early.
This is evident because it maintains the few-late type of mutation
pattern in later generations and rerely (if ever) mutates directly
back to an organization giving the Ds-early type of mutation pattern.
4). In kernels having Ds-eerly, a number of internal

deficiencies arise that include segments sdjacent to the Ds locus. 4

nein

The deleted segments are of various lengths. The most frequent: dele-
tions are short, although some longer deletions occur. Following
such deletions, Ds activity may be retained in the chromosome or it may
be lost from the chromosome during the event that gives rise to the
deletion,
5). In Ds-few-late, the visible mutations, that igs, losses
of segments of the short arm of chromosome 9, often involve some
event that is definitely not dicentric chromatid formation with fusion
of sister chromatids at the Ds locus. The production of internal
deficiencies or the production of dicentric chromatids coming from
fusions of sister chromatids at positions other than Ds, can occur.
6). The Ds action may be lost from the chromosome completely
without altering the chromosome morphology. (The evidence for this
when Ds is in its standard location has not been considered unr to now.
I have obtained some normal chromosomes $ without Ds that have been
derived from Ds Ds Ac ac plants.} This loss of Ds action is inter-
preted to arise as the consequence of removal of Ds from the chromosome
complement altcgether or removal of the Ds loeus from its standarég
position and insertion elsewhere. The removel of Ds action following
the ¢ to C mutations of c-ml and the removal of Ds from its standard
location and insertion into a normal C locus to give c-ml is

Substantiating evidence for this interpretation.
-33-

7). The Ds locus may be removed in tack from its stendard
location and be inserted elsewhere. A spontaneous breakage elsewhere
in the complement is probably responsible for the position of insertion,
as the analysis of case 4306 shows (see "Notes to accompany Annual
Report). In these cases, the Ds locus must be freed from the
chromosome completely because it can enter between the two broken ends
at the position of the spontaneous break; fusions of broken ends then
follow and the Ds locus is now in its new location. The freed Ds locus
must have unsaturated broken ends in order that it saturate other
broken ends by fusion. Therefore, breakage of some kind must be
involved at the Ds locus to give a Ds locus with unsaturated broken
ends. The freed Ds locus need not carry with it a large segment of
chromosome. In its new location, it need not interfere with crossing
over. This is shown in the c-ml case and the 4306 transposition-
translocation case. In both cases, crossing over at the region of
insertion is not altered. Gross chromosomel abnormalities, erising
from various fusions of the several broken ends when spontaneous
chromosome rather than chromatid breakage occurs at ae locus other
than Ds, muy accompany a change in location of Ds. The transposition
of Ds, in these cases, need not involve a segment of chromatin that
earries Da with it; for it has been shown that Ds can move as a
sub-microscopic, independent unit in these cases.

The analysis of changes in location of Ds indicates that the Ds
locus may be removed from the chromosome as a sub-microscopic, indepen-
dent unit and that, as such, it may be inserted elsewhere in the
chromosome complement. It indicates, also, thet the freed Ds locus
has unsaturated broken ends because fusions of unsatureted broken ends

must have taken place in the 4306 transfer of Ds. Such fusions would
-34~

be expected only if the Ds containing, sub-microscopio fragment of
chromatin had unsaturated broken ends. The “early” state of Ds
(Us-early), giving many dicentric chromatids as the consequence of Ds
mutetions, strongly supports a breaksge-fusion mechanism as the causa-
tive factor underlying Ds -Ae mutation phenomena. On the interpreta-
tion that only one kind of event underlies all Ac controlled mutations,
regsrdless of the visible consequences, some form of chromosomel
breakage must be suspected as the primary event responsible for muta-
tion phenomena.
II. Ds, transposed to the normal © locus, hss given rise to the
e-ml mutable locus.
1. In this position, Ds behaves Just as it does at the
standard location or when transposed to the right of I (ease 4206).
a). Da-early type mutations et ec-ml give dicentric chroma~
tlds with fusion of sister chromatids occurring at -r close to the
c-ml locus as the main type of consequence of mutation.
b). This Ds-esrly stete at c-ml may mutate to the Da-few-
late state. When this occurs, dicentric chrometid formation ceases
as the most frequent visual consequence of Ds mutetions. Instead,
the rate of c to © mutations may risc ebruptly. The frequency of
these ¢ to C mutations may be the same “s the frequency of dicentric
chromatid forming mutations of standerd Ms when this Ds is in the
"early" stete.
2. ¢ to C mutations of Ds at c-ml result in morphologically
normal chromosomes 9.
a). The C locus is usually stable efter such an event.
b). The Ds activity ceases followlug such a ec to C mutation.

D3 eaeppeers to have been lost from the locus altogether as the conse-
-35-

quence of the ec to C mutation.

&. In the heavily variegated c to C kernels (the c-ml Ds
locus equivatent tc the standard Ds-few-lste locus) where many
mutations may be observed, a number of abnormal events have been
ovserved. These observations (progeny test made in 1 case only)
suggest that occasionally:

a). The "o-mi" loous (which hes the genes of the normal C
locus) may be removed from the chromosome along with Ds. This results
in a stabilized c locus, no longer capable of mutating to °.

db). The Ds locus may be removed from the c-ml locus and be
inserted elsewhere, often in the short arm of chromosome 9. Ac
phenotype may result but continued Ds activity occurs at the new
locetion, It gives:

(1) Reneated losses of © in the C vectors arising from
such an event if Ds is inserted to the right of C.

(2) Losses of © in the C sectors if inserted to the
left of the C locus. In this case, however, there are brenkage-fusion-
vridge cycles that result in twin sectors of ¢ and deep € ine / o /o-ml
Ae ac ac constitutions.

These various events suggest that the c to C mutetions of c-ml
result from some form of chromosome breekage that usually eventuates
in loss of the Ds locus from the chromosome. Sometimes, however, the
removed Ds locus may be inserted elsewhere if, st the same time that
a Da mutation occurs, e coincidentel break occurs elsewhere. The Ns
locus may then be inserted between these two newly broken ends.

Mutations of c-ml to C most often restore the full expression of
the C locus at one single step, as far as one can state from observa-

tions of the color intensities. produced. There are a number of
~36~

mutations, however, that give rise to much reduced color intensities
or occeslonally to deeper intensities than a normal C locus. These
quantitative chenges are part of the evidence that must be considered
in any hypothesis relating to the primary event associated with Ds
acticn at the c-ml locus (or elsewhere}. The separate hypothesis of
quantitative units et a locus offers no aifficulties, as seen so fer,
to a straight forward interpretation of events underlying Ds mutations.
If the different quantitative expressions of the various mutations

of « single ioscus are related to some inhibitory factor that can
express itself quantitatively, then 4 new as yet unstudied veritable
factor will have to be included in the over-sell hypothesis. It
relates to the nature of genic organizetion resulting from the
primary evert but not necessarily to the primary event itself.

What Kind of a mechanism will yzive rise to these verious events
with the expected relative frequencies? I heve tnought of one kind
of mechanism that does not seem too inconceivable. It involves the
following assumptions and interpretations.

1. The main event responsible for all Ac controlled behavior is
related to the recuplieation of the Ds locus at the time of gene
reduplication,

2. Ususlly, following chromosome recduplication, each new gene
molecule lies adjacent to the mother molecule but is not joined to
the mother molecule by any chemical bonding.

3. Reduplication of gene molecules is orderly in thet «11 the
daughter molecules lie adjucent to the mother molecule. Also, all
coughter molecules lie in a single plune--that 1 Bly the new dauchter
chromcsome 1s not twisted about the mother chromosome. The behevior

of ring-chromosomes during mitosis shows this.
4. During prophase, a repulsion occurs between mother snd
daughter chromosomes. This repulsion occurs simultaneously or nesrly
so along the whole chromosome. The mother and daughter chromatids
become separated by 2 rather specific distence, as a consequence
of this repulsion force. This is geen by an examination of the two
chromatids at somatic prophase. In order to give this precise spacial
relationship between two chrometids, some form of repulsion force,
following chromosome reduplication, is required.

5. When a Ds locus is present in en ac ac constitution, the
Da locus behavee as other loci do during reproduction and thereefter,

6. When both a Ds locus and an Ac locus are present, the mother
and daughter Ds molecules remain chemically bonded together following
reduplication of the Us molecules in certain mitoses (contrcelled by
what Ac is doing at the time).

7. when the repulsion force that separates the mother and
daughter chromatids sets in, the mother ond dsughter Ds molecules
are still tied together. In the adjacent loci, however, the mother

and daughter molecules are free from one another.

Direction a

of repulsion Dp
force q t T f ¢ Tq — Direction of repulsion

 

 

 

 

force

 

 

Ww

 
~38-

8. Because the bonds uniting mother and daughter Ds molecules
are still present, a break must occur in this region. The Ts locus

mey be yanked out of both chromstids:

 

 

 

1 2
ot x [8-8
Direction é x x
of repulsion >
force
A x -
2 & x np Qn me eee
3 4

x = broken ends capable of fusion with other broken ends.

9. Because of the repulsion forces, broken ends ] and 2 and
broken ends 3 and 4 will come to lie near to one another, respectively.
Fusion of these broken ends will occur. The Ds locus will be lost to
both sister chromatids. A morphologically normal chromosome will
result but it will have no Ds locus.

In the case of the few-late type of Ps action, this mechenism
seems satisfactory as an explanation. It is necessary to assume that
the original insertion of Ds into the C locus, in the case of c-ml,
resulted in an inhibition of the action of the C losus. ‘When s is
removed, by the above mechanism, the original organization of the C
locus may again be restored and a normal ¢ action may azain occur.

This will explain why the majority of ec to C mutations at the c-ml locus
give rise to morphologically normal chromosomes 9 with stable 4 loci
showing no further Ds activity.

Tension produced by the united Ds molecules may extend along the
chromosome curing the repulsion of sister chromatids. Breaks may occur
not as neatly as diagrammed but may sometimes result in aéjacent

segments -of-ehromesomes being pulled out of one or both chromatids.
This could give small internal deficiencies in the region of the 5s
locus. This may be responsible for the very lerge number of new
recessive mutants that heave anpesared in theme stocks.

Again, Ds may be pulled out of one chromatid but not cut of the
other. Or, the Ds locus may be pulled out of both chromatids but in the
proceas be itself broken. Two Ds loci could then enter one of the
sister chromatidg if the various broken ends were close to one another.
This could give a new, compound Ds locus. Such compounding could
oontinue to build up a rather complex, compound Ds loci. I believe
this must ocour.

Or, if a coincidental break occurred elsewhere, the free broken ends
of the extruded Ds locus might unite with these cther broken ends.
Transposition of Ds could then oecur. If broken ends 2 or 4 were
likewise close to the new brokea ends, compound, gross chromosomsl
rearrangements could arise. Verious abnorm: lities could be expected to
arise and be visible genetically, when eppropriate genic markers are
present, to make the events realized.

Although this dlagremmed scheme seeme satisfsctory, in general, to
account for the few-late Ds behavior it will not tell why one zets the
us-eurly behavicr, that 1s, dicentric formation because of fusion of
sister chromatids at the Ds locus. As stated above, the few-late tyove
‘f Ds behavior is rather stable but the early s type frequently throws
the few-late type. The early type Ds behavior probably involves ® more
complex organization of the Ds locus, possibly a compound organization
with duplicate Ds loci having, therefore, many bonds following gene

redupliestion.,
~40-

This may be:

 

 

 

 

 

 

 

 

 

The extensive binding action of these reduplicated Ds loci may

result in rather complex tensions and breakages during the repulsion
period that may result in placing broken ends of sister chromatids
close to one another so that fusions readily occur. Also, a reversion

to a simpler form of Ds organization through deletion of some of the

fet,
Sea Tre

Ds loci could be anticipated, that is, a few-late Ds organization
arising from the early-Ds organization.

The various isolates cf Ds standard and of c-ml that show grades
of Ds behavior between the extreme few-late and the extreme Ds-earlv,
may be fitted into this scheme. The more and the wider separated the
Ds loci, the more the chance for aloentric-forming fusions of broken
ends and the legs the chante fcr broken ends of the same chromatid to

fuse. Also, sequences of change in state of the mutable loci should be

anticipated on this mechanism. The high dicentric producers should
be the most unstable in mainteining their states whereas the lowest
dicentric producers should be more stable in their states, rarely, if

ever, throwing a really high dicentric producing state through a
-41l<

single mutation. The states producing intermediate rotes of dicentric
formation should go in both directions: from intermediate to hizh or
to low. Also, the new unstable mutable loci that arise ss the conse-
quence of transpositions of Da may reflect, to some extent, the
state of the locus before the transposition. If a compound locus
is transposed, in tac¥, then the mutstion phenomens observed will be
culte different from that observed if a single Ds locus enters the
new position.

The hypothes!s outlined 1s not complex. It does rot call on
many assumptions of a purely speculative nature. It is rather one that
could well heave been enticipated in sdvance of sny suggestive evidence,
There is no reason to assume that something could not go wrong with the
reduplication process that would lead to fust this sort of association
of daughter molecule with mother molecule. The aberrant mitotic
behavior of ring-shaped chromosomes suggests that something of this
nature may go on as an occasional error during the reproductive staze,
even in nor-al loci. It might likewise be responsible for some of the
spontaneous breskage phenomena known to occur rather frequently in
the maize chromosomes. It is the Ds - Ac combination thet brines this

to frequent expression.
~42-
Part II. The c-m2 locus

Clessification and description of the types of mutations occurring

at the c-m2 locus

Mutations occurring at the c-m2 locus differ from those occurring
at the c-ml locus in several easily distinguishable respects. The
e-m2 locus is Ae controlled; mutations occur only when Ac is
present. Also, the time and frequency of mutations of c-m? respond
to Ac dosages as do ceml and the standard Ds.

The mutations of c-m2 may be classified into several catrgories:

I. The Pink mutations. These give verious grades of pink
to red color in the aleurone with pr pr constitutions or various
grades of purple color with Pr. The term "pink" is laboratory
language. It is not a well chosen descriptive term. Until T know
more about the various classes of c-m2 mutations, I shell not try
to coin specific designations. I shall use the term "pink" to
cover a series of mutations that appear to be similer or related,
Later, it may be necessary to distinguish between various sub-
Classes of this group.

a). There is a wide range in the intensities of the color
produced following mutations of ec-m2 to Pink. Some mutations to
pink sre too feint for the outlines of the sectors to be certainly
defined. Some of these "hidden" mutations may be detected when
special conditions are present. These will be described. Other
pink mutations are quite dark. A quantitative series of pink
alleles are produced by mutations of e-m2. These quantitative
alleles of pink may be obtained and maintained from isolates of

germinal mutations. Only a limited series of such alleles have
~43-

been isolated and studied in later generations but many more are
available for such purposes. The germinal pink isolates that have
been studied so far have been relatively stable in thelr expression.
I suspect, however, that some of these germinal mutations may
respond to Ae by giving variegation in the depth of color. TI am not
too sure of this as yet because the pink phenotype in these isolates
does not show uniform distribution of color over the aleurone even
in ac ac ac constitutions. It is somewhat mottled although the
grades of color contrats in © single kernel are not extreme. The

Ac ac ac constitutions seem to be more mottled and in patterns
suggesting changes at the locus. I don't know now whether this
effect is due to changes at the pink locus or to changes at other,
as yet non-detected Ac-controlled loci.

b). The pink mutants are often associated with the produc-
tion of some diffusible colorless substance. This substance
(substanc: 1) can be used by 4 normal C locus to increase the
intensity of the aleurone color in those cells having a normal
C locus. Apparently, relatively large amounts of this diffusible
substance may be utilized by single cells having a single normal
C locus. when this utilization occurs, the color of the aleurone in
these cells may become very deep, giving a super-C color intensity.
The dosage responses of a normal C locus may be reflections of the
limited quantities of this substance that one normal C locus
produces. The methods of showing the presence of this diffusible
colorless substance produced by cells having mutations to pink,
involves en analysis of the color patterns and distributions in
sectors of kernels resulting from the following combinations of

loci. These combinations will also show that the colorless
~44-

Giffusible substance is not produced by an unmutated o-m2 locus,
(1). o-m2 female x C-normal Ds male; Ac ac ac or Ac Ac ac constitution
(2). ” ” x chromosome 9 with @ broken end. This broken
chromosome carries a normal C locus. Ae ac ac
or Ac Ac ae constitutions.
(3). 7 X G-ml mele; Ac ac ac or Ac Ac Ac.

(4). pink 7 x C-normal Ds male. Ac ac ac or Ac Ac ac,

3

x chromosome 9 with broken end. This broken chromo-
some carries a normal C locus. ee ac ac consti-
tutions as good as those with Ac.

(5). ”

(6). * 7 X c-ml male. Ac ac ac or Ac Ac ac.

The color patterns produced following each of these crosses will be
Gescribed later.

o). The isolated germinal mutations to pink have given
dosage responses. This has not been extensively worked out but
the grads of intensities of pink color in selfed ears of plents
with pink / ¢ compared with intensities given by crosses of the
same plant to c female plants, certainly suggests dosage effects.
More instructive for the dosage studies of pink are the kernels
with o / co / and a chromosome 9 carrying pink that has a broken
end. The resulting breakage-fusion-bridge cycles in the pink
carrying chromosome produce definite patterns of changed color
intensities, obviously assoclated with different doseges of the
pink locus. Too few kernels are available for an extensive analysis
but those examined clearly show dosege effects. They indicate that
the more pink loci present, the deeper the color. The quality of
the color and the intensities in the sectors with various doses of
pink differ from those produced by similar unit doses of a normal

€ locus.
-45-

dad). The pink mutants (some of them, at least) are deficient
for some substance that a normal C locus produces. Two such substances
may be deficient but at least one is a colorless, diffusible sub-
stance (substance 2) that is associated with the activity of a
normal © locus. The cells with the pink mutation can use this
substance to increase the intensity of the pink color. This has
been shown by an analysis of color patterns and distributions
in the sectors of the kernels having constitutions given above,
In addition, the combination of o-m2 female x C-normal ds male,
Ac ac ac or Ac Ac ac, hag been useful,

II, Mutations of c-m2 giving conditions that resemble
the full C genic activity although verying in
quantitative expression of this activity.

In crosses of c ac females x e-m2 Ac Ac males, pink sectors
appear in the kernels of the resulting ear. Within some of these
sectors, mutations to a type of full C expression often occur,
Unless there has been a duplication of the o-m2 locus at the same
time that a mutation to Pink occurred to give two c-mpe loci, each
capable of independent mutation, it may be concluded that e muta=

tion of a single c-m2 locus to pink can be followed by a second

mutation at this locus to give a full C type expression. None of

the germinal pink mutations that have been carried to a second

generation have continued to give these pink to full c mutations in

Ac constitutions. The selection for continued study of germinal

pink mutants with spots of full c activity in them, has come only

from the crosses of e-m2 females xo males. In these cases, the

female contributed two loai to the endosperm (two chromosomes 9).
-46-

A pink mutation may have been present in only one of them. The
other locus might have been a non-mutated c-m2 locus and capable of
mutating to full C. This could give C spots in a pink background.
The evidence for this is suggestive. In 4 of the 7 tested cases
(4451A, 4456-2, 4456-4, 4458A-2) the chromosome entering the egg
nacleus likewise carried pink. In the other 2 cases, the chromosome
entering the egg nucleus carried a non=- mutated c-m2 locus (4456 -l1,
4456-3, 4458A-1). If the mutation to pink occurred during the
divisions of the female gametophyte, just such apparently conflicting
results could be anticipated. Such female gametophyte could have
nuclei with the pink mutation and nuclei with the unmutated c-m2
locus. Because mutations of all Ac controlled mutable loct usually
occur late in the development of the sporophytic tissues or often
not until the gametophyte or endosperm stage is reeched, the period
of origin of germinal mutations could be as late as that just —
described. (It might be mentioned et this time that occasionally
an Ac controlled mutable locus may mutate early in the development
of the spotophytic tissues. It is a very rare event, however,!
Tests of the stabilization of pink mutations must come from the
reciprocal cross: ¢ x c-m2. The germinal pink mutations showing
full C spots must be selected and tested. They occur but this
obviously critical test has not been made. JI wes not sharply in
focus on the c-m2 mutations when the selection of eerminal mutations
was made!

A single (?) mutation of the c-m2 locus to Rive the two diffus-
ible substances or at least a second diffusible substance (substance
2) that is associated with gene activity of a normal C locus can

Occur. well defined sectors, assumed to have substance 1 and known
-47-

to have substance 2, appear on the kernels coming from the following
crosses: .
(1) o-m2 / e-m2, Ac ac; self-pollinated.
(2) o-m2 female x c male. (Ac ac ac or fe Ae ac constitutions)
(3) c female x c-m2 male. ( ” ” 7 re )

(4) pink female x c-m2 male and reciprocal. (Ae ae ac or
Ae Ae ae constitutions).

It is concluded that if not a Single mutation then 4 sincle
event at the c-m2 locus may result in a modification of genic
organization that will produce both of these substences associated
with action of a normel C locus. The amounts of the substances
produced need not be at the same levels as those produced by a
normal C locus. The levels may be considerably lower if the color
intensities shown are any Indication of such levels,

AS stated above, it is possible that successive mutations occur,

first to pink ,which often produces an excess of Substance 1, and
then to a mutant giving a darker color associated with the production
of some substance 2. There is no certain evidence as yet that
mutations giving substance 1 are produced without some accompanying
pink color formation, however faint this pink color may be. There
is some evidence suggesting that mutations do occur that give
substance 2 but not substance 1. The sectors suggesting this are
pink with deeper colored rims in the pink sector on # restricted
part of the border between a pink and a colorless sector; or, within
a pink sector, darker areas with diffuse borders often appear,
T am not at all certsin that this interpretation is the only one
that will explain some of the eclor patterns of these sectors.
Liore obse: vations and thought are required,

“he mutations to full c activity are quantitetively exnressed

in that various grades of color intensities are present. The
~48-

sectors with these phenotypes may be quite light in color or
fairly dark. No germinal mutations to full © color activity

have been detected so far. This is not evidence that they do not
occur, I have not looked exhaustively for them. Unless they
give deep grades of full ¢c color, I might confuse them with pink.
I know now that, often, the two differ in appearance, regardless

of color intensities expressions. It should be possible to
discriminate between the two, in some cases at least. T have not
taken time to find them but will do go,

III. The unmutated e-m2 locus produces no detectible guanti-
ties of either substance 1 or substance 2, This will be shown when
the kernels resulting from the crosses enumerated above sre
described,

IV. Hidden mutations occur at the e-m2 locus.

The frequency of hidden mutations seems to be quite hieh., This
is shown by:

(1) The types of sectors produced in the cross of ¢ ac females
X c-m2 Ac Ac males. hen the variegation pattern in these kernels
is compared with the variegation pattern produced by c-ml (from the
heavy visible-mutation producers) or with those produced by Ds-eerly,
it is evident that the visible sectors in the ec-m? kernels represent
some form of sub-sector arising in a descendant cell of one that had
undergone some primary event. This original mutation event in the
ancestor cell resulted in a visibly changed phenotype only in a
sub-sector or sub-sectors. Either some kind of (a) segregation to
daughter cells occurs following the initial event or (bd) sugcessive
mutations follow after the original mutation or (e) both types

of events occur.
-49-

(2). Mutations of c-m2 to steble o loci occur rather frequently,
I suspect. Some stable ec loci have been isolated from crosses
involving c-m2 / e-m2, Ac Ac or Ac ac plants. I can not say that
all such mutations are completely stable. Sever2l have been carried
for three generations without showing mutation in the presence of
Ac. Several ears have appeured in the crosses of c / o-mZ,Ac ac
females x c,ac males thet show large sectors on the ear with only
colorless kernels. This mey be due to loss of the Ac Jocus (evidence

for loss of Ac will be presented later; it is not uncommon;) or to

stabilization of the c-m2 locus. kvidenee that will differentiate

between these two alternatives will be eusy to obtein. It will be
necessery, in any case, to determine whether mutations of c-m2 to

stable c are associated with some segregation of chrometids, i.e.,
stable c in one chromatid and an altered organization of the e-m2

locus in the sister chromatid. The sub-sectors, mentioned above,

suggest segregations may sometimes occur,

(3) Some "hidden" mutations ere actually pseudo-hidden in that
they are pink mutetions with such faint colors that detection may be
very difficult or not possible, in many cases.

(4) TI suspect that c-m2, as well as c-ml, has a class composed
of several Kinds of hidden mututions that are not easy to detect.
These include changes in organization of the o-m2 locus that will
result in changes in the frequency and type of future mutation;
losses of parts of the c-m? locus; duplication of parts; relocation
of parts, etc. Although c-m2 may illuminate some of the activities
of a normal C locus, a study of its mutable behavior is discouragingly

complex when one has this locus as well as the other mutable loci to

carry along.
 

-50-

V. Mutations at the c-m2 locus have not resulted in many
obvious losses of segments of the short arm of chromosome 9. The
genetic tests for this are inadequate, however. The crosses that
could show this clearly involve o-m2 Wx to:

(1) ¢ sh wx ds ae and to (2) C sh bz wx ds ac. The Wx-m
locus was carried by the chromosome with c-m2. The wx sreas in the
kernels coming from cross (1) are ususlly due to mutations of
Yx-m. In cross (2), only a few kernels with C Bz - C bz sreas were
observed, These areas may arise from events other than those
associated with the c-me mutations. It may be stated, at least,
that the isolates of the c-mZ locus have not given, 4s yet, any

of Raye nT
states that reeularly result in loss of the short arm of chromosome 9.
I believe they will appear, sooner or later.

To summerize:-

The o-m2 locus may be compound in that the mutations
occurring at c-m2 are not asscclated with the quantitative expression
of one type of reaction. Two distinctly different types of visible
mutation occur at c-m2. They involve the production of two different
diffusible, colorless substances, both necessary for full C expres-
Sion. The production of substance 1 is often assoclated with the
appearance of some pink color. Mutations giving both substance 1
and substence @ may occur. Although both substances may be present
in a sector arising from a cell having such se mutetion, full ¢
color need not appear in this sector. quantitative expressions of
full C activity are observed. The mutations giving pink likewise
show quantitative expressions. Some mutations to pink give rise

to stable loci that no longer mutate in the presence of Ac. Others

may be mutable but this is not certain as yet. Mutations to full ¢
-5l-

type activity, regardless of the quantitative level, result in
production of some diffusible substance (substance 2) that the pink
locus can use to increase its color intensity. This seme substance
is likewise produced by & normal C locus.

That the weakened color intensity of pink mutations appears
to be associeted with some specific deficiency of a needed substance
is suggested by the dosage responses of the pink mutetions. This
substence can not be substance 1 for substance 1 is often produced
in excess even in the mutetions ziving very faint pink, It may be
substance 2. If so, then all mutations giving pink must likewise
produce some substance 2. Neither substance 1 nor substance 2
is produced. by the unmutated c-m2 locus, however. If the intensity
of the pink color is an expression of the level of some limiting
substance this substance must be one of the products of mutations
of c-mé. It igs not controlled by other loci, as the dosage responses
of pink indicate. This may be substance 2, as mentioned above,
or it may be a third substance associated with genic action of a
normal C locus. Although this substance-producer interpretation
of mutetions of c-m2 fits the observations so far made, I believe
that one should be canny about accepting it as it hes been presented,
I have that uncomfortable feeling of having mentally over-looked
the missing link that could simplify the whole interpretation.
This is by way of confession and not of retraction.

Before illustrating the color patterns produced in the kernels
coming from the enumerated crosses, some statements should be civen

regarding the origin of o-m2,
~52-
The origin of the c-m2 locus

The c-me locus first appeared on an ear of a self-pollinated

plant--plant 4000B-2. This plant had the constitutions:

m=-2 ea a. m
van ; -Pydig Sh wx | ac Ac. It is only in this
C-normal Sh wx™
one plant of the culture that I have been able to find any evidence

 

for a mutable ¢ locus. Also, I have not been able to determine,
as yet, whether or not a muteble C locus was responsible for the
first appearance of this c-m2 locus, or whether it came fron a normal
GS locus, as did c-ml. All that I cen state is that there is no
certain evidence for its origin from a C-m locus. Culture 4000
was segregating a mutable pale-green locus, Rach plant in the
culture was self-pollinated to determine whether or not the pale-
green muteble locus was present. The origin of plant 4000B-2,
that had the c-m2 locus, will be given starting as far back es the
ral had ceeieoad
eross that cive rise to a kernel with two broken chromosomes S

in the zygote.

 

(1) 2467A-5 x 2476-6
I wx Cc WX,
“oC we
I Wx ‘
Sh wx
oO

One I-C; Wx-x kernel was selected from this cross because both
chromosomes $ had newly arising broken ends terminating the short

arm. This kernel gave plant:
Suse VAY -53-

(2) 42-8, The main ear and the tiller ear of plant B-42 were
selfed. The tiller ear Segregated the new mutable pale-green locus.
The progeny from selfing the main ear did not aive this mutable
locus,

(3) The progeny of the tiller ear gave culture 3592. This
culture segregeted pyd as well as the new mutable pele-green locus,
Cytolozical examination of e number of plants in this culture were
made and genetic tests confirmed the constitution of the tiller of
plant 42-B to have a chromosome 9 with a duplication of the short
arm and carrying I wx and a morphologically normal chromosome 9 with
pyd C Sh wx. Ae was also present in culture 3592--whether homo-
2y@ous or segregating, I do not know,

(4) Plant 3592-9 was selfed. It had a duplication chromosome 9
with I Wx and a normal chromosome 9 with pyd C Sh WX

The progeny
of this self gave:

(5) Culture 4000. 40008 came from the I Wx y kernels,

Selfing 4000B-2 gave the o-m2 locus carried by the morpholoei-

cally normal chromosome 9 of this Plant. Nome of the other plants
in culture 4000 showed the presence of a ec-me locus,

Examination of the selfed ears of other 3592 nlants has siven

no indication of a c-m2 locus. These plants, however, did show other
mutable loci. Those recognized were:

a). ytoY¥

b). Starch consistency change from wx-like to Wx but the wx-like
starch was not associated with wx-staining reaction, Possibly the

areag with Wx-like starch consistency stain a deeper blue than
-54-

normal Wx. I am not certain that ajl these areas show this. It
has received only a cursory examination.

ec). Psle-green seedlings mutating to normal green.

ad). A Ds-few-late type of hehavior in one plent.

e). In plant 4000B-2, a jix-m locus was present. The tests
could not show for certain whether this “x-m locus was likewise
present in other plants of the culture, I think it must be from
the constitution of plant 4000B-2. It could be found out, though.

Obviously, the parent cultureg from which plant 4000B-2 arose
was full of mutable loci. Exeept for e-m?2 and Vx-m, both of
which are Ac controlled, it is not known whether or not these
mutable loci are Ac controlled. b) and 4) above both look like
Ae controlled mutable loci from the patterns of variegation seen in

the examined kernels,