GENETIC AND CYTOLOGICAL STUDIES OF MAIZE Barbara McClintock Further Studies of the Spm System The manner in which the Spm (Sup- pressor-mutator) element in maize con- trols gene action and mutation at the modified A> locus in chromosome 5, desig- nated a2”"1, was outlined in Year Book 57. The alternating cycles of activity under- gone by Spm, and their relation to the patterns of anthocyanin distribution in plant and in kernel, were described. It was reported that the time of change from one phase of activity to the other during development appears to be under genetic control. Some isolates of Spm remain ac- tive during all stages of development, or become inactive in only a few cells very late in the development of plant or kernel; other isolates undergo early change in phase; and still others may be completely inactive during all stages of development, or return only very occasionally to the ac- tive phase. Studies aimed at revealing some of the conditions responsible for con- trolling changes in phase of Spm activity were continued this year, It is now evi- dent that the readily distinguishable dit- ferences between plants or within different parts of the same plant with regard to changes in phase are reflections of different types of modification of the Spm element itself and also that such modifications 0c cur at the time of change in phase. Investigation of cyclical changes in phase of Spm requires examination of its ac- tivity in all parts of individual plants, in- cluding the kernels produced by different ears of a single plant. The type of Spm modification occurring in an individual cell of a plant during development is re- flected in the progeny of that cell. The phenotype of the tissues derived from it indicates not only that a reversal of phase of Spm activity has occurred but also that the modification of Spm responsible for the reversal is likewise responsible for con- trolling the time and frequency of occur- rence of subsequent reversals. Much of the evidence was derived from a study of sequential changes in phase of one particular isolate of Spm whese be- havior was followed through four genera- tions of plants. It was possible to select kernels from ears of plants in which Spm was either unaltered or had been modi- fied in an individual cell during plant de- velopment; and plants grown from such selected kernels exhibited either the paren- tal type of Spm behavior or some modifica- tion of it. Each progeny plant was then examined in the same manner as the par- ent plant. Again, kernels selected from some of the ears were sown, and the plants derived from them were investigated. This method of examining the behavior of Spm has made it possible to draw the inferences mentioned above, namely, that each change in phase of Spm is associated with an event in some cell that alters the Spm element itself, and that each such event effects a particular type of modification of Spm. The type of modification is made evident in the descendent cells by the time and fre- quency of occurrence of subsequent re- versals of phase; and the different modifi- cations of Spm produce a wide range of effects in this regard. An unanticipated aspect of Spm _ be- havior was revealed during the year. It had been observed previously that, when an Spm element in its active phase is in- corporated into a nucleus carrying an in- DEPARTMENT OF GENETICS — 453 active Spm that rarely reverts to its active phase, the inactive Spm is apparently ac- tivated. The pattern of anthocyanin dis- tribution that develops in plant and ker- nels is not the one that is expected when a single active Spm element is present in a plant. Instead, it resembles the one that appears when two initially active Spm ele- ments are introduced into the zygote, or when three are present in the primary en- dosperm nucleus and each of them inde- pendently undergoes inactivation during development. It has now been learned, however, that the inactive Spm element suffers no per- manent alteration by reason of its associa- tion with the active Spm. By means of meiotic segregation in plants having both these elements, their association within a nucleus can be terminated, and the subse- quent action of each can be observed in progeny plants where it is present alone. To identify each of these two elements in the progeny, the relative locations in the chromosome complement of the Spm ele- ments in the parent plant must be known. Therefore, progeny tests were conducted with two sets of plants. In one set, the in- active Spm element was located in chromo- some 9 and was closely linked with wx, whereas the active Spm element was inde- pendently located. In the other, the loca- tion of the two elements was reversed: the active Spm was linked with wx and the inactive Spm was independently lo- cated. In the individual progeny of either set it was possible to determine whether or not Spm was present, whether both ele- ments or only one had been received from the parent, and, if only one, which of the two it was. The progeny tests showed that, as long as the inactive Spm element is carried in a nucleus having an active Spm, it be- haves like an active Spm element. When its association with the active element is terminated, however, it behaves much as it would have behaved if it had never been so associated. In other words, its ini- tial constitution is not altered. 454 Another test, similar to those just de- scribed, was conducted with plants having two initially active Spm elements, one lo- cated in chromosome 9 and closely linked with wx, and the other independently lo- cated, in order to see whether each would exhibit its particular cycle of phase re- versal after they were separated by meiotic segregation. Because one element was closely linked with wx and the other seg- regated independently of it, it was possible in the progeny (except in a few individu- als that carried a transposed Spm ele- ment) to identify each Spm element, and to compare the behavior of each in those individuals that received one or the other. By this means it was learned that the asso- ciation of the elements did not affect their subsequent control of phase reversal: each retained its own type of control. It may be pointed out at this time that there appears to be more than a superficial resemblance between the Spm system of control of gene expression in maize and the system that controls the expression of flagellar antigens in the bacterium Sal- monella, described by Lederberg and Iino. The resemblance is evident in the mode of control of gene action, the changes in phase of activity, the duration of a phase and the changes in duration that may occur, and the “states” of the genes whose action is being controlled. Chromosome Constitutions of Some South American Races of Maize Studies at Cold Spring Harbor were in- terrupted during the winter of 1958-1959 while I spent several months in Colombia examining the chromosome constitutions of plants belonging to different races of maize from Ecuador, Bolivia, Chile, and Venezuela. I was invited to make such examinations by the Committee on Pres- ervation of Indigenous Strains of Maize, of the National Academy of Sciences-Na- tional Research Council in Washington, several of whose members have taken part in identifying and classifying these races. CARNEGIE INSTITUTION OF WASHINGTON The collection and propagation of in- digenous races of maize of the above- mentioned countries have been the respon- sibility of some of the members of the Rockefeller Foundation who are associated with its agricultural program in Colom- bia, namely, Dr. Lewis M. Roberts, Direc- tor; Dr. David H. Timothy, in charge of maize improvement in Colombia; Dr. William H. Hatheway, whose knowledge of maize races is extensive; and Ing. Ri- cardo Ramirez, who also has a broad com- prehension of the subject. Each of these men has contributed his talents to the project of collection, identification, and propagation. I am very grateful for their highly effective assistance, as well as for the many courtesies shown by each of them during my study of chromosome constitutions of some of the races. All chromosome examinations were made at the Universidad Nacional, Facul- tad de Agronomia e Instituto Forestal, located in Madellin, Colombia. Facilities of the Institute were generously and cour- teously extended to me by the Director, Dr. Garcés, and by Dr. Sanchez, in whose department the examinations were made. I am particularly indebted to Sefiorita Rocio Diez P., who worked with me daily in order to learn to identify maize chromo- somes and whose progress was $0 rapid that she was able to make a contribution to the study. The sporocytes of plants whose chromo- somes were to be examined had been col- lected and stored in a deep-freeze unit. Races of Ecuador, Bolivia, and Chile were selected by Dr. Roberts and Ing. Ramirez. to be explored in the limited time avail- able. Pachytene stages of the microsporo- cytes of plants belonging to these races were examined for the presence of knobs in any of the chromosomes; when knobs were found, their location, size, and mor- phological characteristics were determined. If B-type chromosomes were present, the number was ascertained. Note was taken also of any readily identifiable structural modification affecting chromosome organ- ization. There are more than twenty known lo- cations in the 10 chromosomes of maize at which knobs may be present. At any one location, knob morphology may vary with respect to size or shape, or both. Pre- vious studies by maize cytologists had shown that a particular type of knob at any one location is heritable in that it Passes to successive plant generations with- out change. Their work had also revealed that number and distribution of knobs differ among different strains of maize: some strains have no knobs; others have a few, at specific locations; and still others have many, whose types and locations de- pend on the strain. As the data accumulated, it became in- creasingly evident that detailed knowledge of knob constitution would be a useful adjunct to the observation of morpho- logical and physiological properties as cri- teria for characterizing races. Its useful- ness for considerations of modes of origin of races was also evidenced, particularly during the examination of races indige- nous to high-altitude regions in the three countries. At the completion of this ex- amination it was found that plants of all but 2 of the 32 highland races studied were amazingly similar with regard to knob constitution. In these 30 races, a small knob was present at one particular loca- tion in the long arm of each chromosome 7. In some but not all of the plants of these races, a very tiny knob was present in the long arm of chromosome 6. When present, it was always at the same location —an important fact because there are three knob locations in the long arm of this chromosome at each of which the knobs may differ in size or shape. No other knobs were found in the chromosome com- plements of the 30 races, except in 2 plants among the 125 examined. These 2 were derived from different collections made in Ecuador. Each of them carried one addi- tional knob, which in both cases appeared DEPARTMENT OF GENETICS 455 in only one homologue of the chromosome in which it was located. In one plant, the extra knob was in the long arm of one of the two chromosomes 2; in the other, it was located in the long arm of one chro- mosome 8. These 2 exceptional plants probably resulted from an earlier con- tamination with a race having other knob constitutions, The strikingly uniform pattern of knob constitution described above characterized 9 of the 10 highland races of Ecuador that were examined, 11 among 12 highland races of Bolivia, and all 10 of the highland races of Chile included in the study. In addition, B-type chromosomes were found in some plants of many of these races, the number per plant ranging from 1 to 6. In contrast to the highland races, many of the lowland races of these countries dif- fered greatly from one another with regard to number, location, and size of knobs and also with regard to degree of heterozygos- ity of a knob at any one location. Within many of these races, however, there was a consistent pattern of distribution of knobs at particular locations in the chro- mosome complement as well as consist- ency in the type of knob found at any one location. The similarity of chromosome constitu- tion among most of the Andean races of Ecuador, Bolivia, and Chile was so im- pressive that we wanted to learn whether or not it extended to the highland races of Venezuela. Sporocyte collections had been made from plants of only 4 such races, but chromosomes from all the available collec- tions were examined. Knob constitutions in the 4 races proved to be very different from those of the Andean races previously examined. In 2 of them, knobs were pres- ent in all the chromosomes except chromo- some 10, and some chromosomes had two or three knobs; some of the knobs, more- over, were exceedingly large. In addition, a high degree of homozygosity was ex- hibited; that is, in each homologous pair of chromosomes the same type of knob was present at the same location in each 456 member of the pair. Plants of the other 2 races examined had some of the same knobs, but often a particular knob ap- peared on only one of the two homologues of a chromosome. At the time the chromosome examina- tions were being made, I did not know the exact location from which a particular collection had come but only the country and the elevation. After the examinations had been terminated, the collected data were taken to Bogot4, where, with the in- valuable cooperation of Dr. Timothy and Dr. Hatheway, the relations of chromo- some constitutions to exact geographical Jocations were plotted. It then became more fully apparent that there are sig- CARNEGIE INSTITUTION OF WASHINGTON nificant relations between knob constitu- tion and geographical location, and that it would be possible to utilize a knowledge of knob constitutions in attempting to trace the origins and migrations of maize races. It is also possible to draw inferences about the probable contributions of hy- bridization to the development of some of the races. The impression gained from these pre- liminary studies is that present-day maize may have derived from several different centers. Migration from such centers in the past was followed by hybridization. Examination of the chromosome knobs of plants from various geographical locations may help to identify some of these centers. BIBLIOGRAPHY Bal, A. K., and B. P. Kaufmann. The action of deoxyribonuclease on chromosomes of micro- sporocytes. Nucleus, 2, 51-62 (1959). Balbinder, Evelyn. See Campbell, A. Banit, S. Evidence of reverse mutation from streptomycin resistance to sensitivity in Sal- monella typhimurium. Schweiz. Z. allgem. Pathol. u. Bakteriol., 22, 511-514 (1959). Banit, S. Transduction to penicillin and chlor- amphenicol resistance in Salmonella typhi- murium. Genetics, 44, 449-455 (1959). Burgi, E. See Hershey, A. D. Campbell, A., and Evelyn Balbinder. Transduc- tion of the galactose region of Escherichia coli K12 by the phages 4 and \-434 hybrid. Genetics, 44, 309-319 (1959). Demerec, M. Albert Francis Blakeslee. netics, 44, \-4 (1959). Demerec, M. Genetic structure of the Salmo- nella chromosome. Proc. X Intern. Congr. Genet., 1, 55-62 (1959). Demerec, M., and P. E. Hartman. Complex loci in microorganisms. Ann. Rev. Microbiol., 13, 377-406 (1959). Demerec, M., and H. Ozeki. Tests for allelism among auxotrophs of Salmonella typhimu- rium. Genetics, 44, 269-278 (1959). Demerec, M. See also Miyake, T. Dutt, M. K., and B. P. Kaufmann. Degrada- tional action of deoxyribonuclease on unfixed chromosomes of Drosophila and grasshop- pers. Nucleus, 2, 85-98 (1959). Fuscaldo, K. E., and H. H. Jones. Ge- A method for the reconstruction of three-dimensional models from electron micrographs of serial sections. J. Ultrastructure Research, 3, 1-10 (1959). Hartman, P. E. See Demerec, Mz. Hershey, A. D. Bacteriophages. In Viral and Rickettsial Infections of Man, 3rd ed., edited by T. M. Rivers and F. L. Horsfall, Jr., J. B- Lippincott Company, pp. 172-198, 1959. Hershey, A. D., E. Burgi, and G. Streisinger. Genetic recombination between phages in the presence of chloramphenicol. Virology, 6, 287-288 (1958). Hershey, A. D., and others, editors. Bacterio- phages, by Mark H. Adams, 592 pp., Inter- science Publishers, 1959. Jones, H. H. See Fuscaldo, K. E. Kaufmann, B. P. See Bal, A. K.; and Dutt, M. K. Miyake, T., and M. Demerec. Salmonella- Escher- ichia hybrids. Nature, 183, 1586 (1959). Ozeki, H. Chromosome fragments participating in transduction in Salmonella typhimurium. Genetics, 44, 457-470 (1959). Ozeki, H. See also Demerec, M. Sengiin, A. Effect of X-rays on salivary-gland chromosomes during early stages of devel- opment. Nucleus, 1, 162-172 (1958). Streisinger, G. See Hershey, A.D. Tomizawa, J. Sensitivity of phage-precursor nucleic acid, synthesized in the presence ° chloramphenicol, to ultraviolet irradiation. Virology, 6, 55-80 (1958).