CARNEGIE INSTITUTION OF WASHINGTON
DEPARTMENT OF GENETICS

COLD SPRING HARBOR, LONG ISLAND, N. Y.

January 20, 1950

Dear Charlie,

Your good letter of January 4 was not answered before
this as I have been trying to consider just how to get
the information to you in order to answer the questions
raised in your letter. It pleased me to learn that you
had acquired so much about the Ds, Ac story when it was
presented to you in such a rush and with no tixe for
adequate annotation where necessary. Ix dont like to give ym
a short acoount that would be inadequate for a real
comprehension; neither would I wish to burden you with the
long accounts that have been written. As mentioned to you,
these accounts were written, and are still being written,
in order to get the data together ami to have the correlations
indicated in the required sequences. The accounts have
been sent to Marcus regulsrly for he has been following the
story with considerable interest since before he left for
Illinois, He has also received samples of the kernels -
that illustrate the story. It seems to me that it would be
wise for you to have samples of the various types of behavior
if you are to grasp the descriptions with the least amount of
confusion. Suppose, then, that I collect a group of such
illustrations and send them on to you. tf, after examining
them and reading the descriptions that will be sent with
each sample, you would like to see the full account, I should
be glad to send on copies. I warn you, however, it is not
easy reading, not because the subject is difficult to grasp
but because of the number of experiments that have been made,
It is a long chore and I would well understand the emotional
reaction of not wanting to start such a task. If you could
have stayed over for several days, we could have gone over
the material here in short order and clarified. the details
quidkly. Now for your questions, however, or the comments.

Comméatn 1. "The Ds locus was, when first located,
on the short arm of 9 just outside the region with heavy
chromomeres, " This is correct. It lies to the right of
Wxp about 2 to 3 cross-over units away. This is called
the standard location because it can appear at other
locations.

Comment 2. This comment is complex but involves the
mechanism of dicentric formation, Several types of events
occur at the Ds locus as the consequence of ax Ac initiated
mutations, The frequency of one particular type of event
depends on the "state" of the locus. It might be better
worded to state that the "stste" of a locus is reflected in
the frequency of the particular types of events that can occur
at the Ds locus. One of these events is the formation of
an acentric fragment and a dicentric chromatid. This
cE.

occurs at a mitosis -- probably during or just after
division of the chromosome -- and involves both chromatids
of chrorrosome 9. The positions where the fusions to give
the acentric fragment and the dicentric chromatid occur at
the Ds locus: y

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A second type of consequence of a Ds mutation results in the
loss of the Ds locus from at least one of the chromatids of
chromosome 9. ‘the mechanism of this loss is not known
but is inferred from the other events that are known to occur.
It is possibly pulled out of the chromosome:

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A third consequence of a mutational event at Ds results in
the transposition of the Ds locus from one position to
another. The mechanism of transposition is believed to be
chromatid bre kage and fusion, Gross chromosomal 3
rearrangements have accompanied the transpositions of the
Ds locus in some of the examined cases. Two examples may s
be given, Avéh®dmosome 9 carrying I Sh Be Wx De-standard |
gave rise to a chromosome with the following constitution: v y
y
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o i_Des Sh BZ Wx . 2 UZ. a Bz « Sb. Ds —

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In another plant, having a morphologically normal chroxiosome

9 with I Sh Bz Wx Ds-standard, one gamete was found that had

a duplication of the follwwing type: wo Vad
- |

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1 2. a
a De_} °h Bz Wx_Qs_ 1 3h Bz Wx <
As.

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In both cases, two bhromosome breaks (involving both chromatids
at the same locus) are necessary to produce the duplications.
It may be noted that one break occurelat the known position

of Ds and the other break occurred at a position whtch is marked
by the new position or positions of Ds. That Ds events are
associated with chromosome breakage and fusions is indicated by
the usual consequence of Ds events -- dicentric chromatid
formation. That the transpositions of Ds are associated with
chromosome bre:Kages that result in the insertion of Ds between
broken ends could be suspected from the frequent association of
transpositions with gross chromosomal rearrangements where the
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breakage positions can be well established, as in the above

two cases. Either Ds was inserted as the consequence of a
prezk that occurred elsewhere in chromosome 9 or Ds activity
appecred at this new position as well as remaining at its
standard location and the gross chromosomal rearrangement
resulted from coincident Ds events at these two positions.

The latter assumption is unlikely because, where these

gross chromosomal abnormalities have arisen in association with
a new location of Ds activity, no evidence of any sector

having a normal chromosome 9 with Ds at the new position has
been seen, Either one event must foblow the other immediately
or the appearance of Ds in a new position and the production

of a gross chromosomal rearrangement are the consequences of

a single event. Such an event may be diagremmed as follows:

 

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If the above is the mechanism responsible for transpositions of
Ds, then only certain kinds of transpositions will be obtained
from male gametes that may be recognized immediately on
crossing to © sh bz wx ds female plants. The chromatids with
long deficiencies will be eliminated. The translocations
will not be recognized, Many of the duplications will not
pass through the pollen because of the extent of the duplication.
Also, normal chromosomes 9 with a Ds locus in a new position
and no Ds xsmakxinmx locus in the old position or a morphologically
normal chromosome 9 with two Ds loci, one at the new wosition
and one at the old position will be found. Examples of the
latter two types have been fqund, as expected. As far as the
7 evidence shows, there is ng position in chromosome 9 where Ds
activity is, Tiable to appear,as the consequence of trans osition.
The various positions given below have been found so far and
more are being investigateds
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Comment 3. “When Ds shifts to a point near another
gene it causes it to mutate, e.g. ODs = c; amd if Ds is
subsequently lost, a reversion mutetion occurs from c to ¢.*

nis is essentially correct. In the plants having Ds and
Ac, new mutable loci are arising that respond to Ac as does Ds.
One of these has received consicerable study. This is the
mutable locus called c-ml. The designation "m-1" refere to
the first of the mutable c loci that were found. It
appeared on the ear in a single kernel from the cross of a
c sh wx ds ac female by a note plant having two morphologically
normal chromosomes 9 carrying C Sh wx Ds in each chromosome.
The Ds locus in both chromosomes was at the standard location.
The C locus was normal in both chromosomes. It has been in
my stocks for years and has always behaved as expected. In
the cross of this plant to the c sh wx plants, nearly 3,000
kernels were obtained but only in a single kernel on the ear
of one of these crosses did a peculiar type of behavior occur.
Insead of showing colorless areas in a colored background, due
to losses of the © locus following Ds mutations at the standard
location, this kernel showed C spots in a colorless background.
It was just the referse of the expected type of variegation.

|It is from this kernel that the c-ml case originated. It
'proved to be as follows. A change from a normal 0 locus to a
'@ locus capable of mutating back to 0 occurred coincidently with ~

the disappearance of the Ds locus at the standard position and
its appearance at a location that could not be separated from
the mutable C locus. When this c mutates to C, all Ds
activity ceases, except when Ds is transposed to another
position coincident with the appearance of the C phenotype.

The C locus, following a c-ml mutation, is just as normal as it
was before the appearance of Ds activity at this position --
that is, before c-ml arose.

Comment 4. ‘In order that these mutations occur, an
activator (Ac) must also be present. Here are some of the
questions:

a) If the activator is not present (i,e,ac), does Ds
break loose and shift?"

The answer to this is sharp. Ds will undergo breakage
events only when Ac is present. If Ac is not also present
in the complement, there is no evidence whatsoever of am
a Ds locus. To illustrate, if a chromosome has C Sh wx Ds, or
I Sh Bz Wx Ds and is crossed to c sh wx or C sh bz wx, and if
no Ac locus is involved, the kernel types are completely normal.
There is no indication of any abnormal locus in these chromosomes.
As soon as Ac is added, however, the presence of Ds will be
apparent. Again, a c-ml locus in an ac constitution will be
completely colorless and no mutations to C will occur.
Question (b) of this comment: "Your c-ml and c-m2, are
these similar in phenotype, but separate occurrences; or do they
differ in phenotype?®

The answer is simple in only one asvect -- they differ
decidedly. The c-ml mutable locus mutates directly to a full
C phenotype with few exceptions. c-me mutates to two different
basic phenotypes, each of which may be expressed quantitatively.
The two mutable c loci arose from normal C loci, tet quite
independently of one another but in plants having Ds and Ac
loci.

Question (c) of this comment: "Is the Ds locus recognized
cytologically after it as broken loose?"

The answer to this one is also simple. One cannot
recognize any physical basts for a Ds locus. When it is
transposed to a new position, such as between I and Sh, it does
mt interfer with crossing-over between these two loci, Recogni-
tion in a new position follows from the Ds breakage events that
occur in this position. ‘+o illustrate: If Ds is at the
stendard location in an I Sh Bz Wx chromosome, the position will
be to the right of Wx.withxreferenze When a Ds event occurs
at this position, that gives rise to a dicentric chromatid and
an acentric fragment, the marked loci I Sh Bz Wx will be
carried by the acentric fragment. This fragment will be lost
to the cell. Ail the cells arising from this cell will have
no I Sh Bz Wx loci in endosperms that received this chromosome
from the pollen and a © sh bz wx ds chromosome from the egg
parent, A sector would appear in the kernel showing the
C sh bz wx phenotype. If, however, Ds were located between
Bz and Wx, the acentric fragment would carry I Sh Bz, but
Wx would remain in the dicentric chromatid, The sector formed
after a dicentric forming Ds event would be @ sh bz but would
be variegated for Wx to wx because of the breakage-fusion-bridge
cycle initiated by the Ds breakage event. The position of these
breaks may be seen in the microsporocytes. They coincide
with the positions inferred from the genetic evidence (either
link>ge data or sector type or both). Thus, the Ds locus can
not be seen but its position is determined by linksge, by
breakage events observed in the endosperm or plant and by
the breaks observed cytologically in the microsporocytes.

Comment 5. "Ac may shift into #9 or in another chromosome,
Where was it when first located?"

The position of Ac wnen first discovered is not known.
That it may go from one chromosome to another is apparent.
it may go from one unknown chromosome to chromosome 9 and there
be identified because of linkage witn the marked loci on chr osome
9. After it hag come into a position in chromosome 9, it may
move off and into another chroiosome, Over 50 transpositions of
Ac have been observed, or better, investigated. Three of these
entered chroosome 9, two in the long arm and one in the short arm.
-6-

Comment 6. This is related to what happens when two
Ds loci are present. The answer to this depends upon
several considerations. First, if the Ds loci are in different
chromosomes - thet is in homologoues and at the same location
in each homologue. If the two Ds loci have the same frequency
of the consequence of the Ds event ax number of results can
occur depending on the states of these Ds loci. If the
majority of the events give dicentric chromatids, coincident
mutational events can be high and much homozygous defécient
cellg production results. If one Dea locus has a high rate of
dicentric formation as the consequence of the Ds event and the
other a low rate of dicentric formation but a high rate of Ds
loss, then the result is quite different, as can be pictured.
In this latter case, the results are quite nice when the De
loci in the two homologues do not occupy allelic positions.
The case of c-ml and Ds-stendard can be used to illustrate this
If a plant having c-ml (Ds at the C locus to give a c phenotype)
and wx is crossed by a plant having ec (reguler recessive allele,
non=mutsble with Ac) and WR and De-standard the folbowing
constitution is present in the endosperm:

Ty sintp bis i
c-ml wx ds (2 gluse Suns bates AR ALA que Wipe
Mba CWE | -

c Wx Ds wr
If the Ds events occurring at the c-ml in/this selection of Ds
give rise mainly to c to C mutations and/few dicentrics and if
the Ds in the c Wx Ds chromosome gives mainly dicentrics and
few losses of Ds, coincident mutations are readily observed.
A mutation of c-ml to C will give a cell havey,the normal 0
phenotype. The sector produced after such an event will be
C. If a coincident mutation occurs at the Ds locus in the
c Wx Ds chrom-scome, a dicentric chromatid and an acentric
fragment will be formed, The acentric fragment will carry the
Wx locus. Loss of this fragment will result in a sector with
no Wx. It will be wx in phenotype. Coincident mutations of
c-ml and Ds at the standard location would give sectors that
are C and the underlying starch should be wx. The borders of
the © and wx areas should coincide. Theat is, aC sector
should heve underlying wx starch. When the combination is
made, as given above, just such sectors are very frequent.

You mentioned something about the sticking together
of Ds loci --— the more loci, the more sticking. Tnis was
mentioned in our conversation because of the following observ-tions.
The Ds locus is able to change from one "state" to another "state",
As mentioned above, the states of the Ds loci are identified by
the frequency of alternate Bete That Can occur ag—the-consequence
of a mutétional event occurring at the Ds locus. Ds loci
that are in a stete giving dicentric chromatid formation as the
consequence of the majority of the events can change, by a single
event, to a st:te that gives few dicentric events and many losses
of the Ds locus without noticable alterations in the chro sosome,
Mery four duets WO Ag tout,
A Ds locis in this latter state does not return to one giving)
ahigh frequency of dicentric chromatids by a single event,
A sequence of events must take place, each event increasing
the frequency of dicentric chromatid formation, ¢ It
this observationg that has led me to suspect that the Ds
loci giving high rates of dicentric formation have more Ds
units than those giving low rates of dicentric formation on
the assumption that it is easier to lopse Ds loci by the
mechanism associated with Ds breakage events than to gain them,
As you can imagine from the above brief discussion of
the Ds Ac story, it is a very large one. Extensive examinations
of a number of aspects of this story have been conducted.
I have to:d you very little about Ac itself. Even though
the details are manifold, obviously, there is a consistency
that does not fail. You can see why I have not dared publish
an acount of this story. There is so much that is completely
new and the implications are so suggestive of an altered
concept of gene mutation th:t I have not wanted to make any
statements until the evidence was conclusive enough to make me
confident of the validity of the concepts. I feel thst I
have made considerable progress and much that formerly was
confusing is now quite sharp and clear. The size of the job
ahead is staggering to contemplate, however. I dont know just
what to do about it. So much needs to be done that only
selections of the most important aspects can be considered.
This involves a constant digest and reevaluation of the
requirements. o

Gy tu sth foud, 0 do Loews Can es ft ove Query aus ebreatbiacn © one § ora

Thank you for sending me the extra sheets of the manuscript.
*he past three weeks heve been filled from early morning until
late at night with no bresks.; I shall have the required time
this coming week-end and will send your copy with comments
sometime next week,

Sincerely,