CARNEGIE lNSTltUTlON OF WASHINGTON DEPARTMENT OF GENETICS COLD SPRING HARBOR. LONG ISLAND. N. Y. January 20, 1950 Dear Charlie, Your good letter of January 4 was not answered before .this as I have been trying to consider just how to get the information to you in order to amwer the questions raised in your letter. It pleased me to learn that you had acquired 80 much about the De, AC story when it was presented to you in such a rush and with no ti.:e for adequate annotation where necessary. I$ dont like to give yol a short amount that would be inadequate for a real comprehension; neither would I wiah to burden you with the long accounts that have been written. As mentioned to you, these account8 were written, and are atill being written, in order to get the data together ati to ha.ve the correlations indicated in the required sequences. The accounts have been sent to Marcus regularly for he has been following the story with consiaerable interest since before he left for Illinois. He ha8 also received samples of the kernels' that illustrate the story. It seems to me that it would be wise for you to have samples of the various types of behavior if you are to grasp the descriptions with the least amount of confusion. illustrations Suppose, then, that I collect a group of such and send them on to you. rf, aftr examining them and reading the descriptions that will be sent with each sample, you would like to see the full account, I should be glad to send on copies.' easy reading, 1 'warn you, however, it is not but because of not because the subject is difficult to grasp the number of experiments that h?ve been made. It is a long chore and I would well understand the emotional reaction of not wanting to start such a task. If you could have stayed over for several days, we could have gone over the material here in short order and clarified-the details quidkly. Now for your questions, however, or the comments. qolnmeint~l 1. "The Ds locus was, when first located, on the short arm of 9 just outside the region with heavy chromomere8.1i This is correct. It lies to the right of WX~JI about 2 to 3 cross-over unit8 away. This is oalled the standard loca.tion because it can appear at other locations. Comment 2. This comment is complex but involves the mechanism of dicentric formation. Several type8 of events occur at the Ds locus as the consequence of 8~ AC initiated mutations. The frequency of one particular type of event depends on the Itstate" of the locus. worded to state that the l~state~ It might be better of a locus is reflected in the frequency of the particular types of events that can occur at the Ds locus. One of these events is the formation of an acentric fragment and a dicentrio chrozatid. This OCCUrS at a mitOSi8 -- probably during or just after division of the Chromosome -- and involves both chromatide of chro:cosome 9. The positions where the fusions to give the acentric fragment and the dicentric chromatid occur at the Ds locus: si4hdgouMLtl;Bd : h I " - -- -. " ti 4 A second type of consequence of a Ds mutation results in the loss of the Ds locus from at least one of the chromatide of chro!nosome 9. The mechanism of this loss is not known but is inferred from the other events that are known to occur. It is possibly pulled out of the chromosome: M 6 \ _ 23 '( h- +d ipj& &&if3 "7 - -a-----. I-.-. -_.__ L ca A third conseouence of a mutational event at Ds results in the transposition of the Ds lOCU8 from one position to another. The mechanism of transposition is believed to be chromatid brecka.ge and fusion. Gross chromosomal rearrangements ha.ve accompanied the transpositions of the Ds locus in some of the examined cases. Two examples may be given. gave rise to AF8??i%nosome 9 carrying I sh Bz Wx Ds-standard a chromosome with the following constitution: 0 I Ds\ Qh Bz Wx I i -- __. _---. .-.I . . .._ ._._. CL . e ~~~.iT\M~*eh$a8~. In another plant, having a morphologically normal chrozosome 9 with I sh Bz Wx Ds-standard, one gamete was found that had a duplication of the follvwing type: ,,A ..----.-- .-`- CL 2 -,.--__r In both cases, two bhromosome breaks (involving both chromatids at the same locus) are necessary to produce the duplications. It may be noted that one break occur&at the known position of Ds and the other break occurred at a position wh&ch is marked by the new position or positions of Ds. That Ds events are associated with ChromOSOme breakage and fusions is indica.ted by the usual consequence of Ds events -- dicentric chromatid formation. That the transpositions of Ds are associated with c'hromosome brezkages that result in the insertion of Ds between broken ends could be suspected from the frequent association of tranSpo8itionS with gross chromosomal rearrangements where the -3 - breakage positions can be well established, as in the above two cases. Either Ds was inserted as the consequence of a break that occurred elsewhere in chromosome 9 or Da activity appeared at this new position as well as remaining at its standard location and the gross chromosomal rearrangement resulted from coincident Ds events at these two positions. The latter assumption is unlikely because, where these gross chromosomal abnormalities have arisen in association with a new location of De activity, no evidence of any sector having a normal chromosome 9 with Ds at the new position has been seen. Either one event must foiblow the other immediately or the appearance of Ds in a new position and the production of a gross chromosgmal rearrangement are the conseauences of a single event. such an event may be diagremmed as follows: If the above is the mechanism responsible for transpositions of - Ds, then only certain kinds of transpositions will be obtained from male gamete8 that may be recognized immediately on crossing to C sh bz wx ds female plants. The chromatids with long deficiencies will be eliminated. The translocations will not be recognized, Many of the dUpliCatiOn8 will not pass through the pollen because of the extent of the duplication. Also, normal chromosomes 9 with a 13s locus in a new position and no Ds fwxhaxxw~x locus in the old position or a morphologically normal chromosome 9 with two Ds loci, one at the new position and one at the old position will be found. Examples of the latter two types have beencfLqund, as expected. As far as the ? evidence ShoWS, there is no position in chro:Ijosome 9 where Ds activity isFable to appe&$as the consequence of trans-osition. The various positions given below have been found 80 far and more are being investigated: m + & ;. ! ! : *- -___ f-- --.__ 3 -_._.__ -+---".-~- 3 &by' T*#@ Vhen Ds shifts to a point near another gene it c6u6es it to mutate, e.g. CDs = c; amd if Ds is subse 9 uently lost, a reversion mutation occurs from c to C." his is essentially correct. In the plants having Da and AC, new mutable loci are arising that respond to AC a6 does Da. @ne of these has received consirerable study. This is the muts.ble locus called c-ml. The designation %-la refere to the first of the mutable c loci that were found. It appeared on the ear in a single kernel from the cross of a c sh wx ds ac female by a male plant having two morphologically normal chromosomes 9 carrying C Sh wx Ds in each chromosome. The Ds locus in both chromosome8 was at the standard location. The C locus was normal in both chronosomee. It has been i:: my stocks for years and has always behaved a.s expected. the cross of this plant to the c sh wx plants, nearly 3,000 kernels were obtained but only in a single kernel on the ear of one of these crosses did a peculiar type of beha.vior occur. Insead of showing colorless areas in a colored background, due to losses of the C locus following Ds mutations at the standard location, this kernel showed C spots in a colorless background. It was just the reverse of the expected type of variegati;n. It is from this kernel that the c-ml case originated. proved to be a6 follows. A change from a normal C locus to a c locus capable of mutating back to C occurred coincidently with the disappearance of the Ds locus at the standard position and its appearance at a location that could not be separated from the mutable 0 locus. When this c mutates to C, all De activity ceases, except when Ds is transposed to another I I _' position coincident with the appearance of the C phenotype. The C locus, following a c-ml mutation, is just as normal as it was before the appearance of Ds activity at this position -- that is, before c-ml aro6e. Comment 4. flIn order that these mutations occur, an activator (AC) must also be present. Here are some of the questions: a) If the activator is not present (i,e,ac), does Ds break loose and shift?" .,`_ ,.a . `., .j %c' ! \- \. The answer to this is sharp. Ds will undergo breakage events only when AC is present. If AC is not also present in the complement, there is no evidence whatsoever of zw a Ds locus. To illustrate, if a chromosome has 0 Sh wx Ds, or I Sh Bz Wx Ds and is crossed to c sh wx or C sh bz wx, and if no AC locus is involved, the kernel types are contpletely normal. There is no indication of any abnormal locus in these chromosomes. As soon as AC ie added, however, the presence of Ds will be apparent. Again, a c-ml locus in an ac constitution will be comple?;ely colorless and no mutations to C will occur. - ;I - Question (b) of this comment: these similar in phenotype, "Your c-ml and ~412, are differ in phenotype? w but separate occurrences; or do they The answer is simple in only one ast>ect -- they differ decidedly. The c-ml mutable locus mutates directly to a full C phenotype with few exceptions. basic phenotypes, c-m2 muta.tes to two different each of which may be expressed quantitatively. The two mutable c loci arose from normal C loci,- quite independently of one another but in plants having Ds and AC loci. Question (c) of this comment: u6 the Ds locus recognized cytologically after it &IS broken loosePa The answer to this one is also simple. One cannot recognize any physical bash6 for a Ds locus. When it is transposed to a new position, such a6 between I and Sh, it doe6 at interfer with crossing-over between these two loci. Recogni- tion in a new position follows from the Ds breakage events that occur in this position. lo illustrate: If Ds is at the standard location in an 1 Sh Bz Wx chromosome be to the right of Wx.wi*hxr~frrwnew the position will When a'Ds event occurs at this position, that gives rise to a dicentric chromatid and an acentric fragment, the marked loci 1 Sh Bz Wx will be ca.rried by the acentric fragment. to the cell. This fragment will be lost All the cells arising from this cell will ha.ve no I Sh Bz Wx loci i n endosperms that received this chromosome from the pollen and a C sh bz wx da chromosome from the egg parent. A sector would appear in the kernel showing the C sh bz wx phenotype. If, however, Ds were located between Bz and Wx, the acentric fragment would carry I Sh Bz, but Wx would remain in the dicentric chrcmatid. The sector formed after a dicentric forming Da event would be 0 sh bz but would be variegated for Wx to wx because of the brea.kage-fusion-bridge cycle initiated by the Ds breakage event. breaks may be seen in the microsporocytes. The position of these They coincide with the positions inferred from the genetic evidence (either linkage data or sector type or both). Thus, the Ds locus can not be seen but its position is determined by linkage, by breakage events observed in the endosperm or plant and by the breaks observed cytologically in the microsporocytee. Comment 5. "AC may shift into #9 or in another chromosome, Where was it when first loca.ted?lf The position of AC wnen first discovered is not known. That it ma,y go from one chromosome to another is apparent. It may go from one unknown chrohlosome to chromosome 9 and there be identified because of linkage with the marked loci on chr;*,osome 9. After it has come into a position in chromosome 9, it -may move off and into another chroil!osome. Over 50 transposition6 of AC have been observed, or better, investigated. entered chro:l:osome 9, Three of these two in the long arm and one in the short arm. -6. Comment 6. This is related to what happens when two Ds loci are present. The answer to this depends upon several considerations. First, if the Da loci are in different chromosomes - that is in homologoues and at the same location in each homologue. If the two Ds loci have the same frequency of the consequence of the Da event 6,# number of results can occur depending on the states of these Ds loci. If the majority of the events give dicentric chromatids, coincident mutational events ca.n be high and much hemczygous deBcient cell& production results. If one Da locus has a high rate of dicentric formation as the consequence of the Ds event and the other a low rate of dicentric formation but a high rate of Ds loss, then the result ia quite different, as can be pictured. In this latter case, the results are quite nice when the Ds loci in the two homologues do not occupy allelic positions. The case of c-ml and Da-standard can be used to illustrate this If a plant having c-ml (Ds at the C locus to give a c phenotype) and wx is crossed b a plant having c (regulsr recessive allele, non-mutable with AC 3 and Wg and Da-standard the folbowing constitution is present in the endosperm: C Wx Ds f3w If the Ds events occurring at the c-ml in this i' selection of Ds give rise mainly to c to C mutations and few dicentrics and if the Ds in the c Wx Da chromosome gives mainly dicentrica and few losses of Ds, coincident muta.tions are readily observed. A muta.tion of c-ml to C will give a cell havhthe normal C phenotype. The sector produced after such an event will be c. If a coincident mutation occurs at the Ds locus in the c Ix Da chrome-scme, a dicentric chromatid and an acentric fragment will be formed. The acentric fragment will carry the wx locus. Loss of thi6 fra.gment will result in a sector with no Wx. It will be wx in phenotype. Coincident mutations of c-ml and Ds at the standard location would give sector6 that are C and the underlying starch should be wx. The border6 of the C and wx areas should coincide. Tha.t is, a C sector should have underlying wx starch. When the combination is made, as given above, just such sectors are very frequent. You mentioned something about the sticking together of Ds loci -- the more loci, the more sticking. This was mentioned in our conversation because of the following observ=tionS. The Ds locus is a.ble to change from one flatate" to another Hstste". As mentioned a.bove, the at e Ds loci are identified by at can occur *me w the frequency of alternate m of a mutztional event occur he Ds 1~~s. Ds loci - that are in a state giving dicentric chromatid formation as the consequence of the majority of the event:, can change, by a single event, to a st