Memorandum to Marcus Rhoades. First Section. Jantlary 1949 Outline of topics discussed. Introduction. Part I. The c-ml and standard Ds loci compared. Page 4 1. 2. 3. 4. 5. 6. 7. 8. 9. The c-ml locus: General considerations. Page 4 Stabilization of the mutated c-ml locus. PRge 13 Evidence for the conclusion that no Ds locus waa present at the standard location in the original c-ml carrying chromosome. Page 15 The standard Da locus: TN0 contrasting types of mutation behavior. Page 18 The relation between the two types of behavior of c-ml and of the standard Ds locus, Page 20 Comparison of rates of mutation of c-ml and of the standard Ds. Page 22 The AC control of mutation during deveiopment. Page 23 The mode of origin of mutable loci. Page 26 Possible mechanism responaible for mutable phenomena. Fage 31 Part II. The c-m2 locus. Page 42 1. Classification and description of types of mutations occurring at the c-m2 locus. Page 42 2. The origin of the c-m2 locus. Page 52 'x .- . Illustrations of the phenotypic changes that are associated with c-m2 mutations. Appendix - Extra Tables. Not explained in text. Memorandum to abrous. Jmmary, 1949 Introduotion The orno locrus (hereafter written o-ml for speed in typing) appears to have arisen as the oonssqusnoa of a ohange in loostion of Df3: r8mOVe1 from its fonner position and Insertion into for close to) a norm::1 0 loous. In this new position, it behavea just RPI it did in its former position. The o to 0 mutations appear to be assouistad with its behavior in this new position, aa I ehall try to point out. In this new looation, more has been learned about IIs behavior than oould have been learned from Da in its "standard" location. The position in crhrozaosome 9 where De was ffrst discovered will be Galled the Hstandardw looation. A position of' reference is needed beclause Ds does change its looation. In Its standard position, it ia known that Da changes its mutation pattern from one general type of behavior to another, i.e., the Qarly" loss type and the Yew-late" loss type ot behavfor (for details of these two general types, see below). Previously, the behavior of the Ds loous in the mfew-late" state had been difficult to analyze and interpret. In its new position at the C locus, various types of behavior of Ds are subject to analysis, as will be indioated. Becrondly, ohromoaome breakage of' a type not previously recognlved may be rseponslble for the mutation phenoaena associated with mutable lo&.. The type or breakage and aubsequent fusion of' broken enda that will ocmur in any one "state" of a mutable 100us my be controlled by the particular organization of' the inserted loous reaponslble for thi 8 breakage phenomenon, at the time of Its Insertion into a new looation. Thfs breakage prooess need not lead to the simple criss-oross type of fusions as previously suspeotad, nor to the formation of U-shaped dioentrio ohromatids already known to arise from mutations occurring In one "state" of the Ds loous. rt Is probably something quite different. This breakage mouhanism will be outlinsd later. This meohanfsm should sometimes alter the organization of the inserted locus so that new types of organizations would be bormed. Thess altered organizations will give rise to nsw "states" of the mutable locus, that is, dlffsrent rates of producrtion of visfble mutations, of stabilization of loci, rend of the typas of' fusions that will ocour following breakage. The state of a nutable loous is something more than a quantitative change at a loous. Starting with any one organization that gives a particular series of breakage-fusions events, the types 0r ohanga to new organizations giving an altered series of evsnts may follow rather definite sequsnoes. Thirdly, stabilization of a mutating loaus may follow as a consequenoe of lossiduring a mitotia ayole,of the Inserted locus responsible for the mutation phenomena or of Its aotlve component. #ith some organization8 of the inserted locus, each mutation of the mutating locus,with some exoeptions,will result in stabilization of the mutating locus. '#ith other organizations, some of the mutstions will not reault in elimination of the acltivs loous. In these oases, continned mutations will ooour. This mutating locus may be a dominant, an intermediate allele or a recessive but mutwtlons will oontlnue. Fourthly, I suapeet that many loei, such as the C loous and the AL and A2 looi are crompound loe,i involving ganic aotion assocla~ted with SUOO~SS~VS or related steps in the oonverslon of substrate to produot. The type of mutations ooourring followin$ insertion of a "Da" loaus into suoh looi will depend upon (1) the posltlon of insertion, (2) the organization of the inserted louus at the time of its insertion and (3) the changes in organization of the Ds locus that will oa~lur subsequently. If the aoaumulating data has been lntarpreted correctly and continues to give further supporting evidenoe, it will be neaessary to ahange the symbol "Dew to something more expressive of its basic aotion. The observed dissooiation phenomenon (dioentric ohromatid formation) is only one of the ooneequenoes of mutationa of Ds that ooour. The various typss of Ds mutation behavior are probably expressions of one basic type 0r event. This. is the important event underlying all expressions of mutability of mutable loci. The newer evidenae has made it possible to consider this event more specifl- oally even though there is muoh that is purely speculative. Neverthe- less, the expanded knowledge allows for better dlreated experimen- tation and observation. Fifthly, there are probably a number of unstable dominant looi in my material. These wfll eventually throw unstable reoesstves. The wx-ml loous arose from an unstable dominant locus, designated Vx-m. This unstable dominant loous has now been isoltlted. It will throw more unstable reaessives. One suoh recessive, a new*-m, is growing in the greenhouse bgrt many more should be detested following suitable crosses now that the unstable dominant that throws unstable recessives has been isolated. The instability of dominents and the unstable reoessives they throw are probably ~11 expressions of the one kind of event, i.e., ohromosome breakage. Mutable expressions go in a aycle from unstable dominant to unstable reoessive to unstable dominant, eta., or to various quantitative levels between full dominant and full rsoessive. (Full dominant is a relative concept. Super dominants can be isolated.) Stabilization may occur at any quantitatively eipressed level, however. Part I. The c-ml and standard Ds loci oompared. The o-ml loous; general aonsiderations For the details, the c-ml 0888 may be oonsidsred first. A brier review of the behavior of this loous will give the essential raots neoessary to an understanding of the discussion. The a-ml locus L,,l 0 37&,l,'"' arose in the ~lros5 of a yg a sh Bz wx ds ae female plant b a mele J I j j J i* 0 .i , _ .,_< ..-. . -2' plant that was Yg C Sh Bz wx Ds / Yg C Sh Bz wx Ds, Aa ac.: Ten crosses of this male parent were made to e sh ds aa famnle plants. The ratios were: 2077 C, non-variegated (ac ac ac) : 1199 C to c variegated, early losses (AC ao ac) : 317 I: to 0 then classified as having the few-late type of Ds action (mainly Ao ac ac) : 1 c to C variegated kernel. This last kernel W~'IS obviously not the regular C to c variegation expected as the oonsequenoe of Ds aotion. It showed the opposite type of variegation, from o to C. A plant was grown from this aberrant kernel in the summer of 1947 (plant 4204). The plant was variegated. It showad streaks of yg. This variegation was not unexpeotad beaause it was known that both chromosomes 9 in the male parent oarried Ds. If Ac were present, just such yg streaks should .appear. Xt was only necessary to determine that AC was also present in this plant; the later tests proved that plant 4204 was AC ao. The aonstitution of this plant was then believed to be Yg 0 sh wx ds / Yg c-ml Sh wx Ds, Aa ao. The data and analysis given below shows that the looation of Ds is not as given. Instead, it is probably at, within or oloee to the C locus. In this plant (4204) no ,evidenoe has been obtained for a Ds in the "standard" Plant 4204 was orossed to: (1) yg o sh wx ds ao (2) C sh ba wx ds ae (3) C 3h wx De / C Sh wx Ds, do ac (4) pyd c Sh tix da / yg c Sh WI de, ao ac. From aross (11, It was clear that the male parent possessed two ahromosomes $,ons with Q o-ml 812 wx and one with yg c ah wx. (For kernel taunts, sac Table I f Appendix.) From cross (2) it was olear that a D s loous was likewise present in the o-ml 9h wx ohromosome. . of the 8h kernels (the Ao oontaining kernels) were C B-C bz variegated. (See Table 2 , Appan- dix.) There were few suuh kernels in the sh class. (This latter observation should have made me suepicious about the looation of De but 3 failed to register a reaction. At the time, I did not know that Ds oould ohange its lactation. Realization of this did not enter my oonsoiousness until late this spring, following the harvest of .1 ,c : r*: I the greenhouse crop. From oross (3) it was likewise olear that a Dr loous was present in the o-ml. Sh wx ohromosome. Some ot the kernels were C to c variegated following Da mutations in the G Da ohromosome contributed by the female.parent; in some of these kernels, the o areas showed o ,I,. * I. q ')'i f I, 'A to C m&atfoti$ due to o to C mutations ooourring at the o-ml locus in the ohromosomes 0 oontributed by the male parent (4204). These latter kernels most always showed homozygous deficient tissues of the type oocurringS when three Ds loci are present, one in each chromosome 9. The oombined evidenoe indioated quite definitely that a Ds locus was present in the o-ml oarrying chromosome of plant 4204. From oross (1) it was obvious that the expresoed mutation rates of o-ml are not alike in all the kernels. The majority of the kernels show relatively few o to C mutations. A small number of Sh kernels, however, had very many o to C mutations. I assumed from this that the kernels showing only a few c to C mutations were either (1) those having a o-ml Sh wx Da constitution and that the Ds mutations were eliminating the segment of the abort arm oarrylnR the a-ml locus from many sector8 of these kernels so that the o to C mutations oould not ooour In these sectors; or (2) that the state of the o-ml locus was low, i.e., few o to C mutations \bWttr ooourria@; even in those kernels with o-ml and no Ds (those derived from cross-over ohroma- tias). Again, it was assumed that those kernels with many o to C mutations probably possessed no Ds beclause Ds oould have been removed from the chromosome as a oonsequenoe of arossing over between Sh and the assumedhDs loous in the parent plant (4204). Saoause both uhromosomea 9 in thin&rent carried wx, there was no immediate genetio determination of such a cross-over. The sh kernels showing o to C variegation obviously oarried a cross-over ohromatid. Barrin double crossovers, which are rare, the e-ml chromosome in these kernels should have no Ds locus. These kernels, however, did not show high rates of c to C mutation. That a changed looation of Da had ooourred when o-ml arose oould not be clearly FeveRled from an analysis of the crosses made during the summar of 1947. That something was wrong soon beoame obvioua. The nature of the disorep- enofes ~111 be indicated. -I- The seleotion of kernels for tests In the greenhouse last winter,, oentored on eetablishing the, sontrol of c-ml mutation phenomena by Ao. From this crop, it wc98 determined that Ao OontrOll8d the c-ml mutations; without Ao, no mutations ocourred. It alao showed that the time in development when mutations would oocur depended upon the dosage of AC. In this respect, it was quite similar to Ds in response. In the progeny derived from crosses of those plants that were drown from the highly o to C variegated kernels, this greenhouse orop also showed the exact oorrespondsnoc in pattetrns of mutation to those already known for one typa of Ds. You will remember aeeing, this aimllarlty in comparing kernels of the oonstitutlon C da / C ds / I ?7s, Aa ac ac (and AC Ao ac) with those kernels having o ds / c ds / c-ml, AC ac 80 (and ho AC a~). The two sets of kernels ware indistingulehable, respectively, in ooior pattern of variegation. The mmmer planting (1948) included a selected number of 'sx kernels showing c) to C variegation from oross (4) above. The Vg df co< f+fv plants arising from these kernels were till variegated for pyd. This ? was not antioipated for 1 had expected a few of these plants to have a o-ml chromosome with no Ds beaause of crossing-over between o-ml and wx in the male parent. It did not bother me too much as the sample was small. In order to be sum to eliminate Ds from a c-ml oarrying chrol[Dosome, some of these plants were crosses (1) to o sh wx ds ao, [2) to 0 sh bz WI da ac, and (3) to plantls having the rearranged chromosome 9 with o sh %K and a female-transmissible deficient chromosome 9 oarrylng 3h and wx but deficient for the c locus. There were other reasons for these orosscs but mainly it was to observe the efteots of c-ml mutations when Ds was certainly removed f mm ths ohromosom. No Ds (standard location) should be present in most of the cr-ml Wx ohromatids. Only double croBsovera ~0uld give the 00nstitutPon c-ml lrJx Ds and these are rare. The rsaults from cross (I) were Strikingly different from 0xpsatat~on. E'early all the -$r kernels thet showed o to C vnrie@- tion were also heavily W Lo wx varfsgst.ed; this :;$x to wx variegation resembled ehe usual Ds typs'variegation. In these kernels, the rate of c to C variegation was low, except for Sectors in Sorie of the kernels, A few of the '#x kernels, however, Were heavily VariegEited for c to C. These kernels showed only a faw wx areas and they uauc.lly were quite small. A tabulation of the olasses of kernels resulting from oross (1) mty interest you for they tell the full story much better than words (table 1). Because the male parent was heterozygous for Ao, only half of the kern818 having a o-ml locus could show c to C variegation. Also, the kernels that received the stable o locus (from the perentel c Sh Ix chromosome) will not show c tc G variaga- tion with or without AC. You will note that only 14 of the $90 o tix kernels (no visible c to C mutations) Showed 'iix -wx variega- tion whereas all of the 140 c to C variegated kernels that were `#x likewise showed 3x to wx variegation. The prasenae of deteotible sh sector8 in some of these heavfly Wx-wx variegated kernels made it clear that 1088 of a segment of the short arm of ohrOmO8Om8 9 was responsible, or aften associated -Jiith, the appearance of the wx sectors. Some kind of Da-like action %a8 occurring in these kernels -Mith a c-ml :Vx chromoa0ma. This Da action waS not associated with a standard Ds loous in the c Sh ~Yx chromosome of the male parent. If' R 11s locus had been present in this ohromosome, many of the o 'Sx kernels shouL6 have been #x - WL varfEqted but they were not. Secondly, all the ax kernels showing o to C variegation likewise 43504 x 44343-3 46 90 141 27 3 1 1 4346-18 x w 40 80 146 10 1 cl" 0 4349-2 x 34 03 X48 22 0 0 1 Totals 120 253 4s 67 4* 1 2 c 2 .C c7C c CrbSS c wx c .I:X s 9x c: wx wx 3x 3 wx i~X+wX 4346-16 x 4434h5 27 57 73 u 2 ? Cl 2 ----m-e 4340-31 x rl * 29 3 1 3 1 1 * -26 x iv w 37 99 148 28 0 2* 2 f' -34 x '1 ?' 34 100 134 15 4+ 0 3 Totals 1.37 367 75 1ov 3 11 - - Bhbwed 3~ tb WI: variegfitibn. These represent the kernela receiving a crossover chromatid that brought o-ml and #x onto the same chromosome. Since all of these ohromosomes show this Ds-like action, a Da-like locus must have been present in the o-ml 3h wx chromosome of the male parent. Its location must be in, close to or to the left of the c-ml locus. Thirdly, of the 16 C kernels arising from germinal mutation of a-ml, only one showed any C to c variegation and this was Q peaulhar type. (It will be polinted out later that just such new variegations are to be expeoted. They represent changed looation or changed organizations of' the inserted locus responsible tar mutable phenomena.) Unless all such C kernels heve an ac ac ac constitution, a Ds type variegation should be present in some of' them, on the assumption that a Ds is in some ponition other then the c-ml lacus. Fourthly, extimination of the C areas in both the c to C, ViX - wx kernels and the c to C wx kernels were likewise instructive. In these kernels, with aotne expectted sxceptions, the C areas were nut v8riegated for C to c. In the c to C, Wx - wx kernels, the C areas were Yx, not ';r'x -wx variegated as would be expected if Ds were present to the left of the C locus. Obviously, then, when k mutation to C occurred, the N-like action In this chro?nosome ceased (with some sxoeptions, to be consSdered later). The oombiaad observations made me suspsot that the Ds action IRS aseociated with the presenue of Es in the o-ml locus and that the Da locus was usually lost from the chromosome when a mutation to C occurred. The following conclusions were formulated: that (1) the o-ml loaus tlrom f'mm 8 c Sh wx IIs chromosome fcllowing removal of the Da locus from its standard locaticn and insertion into the normal C locus; (2) when a mutation or o-ml to C occurs, Da disappears; (3) the insertion of Ds into the C locus brought about an inhibition in the expression of the genea at the C locus; consequently the modified C loous resembles o in phenotypic expression. (4 Yhen this 953 locus is removed from the ohromosome, the normal qenio action of the C loous cm reappear. (5) Two contrasting types of Ds action a*,,,., MM be reoognized when Ds is in this new position: (a) one giving the typloal. diosntric ohromatid through breakage snd fueion at this new location and (b) one giving tha c to C mutations that u+ally result in no visible alteration of the chromosome. For an understanding of the events underlying mutability phenomsno, conclusions (4) and (5) above are most important. They bring up the question: Does Ds in its standard location show these same two contrastine types of behavior? A positive answer to this question is indicatert. Two dissfmilar types of behavicr of Ds in its standrrrd location have long been recognized. The extreme of one type gives many dicentric chramatids RS the visible conaecuance of Ds mutations. The other extreme gives the *few-late".pattern of chromo- some segment losses, i. 0., few dicentric-forming mutations occur. The relationship between these two contrasting types of bb mutation behavior of' Ds in its standrsrd location will be indiaated after 8ome of the supporting evidence for the conclusions (1) to (4) are given. Cross (2) -- C sh bz wx ds au female by o-ml Sh Rz wx / c Ph Bz !ivx, AC ac male -- will be mentioned first. On the ears resulting from cross (2), a namber of kernels were vsriegated for Be -bz. In those that had the YX locus, the C b-r Areaa most always were variegated for 3x -wx, i.e., $3~ was lost before 'pix. This would b8 expected if dloentric chromntid formntion rasul.ted from -110 Ds mutations at the C looue. (If Ds wem in its standard position, the bz areas would but wx.) The results from one such uross are a8 follows: C sh bz wx da ac x (Plant 4684-7) o-ml 3h Bz wx / a Sh Bz Wx, AC ao [Plant 4434D-5) c Bz $i'x non-var. C Bz - C bz c Bz" C Bz -C bz #x .Am wx wx not obviously VEtI?. 124 12 89 41 The kernel@ resulting from crosses of type (3) were likewise useful because the classes sre those expected on the basis of the comlusione given abave. 088 such cl-089 gave: ac! acz x c-ml Sh wx / o Sh Wx, AC ac (Plsnt 4642-6) ( Plant 4434D95) S C Sh Wx non-varhegated. 1 C Sh wx non-variepted 11 o to C lightly variegated; Wx-#i;r variegated; homozygous deficient tissue8 (from dioentrio crhrormtids following Da mutations). 19 c to C lightly variegated; wx. Homozygous defiolent tissues present (front dioentrio ohromatid formation resulting from Da mutations). 5 c) to C, heavil variegated; wx. tiid 370 obvious hoaozygoua defioient Yuoh tissuea would not be detected if dicentria ahroarat2d formation oarrurred late.) 63 o sh wx; no homozygous defioient tissue. wwwwwww"w-wwwwwwww-wwwwwwww--wwwwwwww~www"wwwww"--wwww----w-----w--w * Unless early losses of 32 oocur, it is often not possible to detect C Bz-C bz variegation. Bz substame from the surrounding Bz Areas diffuses into the bz areas giving them the Bz phenotype. A few of the kernels in this *may belong in the C Bz-C bz class. -120 43 ct to C, ve&eg~&tjd mostly lightly variegated; Wx; no homozggous de- fioient tisaua 240 c Sh Wx; non-variegated; no homozygous deficient tissue. The plant arising from the original kernel showing c to C varla- gation (plant 4204) ia now &$%.-to have had the constitution gg c ah Bz wx da / Yg o-ml Ds Sh Bz wx ds, AC ac. Before th8 trans- position af Ds to the C locus was re:ilized, the aelection of sh kernels that showed c + C variegation in the aross of this plant to CI ah wx da ao plants, was expected to give kernels having a e-ml loous but no Ds lccus. Suoh cm-l carrying chromosomes represent orosa-over ohromatids and if Dig had been present at its stsndard location in the a-ml Sh wx chromosome of plant 4204, they should heve no Ds loaus. Plants were grown from some of these o + C eh kernels. Croasss of' these plants were made to (1) C sh bz wx da ao plants and to (2) plante having a deficient chromosome 9 (the d8fiCienCy included the C locus) Sh wx and a resrranged chromosome 9 with c sh Wx [es wall as to c sh de ac plants) Following cross (11, C Bz - C ba variegated kernels appeared on the ear. Following Ctross (2), homozy- gous deficient tisrsus appeared in the o --i, C Sh wx kernels. Obvicuely, the Ds aotion had not been removed from the c-ml ohrOmOSOm8 by crosaina-over between o-ml and Sh. Beoause the transposed position of Da in the o-ml ohromosome was not suspeoted this past summer, the most favorable types of orosses to illustrate a new position of Ds were not spst8matioally made. Nevertheless, some additional ClrosseB, made for other purposes, have been helpful beaause they are eonsiatent with the above interpretation, An illustration of one of auoh oade is given In the aross of a c-ml sh `-4X / c-ml sh wx, Au ao male plant to an ac ac female plant that had -x3- a terminal defialent ohromosome 9 with C Sh wx. This croaa gave: 90 I @lx kernels 3 c A% I&ml 34 C wx normal; no homozygous deficient tissue 20 C WI, with large amounts of homozygous deficient tissue. %The horaogygous deficient tissue in the 20 C wx kernels srlaes as the ~-.l~v. 37 .d consequence of dioentsic'formntfoa assocfatec! with !M mutetfnns at the o-ml locus. There are several points that require further oonsfderatlon. (1) Why does a stable C loaus arise when a c to C nutation occurs9 '&hat happens to the Ds locus durin@ such ~tn event? (21 dhet is the relation between the two visibly different types of events thnt cm occur at the c-1111 loaua; (a) diaentric chromRtid Formation snd (b) the c to C nutntione that do not alter the chromosome mornholoqgQ (7) Does Ds in its standard looation ahow these game two mein types of behavior? (4) #hat i t n errelations exist between these two contrasting behaviors of Da? (5) iiiihat is the evidence Car the oon- elusions that no II3a locus is present at t3e standard locatIon in the original. c-ml 9h wx chromosome? _tabili?ation of the C locus fcllowi~ a o to C mutation of o-ml will be uonsidered first. Stabilization of the mutated o-ml loous. On examining the variegated kernels arising from the ffrst crosses (c sh wx ds aa x 4204), it was noted that the mefority of the C areas srising irOle mutations of' c-ml to C were not varieRa+,ed for C to c, as could be expected. This expectation :vas based on the assump- tion thut a Us locus was present in its standnrd loaation in the c-ml carrying c3hrozPoeome of the naale parent. ff this had been true, TO to 80 paroent of the e to C variegated kernels should hove had this Ds locus. Consequently, this absence of C to c varlegotina in the S preae waB unexpected. I wondered at the time whetherBin this case, a c to C mutation was associated with a change in AC activity co that no further AC controll& mutations would occur in the sector. ?he evidenae for suah a conclusion was not substantiated in other crosses. Tt was known that c to C mutations in one chromosome could occur following i3a mutations Ln the hamol~gous chrolatosoaee. This wa:; revealed in kernela resulting Prom the orom of C wx DE? / C wx Ds ac ec female x 4204. It was obvious hem thet dicentric- chrom&tid-forming Da mut;ztLons aould occur in the fermtle chl*omsomeEl: 9 early in dev~lnpmnt and that these could be fo2lomd in Zzlter eel.1 generatfons by c to C muttl t ions clit the o-n1 locus in the chronosorne 9 contributed by the lU~.10. In the croaseh~ or Q sh XX ds ac x 4204, a nuaber of geruinal mutations of c to C appeared on the ears. )rTone of these C kernels showed an.y C to c variegation of the Ds type. If a sttindard Ds locus were prezerit in the C chromosome of these kernels, many of these C kernel8 should have shown the Ds activity by giving C to c variega- tion, for ha31 of thalm 83ould have an AC HC ac constitution. 29 c, non-variegated kernels arising from these crosses, end from some similar irnt>s mado in the greenhouse last winter, were removed from the ears and planta grown from them in the field this past summer to determine (1) if an Ac locus were present, (2) If Ds were present and (3) if the G locrua arising from a o-ml tmtcation was stable in later ge:zcrc?;x kernels (ge,rmSnal mutations) are C ho c variegated with the o areas being wx:typioal DY type. There oan be no question, then, that Ds mutations oari occur in a o-ml carrying chromosome following a c to C mutation if Ds is in its standard locntion. Gith Ds at the C locus (c-ml) a mutation of c to C usually eliminates the I)s locue or its action and the newly srieine C producing locus is stabilized against further mutation phenomena. Mutations OP c-ml to C do not uaucllly result in mor?hologiaally altered chromosomes 9. One obtains a normal ohromosome 9 with a nora? C loaus action.* Nevertheless the Ds loous at c-ml shows two main contrasting types of behavior (1) the production of diaentrio chromatids and (2) the mutations giving rise to a C phenotype without alteration of the chromosome and usually with elimination of further Ds activity. ---------"cI--l-"-----.--------------------------"------------------- * The use of *normal C loouat* must be qualified. "Jlutations at the o-ml loous have given many apparently normal reaoting C loci. There are a number of sub-levels and some super-levels as well as a few resembling the pink mutations (very rare) of; c-m2 arising, as the consequence of mutation8 of c-ml. These represent both quantitatively different reaction oapaaftise a8 well AS qualitatively different reaation oagaoities. Aooordlng to my grasent interpretation, suuh quentitati ve levels of expression of C aotivity are to be expected as well as very much less frequent ahclngee that will incorporate adja- cent looi (the pink reaatlon). It would be more profitable to post- fwciabl poue this disoussion until after & mechonia,,responsible for mutable phenomena haa been discussed. -18. The standard Ds looua: Two contrasting types of behavior As the last senI;t..noo above indicates, it is concluded that 33s activity is responsible no S only for the dioentric chromatid ?orma- tion at the o-ml, locus but also for the c to C mutations of this locus. This conclusion LY based upon the striking resemblance between two contrasting tgas of Ds activit,y when Ds is In its standard J.ooRtion and the two contrasting types of events occurring at the c-ml loous. The nature of the evidence for this general conclusion will be considered. That the 119 locus in Ita standard pcsition gives ~H'G Tain type8 of recognizable behavior,has been known for a Zong time. In sn AC aa a0 constitution, Do looi have been isolated thet give meny dicentric chromatids as the conseguenos of mutations of l?s. Kernels with appropriate genie oonstitutfone reveal this clearly. Sny of these mutations occur ear3y in development. Such Ds loci have been iiesignatsd Vs-early". A contrasting type of Ds behavior results in only a few losses of segments of the short arm of chromosome 0 and they usuilly OCJCUF late. Such Ds looi have been designated '3s-fe+ 1Hf;tt" * liz the kerneiu 4th a. few-late I)s locus, many aberrant types of chromosome breakage ocour; ,y:, b \' the brecnklsg8fior%ginate at positions other than the P>s Iccus. The significance of this will be discusser! 1Eiter. uy, LO show that the fey;-late type of 9s behavior F3 associated with ti particular state (orgenizntion) of 2he D3 loous ani! the*: it is not due to inodff'iers loctated e3ssv:here in ti-,e chromoaone complement, the fallo-9ing ~ros3es were n;uds. Plants were obtained that ha:! the Gcdristituiisn: I ra Ez 1-d Es-early / 1 3h Rz wx Us-fsirv lute, kc ac. These plants 'vvere orossed to female plants with C sh bz wx ds ac. -19- The types of variegated karnels,with respect to &'x and wx constitu- tlons.wer8 tabulated. The results of these oroases are given in ., . table 3. Crossing ov8r between W 8Dd Ds is low, only 1 to 2 or 3%. The 10 I - C bz, wx kernels showing early losses probably 8ro98 from urossing over between wx and Ds. The 34 I - C bz, Wx - wx k@rn8lS, with the few-late loss pattern of Ds,r8prasent oross-overa between %r end Ds together with newly arfsing few-late Ds loci derived from early Ds looi. The few-lfite type of Ds loous ariasa rAther L ~6 he 0breyoed as yet) frequently 819 a mutation of the early Ds COCCUS but only rnrely4doee a Ds-early arise from 8 DS-reW-18t8 locus. The evidenae from table 3 strongly supports the Bonolusion that the early and the few-late types 0r variegation refleut two ditferent Qtates" of the Ds locus. Other observations make this COnd.USiOn even stronger. One pertinent observation concerns the apgearanoe of sectors showing the r8W-hit8 type of Ds behavior in kernels that otherwise show the early type of Ds behavior. If the f8w-late type of Ds behavior arises rather frequently from the early type of Ds behevior, then mob sectors should be seen. The most instructivs 8vid8no8 for the prssenm of two suuh contrasting types of Ds behavior can be obtained from uomparisons of these two types of behavior of Ds at the standard location with the smie two types of behavior of Ds When at the a-ml locus. I shall attempt now to point out the sinailarlties. 4363-B x 4679-2 46 4 4684-10 x * 1 73 0 51. 4362-23 x 4679-3 126 93 15 117 6 114 4363-35 x 4679-5 77 72 1 63 2 87 4 4364-1 x 4679-5 120 92 13 120 ) 2 79 5 Totals 361 -20. The, relation between th8 two types of behavior of o-ml and the two 1 types of behavior of DS at its standard looatlon. In the or088 of a female c1 sh wx da 80 plant by a male e-ml 3h wx o Sh '#I, AC aa plant, the Ix kernels showing a to C variegation were of two distinat types. These were Oonsidered earlier but wfll be further uonsidered here. On8 type was most frequent. It @IV@3 Very f8W Viaibl8 o to C mutations but many mutations leading to dioentric pi&:,. 5:. &(;,l ohromosomes. There were a few #cJr kernels, hOWeVer, wfth V8I7 many c to C rautations. In the88 kernels, few dioentrio forming mutationa oaaurred r0r very few wx spots were preerent. In general the pattern of the88 WI Spots resemble in distribution and si.28 the patterns given by the rteW-ht8 5)s in its Standard looation. In both camet, losaea of Segments of the short arm of ohromoeome 9 occurred infrequently I: i : .;.l.J 9' ': ;' and,late in d8VelOpm8nt. ', Mth respect to typee and frcquenulee 0r loss of segment8 of the short 8x7~ of ohromosome 9, both ban b8 alassi- fied 8S NDs-f8w-loteR States. Again, a Similarity is found between those c-ml blrx kernels Showing low rates of a to C variegation but high rates of dioentric formation with th8 pattern produced by Da-early. Furthermore, in some of these kernelS, distinct Seaters appear with high rates of o to C mutatione. These sectors are Wx and have few if any wx spots In them. To review: The k833818 with high ratasl of 0 to C mutations give low rates of cbegmental losses in the Short arm of chromosome 9. The pattern of these losses r888mble the pattern8 of losses obtained from the f'ew-late DS in its standard location. The patternS of losses in the kernels with high rate?.of 1oSsrr of Segments of the short arm of the o-ml chromosome~resemble the patterns of lospleg obtained from the standard Da-early loous. In the forms? kernels, the seotors with high rates of' o to C mutations resemble in frequenay and ooaurrenoeYtha motors in the latter kernels that show low rates of segmental loss (Ds-few-late sectors). Again, o-ml loci giving high rates of aeignental loss throw a-ml loci giving the low rates of se@nental loss. Mmy of these, in turn, have a high rate of c to C mutation. It is concluded, then, that the state of De-early in the standard looatlon and the stete of o-ml giving high segmental losses ropresen0_ the same mutational state of Ds whereas the Ds-few-late In the standard position and the o-ml with high rate8 of Q to C mutations represent the sme mutationcsl state of Ds. From this, it Is conclutisd (388 figure 1) that the Ds-few-late, in its standard looetion, is undergoing many mutations ah are hiddsn because n+o visible phenotypio Cfects 70110~ from such mutatPons. In arder to see this type of mutation behavior of Ds, the Ds loous must be inserted into or olose to a loous that will give a phenotypicallg deteotible etfeot, following such a muttstion. This oucurs when Ds is In the c-ml locus but does not occur when inserted in Its standard looation. One more resemblance between o-ml and DEJ (standard locationjshould be mentioned. Xhen kernels with high rates of o to C mutations of c-ml are seleoted and plants grown from them, the high rate of mutation of c to C is retained in later generations. Again, In this respect, the high rate of o to C mutation pattern of c-ml resembles the few-late type of Da behavior (stzndard position) because in both cases, mutations back to the dicsntric chromatid forming type of Da behavior occur rarely. The "few-late" type of Da behavior represents a relatively stable stats of a Ds loous. Figure 1 Comparison of Ds activity at the standard looation and at o-ml Standard Looation Ds behavior o-ml Ds behavior Ds -early Same erant 1) low rata of hidden mutations i St )l) lowratesora~c mutation a) high rate of visible mutations (segmental losses) \ ., 2) high retes of segmental losses Ds-few-late Same event 1) high rate of hidden mutations n 1) high rate of o -. C mutationtj: 2) low rate of segmental losess w 2) low rate of semental \ lossas -220 Comparisons of rates of mutations of c-ml and standard Ds. St follows from these observations and conclusions that one is in a position to interpret the mutation rates in the kernels with C de / C ds / 1 Ds-early, AC ac ac (or AC AC ac, AC AC AC) with thoss having a / c / c-ml, Ac ac ac (or At! AC ac, AC AC Aa). Because the color pattern produced by losses of I' that occur as a consequence of dicentric forming Ds-eerly mutations are the same as the color pattern produced by I: to C mutations, direct comparisons of mutation rates and patterns can be node. Yith these particular constitutions, the kernels showing high rates of c to C mutations and thoae having a Da-early (standard location) may be indistinguishable in phenotype. Since c-ml loci giving high rates of a to C mutation show reletivelg few segmental losses and since it has been concluded that thel'loci have a few-late type Ds locus, it is further concluded that the rates of Ds mutation in both the early Da state and the ft:w-late Ds state are really the same. AC controls the time and frequency of some event that occurs at a Da locus whether or not the consequence of thI.s event lea&to a detectable change in phenotype. The mutations resulting in no C&actable change in phenotype may be called "hidden" mutations. The presents of hidden mutations will remain a factor aausinq difficulty in the study of all mutable loci. Their presence Is suspected in all the Ac controlled mutable loci. They are a factor that will interfare with any attempt to evalutlte mutation rates of any mutable locus. ,Means of detectinq and cltiasifyin% the types of hidden mutations Is absolutely necessary if an understandin& of the factors involved in bringing about visible mutF-tions Is to be realized. The Ao control of mutation during development The sirail:-$rity in rr?tes of mutations at the a-ml locus, when the tz -$ C rates are high, to segmental losses rates at the standard Ds loous when these are high, has presented an opportunity to determine something about the nature of AC action in particular cells during development. jYhy do mutations occur in some of the cells during development and not in others? If two &z-controlled mutable 1001 are present in a cell, will Q mutation occur at each locus at the some time in the same cell? I3ow could this be determiaed~ Crosses of plants having e-ml wx ds in chromosome 9 (high rate of c 7 CT mutations) bg plants having c (stable) *!r Ds-early (stenderd loaation) in chromosome 9 should make this relationship ene'pznble. If, in the resulting kernels, the c-ml locus in a c-ml wx ds chrorna- -some mutates to C, a C urea will arise, the size Rnd position de?endinq upon ilrhen and where the u to C mutation occurred, that is, in what cell this occurred during development. If', in this same cell, a mutation ocourred at the Da (standard) locus in the c .7x 3s chromosome, the 8x locus will be deleted and the oells arising following this mutation will be wx. By examining the starch constitution of the cells underlying the C areas in the kernels resulting from suah a cross, it should be possible to determine whether or not particular conditions,- controlled by Ao, arise in oertain ~~11s and whether these aonditions affeut all Ac controlled mutable loci elike - that is, produue a mutation in all Ao-oontrolled mutable looi. If so, the C areas should have underlying wx staroh constitutions and an exact correspondence in sectors should appear; or some relationship should be visible between t&c :::,a typos of mutations that will tell whether such a coincidence :t mutations occurred, (that is, whether one-strand or two atrends are involved in the muttltion process in a particular cell). Fortunately, in some of these crosses, the frequency of coincident mutations of o-ml to C in a c-ml oarrying chromosome and of Ds muta- tions in the c Vx Ds chromosom8 is remarkably high. A large propor- tion of the C areas have underlying wx starch. This is easily seen in the Ao ac 80 oonstitutione but it is better observed in AC AC ac constitutions when a relatively early mutation occurs. In this latter case, the coincidence mutations are very frequent. There are a number of exceptions but suoh exceptions should be anticipated. This is beoause several types of hidden mutations occur end also because one of these changes the state of the locus. These newly arising states will give cthanged rates of visible mutations. Also, the mutation grooess itself frequently gives rise to exoeptionel conse- quences some of which are visible others of whiuh are hidden, as will be shown later. The high rates of coincident mutations, mentioned above, might lead one to conclude that the Au control of mutability is related to some particular &-controlled condition that arises in particular ~11s during development. llihen this condition arises, it will affect a mutation at all AC controlled mutable 1001 in this cell whether or not the mutation occurring at any one pertfoular loous will result in a changed phenotypic expression. The Ac locus Itself may mutate at the same time also. All of this may well be true but the diffiaulties in proving it are great. The crosses described above certainly suggest this conolusion regarding AC control of mutable loci. Nevertheless, other oroa885, originally thought to be similar, do not give such a striking coincident rate of visible mutations of these two Ao controlled mutable loci even though many ooinoident mutations did occur in the -2% development of the kernels on thelre ears. I am inclined to believe, from observing patterns end rates of' visible mutations, that there oan be a limited range of statea pf Ds loci, from ones giving few hidden mutations and many viaible`mutationa to othera giving many hidden mutations and few visible mutationa. Combinations of c-ml ds / o Wx D8 that are not reolprooally balanced In this respect would show a number of non-coincident visible mutations. This would not mean, however, that aoinoident mutations did not oocur. Rather, it was not possible to see them. A possible method is available for examining whether or not a range exists in the rates at which these two classes of mutation will occur. It involves various isolates of the c-ml locue. Here, two of the types of' mutations of a single Ds locus c1an be distinguished in c ds/ c ds / a-ml Wx oonstituticns -- the o to C mutations and the segmental loss mutations. Also, some of the mutations leading to changes in state niap be recognized. Selection of o-ml loci giving intermediate frequencies between high c to C and low se*gmental losses vs. low o to C mutation and high segmental loss can be made. As yet, X do not have available for examination a seleotion of such c-ml looi with the proper constitutions. However, a comparison of the behavior of the c-ml locus derived from single plants In the oroa9es of these planta to both (1) c sh wx ds ac and (2) C sh bz ds ac certainly presents su&eative evidence for the preeence of' such intermediate states of Ds. The o to C rate of mutation following cro8s (1) hae been compared with the C Bz - C ba variegation rate following cwos9 (2). Medium rates of o to C variegation appearing in the kernels followinhz; cross (1) are correlated with medium rates of loss mutations in the kernels arising from cross (2). If this proves