medicine

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TIBS - December 1983

Barbara McClintock: Nobel Prize in Physiology/Medicine The recent award of the Nobel Prize in Physiology or Medicine to Barbara McClintock is a long overdue acknowledgement of a brilliant scientist. Although she was cited for the discovery and elucidation of transposable genetic elements, Dr McClintock has made many other fundamental contributions to the field of genetics during her long scientific career. In this article we would like to present a brief chronicle of her accomplishments that preceded the work on transposable elements. Additionally, we hope to indicate how her understanding of mobile elements anticipated many of the results that came later from prokaryotic and molecular genetics.

Corneii and the beginnings of maize genetics McClintock decided her scientific direction while still an undergraduate at Cornell University. Genetics was in its infancy at that time and she became attracted by the new and exciting work of the plant breeding group headed by R. A. Emerson. Recognizing the considerable advantages that the maize plant offered, the Emerson group quickly established it as a favored system for plant genetic studies. Linkage groups were identified and genetic maps for a number of phenotypic markers were constructed. Interactions between certain genes were demonstrated and the first unstable locus was described. This intensive activity at Cornell was paralleled by work of the Morgan group on Drosophila at Columbia University. McClintock's first breakthrough was the demonstration, contrary to popular belief, that meiotic prophase chromosomes comprised excellent material for the study of chromosome morphology. Unlike the highly condensed mitotic metaphase chromosomes, each member of the meiotic complement of ten chromosomes in maize could readily be distinguished by characteristic arm lengths and morphology ~. Many years later McClintock was to extend these observations to the study of chromosomes in Neurospora. Her first exploitation of these morphological differences was to associate linkage groups with specific chromosomes. To accomplish this she first constructed trisomic plants - plants in which any given chromosome is represented in triplicate rather than in 1~83. tlse~ier ~icncc publisher,; B ~ ,

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duplicate. Cytological examination revealed which chromosome was present in extra dosage. These plants were crossed with tester stocks for the different linkage groups and then backcrossed to the recessive tester stocks. When the markers were associated with the triplicated chromosome, 2:1 rather than 1:1 ratios were obtained in the backcrosses 2. McClintock also used the cytogenetic features to localize breakpoints of translocations between different chromosomes and thereby was able to place specific genes at defined positions on the physical chromosome map 3. Creighton and McClintock 4 took advantage of genetics and chromosome morphology to show that cytological interchange could be correlated with genetic recombination. They used plants that were heterozygous for a normal chromosome and one having a terminal knob at one end and a large translocation on the other half. The chromosomes were also distinguished by two genetic markers borne between the two morphological characters. They found that recombination between the two genetic markers resulted in the distribution of the morphological markers to the same gametes carrying the most closely linked genetic trait.

Another of McClintock's contributions during this period was the recognition that a particular region of the maize chromosome complement was responsible for the formation of the nucleolus. She demonstrated that this region, which she named 'the nucleolar organizer', had a discrete chromosomal location and that it could be functionally subdivided -~. Rhoades 6 in reviewing this period of intense productivity cited McClintock's use of meiotic chromosomes to resolve genetic problems as the founding of maize cytogenetics. In the six years that followed he listed seventeen major accomplishments and McClintock had figured in nine of them.

Variegation and unstable loci By the 1940s a number of unstable alleles had been identified in Drosophila, maize, and other plants. Rhoades had demonstrated that the unlinked Dotted gene controlled the mutability of an otherwise stable recessive allele at the A locus ~. The relevance of these genes to the study of mutation was debated as some geneticists believed that unstable genes were fundamentally different from stable genes. In retrospect both views were correct, for spontaneous mutations (sometimes unstable) may be the result of insertional rearrangements of a locus mediated by transposable elements. McClintock's first experiments with variegation in-

Maize cvtogeneticists at Cornell University, 1929. Left to right: Charles Burnham. Marcus Rhoades, George Beadle (kneeling). Rollins Emerson and Barbara McClintock. (('ourte~ v ~! Marcus Rhoade~.

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430 volved unstable ring chromosomes. In heterozygous combination with linear chromosomes carrying recessive alleles, the ring chromosomes produced somatic sectors on plants when they lost material or were asymmetrically distributed to daughter cellss. The behavior of ring chromosomes suggested to McClintock that broken chromosomes could fuse: she tested this idea by examining a heterozygote between a normal chromosome and one with an inverted terminal duplication. When a crossover occurred within the inverted region as it paired with its homologue, a dicentric chromatid resulted and the fused arms formed a continuous bridge as the two centromeres separated at anaphase. Ultimately the bridge broke: however, the broken ends did not remain free but following chromosome replication rejoined to reconstitute a new bridge. This process, which McClintock named the 'breakagefusion-bridge cycle', continued in the endosperm but not in the embryo '~. She realized that if multiple breaks had occurred and the broken ends had healed, the result would be a rodshaped chromosome with internal deletions. She therefore set out to find mutations caused by the breakagefusion-bridge cycle using the short arm of chromosome 9. In addition to identifying the predicted mutations, she also found unstable mutations and spontaneous losses of the whole arm ~. The latter observation was the result of breakages that mapped to a specific locus near the centromere. She also observed that there was a similarity between the pattern of breaks during endosperm development and the mutability of the unstable loci. In mapping the locus of chromosome breaks, termed Ds for Dissociation, she identified over 20 instances of its appearance in new locations on the same chromosome arm or occasionally on different chromosomes. The key observation was the finding of a new mutable c allele (a determinant of aleurone pigmentation) when Ds activity had also been transposed to the C locus. Upon reversion of c-mutable to wild-type expression Ds activity was lost from the C locus. H e r conclusion was that both chromosome breaks and c-mutability were the result of Ds action. McClintock also found evidence indicating that Ds induced adjacent deletions, inversions and duplications. She soon recognized that neither the mutable loci nor Ds were behaving autonomously but were responding to another element in the genome which she called A c (Activator). A c itself was

T I B S - D e c e m b e r 1983

Other geneticists working with transposable elements

Barbara Mc(Tintock at ('old Spring ttarhor, 1947. ~Courte~'y of Marjorie M. Bhavnani.)

transposable. If A c was removed from the genome by segregation, all the unstable alleles became stable recessives, but upon reintroduction of an Ac the mutant alleles re-expressed mutability. These observations were formally presented in 1951 ~ but it is clear that McClintock understood the basic principles by 1948 ~2. The 1951 paper also included an extensive analysis of the mode of Ds transposition. It should be emphasized that the felicitous property of some alleles ('states') of Ds to break the chromosome in response to Ac has allowed Ds to bc mapped even if it is not inserted in a known gene locus. McClintock used two aberrant transposition events to show that the transposition probably occurred during chromosome replication and was accompanied by its excision from the original location. This conservative mechanism contrasts with the replicative model that has been proposed for prokaryotic elements. It remains the best evidence for the mechanism of transposition of any eukaryotic transposable element, with the exception of the retroviral-like elements. Two element systems are characteristic of maize transposable elements and six families have now been recognized. In a superbly illustrated article, McClintock ~3 described the three systems she had worked with. She discerned the topological relationship between the nonautonomous and the autonomous elements when she found examples which indicated that the former were defective derivatives of the latter H. She also realized that active elements could be derived from quiescent ones present in any genome. A 'genome shock', generally any process resulting in chromosome breakage, could elicit the appearance of known transposable elements from stocks with no history of instability ~5.

It has been said that McClintock's work went largely unappreciated until the discovery of transposable elements in bacteria. To the extent that its importance was lost for most geneticists moving toward microbial and molecular subjects at that time this is probably true. However, her work was soon extended by other members of the maize genetic community. In addition to Rhoades, several other members of this group deserve mention: R. A. Brink and his students t~' quickly applied McClintock's findings to the study of variegated pericarp and reported much about the transposition of Ac that complemented McClintock's work. P. A. Petcrson 17 independently discovered the Spin family of elements and later with his students described two other families. M. G. Neuffer tx obtained information indicating the transposition of Dt and discovered new alleles controlled by Dt and Ac.

The control of gene action by transposable elements If it took an inordinately long time for geneticists to accept transpositkm, it has taken at least a decade longer for them to acknowledge the idea that these mobile elements can operate as units of gene control. From the analysis of several systems in which transposable elements exerted control over previously stably-expressed loci, McClintock perceived that the elements did not perforce modify gene action by interrupting gene structure, but could act by exerting regulatory control. In so doing McClintock I~ made the distinction between 'gene elements' (structural genes) and control elements, whether mobile or not. This proposal preceded the genetic dissection of the lac operon of E. coli by several years. In one of the most persuasive demonstrations of this principal, Dooner 2~) showed that when the element Ds transposed to the B r o n z e locus an unaltered gene product was synthesized at a new time during development, in different tissue, and that now the locus was no longer under the epistatic control of normal regulatory genes. MeClintock's findings that a transposable element can effect new regulatory controls over a structural gene when it is inserted at the locus have subsequently been shown to be the case for the R O A M mutations caused by Ty elements under the control of the mating type locus in yeast, and by retroviral activation of oncogenes in mammals.

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McClintock 2~ has pointed out that once such a controlling element becomes stabilized, new alleles with altered patterns of expression during development are generated.

Races of maize

At the outset we mentioned that McClintock's first important contribution was the demonstration that the cytogenetic features of the pachytene chromosomes at meiosis were a reliable method for distinguishing the maize chromosomes. In the years that followed, McClintock and others noted that some of the structures were subject to genetic variation. Knobs could be found at defined positions on the chromosome arms and various races could be classified by the presence or absence of knobs at each of these positions. A strain-dependent variation for an elongated or 'abnormal' chromosome 10 was reported as well as highly heterochromatic accessory, or 'B-type' chromosomes. During the 1960s McClintock turned to an extensive examination of the chromosomes of the races of maize in South and Central America. She was joined in this effort by T. A. Kato and A. Blumenschein 2~. In all, the karyotypes of at least six individuals from approximately 1 400 strains of maize and teosinte from North, Central and South America were examined. In clearly demonstrating the affinities between Mexican teosinte and maize, McClintock and her colleagues provided support for the current hypothesis that native Americans began selecting modern-day corn from teosinte about 5 000 years ago on the Central Plateau of Mexico. Maize did not originally exist in the wild but owes its distribution to man. Greater and lesser similarities were seen in the karyotypes of the many local varieties examined. These relationships suggested

the migration routes and paths of commerce probably used by pre-Columbian civilizations. A tribute

In retracing the progress of McClintock's productive career, the connections between each step of her research become astonishingly apparent, Each new phase seems to have been a logical outgrowth of one which preceded it. This is not surprising considering her meticulous and intensive attitude toward research. She also possesses the unique gift of being able to perceive things unknowingly passed over by others, Her unrestricted mind has always allowed her to recognize both the unexpected and the contradictory and she has used her insight and persistence toward understanding them. Her proofs have often been so meticulous and exhaustive that her publications frequently only hinted at their actual depth. McClintock continues to be an inexhaustible font of knowledge on the maize controlling elements and other genetic phemonena. Her knowledge of intriguing scientific problems has astounded and delighted many of her visitors who have always left her laboratory feeling inspired and more aware. She is a prodigious reader who continues to integrate new findings in molecular biology into her concepts. Above all she continues to watch for the inception of those new problems that may profoundly alter our conceived notions of science. We are sometimes asked what is so special about the maize plant that led McCfintock to the discovery of transposable elements. What was so extraordinary was probably less the organism than the perceptive maize geneticists, McClintock foremost among them, who with their keen powers of observation and vigorous intellects made those discoveries possible.

References 1 McClintock, B. (1929) Science 6% 629 2 McClintock, B. and Hill. H. E. (19311 Genetics 16, 175-190 3 McClintock, B. (19311 Proc, Natl Acad. Sci. USA 17, 485-491 4 Creighton, H. B. and McClintock, B. 11931) Proc. Natl Aead. SoL USA 17. 492-497 5 McClintock. B. (19341 Z. ZelIJ'or~ch. Mikrosk. Anat. 21,294~328 6 Rhoades. M. M. 1"he Golden Age of Corn Genetics at Cornell as Seen through the ,~ves of M. M. Rhoades, unpublished speech given at the 75th anniversary of Synapse. June 1982, Ithaca, NY 7 Rhoades, M. M. (19381 Genetics 23. 377-397 McClintock, B. (1932) Proc. Natl Acad. Sci. USA 18. 677-681 McClintock B. 119391 Proc. Natl Acad. Sci. 9 USA 25,405-4 16 10 McClintock B. (1945) ( "arnegie Inst. Wash. Year Book 44, 108--112 11 McClintoek, B. (1951) ('old Spring Itarbor ,~vmp. Quant. Biol. 19, 13J,7 12 McClintock. B, (1948) ('arnegie hist. W~L#I. 13 Year Book 47, 15,5-169 McClintock, B. (1965) Brookhaven ,~vmp. Biol. 18, 162-182 14 McClintock. B. (1962) Carnegie hist. Wash. Year Book 61, 44~461 15 McClintock B. (1950) Proc. Natl Aead. Sci, USA 36, 344-355 16 Brink, R. A. and Nilan, R, A. (19521 Genetitw 37, 519-544 17 Peterson, P. A. (1953) Genetics 38, 082 18 Neuffer, M. G. 119551 Science 121,399~110 19 McC[intock, B. (1956) Cold Spring ttarhor Syrup. Quant. Biol, 21. 197-216 211 Dooncr, H. K. 11981) Coht Spring ttarbor ~'~vmp. Quant. Biol. 45,457-462 21 McClintock, B. ( 19781 Stadler Syrup. 10, 2,5-47 22 McClintock, B., Kato, T. A. and Blumenschem, A. ( 1981 ) Chromosome Constitution ~ff'Races o f Maize, Colegio de Postgraduados. Escucla National de Agricultura, Chapingn, 517 pp.

BENJAMIN BURR and FRANCES A. BURR Biology Department, Brookhaven National Laboratory. Upton, N Y II9Z~, USA.