to b8 ~8Il8lXilly tl3l8, it will explain why the oroeses combining

dSZfsrsnt o-ml wx 4s ohrOmOsOm8e with 6 Wx Da ohromosonms showed

differcent colnoident rates of visible mutations or the two 1001.

If the observed oolnoidsnt ratee are relatively low, the rates of

hidden and visible mutations, in the two loai, may not be properly

balanoed to show the rates of oolncident mutations that aotually

ooourred.

The mode Of origin Or mutable loOi

   The analysis of the o-ml oaasa has been of oonsiderable interest

beOaUS8 It point8 towards the mod8 or Origin of mutable looi.  It la

only neoe88ary, it would appear, to insert a Ds loous into (or adjaoent

to) a normal dominant loous.  Inhibition of the dolainant looue, muert be

aasooiated with this insertion, if a changed phenotypic expression

is to be reoognized following insertion.  If the organization of the

inserted Ds loous gives the "h3W-lat8f pattern of behavior, which

uaually involves 108s of the Ds locus as a oonsequenoe of a mutation,

the inhibitor aotion will be removed and re8BtabliSbment of' the normal

organization and genie action may occur.  Suoh mutatlons will occur,

of oourae, only when Ao i.8 pr888nt.  If this projected mode of' orl~in

of mutable 1001 ia the oorreot one, many newly arising Ao oontrollsd

mutable loci should appear in Ao ao and Ao ko planta that also have

DY loui somewhere in the ohromosomal complement.  That such newly

arising Ao-controlled mutable loci are oontinually appearinq in the

AC AC and AC ao plants is well known already.  The rate at whiah

such new mutable loci oould arise would depend upon the frequenoy

of tranapoaition of Ds loci from one position in the chromosome

3amplsment to another position and also upon the relative number of


loci present in the ooaaplement thaf oan give a ohanged phenotvplo

expression mi0wing such Insertiona.  The type Of mutable phenomena

to be first observed aftar suoh insertions of Ds 1001, would Aepend

on the organization of the Ds loaua at the time of its inaertlon.

There is suffioient evidence now to indioate that the rate of trans-

position of the Des loous is rather high. This adds ooneiderable

strength to the plausibility of this Da-insertion oonaept of the

origin of new Ao aontrolled mutable loai.

   With regsrc? to changed looations of Ds, the c-ml case has been

particularly fnatructive.  It arose in a C Sh wx Ds / C Sh wx Ds, Ao ab

plant.  This plunt had besn examined cytologioally and wss known to

have two morphologically normal ohromosom8a 9 (exoept in thoae cells

that had undergone a Ds mutation).  Among the 4000 teated msla gametes

of this plant, one pessessed a new mutable loous--o-ml.   When this

chromosoms, oarrying o-ml, was tested, no Ds aotivity oould ha

detaoted at the standard looation--the posftion it was known to occupy

in the two chromosomes 0 of the parent plant. Instead, typical De

aotivity oould be detected at a new looation: 01088 to or at the newly

arising o-ml locus.  No obvious ohromoaomal alterations sppear to have

accompanied this change in looation of Ds.  The ohromosomes 9 in plant

4204, :,weFe naorphOlO~iO,ally normal.  Also, the a-ml carrying chrOmO8Om8

$ave no svidenoe of rsduoed transmisafon through the pollen.

   Gross ohromosomal alterations may aooompaay a change in location

of Ds.  One suoh 0888 has been analyzed (from plant 4306; for 8 summary

of this analysis, see "Notes to acoompany annual report" included with

this memorandum).  This analysis points towards the mechrnnism undar-

lying suoh ohangers in looation of Ds and also supports the conolusion

that Ds mutations are associated with Some prooess that often results


-20.

in tearing out of the Ds laoua from the chromosome. This tearinpout
prooess produoes broken ends uapable of fusing with other broken
ends, not only fn the torn chromosome but also in the torn-out Ds

loous.  The torn-out Ds 10~8, with broken enda oapable of fusion with
other broken ends, may be inserted into a new location ff a coinoi-
dental break occwss elsewhere in the chromosome complement.  Fusion of

unsaturated broken ends, a well established phenomenon, is all thet
is required to complete tke promso of change in location of 3s.
A seoond aase, possibly quite similar to this one, is now rece!lvinq!
analysis.  Other cases involving gross chromosamal alterations that
aooompsny transpositions of Ds whioh should also add to the evidence
on the nature of Ds mutation and transpasition, will certslnly be
found now that I am sensitized to recognize them.

  Because of the ohanged nature of variegation appearing in
individual kernels, numerous bases of changes in location of Ds
have recently been rsaognized.  Such chen%ss in location of Ds are
readily deteclted in crosses of C sh bz WC ds ac x I Sh Rz TX Ds, AC.
These transpositions lmluda insertions of Ds to the left of I,
between 3 end Bz or between Bz and '8x to give Ds I Bz Xx, T Ds I& TX
and I Bz Ds 8x constitutions, respeatfvcly.  Some of these kernels
have been germinated in the greenhouse this winter to obtain ilnes
with these various altered louations of Ds.  Unfortunately, germlna-
tion of some of them did not occur.  This suggests that gross chromo-
somal abnormalities affeating germination oapaoltfes may have
aoooglpanied the transposition of Da in some of these cases.
  Before considering a possible mechanism responsible for all of
the Ga mutation phmomena, it would be profitable to presentJbrleflyJ
some observations of relatively infrequent but nevertheless very


-EQ-

important types of events associated with Ds behavior. These are
most easily deteoted in crosses of C sh bz wx ds ac by I Sh Bz :tTx Ds-
early or Ds-few-late, AC aales.  A change in location of Ds, a8
mentioned above, is one of these relatively infrequent events. These
infrequent events are additional cluea to the fundamental mechfinism

underlying the Ds mutation phenomena.  In examining the variegated
kernels of the constitution C 3h bz wx ds / C sh bz wx ds / I Sh Bz Wx

Da-early, Acr au ac, some very clear cases of internal chromosome
deletion in the Ds carrying chromosome have been observad.   These

'involve (1) deletion3 of the Xx locus.  The resulting sectors are I Bz wx
Typioal dicentrio Ds behavior may or may not be present in such sectors.
(2) Th8 deletion may inolude both Bz and jYx. The resulting seotors
are then I bz wx in phenotype.  Such sectors may ahow typical Da
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-j,ate instend of the

Ds-early, this aberrant 't;ehavior is much more frequent per visible
mutation then occurs when Ds-early is present.  I believe that many
of the observed visible mutations produced by the Da-few-late state
are due to such aberrant consoqucnces of 3s mutations.

   ';r'ithout question, the presence of a P3 locus, whether in the Ds-
ec"rly or Ds-few-late 8tate, introduces chronosomal abnormalities other
than those resulting from the zore usual types of Ds action.  Any hypoth-
esis of Ds behavior must consider the origin of these numerous ce3es
of well defined types of chromosomal abnormalities that occur .*hen Da


and Au are present but do not oomr with Ds aa, da P.c,or ds 80~  The

frequenrry of appearmoe of these various classes of duplioetion,

deffoiency etnd transposition is too greet to be neglected.  They muat

represent some of tha oonsequencea of the underlying meohanism

assoaiatad with Ds mutation phenomena.

   The genetic observations of odd types of ahromosomal ebnormali-

tieta accompnnying Ds mutations have received cytological confirmation.

Ic plsnta that were Ds Da AC 80, clusters of sporocytes or lndividus.1

sporoeytes have been observed with duplioatfons or interns1 defician-

ciea in the short arm of chromosome 9.  A few have shown inverrJlons

Csome periceatrio).  In fmm! oases * only one full chromosome 9 -was

present.  The other chromosome 9 was raprsarnted by a rln+shaped

ohromosoma, usually relativel,v small.  fn Ao ac plants having a da

cerrying chromosome 9 with 8 heterochromqtic .extenaion eg the end of the

short arm, and.a morphologically normal chromosome 9 carrying Ds, it
crould be determined that th&sberrant chromosome events involved the

I9s oarrylng chromosome and not the (3s carrying chromosome.   These

observations support the oonclusion that the aberrant types of

chromoeomal events are asaooieted with the Da mutation phenomena

itself--something must ocuur at the fls locus Itself before the

observed typea of ahromosomal rearraneenenta will arise.

   These cytological observations were made early in the study of

Ds behavior.  They had caused me much concern, even though they were

relatively infrequent.  Hera, again, their frequency was auoh too high

to be ignored.  It is realized now that just euoh conditions are to

be expaoted.  It should be possible to obtain from the Da Ao plants

a number of strains with varI.ous chromosomal abnormalities.  Al.raQdy,

1. have obtained plants with duplioations and plants with deficianoiea


-3l-

in the short arm of ohromosome 9 that arose from Ds .GC plants having
morphologically normal ohrOmOSom8s 9.  The methods of detecting
suoh plants pertain mainly to abnormalities within the short arm or

chromosome 9.  However, I do have a 0888 of the involvement oi the
short arm of chromosome 9 with the long arm at chromosome 8.  I have

not teen looking for such CWWS.  @ore should he found.

Possible meohanism responsible for mutable phenomena

  Enough information has been oollecrted on De Ao behavior (1) at the
standard looation, (2) when transposed to the right of T (case 4306)
and (3) when at the C locus (c-ml) to allow cons!deration of a possible
meahanism that could account for the observed types of behavior.
These various types of behavior may be enumersted.
  In the presenoe of Ao, aad onl.y in the presenae of AC, the Da locus
is associated with different kinds of ebnomal ahromosome. or sene

behavior.  These various kinds of abnormal behavior surely must arise
as the consequenoe of one fundamental, primary type of event ocourring
at the Ds locus.  It would be diffioult to picture a number of
different kinds of primary events but it is rs1ativel.y easy to picture
a humbar at different oonsequenaes of one primary type of event.
This event must aocount for the following behavior:

    I.  Ds at its standard looation gives rise to:
      1).  Dioentria ohronratids: the fusion of sister chronstlda
ooeurring at the Ds locus.
  ( y,< jyc-;           This is the usual event with the Ds-early
organization of' the Ds loous.

   Z).Jh,Ds-early state oan mutate to the Ds-few-late state by a
ohange et the loaus that arises in one aall (at a mitosis?).  This is
shown by the sectors of Ds-few-lute appearing in Ds-early kernels.


These sectors are sharply Qelimitad.

     3). Ds-Pea-late is more stable in organization than Tss-early.

This IS evident because it maintains the Petit-late type ot mutation

pattern in later generations and rarely (if ever) mutates dfrectly

back to an orgnnizntion @.ving the Dcj-early type of mutation psttern.
     4). Xn kernels having Ds-early, n number of interm
defZcieacies arise that include segments Rdjacent to the 2:s Lo~us...~,
                                                                           ,4.L-
The deleted segments are of various Iztngths.  The most f requent:,.hele-
                                                                           I
tions are short, althcsugh some longer deletions occur.  Pollowing

such deletions, Ds activity may be retained in the chromosome or it may

be lost from the ohromo.some during the event that gives rise to the

deletion.

     5). In Ds-rew-late, the visible mutations, that is, losses

of' scfpents of the short arm oi chromosome 9, often involve some

event that is defi.nitely not Ufoentric chrometid formation with fusion

of sister chromatida at the Ds locus.  The production of internal

deficienoies or the production of Elicentric chromatids coming from

fusions of sister ahromatids at positions other than 3, CRT! occur.

      0).  The Ds aotion may be lost from the chromosome completely

without uZtering the chromosome raorphology.  (The evide nce for this

when Ds is in its stfindard looatlon has not been considered up to now.

I have obtained some normal chromosomes 9 without Ds that have been

derived rrom Da Db Aa ao plants.)  This loss of Ds aatioa is inter-

pretad to arise as the oonsequenoe of removal of 3s from the chromosome

oomplomeot altogether or removal of the 3s locus rrom its stendrlrd

positton ana insertion elsewhere.  The removal or Ds sctlon rollowing

the c 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 18

substantiating evidanas f'or this interpretation.


-aa-

    7). The Ds locus may be removed inxtac&.$rom Its standard

location and be Inserted alaewherc.  A spontaneoua breakage elsewhere
in the oomplement is probably responsible for the position of insertion;
as the anslysis of oaae 4306 shows (see *Notes to aocoatpany Annual
Report').  In these cases, the Ds locus aust be freed from the
chromosome completely because It oan enter between the two broken ends
at the position of the spontaneous break; fusions of broken ends then
follow find 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 et the Ds locus to give a Ds locus with unsaturated broken

ends. Th8 freed Da loous need not carry with it a lerie segment of

ohromosome.  In Its new location, it need not interfere with crossing

over.  This is shown in the c-ml ease and the 4306 transposition-

trhnalooation case.  In both oases, crossing over at the region of

insertion is not altered.  Gross chromosome1 abnormalities, arising

from vttrious fusions of the several broken ends when spontaneous

chromosome rrithes than chrowetid breakage occurs at a locus other
than Ds, my accompany a ohnnge in looatlon of Ds.  +?he transposition

of 93, in these cases, need not involve a segment of chromatin that

oerriss Ds with it; for it has been shotlrn that Ds can   move as a

sub-miaroscoplc, independent unit in these cases.
  The analysis of ohangee in location of Ds indicates that the Ds

locus may be removed from the chromosome as a sub-microscopic, indepen-
del?t unit and that, as mch, it my be inserted elsewhere in the

chromosome complement.  It indicates, also,  that the freed Ds loaus

has unsaturated broken ends beaause fusions of unsaturated broken ends

must have taken plaae in the 4306 transfer of Ds.  Such fusions would


be expeoted only if the Ds cootstining, sub-mimosoopio fragment of

chromtin had unsaturated broken ends.  The "early* state of 3s

(Es-early), giving many dloentric chromtids as the consequence of Do

mutationa, strongly supports a breakage-fusion mohenism as the causa-

tive factor underlying Ds -AC muttrition phenomena.  On the interpreta-

tion that only one kind of avant underlies all AC controlled mutations,

re.Tr:rdless of the visible consequences, some fom of chrolnoaone.1

breskags must be susgected as the primary event res:;onsible for nuts-

tion phenomena.

    IX.  Ds, transposed to the normal Z locus, hf+s given ripe t,o the

c-vi1 mutable locus.

        1.  In this position, Da behaves just 83 It does at the

standard lomtion or xhe:I trwnsposet- 3 to the right of I (CRSB 4706).
      a). Da-early type mutations et c-&L give dicmntric rthroma-

tids with fusfon of sister ehrcmatids occurrfn:? at  r close to the

c-ml locus 8s the min type of consequenoe of nrutation.
      b). This i3s-emly stete r:t c-ml. say autate to the Ds-few-

lht&? strta.  &hen this occurs,  dicentric chromatid forztatioh ceases

as the most frequent visual consequccce of 3s rwt?tions.  Instead,

the rote of' c to C aututions r,l,ay rise: cbruptly.  The frequ~ncp of

these c to C Hiutations my be the sazw AS the frequenclr of' dicentric

ohromatid forming mutations of standard 11s when this 03 is in the

'Tearly** stete.

      2. c to C matations oi DES RX c-tel result in ~~orpholopicnlly

nomml chromosomes 9.

      a). The C Loous is usually stable efter such nn event.

       b). The Da activity ceases follotiin~ such a c to C mutation.

i?st sppeers to hem been lost from thr+ locus altogether tis the conse-


-as-

quence of the c to C mutation.

         9
             s. .  In the heavily veriagated c to C kernels (the c-ml D8
locus aquiva*lent to the standard Du-few-late locus) where many

mutations may be observed, a nLrnber of abnormal events have been

observed.  These ohservstions (progeny test m&e In 1 case only)

suggest that occasionally:

      a). The n~-~lW loous (whloh has the genes of the nor-1 C

locus) my be removed frolrz the ohromosome along with Da.  This resultrr

in a stabilized c locus, no longer capable of' matating to 9.
      b). The 3s locue may be removed from the c-rr.1 locus and be

inserted elsewhere,  often in the short arm of chromoao?ne 9.   ~.i, rl

phenotype rztuy result but continued 11~ activity occurs at the new

location.  It $,ioes:
       (1) Rer)er;ted loages of C in the C Peotors rarisinq from

such an event if Ds is inserted to the right of C.

        (2) Losses of C ia the C sectors if inserted to the

left of the C locus.  In this cane, ho'ticver, there are brenkflge-fusion-

bridge cycles that result in twin Hectors of c 8nC deep G in c / a /a-ml

Ro nc 80 constitutions.

   These various events suggest thftt the c to C mutetiona of o-ml

result from sozne form of chromosome breekage that usually eventuates

in loss of the 3s locus from the chromosome.  Sometimecj, however, the

removed I)a locus may be inserted sbsewhera if, fit the 8ame time that

R P)B mutation ocours, e coinctdantsl break occurs elsewhere.   The P8

locus may then be Inserted between these two newly broken ends.

  Mutations of c-ml to C most often restore the full crtpressfon of

the C locus at one single step,  as r'ar as one can state from obssrva-

Sian3 of the color intensitiea.produued.  There are a number of


Eutations, huvmvm?, that give rise to much reduced color intensities

02 occevfoually to cieeper intsnsitiee than a normal C loous.  These

quantitative changes are part of the evidence that roust he oonsidared

in my hypothesis x-slating to the primary event associated with Ds

action at the c-n1 iocue (or Flsewhere).  The separate hypothesis of

quantftatfve units at a loclue offers no difficulties, as see11 80 rar,

to a s'kraight i'orwurd interpretation of events underlying 3s mutations.

If the different quantitative expressions of the various mtstions

of a single loous are related to some Inhibitory factor that can
expred:; itr;elf'quantitatively, then a new a$ yet unstudied wr5ahle

rnctor will have 50 be included in the over-all hypothesis. It

relates to the nature of genicr organization resulting from the

primary evmt but not necessarily to the primary event itself.

   Yhst kind of a mechcaissl will give rise to these various events

with the expected relative Frequencies?  I have thought of one kind

of secbanis~ that does not see.n too inconccivabfe.  It involves the

fcllo3ving a.ssu=lptions and ictwpretations.

    1.  The main event; responsible for all Ao controlled behavior is

related to the reduplication of the 3~ locus at the Lime of gene

re~3uplicntion.

    2.  Cruelly, following chromosome reduplication, each new gene

mlscule lies adjacent to the mother molecule but is not joined to

the nather molecule by any chemical bonding.

    '1
      c .  Hedu;jlfcatfcn of gcrne molecules is orderly in that a11 the

dau;hter mol8cLiles lie Eidfc:cent to the mother molecule.  Also, all
dc:ughtez' aoleculeo lia in a single plane--that i23 the new dau;zhter
chroxi~~some ia not twisted about t;hrj mother chromosome.  r'he behavior

of ring-chromosomea during mitosis ShOWS this.


-as-

                          . _.
4.  During prop)mse, a repulsion occurs  between mother snd

daughter chromosomes.  This repulsion occurs simultaneously or penrlp

so along the whole chromosome.  The mother and daughter chromtlds

become separated by a rather specific distence, as a consequence

of this repulsion force.  This is seen by an exmination of %h.e two

chromatids at somatic prophasc.  In order to give this srecfse spatial

relationship between two chrormtids, some form of repulsion force,

following chromosome reduplication, is required.

    5.  '#hen a Ds locus is present in an ac ac constitution, the
Ds locus behaves rts other loci do during reproduction nnd thereafter.

    6.  tihen both 8 Ds locus and an Ac locus are gressnt, the mother
end daughter Ds molecules remin chemically bonded together following
reduplication of the Ds molecules in oert%in aitoses [contrclled by
what Ac is doing at the time).

    7.  'khen the repulsion force that separates the mother and

daughter chromatids sets in, the mother fjnd dsughter Ds molecules

are still tied together.  In the adjacent loci, however, the rnother

and daughter moleaules are free from one another.

Tllrection
of repulsion
force

        n        n t-4
J-T- $+- Direction of repulsion
                         fC?TYitt
        " " " "
Ds


-33-

a.  Because the bonds uniting mother and daughter Ds molecules

are still present, a break must occur in this region.  The CR locus

mc~y be yanked out of both chromotids:

                        1      2
          -v        X     .a

Direction              i
of repulsion.~>
force  ~'I
                 \i

                            Y     -4       X
                        3      4

x P broken ends capable oi fusion with other broken ends.

    9.  IIecause of the repulsion forces, broken ends ! and 2 and

broken ends 3 and 4 will come to lie neer to one another, respectively.

Fus&on 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 Pew-late type of Ds action, this mechanism

seems satisfactory as an explanation.  It is oecessery to essu~~e 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 106~s.  Xhen 3s is

removed, by the above mechanism, the original orgnnizatiDn of the C

locus may again be restored and a normal. C action may nqsin occur.

This will explain why the majority of c to C mutations at the c-ml l.ocus

give rise to morphologically normal chromosomes 9 with stable ': loci

showing no further Ds activity.

   Tension produced by the united Ds molecules m;sy extend along the

chromosome during the repulsion of sister chromatids.  Breaks mey occur

not as neatly as diagrammed but mriy sometimes result in %Cjscent

segments

~~being pulled out of one or both chromatids.


                        -39-

This could give slmall internaf aef$ai?naies in the re,-iLox? of the 3s

o???? o  This a~'~y be responsible far tha very l.tirgc number of new
                                h<-
rece~sfve mtants that have ny;rpesred in thmm,atooks.

   AgAn, Ds my be pulled out of one GhI'ozatid but not out of- the

other.  Cr, the Ds locus may be gull&? out of bzth chrornatids but in the

process be itself broken.  Tivo Da loci could then enter one of the

sister ohromatid+$ if the varlouo broken ends wepe close to one another.

This could give u ne:s, compound 3r; locus.  3~ch compounding cmld

oontinue to build up a rather complex, corngound 9:~ locf.
                                                                         ,I`_
                                               I believe
                                                                         .
ihS8 must ooour.
LI
   Or, if a coincidental break oclcurrad elsewhere, the free broken ecds

of the extruded Ws locus might unite with these other broken ends.

Transposition of Ds could then occur.  If broken ends 3 or 4 were

likewise olose to the new brokm ends, compound, gross chromosoael

renrrnngements could arise.  Various abnornrlities could be expected to

arise and be visible genetically, when appropriate genie markers UC

prenentp to maks the events realized.
   h;.thou@, this CIctgmrtmed scheme aeemz sstisfsctory, in q~:qeral, to

account for the few-late 33 behavior it will. not tell -*hy on6 sets the

3s-curly behavior, that is,  dicentric forztisn because of fusion of

sister chromatfds at the 2s 2.ocus.  As stateC ebove, t,he few-late tyoe

,I?' Da behavior is rather stable but the early .J f 9 type frequently thrmva

the few-late type.  The early type Ds behavior probably involves P. zore

complex organization of tho 3,s locus, ptjSSSbl:r a cowp~~i;d organizotioa

with dui;licate Dr3 loci having, thcref~m, mny bcrnda following gene

rcdu;~lication.


This may be:

Or, other gene3 may bEt pressEat between some of the reduplioatsd Ds loci:

The extensive binding action of these reduplioated Ds loci may

result in rather complex tensions and breakage8 during the repulsion

period that mny result in plaoing broken ends of sister ohromatids

alose to one another so that fusions readily ooour.  Also, a reversion

to a simpler form of Da organization through deletion of some of the
                                                                          ..- Y
                                                                         5 . .a ',
Ds loci could be anticipated, that is, a few-late Ds organization
                                        , ..`

@rising from the early-Do organization.

   The various isolates of Ds standard end of o-ml that show grades
of Ds behavior between the extreme few-late and the extreme Ds-early,

may be fitted into this scheme.  The more and the xider separated the

12s loci, the more the chanae for dioentric-forming fusions of broken

ends and the less the chance for broken ends of the same ohromatid to

fuse. Alao,  sequences of change in state of the mutable loci should be
ayiticigated on this meahanism.  The high dioentric produoers should

be the most unstable in maintcining their ststee -#here&s the loWest

dicentric producers should be more stable in their states, raraly* if

eter, throwing a really high dioc:ntric prociuoing state through a


single mutation.  The states producing IatermsAiRte r-Rtes of Eicentric
                                            ,

formatton should go in both directions: from intermediate to hi?h or

to low.  Also, the new unstable mutable loci that arise ns the conse-
q,uence of transpositions of Ds may ref3eat, to some extent, the
state of the locus before the transposition.  If a compund locus

is transposed, in tact, then the mutation pheno;rlreny observed ~3.11 be
suite different from that observed if a single 9s locus enters the

new position.
  The hy?othesls outlined Is not oomplex. It does rot call on
5eny assumptions of a purely speculative nature.  It is rattier one that
could well have been Hnticipated in sdvence of sny sugg,estfve evidence.
There Is no raason to assume that something coul.d Dot go wrong rvith the
reduplication process that would leeid to just this sort of association

of daughter molecule with mother molecule.  The aberrant mitotic
behavior of ring-shaped ohromosomes auggeets that somethlnq of this
mture may 80 on as an occasional error during the reproductive strlge,
even fn nor:ul loci.  It might likewise be responsible for some of the
epontaueous breakage phenomena known to occur rntber frequently in

the Maine chromosomes.  It is the Ds - AC combinatic?n that brinq thiR

to frequen t expression.


-42-

Part II.  The c-m2 loous

Classification and description of the types of mutationa occurring

               at the o-m2 loous

   Mutations ocourring at the o-m2 loous differ from those occurring

at the c-ml locus in several easily distinguishable respects.   The

c-m2 locus is AC controlled; mutations occur only when Bc is

present.  Also, the time and frequency of mutations of c-m2 respond

to AC dosages as do u-ml and t!:e standard Ds.

  The mutations of c-m2 may be classified into several cat>gorios:

    I.  The Pink mutations.  These give various 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 ia 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 rppinkV! to

cover a seriaa of mutations that appear to be similar or related.

Later, it may be necessary to distinguish between various sub-

classes of thls group.

    a).  There is a wide range in the intensities of the color

produced lollowing mutations of' c-m2 to pink.  Some mutations to

pink are too f'aint for the outlines of the seotors to be uertainly

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 o-m2.  These quantitative

alleles of pink may be obtained and maintained from isolates of

germinal mutations.  Only a limited series of' suah alleles have



-439

been isolateU and studied in later generations but many more are

aPailabf8 fOX' 8UCb JRll?pOSCaS.  The germinal pink isolates that have

been studied so far have been relatively stable in their expression.

I suspect, however, that some of these germinal mutations may

respond to Ac by giving variegation in the depth of color.   I 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 ao ac ac constitutions.  It is somewhat mottled although the

grades of color contraits in e single kernel are not extreme.   The

AC se] ac3 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 ohanges at the pink locus or to changes at other,

as yet non-deteoted Ao-controlled loci.

    b).  The pink mut&nts are often assooiated with the produc-

tion of soxe diffusible colorless substanoe.  This subseance

(substanc. 1) can be used by a 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 norms1

c locus.  &hen this utilization occurs, the color of the aleurone in

these oelle may become very deep, giving a super-C color intensity.

The dosage responses of a normal C locus may be reflections of the

limited r,uantlties of this substance that one normal C locus

iJroduces.  The methods of showing the presenoe of this diffusible

colorless substance produced by cells having mutations to pink,

involves an analysis of the color patterns and distributions In

sectors of kernels resulting from the following uombinatfons of

loci.  These combinations will also show that the colorless


diffusible substance is not produced by an unmutated c-m2 locus.

(1). o-n12 female x C-normal Ds male;  Ao ac ac or Ao AC ac constitution

(2). r, *    x chromosome $3 with a broken end.  This broken
                ohromosome aarries a normal C locus.   A0 ac 8c
                or AC AC ac constitutions.

( 3 ) . "        "   x o-ml male; AC ac ac or AC AC Ao.

(4). pink *    x C-normal Ds male.  AC ac RC or AC AC ac.

(5). rt n    x chromosome 9 with broken end.  This broken chromo-
                some oarries a normal C loous.  ac ac se oonsti-
              tutions as good as those with As.

( 6 ) . *'        "    x c-ml male.  AC ac ac or AC AC ac.

Ths color patterns produced following each of thsse crosses will bs

desuribed later.

0).  The isolated germinal mutations to pink have given

dosage responses.  This has not been extensively worked out but

the grades of intensities of pink color in selfed ears of plants
with pink / c compared with Intensities given by crosses of the

same plant to c f8male plants, certainly suggest3 dosage effects.

More instructive for the dosage studies of pink are the kernels
with CI / o / and a chromosome 9 carrying pink that has a broken

end.  The resulting breakage-fusion-bridge cyolea In the pink

crarrying chromosome produce definite patterns of changed color

intensities, obviously associated with different dosages of the

pink locus.  Too few kernels are available for an extensive analysis

but those examined clearly show dosage 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

c lOOUS.


-4s

    d).  The pink mutants (some of them, at least) are deficient

for some substsnce that a normal C locus produces.  Two such substanaes

muy be deficient but at leaat one is a colorless, diffusible sub-

stance (substance 2) that is associated sith the activity of a

normal C 10cus.  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 c-m2 female x C-normal ds male,

AC ac ac or AC Ao ac, has been useful.

II.  Hututions of c-m2 giving conditions that resemble

the full C genie activity although varying in

quantitative expression of this activity.

   In crosses of o 80 females x c-m2 AC AC males, pink sectors

appear in the kernels of the resulting ear.  Xithin some of these

sectora, mutations to a type of full C expression often occur.

Unless there has been a duplication of the o-m2 locus Rt the same
time that a mutation to pink occurred to give two c-m2 loci, each

                                                                           _a
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 ~3. full C type expression.  Yone of

the germinai pink mutations that have been csrried to a second

generation have continued to give these pink to full C mutpitions 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 clrosses of e-m2 females x o males.  In %hese cases, the
female contributed two loui to the endosperm (two chromosomes 9).


-460

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 evidenoe for this is suggestive. In 4 of the 7 tested oases

(4451A, 4456-2, 4456-4, 4458A-2) the chromosome entering the egg

nucleus likewise oarried pink.  In the other ? cases, the chromosome

entering the egg nucleus carried a non- mutated c-m2 locus (4456 -1,

4456-3, 44568-1).  IQ the mutation to pink oucurred during the

divisions of the female gametophyte, just such apparently conflicting

                                               w
results could be anticipated.  Such4female gametophyte could have
nuclei with the pink mutation and nuclei with the unmutated c-m2

locus.  Because mutations of all AC controlled mutable loci ususlly

occur lste in the development of the sporophytic tissues or often

not uqtil the gametophyte or endosperm stage is reached, the period

of' origin of germinal mutations could be as late us that just

described.  (It might be mentioned at this time that occasionally

an jIc controlled mutable locus may mutate early ir, the development

of the spotophytic tissues.  It is a very rare event, however,)

Tests of the stabilization of pink mutations nlust come from the

reciprocal cro3s: c  x c-In.2.  The germinal pink mutations 3howIng

full C spot3 must be selected and tested.  They occur but thfs

obviously oritical test has not been made.  I was not sharrlp in

focus on the c-m2 mutations when the selection of germinal wtstions

wus made!

A singl.e (?) mutation of the c-m2 locus to give the two diffus-

ible substances or at least a second diffusible substance (substance

2) that is associated wfth gene activity of a normal C locus can

occur.   iiel.1 defined  seotors assumed to have substance 1 and known
                                  ,^


to have substance 2, appear on the kernels coming from the following

                                                                           /_
crosses:

(1) o-m2 / e-m2, AC ac; aelf-pollinated.

(2) o-m2 female x  c male. (AC ac RG or AC :?c ac constitutions)

(3) c female x c-m2 male. ( w         *' "              ,t      1

(4) pink female x  c-m2 male and reciprocal.  (AC RC ,uc or
                   AC AC? ac constitutions).

It is concluded that if not a single mutation then a sinale

event at the c-m2 locus may result in a modification of genie

organization that will produce both of these substences associe.ted

with action of a normal 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 u mutsnt 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 muttitions do occur that give

substance 2 but not substance 1.  The sectors suggesting th!s are
pink with deeper colored rims in the pink sector on p. restricted
part of the border between a pink and a colorless sector; or, within

a pink sector, darker areas with diffuse borders often wppeisr.
I am not at a12 certain that this interpretation is the only one
that will explain some of the color patterns of these sectors.

Xore obse.vatians and thought are required.

  The mutatioils to full C activity are quantitfitively expressed
in that various grades of color intensizies are present.  The


sectors with these phenotypes may be quite light in color or

fairly dark.  No germinal mutations to full C color activity

have been detected so far.  This is not evidence that they do not

OCGUI'.  I have not looked exhaustively for them. Unless they

@vs deep grede3 Of full !: color, I might confuse them with pink.
I know now that-:often>the two differ in eppearnnce, reaerdlsss
of color Fntensit& sxsressions.  It should be possible to

discriminate between the t*o, in some cases et least. I have not

taken time to find thorn but will do so.

    ?II.  The unmutated c-m2 ~GGUS produces no detectibls quanti-

tiea of either suhstunce 1 or substance 2.  This will be shown when

the kernels resulting from the crosses enumerated above Ere

described.

    IV.  Ridden mutations occur at the c-m2 locus.

   The frequency of hidden mutations seems to be quite hlch. This

f~ i?hown by:

   (I) The types of sectors produced in the cross of G ac females

X  O-82 i'iG AG IIlQlt'33.  ;;hen the variegation pattern in these kernels

is oompared with the variegation pnttern produosd by c-ml (from the

heavy visible-mutation producers) or aith those produced by DB-enrly,

it is evident that tLle visible sectors in the G-PC? kernels rr-!:>resent

some form of sub-sector arising in 8 descendtint cell of one t3nt had

undergone some primary event.  This orininal mutation event in the
                                                    .a

nnGestor cell resulted in a visibly ohanged phenotype only in a

sub-sector or sub-sectors.  Xithsr some kind of (a) segregation to

daughter aells occurs following the initial event or (b) su~cessiv3

mutatfons followr  after the original mutation or (c) both types

Of eV0lltS OGCUI'.


-49-

   (2).  Mutations of c-m2 to stable o loci occur rather frequently,

? ???????? o  Some stable c loci have been isolated from crosses
involving c-m2 / c-m2, AC AC or AC tic plants.  I can not say that

all such nutations are completely stable.  3eversl have been carried

for three generations without showing nlutatLon in the presenue of

AC.  Several ears have apperlred in the crosses of c / o-m2,Ac ac

females x  c,ac meles tht+t show large sectors on the ear with only

colorless kernels.  This may bo due to loso of the i?c locus (evidence

f'or loss of AC will be presented later; it is not uncommon;) or to

stabilization of the c-m2 locus.  Evidence that will diff'erentiate

between these two alternatives will be easy to obtsln.   It will be

necessary, in any case,  to determine whether mutations of c-m2 to

stable c are associuted with some segregation of chromatids, i.e.,
stable c in one chromat,id and an altered organization of the c-m2
locus in the sister chromatid. The sub-sectors, mentioned above,

suggest segregations may sometimes occur.

   (3) Soma "hidden" mutaticns fire actually pseudo-hidden in that

they are Fick nutet-lons with such faint colors that detection may be

very difficult or not possible, in many cases.
  (4) I suspect thnt 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 c-n2 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, 8tC.  Although c-m2 may illuminate some of the activities

of a normal C locus, a study of its mutable behavior 13 discouragingly

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

carry along.


  --
I

-5O-

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 inrrolve o-m2 A'x to:

(1) c sh wx da ac and to (2) C sh bz wx ds ac. The ;tix-m

locus was carried by the ohromosome with c-m2.  The wx areas in the

kernels coming from cross (1) Sire usually due to mutations of

lVx-m.  In or058 (21, only a few kernels ;yith C Ez - C bz tireas were

observed.  These cress may arise from events other than those

associated with the o-m% nutnticrns.  It may be stated, at least,

that the isolates of the c-m2 locus have not given, as yet, eny
                    4 m%P-

states that regularly result in looa of the short arm of chromosome 9.
                                        4

I believe they will appear, sooner or later.

To summarize:-

The o-m2 locus may be compound in that the mutations

occurring at c-m2 are not asscciated 5sitb 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, colorleso substa.nces, both neoessary for full C: expres-

sion.  The production of substance 1 is often associated with the

appearnnce of some pink color.  llutations giving both substtince 1

and substsnce 2 may occur.  Although both substances may be present

in a seotor arising from a cell having such a mutetioa, full C

color need not appear in this sector. juantitative 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

rniiy be mutable but thiB is not oertain as yet.  Mutations to full C


-510

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 3mc? substance

is likewise produced by a normal C locus.

   That the weaksned color intensity of pink mutations appenrs

to be associatad with some specific deficienoy of a needed substiince

is suggested by the dosage responses of the pink mute+,ions. This

substance can not be substance 1 for substance 1 is often produced

in excess even in the mutcticns aivinq very foist pink.  Tt my be

substance 2.  If so, then all mutations giving pink must, likewise

produce some substance 2.  Neither substance 1 nor subs%ancF: 2

is produced.by the ucmutated c-m2 Incus, however.  If the intensity

of the pink color is an exprsssion of the love1 of some limiting

substance this substance must be one of tho products of mutations

of c-I&2.  It is not controlled by other loci, as the doswge responses

of pink indicate.  This may be oubstence 2, u3 mentioned above,

or it may be a third substance associnted %ith ~enic action of a

normal C locus.  Althou@ this sub&awe-producer interpretation

of .mutations of c-m2 fits the observations so far made, 1 believe

that one should be canny about accepting it as it has been presented.

I have that uncomfortabla 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.

   Bsfors illustrnting the color patterns produced in the kernels

coming from the enVxuerated crosses,  some statements should be given

regarding the origin of c-W.


-52-

The origin of the c-m2 locus

The o-m2 locus first appear8d on en ear of a self-pollinated

plant--plant BOOOB-2.  Thic plant had the constitutions:

I

   ,
,&. / *pyd I: m-2       m
                       3h 'Xx  ) AC AC.  It is only in this

one plant of the culture that I hrjve been able to find nny evidence

for a mutable c locus.  Alao, I hnve not been able to determine,

as yet, whether or not R mutable C locus was responsible for the

first appeoracce of this o-m2 locus,  or whether it came froa a normal

G locus,  ~3 did c-ml.  All that I ten state is that there is no

certain evidonoe for its origin from a C-m locus.  Culture 4000

~8s segregating a nutable pale-green locus.  Each plant in the

culture *#as self-pollinated. to determine whether or not the pale-

green mutable locus ~8s present.  The origin of plant 4000B-2,

that bed the c-m:! locus, will be given starting as far back 19s the
                          &&j &A r,;it: t +4.

cross that Five rise to a kern81 W&S& two broken chromosomes S

in the ZygOt8.

(1)       2467A-5               X         2476-6
   I Ax;               G "x/
           ,- ,a'       i
                 I             /
         I ;Jix          @---t--
                                                             I
                                   \Sh wx

4  G ' :d?r

One I-S;  *h'x-x kernel MS selected from this cross because both

chromosomes S had newly arising broker! ends terminriting the short

arm.  This kernel gave plant:


     S~-p~.-,~-~ _. i,;`j$i
      t- -`.,     -530

     I      ;6' a'. f
(2) 42~B.  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 nslfing the main ear did not give this mutable

locus.

   (3) The progeny of the tiller ear gave culture 3592.  This

culture soaregeted pyd ee well as the new mutable pale-green locus.

Cytological examfnnt,ion of a number of pl%nts in this culture were

made and genetic trsts confirmed the constitution of the tiller of

plant 42-B to hsve % chromosome 9 with 8 duplication of the short
%rm and carrying I 8x and a marphologically normal chromosome 9 with

pyd C Sh wx.  Ao was also present in culture 3592--whether homo-

zygous or segregating, I do not know,

   (4) Plant 3592A-9 was selfed.  It had a duplication chromosome 9

with I gx and a no-1 chromosome 9 with pyd C Sh wx.  The progeny

of this self gave:

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

  ijelffng 400QB-2 gave the c-m2 locus carried by the aorpholo~i-
cs11.y normal chromosome 9 of this plant.  Mofie of the other plants

in culture 4000 showed the presence of a c-m2 locus.
  Examintiti~n of the solfsd ears of other 3592 plants hes given

no indication of a c-m2 locus,  These plants, however, did show other

mutable looi.  Those recognized were:

   a).  y to Y

   b).  Starch consistency change from wx-like to i<x but the wx-like

starch was not associated with wx-staining reaction.  I'ossibly the
areas with %x-like starch consiotenoy stain a deeper blue than


-549

normal Wx.  I am not certain that all these areas show this. It

has received only a cursory examination.

Q).  .X%le-green seedlings mutating to normal qeen.

   d).  A B-few-late type of behavior in one plent.

   4.  In plant 4000%2, a ,$x-m locus `t:u'as present.  The tests

could not show for certclin whether this Y'x-n locus was likewIse

present in other plants of the culture.  I think it must be from

the constitution of plant 4000%2.  It could be found out, thouph.

   Obviously, the parent culture1 from which pl.ant 4000R-2 arose

was full of mutable loci.  Except for c-n2 and :Xx-m, both of

whioh are Ao ooctrolled, it is not known ??:hether or not these
    I

mutable loci are AC controlled.  b) end d) above both look like

Ao oontrolled mutable loci from the patterns of variegation seen in

the examined kernels.