Transposable Elements in Maize: Their Role in Creating Plant Genetic Variability

TRANSPOSABLE ELEMENTS IN MAIZE: THEIRROLEIN CREATING PLANT GENETIC VARIABILITY Peter A. Peterson Department of Agronomy Iowa State University Ames, Iowa 5001 1

I. Introduction: The Heterogeneity Question 11. Maize Breeding Accomplishments: What the Plant Breeder Has Wrought A. The Maize Genome: How Much Has Been Manipulated? B. Yield Components 111. Transposable Elements A. Their Phenotype Variegation B. Transposable Elements: Their Discovery C. Components of Transposable Elements D. Systems E. Genetic Resolution of a Transposable Element F. Transposition G. Effects on Gene Expression H. Presence in the Maize Genome I. Neoteny and Realignment of Resources for More Efficient Networks J. Summary of Effects Definitions of Terms and Symbols References

I. INTRODUCTION:THE HETEROGENEITY QUESTION The focus in this review is on the manipulation of plants by plant breeders that has resulted in a striking improvement of crop plants during the twentieth century and especially in the last three decades. Transposable elements associated with maize (Zea mays L.) breeding are the primary focus, although this subject is applicable to other crop plants. Breeders’ efforts with maize have uncovered a 79 Aduanrrs in A p m y , Vdumr Y J

Copyright 0 1993 by Academic Press, Inc. All rights of r e p d u d o n in any form reserved.

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highly heterogeneous genotype (Goodman and Stuber, 1983a,b) that can be manipulated in most directions to achieve the desired goal (Russell, 1991; Hallauer, 1992). Plant breeders have used numerous breeding schemes with a varied measure of success. When one then examines the Iowa recurrent selection program with the BSSS populations, from which successful inbreds have been uncovered (Hallauer, 1990a,b), an immediate question is where the variability leading to improvement originated. There are three possible options: (a) This heterogeneity is the assemblage of favorable alleles arising from the recombination of existing heterogeneity in the populations during selection protocols. According to this scenario, the existing variability in the genomes of current maize populations is recombined or resorted to fit the breeder’s needs as selection proceeds. (b) In a second option, new variability (new alleles) is continually being generated (Peterson, 1986a,b). According to this scenario, maize is possibly unique in its potential to generate changes at a high rate in the genome, some that are favorable alleles and others that are unfavorable. Thus, breeders’ selection procedures incorporate these new favorable changes and discard the unfavorable types. (c) In the final scenario, the heterogeneous genotype that the maize breeder has available and manipulates to make better inbreds is derived from a combination of (a) and (b). Thus, (a) + (b) = (c). This review is biased toward option (c), with a major emphasis on the genetic input found in (b) in this formula. Specifically, (b) is enhanced by transposons prevalent in most plants but highly visible in maize. These transposons will be described, and their effect on individual genes will be illustrated. Further, their pervasiveness in maize populations will be examined.

II. MAIZE BREEDING ACCOMPLISHMENTS:WHAT THE PLANT BREEDER HAS WROUGHT

A. THE MAIZEGENOME: How MUCHHAS BEEN MANIPULATED?

There has been considerable progress in maize improvement. W. A. Russell (1991) has summarized concepts and results of maize breeders during the eras of scientifically driven maize breeding. In this review, Russell examined the genetic gain that resulted in maize improvement. (Genetic gain is that improvement derived from genetic changes. These changes, though, accommodate husbandry practices that advance maize culture.) He reviewed his own extensive research on this problem as well as that of others, notably Duvick (1977, 1984, 1986, 1991) and Castleberry et al. (1984). The reader is referred to his review (Russell, 1991), but a few principles will be summarized.

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1. Some General Concepts a. Exotic Germplasm Exotic germplasm has had a relatively insignificant role in Corn Belt maize breeding programs (Hallauer, 1990a). Though many attempts have been made to incorporate nonadapted germplasm, this resource has not been utilized, despite the multitude of available diversity (Goodman, 1985, 1990; Goodman and Stuber, 1983a,b). This feature will be discussed in a later section. b. Genetic Gain There has been genetic gain over eras of maize breeding: in the decades between 1930 and 1970, there has been a marked increase in yields of maize cultivars at several plant densities but most notably at the higher plant densities. What is very evident is that the 1930s material could not sustain its yield potential at the high plant densities when compared with current materials. It could be concluded from these studies that 79% of the increased yield is attributable to genetic gain. This attribution to genetic gain is substantial. These results are supported in a second study (Russell, 1984) that covered the 1930- 1980 period. The Duvick studies (1977, 1991). using different materials, showed somewhat similar results with genetic gain of 60%. What can be concluded from the genetic gain findings involving higher plant densities is that the maize breeder manipulated the genotype that led to the newer hybrids and, in doing so, accommodated higher plant densities. The plant breeder met the challenge of new husbandry practices of fertilizer, pesticide control, and advances in machinery. Sustaining high yields at higher plant densities includes changes in many plant components that “tailor” or “fine-tune” the genotype for this new level of plant culture. Some changes could include “downgrading” some gene functions. We will return to this aspect in a later section. c. Maize Hybrids and Stress Since final yield is a complex process attributable to a number of components, one significant item is resistance to stress. This is a dominant characteristic of the newer hybrids. The capacity of plants to withstand the debilitating effect of either drought or heat leading to stress is a significant genetic gain. The 1980 hybrids show a decided resistance to a stress environment (Russell, 1984; Duvick, 1991). In the evolutionary development of our progenitor maize plant, the plant accumulated many fail-safe systems such as leaf rolling to avoid moisture loss-a feature used differently in modern maize culture with the numerous amendments. d. Efficiency in Nitrogen Utilization Modern maize growing practices have prompted the selection of hybrids that show a greater capacity to utilize nitrogen by expressing greater nitrogen effi-

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ciencies. This must be a complex trait that would accommodate the more available nitrogen into the maize plant, affecting sink, kernels, leaves, membranes, and other structures. A number of other traits have been improved, such as root and stalk lodging resistance, disease resistance, reduced barrenness, and a simplified smaller tassel. These are changes in the genotype, and in Section I11 the origin of such changes will be discussed. e. Recent Hybrids Are Able to Maintain Yields at High Densities The sustained yield at higher densities of modern-day hybrids is a necessary characteristic. How, then, could plant breeders successfully breed this characteristic into their hybrids? Eliminating excess vegetation such as tassel size, leaf excess, and other undiscovered physiological changes would be possible. In tassel size reduction, the limitation of pollen starch development would save resources that could be directed elsewhere, such as to the ear. Molecular investigations on comparative studies of drought-sensitive versus drought-resistant types will likely identify specific genes that distinguish between the two types.

B. YIELDCOMPONENTS Components of the genome that contribute to yield have been investigated in maize. What would qualify as a component that would have an effect on yield? Studies on this theme have been investigated by the Minnesota Agricultural Experiment Station, Geadelmann and Peterson (1978), and the Iowa Experiment Station by Russell’s group (El-Lakanyu and Russell, 1971; Prior and Russell, 1975). The general objective of these experiments is to maximize a “plant’s opportunity” to increase yield. Would the selection and incorporation of deep kernel (D),long ear (L), and multiple ear (M) (prolificacy) components add to the total yield of a plant? Or would combinations of each of these individual units such as DL, DM, or LM when incorporated in a backcross-selection program substantially affect yield? Geadelmann and Peterson ( 1978) concluded that selectively adding these double components in a backcross-selection program did not exceed the normal type at a high plant density-at least, those densities common to those in the Corn Belt. The modified types did perform well at low plant densities as might be expected, for example, prolificacy where the genomes would maximize its potential. This was true for the other single components. In general, according to a cytogenetic view, these results are not a surprise. At best, a backcross program would entail considerable “drag.” The number of genes in a two-component strategy would be numerous and downstream linkages

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would be difficult to dislodge. Further, a two-component genome contribution might be at cross-purposes. Genes for prolificacy (M)might not be positive for those with ear depth. The genome contribution might be considered as a cassette, a subject that will be discussed in Section 111. Thus prolificacy by itself was successfully utilized at low plant densities whereby this trait “capitalized” on the unused resource potential of the maize hybrid.

111. TRANSPOSABLE ELEMENTS

A. THEIR PHENOTYPE VARIEGATION Transposableelements are pervasive in a wide assortment of organisms (Fig. 1) (Peterson, 1987), and they have been found, often unexpectantly, in most genes that have been molecularly analyzed (Schwarz-Sommer et al., 1984; Wessler and Varagona, 1985). When genes are isolated, inserts are often found that are transposable elements. Mobile elements (mobile and transposable will be used interchangeably) transpose (move from one position to another), and when they insert into genes they interrupt gene function (Figs. 2A and B). This results in a null phenotype such as colorless kernel (disrupts gene transcription at a stop codon or out of frame sequence). This disruption continues until an active element excises the insert (Fig. 2B). The subsequent excision of these inserts, namely, their transposition out of the gene, restores gene function, and this activity on different tissues leads to variegation (Fig. 1A). This is most prominently seen in kernels of maize as kernel color variegation or starch differentials. However, it can also be seen in leaves and in other plant parts (Figs. 1A-D). Though variegation is a prominent part of the expression of mobile elements, these elements can only be seen as variegation when they are inserted into genes that are visually expressed, such as those controlling color or starch type in kernels of maize. That is, there must be a ”reporter” that can be expressed. If these transposons are in an anthocyanin gene controlling kernel color and the plant genotype is not expressing kernel coloration, they will not be evident to the observer. Commercial corn, for example, lacks anthocyanin coloration (two of the genes necessary for color are almost always recessive; consequently, color cannot be observed), and an insert in one of the other color genes is not expressed. 1. Transposons since the Beginning of the Origin of Maize

Though transposons may be prominent either in a genotype or in any population, they are not recognizable to the observer because they have not inserted into recognizable genes; they are still transposing and affecting functions not

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Figure 1. Expression of transposable elements in four species. (A) Zea mays, a2-m (Peterson, 1978). (B) Petunia hybridu (Wijsman, 1986). ( C ) Dahlia, horticultural variety. (D) Glycine m u . 4-m18 (Peterson and Weber, 1969). (FromPeterson, 1987, with permission.)

easily discernible. Transposons have been in maize populations since the beginning of prescientifically driven maize breeding (Blumberg vel Spalve et al., 1990), but they escaped detection in populations of maize because transposable elements themselves do not have an observable phenotype. Transposable elements can only be recognized when they interrupt a gene function. For this, it is necessary to have prominent observable gene functions impaired, such as those affecting coloration in flowers (Fig. lB), leaves (Fig. IC),and kernels (Fig. 1A). These genes with an insert would act as reporter alleles. An insert in a gene for a trait that is part of a complex set of genes (e.g., stem elongation) would not easily be recognizable. Surprisingly, one of the first genes to be experimentally

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examined in the beginnings of the science of genetics, namely, the wrinkled peas of Mendel, was identified more than 100 years later (after Mendel’s work) to be caused by an insert (Bhattacharyya er al., 1990). Thus, the absence of transposons in certain crop species may be a consequence of the lack of observable options. A transposable element phenotype has been uncovered in rice (Oryza sariva) (Reddy and Reddy, 1992) and soybean (Glycine m a ) (Groose et al., 1988). One only has to reflect that 50 years of intensive maize genetics preceded McClintock’s (1947) genetic studies before transposons became resolved (McClintock, 1948). These concepts will be demonstrated subsequently.

B. TRANSPOSABLE ELEMENTS: THEIR DISCOVERY 1. A Short Synopsis of the McClintock Experiments

Barbara McClintock arrived at Cold Spring Harbor in 1941. Before going to the Carnegie Laboratory, her studies included chromosome aberrations and rings in maize (McClintock, 1931a,b, 1944, 1945), and before that, she had singlehandedly established the chromosome ideotype of maize that gave maize genetics a sound foundation early in the science of genetics (McClintock, 1929). In the period just preceding these transposon studies, she induced homozygous minute deficiencies by the induction of crossing-over (step A in Fig. 3A) in certain aberrations that led to bridges that yielded mutants (McClintock, 1932, 1944). The breakage events that yielded a number of these instabilities are illustrated in steps B and C of Fig. 3A. The aberration that McClintock used was on chromosome 9 (Fig. 3B). Because chromosome 9 was being used for numerous other studies, McClintock was very familiar with its genes, namely, the color ( C l ) locus, shrunken (Shl) locus, and waxy (wx)locus. She made a detailed analysis of the yellow-green locus at the end of the short arm of chromosome 9. In proceeding with this bridge-breakage-fusion cycle (Fig. 3A), it was clear that the chromosome breaks were at random on the short arm of chromosome 9. Sometimes the Cl and S h l shrunken genes were lost and other times the loss included the Wx gene. Or it was sequential, with the Cl gene lost first, followed by the Shl and then the Wx gene. This random breakage event was expected. However, in the pursuit of these studies, she observed that in one case the breaks were always occurring at a position proximal (direction of Wx toward centromere) to the wx locus (Fig. 3B). Why did the chromosome 9 break at this specific point? She concluded that this specific chromosome site was weakened and had the property to dissociate, breaking rather readily at that specific point. Thus, she named that site Ds for dissociation. As she progressed with crosses in further studies, she observed that this breakage event segregated. That is, this site showed breakage

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Figure 3. (A) The bridge-breakage-fusion cycle and illustration of some of the events during meiosis leading to transmission of the products of this meiosis to the fertilization event. The crossover from the duplication arrangement (1) leads to a bridge and breakage (2) with a rejoining and bridge and breakage and microspore products (3, 4, 5 ) . (From Peterson, 1987, with permission.) (B) Chromosome 9 in maize showing a break at Ds.

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because the site segregated like a Mendelian event. In reconstitution crosses, she uncovered a second factor that was needed for breakage to occur, indicating that the breakage event required two factors-a breakage site Ds and a second factor, Ac (McClintock, 1945). This is what was suggested. She reisolated these lines in appropriate crosses and reconstituted the experiment. Starting with a line (the Ds line) that did not show breaks, she introduced the second factor to this line. From this cross, breakage again resulted. She was able to demonstrate that, indeed, there was a second factor that activated the Ds breakage event. She called the second factor Ac for activator. There were more surprises to come. In subsequent crosses, McClintock found that this breakage event now was located at a position different from the original one on chromosome 9. Instead of losing all the material distal to wx (Fig. 3B) (chromosome 9), the site was now located in a place whereby the Shl and the C1 loci were lost (McClintock, 1948). Could it be that the Ds locus moved from one position to another and was now located just proximal to Shl? Or was a new site activated to become Ds while the other site was deactivated? Further experiments would clarify this observation. The unexpected observation came in the cross illustrated in Fig. 4A, in which an exceptional kernel appeared (part C of Fig. 4A). The two expected phenotypes resulting from the cross are shown in part B of Fig. 4A. The expected genotype C l cl cJ in the aleurone of the cross ( c l lcl X C1lC1) should show the loss of the C1 locus to yield a colorless region when the cross was made against a recessive c line. However, there was an exceptional kernel in that the pattern of the coloration was reversed. Instead of colorless sectors on a colored background (loss of C), the pattern showed colored spots on a colorless background. This particular kernel was identified as cl -ml . A dominant C1 gene had seemingly changed to cl -m. When pursued further in backcrosses, c l -ml lcl x c l I c l , she found that the variegated phenotype was dependent on a second factor (McClintock, 1948). The segregation told her that. Further, in making crosses to Ds lines with the same second factor, she found that this factor indeed was Ac (Fig. 4B). Thus, if Ac was activating this variegated phenotype, it must mean that Ds had inserted itself into the C1 gene, changing it to a cl-ml allele. This was truly transposition from one point of chromosome 9 to the C1 gene. This was also creditable deductive reasoning, reasonable in hindsight but difficult to envisage. There were yet more surprises. Many other loci became unstable and were shown to be controlled by Ac. It seemed that Ds elements exploded and were transposing into genes (a significant novel concept), inactivating many genes, and responding to Ac. Thus, with these observations over a number of years and with many intricate and delicate experiments, there was established the notion of transposition of genetic material that resulted in the control of the expression of genes (McClintock, 195 1). Variegation, therefore, was caused by elements in the genomes that move.

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Although variegation has been identified in historical references since botanists first reported “eversporting” varieties in their study of plants (e.g., Darwin, 1868; Lecoq, 1862; Knight, 1808), these transposable elements were not available for analysis until McClintock (1951) found them in her Cold Spring Harbor nursery. And not because new tools were available in the 1940s. This could equally have been analyzed in the 1920s if the same experiments were conducted with the bridge-breakage-fusion cycle. The first clue was the observation that breaks were at a specific site and regulated by a Mendelizing second factor. This was followed by the observation that these elements could be found at a new site

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and then in a gene cl -ml that showed the variegation that resembled the eversporting varieties of an earlier era. Now transposition of genetic material can be associated with variegation.

2. Concepts of Unstable Genes in the Pre-McClintock Period Such observations require a concentration on detail and precision in experiments, truly a McClintock trademark. But what image did geneticists have of unstable genes during the 1940s and early 1950s? During that period, unstable genes (as they were called in the 1930s) had a different image than they did later. Some of the early history on the concepts of mutable genes in a variety of plant species has been reviewed by Demerec (1935). At that time, they were considered to be unstable genes. Thus, up to the time of McClintock’s experiments, this variegation of plants was attributed to unstable, mutable, or sick genes. The early studies in maize by Emerson, and subsequent studies in the decades of 1910 to 1930, came very close to a genetic analysis. The key component of demonstrating transposition evaded Emerson’s studies, as well as later studies by Rhoades, because of the lack of evidence for transposition. Given more extensive crosses, especially by Emerson (1914, 1918, 1929) with his unstable pericarp, he would have been mystified by the results if transposition was observed, especially at a time when concepts in genetics were being established. If a reporter allele was not present or available, there would have been a difficulty and, especially working with the P allele, most of the possibilities would have evaded Emerson because the P-vv locus (unstable pericarp color) would have covered up some of the mutants that might have occurred in various aleurone genes. Thus, the discovery waited until the mid- 1940s when McClintock experiments with another goal unexpectedly uncovered mobile elements. It is appropriate to set the stage for the mid-1940s when McClintock, working at the Carnegie Laboratory at Cold Spring Harbor, uncovered a large number of unstable mutants while studying the bridge-breakage-fusion cycle (BBF). The gene at that time was vaguely conceptualized as beads on a string. This was amplified by the consideration of the Drosophila banding patterns. Further, McClintock (1942, 1944) had shown in previous work with the BBF cycle that some of the maize chromosomes contained chromomeres that appeared like beads on a string, which could be “fractured,” and the resulting products showed mutations of various genes on that chromosome.

3. The Concept of Genes at the Time of the McClintock Experiments How did McClintock conceptualize gene material when she conducted her experiments? Actually, it didn’t matter because she was driven by her own acute observations, and this led her in pursuit of further experiments. Her colleagues

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at Cold Spring Harbor on Long Island, New York, were very active in the forefront of genetics. Demerec was pursuing Drosophila and Salmonella genes, Kaufmann and McDonald were studying Drosophila, and MacDowell was studying mice (Mus) (Demerec, 1946, 1947). Evelyn Witkin was working with repair mechanisms in bacteria. This was the core group at the Camegie laboratories. Bruce Wallace with his Drosophila populations joined them in the late 1940s. McClintock let her experimental observations drive her research rather than be biased by preconceived notions. Yet, in the genetic community, and especially in the New York area, there was extensive speculation on the genetic material, although the experiments of Avery et al. (1944) had not yet been coupled with those of Hershey and Chase (1952). Also the Watson and Crick (1953) model for the molecular structure of nucleic acids was not available. What was the prevailing understanding of genes there in the late 1940s? Visual observation of the Drosophila chromosome indicated a banding pattern and maize chromosomes showed chromomeres, suggesting beads on a string. Furthermore, the Drosophila work in the late 1930s and 1940s uncovered a phenomenon called position effect. This concept was strongly supported by GoldSchmidt (1953, who proposed that mutations were caused by interruptions in the chromosome by some rearrangement. Why did he support this notion? A number of genes were found, such as the genes of the Notch locus in Drosophila, that by all observable features were an unchanged banding pattern and seemed to be normal. Thus, it could be concluded that there must be some rearrangement of the chromosome material. The banding pattern in Drosophila was the key to gene arrangement. Further, other genes were found to cause mutation by a rearrangement that was not at the gene affected but at a short distance away from the gene site. How could gene expression appear and disappear in development? One could not assume changed genetic material, but one could assume affected genetic material. Thus, there was strong support for position effect, meaning that the normal locus, near a rearrangement break, seems to have changed expression to a mutant action, making the chromosome as a whole the primary focus of gene action. There were many cases of this type. The yellow locus in Drosophila was an example. The BAR duplication (Bridges, 1936) had a quantitative effect when located in a specific arrangement as visualized with the banding pattern. Other significant research was Beadle’s (1932) work with the sticky gene. Here, all the chromosomes were affected by a mutation in only one gene. Trans effects were not part of the dialog. The sticky gene by its action led to an increase in both chromosome rearrangements and point mutations. Thus, it appeared from the phenotype of the sticky gene that it, too, was caused by rearrangements that resulted in a position effect. These observations by Goldschmidt related to his concepts of chromosome structure and integrity. This was similar to thephl gene in wheat (Triticum vulgare) that dominates chromosome pairing between genomes (Gill and Gill, 1991).

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Since position effects could be reversed, it seemed that the genes were unchanged in content but rearranged, and thus could be re-rearranged. There was no support for the idea that there was any difference between position effects and point mutations. Now we come to the so-called repeats. This was a case where mutants (two or more) behaved like multiple alleles, for example, the Drosophila studies of Bithorax by Lewis (1945), lozenge (lz) by Green and Green (1949), and, in maize, the Ab locus by Laughnan (1949). These fit Goldschmidt’s theory of genes and mutation, and although there was limited support for his views, they could not be ignored. Thus, Goldschmidt hypothesized that mutants are rearrangements in the chromosome that cause mutation. This hypothesis supported the idea that the entire chromosome was the basis of genic action, and the interruption in this chromosome led to these mutant loci. This, then, was the status of genes when McClintock hypothesized about her observations.

c. COMPONENTS OF TRANSPOSABLE ELEMENTS 1. Genetic When a gene controlling color, such as a l - m , shows variegation, and is outcrossed to a standard full color line and the subsequent F1 (Allal-m) is backcrossed to a standard recessive ( a l - o ) , two possibilities may be visualized (see Fig. 12). If all or most of the noncolored progeny are variegated (Fig. 12A), one could conclude that the element is expected to reside at the gene locus, and the unstable allele would be considered autonomously mutable. This would indicate that mutability is coupled with the a l - m allele. If, on the other hand, only half of the noncolored kernels are variegated (Fig. 12B), then the control of mutability is independent of the al-m allele. In this second category, independent control, the insert at the locus (such as McClintock’s Ds in our previously described example) was nonfunctional but could respond to an active element that was segregating independently. Specificity: When the second component (identified as Ac) is introduced to variegation in the form of the line with a gene with an inserted component (Ds), spotting results (Fig. 4B). This resulting variegation is derived from a very specific interaction. For example, when this colorless kernel (al-Ds, Ds is an insert in the A1 gene, eliminating A1 expression and therefore a1 -Ds)was crossed to a previously described unstable element, namely, the Dt element (Rhoades, 1936, 1938), nothing happened. There was no instability and the kernels remained colorless. Thus Dt induced spotting on the al-dt allele but showed no spotting with the al-Ds allele, though the inserts are at the same locus. Similarly, when this same al-dt allele that responded to Dt was now exposed to Ac, again

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nothing happened, the kernels remained colorless (Fig. 4C). Thus, though these two alleles resided at the same locus, there was something unique about the interaction of each of the elements with the introduced functional element. The resolution of this specificity of interaction awaited molecular investigations (Fedoroff et al., 1983; Pereira et al., 1986). This was further substantiated as new instabilities were uncovered. For example, when another a1 allele, one of the En system, was tested with Dt, again no variegation resulted although it expressed variegation with En (part A in Fig. 4C ). Similarly with Ac, no variegation resulted (Peterson, 1961). Thus there appeared to be a specificity of the active element such as Ac, Dr, or En with something specifically unique to it at the locus. What was unique at each allele could be recognized only by the specific element. From a one-component autonomously mutable allele, a two-component element system arose. Thus, there was a relationship with the initial insert and the resulting allele when a onecomponent mutability changed to a two-component mutability (independent control). As was earlier indicated by Peterson (1970), the visitation at the locus by a functional element caused a change to an allele with resulting loss of mutability. In this change from a functional element to a receptor element, something was “left behind.” What was “left behind at the allele” awaited molecular investigation. 2. Molecular TNPA and TNPD With the uncovering of the Ac transposable element by the Fedoroff group (Fedoroff et al., 1983; Pohlman et al., 1984), these mysteries regarding specificity quickly dissipated. It was clear that with the investigation of the wx-m9 allele, a comparison was possible with the derivative wx-m9 receptor allele. When the one-component wx-m9 autonomous mutable wx was compared with the wx-m9-derived Ds-containing unit, it could be seen that the wx-m9 Ds was derived directly from the wx-m9 Ac by deletion of part of Ac in the original allele that incapacitated its function. What Ds lacked was part of Ac (now we know it as the transposase-determiningportion of Ac), which is an active component that can recognize the insert that it left behind, namely, the Ds. All the other parts remained intact. Research on the elements uncovered that they had a structure and definition that could be analyzed. The Ac element consisted of 4000+ nucleotides (bp unit), and it was found that there was a definitive structure to the insert. From sequence analysis, a terminal inverted repeat (TIR) of a certain length could be seen as well as a target-site duplication (TSD) (GTT in Fig. 5 ) . Was there a pattern to element structure? Were TIRs and TSDs part of other elements? When EnlSpm was uncovered and molecularly analyzed, the TIR and TSD were also there but were different in length and content. This fit the geneticists’ receptor

PETER A. PETERSON

94

i\ A

I I 5 5 I I

49

;:“ T

A

C A T C -C wxm8 5’ C G T G G T C A A m wxt

CGTGGTCAA

m C A A C G C G G C 3’ CAACGCGGC

Figure 5. Part of a transposable element illustrating the TIR (13 bp) and the TSD (GTT) (3 bp). The motifs (the lines between 2 5 , 29, etc.,) are aligned and continue for 200 bp.

element as a fractionated part of the active element when a comparison was made with the w x - d element (Schwarz-Sommer et af., 1984; Pereira et al., 1986). It was also clear that the identifying mark of the element had a specific TIR and TSD like the En. Thus, what was determined genetically on the specificity of interaction became clear. The elements of a specific group fit into a specific category, namely, identifiable TIR and an identifiable TSD. The functional element Ac or En only recognized its own motifs, and the next several years were needed to verify this. These TIRs and TSDs are illustrated in Table I. So, what was “left behind” as conjectured in 1970 (Peterson, 1970) was a fractional part of the original element. These transposable elements have been dissected and reexamined in more extensive detail with more definitive molecular examination in the last decade. Since the EnlSpm is most fully investigated, it is appropriate to examine this element. The EnlSpm transposable element has 8237 bp and contains a definitive structure (Figs. 6A and B). By examining the full-size sequence of the element and comparing it to the cDNA, it was evident that there were 11 exons and 2

TRANSPOSABLE ELEMENTS IN MAIZE VARIABILITY

95

Table I Molecular Characteristics of Some Examples of Plant Transposable Elements“

Element Dsl ACl En I Spm-I8 MuI-MuS BR rbR rcy:Mu7 Mu RI Tam1 Tam2

NAU Au Au NAu NAu Au

NAu NAu Au NAu

Plant Zea mays L . Zea mays L. Zea mays L. Zea mays L. Zea mays L . Zea mays L. Zea mays L. Zea mays L. Zea mays L. Antirrhinum Anrirrhinum

kb 0.405 4.563 8.287 2.241 4.869 2.2 4.0 17. Varies

DuplicaTerminal in- tion of verted repeat target (bp) site I1 11 13 13 + 200 5 5 200 220 13 13

+ +

8 8 3 3 9 8 8 9 9 3 3

“From Peterson (1987). Hartings e r a / . (1991). and Chomet er al. (1991) bNonautonomous. ‘Autonomous.

open-reading frames. It was also evident that there were TIRs on the end and, of course, a TSD of 3 bp. In subsequent genetic studies following the original description of En at the p g locus (Peterson, 1953, 1960), an al-rnl allele was found to be colorless in the absence of EnlSprn but colorless with spots in the presence of EnlSprn. McClintock found an al-rnl(5729)allele that was colored in the absence of En/ Spm but colorless with spots in its presence. This was different. It is readily evident that the functional EnlSprn suppressed coloration with a Suppressor (S) function and induced spots (excision) with a Mutator (M) function. This is what made EnlSpm so attractive for study. It showed two clear functions, S and M. But, how did these genetic functions relate to the molecular structure? Two functions could be determined in genetic investigations. These interactions are illustrated in Fig. 7. It is now necessary to dissect the molecular sequences and determine the boundaries of the functional components. In Southern blotting and probing with En, two bands are recognized, a heavystaining 2.5-kb (Fig. 2B) and a lighter 6.0-kb band. These two transcripts were related to the En structure (Fig. 6A). And, from the sequences, two proteins could be visualized, TNPA and TNPD (Fig. 6A). Several studies demonstrated the functional domains of these two proteins (Frey et al., 1990a,b; Masson et al., 1991a,b). TNPA is a DNA binding protein and recognizes the IZbp sequence motif of

96

PETER A. PETERSON

P

AUG

P

B

UGA

4

En1

C

D 1-102

P

4

AUG

4

u

PMW-1ERMWAL EXONS

UGA 4590 bp

CMBOXY-TERMINAL EXONS

Figure 6. Structure of the En1 element. The ORFs (dotted in A) and 1 1 exons (open boxes, shaded in A) are shown in B. The origins of TNPD and TNPA are also shown in A. A derivative En2 is illustrated in C showing the deletion of OW2 and part of ORFl. The alm(r) deletion derivative (1102) is shown in D.

the TIR and adjacent motifs. The S function was genetically defined as the suppressor function of the EnlSpm system (Fig. 7 , bottom) by McClintock (1961). It should be emphasized that this suppressor component can only be visualized in alleles such as al-m1(5719),where the insert allows gene expression to occur, and this gene expression can be suppressed by the introduction of an EnlSpm. Other alleles such as al-m(r) (Fig. 6D) (Peterson, 1961) are colorless and, therefore, not S expressive. It has finally been shown that this suppressor function is

TRANSPOSABLE ELEMENTS IN MAIZE VARIABILITY

97

Figure 7. The a-ml allele (a-m157l9A-I) without (top) and with (bottom)En (colorless with spot). It is shown with the linked sh2 gene and a weak M action but a strong S (fully suppressed).

caused by the TNPA protein that blocks transcription read-through (Gierl et al., 1985, 1988a,b). At this point, there are two genetic functions, and they relate to the two transcripts tnpA and tnpD. Also known is the structure of En (Fig. 6). What is not known is how the structure relates to the genetic and molecular observation. What is the extent of the En sequences that govern these functions? How many of the exons (Fig. 6A) are related to the S function, and how many are part of the M component? An attack on this problem would come from a deletion analysis. Two mutants were available, En2 (Gierl er al., 1988a) and Spm-w8011 (Masson et al., 1987), to determine the sequences governing these functions. The En2 mutant was molecularly analyzed and shown to be a fractured En and to have all the exons intact (Fig. 6C). There was, however, a 1126-bp dele-

98

PETER A. PETERSON

tion that included all of ORF2 (open-reading frame) and part of ORFl . In genetic tests, En2 had a full S function. Because the S function was expressed in genetic tests, it could be concluded that the exons were responsible for this function. But the M function was impaired. Only a few spots appeared in genetics tests with appropriate reporter alleles (Fig. 8). The M function must therefore be controlled by the ORFs, which showed a deletion. The few spots could come from residual En activity in the genome according to these investigators (Gierl er al., 1988a). Yet, given another reporter allele with En2, a high expression of M is observed (lower ear in Fig. 8). It is clear from this observation that the deletion in En2 did not eliminate the M function. What of the rest of the structure of the En element? The 13-bp perfect TIRs in the ends of the element and the sequence motifs in the subterminal regions of EnlSpm (Figs. 5 and 6) define the critical cis-determinants needed for excision of IIdSPM elements. Further, the subterminal 12-bp TNPA binding motifs (Fig. 3,though quite similar, must be in an ordered orientation, that is, head to head or tail to tail with a prescribed distribution and repetition for proper excision. Nature is very precise. The trans-effect is derived from the TNPA protein (the S function) that brings the ends of the element together like a zipper such that the

Figure 8. The En2 element with a l m 15719 on top and alm(Au)pule-(mr) on bottom. The difference in spotting is due to the receptivity of the reporter alleles.

TRANSPOSABLE ELEMENTS IN MAIZE VARIABILITY

99

TNPD protein (the M effect) could cut the element where the 13-bp TIRs intersect with the TSD (Fig. 5 ) . These two products of EnlSpm, namely, TNPA and TNPD, were critically analyzed by using transgenic systems. Each was inserted into transgenic tobacco (Nicotiana tabucum) with a GUS insert (Frey et al., 1990b; Masson et al., 1991b). By making several definitive constructs, it was found that with TNPA alone, no effect can be seen. Nor with TNPD alone. However, when the two are combined in the same transgenic tobacco plant, GUS expression (expression of P-glucuronidase) is evident, indicating that excisions are taking place. An I element (dSpm) is inserted in the GUS coding sequence and thus inhibits GUS enzyme activity. Excision of the I element insert permits GUS activity, which is expressed as blue-staining tissue upon histological staining with X-Gluc (Jefferson et al., 1987). With these experiments it was clearly obvious that the S function was associated with TNPA and M was associated with TNPD. The previous concepts of the interaction of elements in causing excision are confirmed as earlier envisioned, namely, that TNPA binds and brings the inverted motifs together (Fig. 5 ) and the TNPD along with it will cause excisions to take place. Thus, with these experiments, it is shown that TNPA is necessary for TNPD to function.

D. SYSTEMS Early in the genetic investigations of mobile elements, it was obvious that these various elements fit into systems. This was based on the exclusion of interaction in some cases and the inclusion in other cases. This is illustrated in Fig. 4C.Here the Ds or the I element is identified as the receptor, and the Ac or the En element is the regulator. When receptor 1 shows a response to regulator 1, it is part of the same system. However, it does not show response to regulator 2. This is the case of an En element with a Ds receptor. Similarly, receptor 2 does not respond to regulator 1, but it does respond to regulator 2. This would then account for the Dr not activating the Ds elements as well as the En not activating the a-dr allele. Relating back to the molecular investigations, the Dt protein does not recognize the /-element motif nor does the En protein (TNPA) recognize the Ds motifs. In past decades with genetic studies of transposable elements, eight systems (Fig. 9) have been identified. Each system shows a specific genetic interaction, and those analyzed genetically show a distinctive molecular DNA sequence. These systems are identified with an exclusion pattern as well as an inclusion as indicated, and when their molecular definition is determined, they show unique TIR and TSD configurations (Table I). A question that will be considered is why

100

+

*

PETER A. PETERSON

= responds as mutability; absence of = Au = Autonomous element

+

indicates no response.

Figure 9. Transposable element systems. Regulatory elements are specific in their activation receptor elements. There are three cases of overlap in activity. Ac activates all Ds’s, but Llq activates only Dsl. Fcu activates rcu and R-r#2 but Spfis limited to R-r#2. Cy and MulR appear to have homologous activity. ( + ) of

have these various elements survived in maize populations and why are there so many diverse systems?

E. GENETIC RESOLUTIONOF A TRANSPOSABLE ELEMENT First, let us examine the nature of their survival in maize populations. Variegation in flowers and leaves as well as in kernels was seen in botanical investigations long before the geneticists realized they were associated with elements (transposable) in the genome that transpose. Further, there was clear evidence that newly arisen variegated loci arose in experiments conducted with mutable loci (Demerec, 1935). Why, then, were these variegated phenomena not assignable to transposable elements? The clue to this has already been given. Reporter alleles had not been isolated that would have provided the tangential evidence needed to establish a relationship. Such a case will be given of a newly arisen variegated locus. In northwestern Colombia, in the swampy region near Panama, the Cuna In-

TRANSPOSABLEELEMENTS IN MAIZE VARIABILITY

101

Figure 10. Cuna tribal corn. (FromGonella and Peterson, 1977.)

dians grow a maize with a striking wine-red variegation symptomatic of a transposable element (as opposed to a mottling phenotype often seen in some crosses) (Fig. 10). If such maize seed were brought into a laboratory, one would ask several questions (Peterson, 1978). What gene is involved? To what transposable element system does it belong? And what is its inheritance, whether it is autonomously mutable or whether it is independently controlled? A crossing protocol as illustrated in Fig. 11 would be developed to answer these questions. The crosses in the Y pathway in Fig. 11 would establish that an anthocyanincontrolling locus was involved. In the Cuna example, the variegation was assignable to the r locus and therefore is r-m(Cuna) (Gonella and Peterson, 1977). The X pathway in Fig. 1 1 is followed to answer two of our other questions-the nature of inheritance and system relationship. The results seen in X-1versus X2 illustrate the inheritance pattern. Because the segregation does not agree with results expected for X-1 but was like X-2,variegation is controlled by an independent factor. Using the variegated kernels from the x-2cross progeny, a system relationship could then be tested. If any variegation appeared in the crosses (X-2I ) , then r-m(Cuna) is assignable to the system represented by that reporter allele. Because none of the reporter alleles showed any variegation in the crossing protocol, it could be concluded that r-m(Cuna) is a new transposable element system and is controlled by an independent factor. This was finally identified as the Factor-Cuna system (Fcu) (Gonella and Peterson, 1977, 1978).

F. TRANSPOSITION 1. Genetic Detection McClintock (1948) established that Ds had moved. From a position proximal to the wx locus in her initial studies, Ds was now near the CI-ShI region. In a

102

PETER A. PETERSON Variegated Cuna

x

X pathway

V pathway tester lines of anthocyanin genes. al, a2, c, r, c2, etc.

only the r tester uncovers the mutant. All the other Fl’s are colored. Conclusion: rlt Is r-m (Cuna)

on to color line &

colored F1 seifed 4

w X-1 - if co1or:variegated -

X-2 if co1or:variegated:colorless

=

31 variegation is autonomously controlled

=

12:3:1 variegation is controlled by an independent factor

determlnatlon X-2-1 Cross r-m (Cuna) to assorted reporter alleles c-m(r), a-rcy, a-Ds., etc. r-m(Cuna) x

reported alleles--ex c-m(r)

b

Colored F1 X-2-2 R/r-m (Cuna) C/c-m(r)

x b

No varieaation

reporter alleles-ex c-m(r) if variegation appeared with one of the reporter alleles, then r-m (Cuna) would be of that system. Result is negative for c-m(r).

-,

Conclusion: r€n is not implicated. The same with all other reporter alleles. A new system Is estabtlshed, Fcu-rcu

Figure 11. Determination of the gene locus, heritability, and transposable element system of a newly discovered variegated allele. Described in text.

strict sense, one could question whether this was the transposition of material. All evidence suggested that it was. For the purist, however, there was a nagging question. Could the events observed be a correlative deactivation followed by activation elsewhere? The geneticists of that period were willing to consider any option but concede that genetic elements moved. That is, where Ds was found in the position proximal to cl, could that be an activation of an element near the cl position followed by deactivation of the one proximal to wx? Similarly, with the origin of c1-mf, could that be from an activation of an element subsequent to deactivation elsewhere? Although this seemed like a remote possibility, it was a legitimate question at the time, especially if one was not prone to accept the possibility that genetic elements can transpose.

TRANSPOSABLE ELEMENTS IN MAIZE VARIABILITY

103

This could readily be obviated if there was a correlated event. If it could be clearly established that the loss at one place was followed by the discovery of the element at a new position, especially in the same tissue, this would be most appropriate. Such a demonstration was possible with a variegated P locus ( P RR-Mp), where a variegated (coarse) phenotype was associated with the original mutable pericarp. This variegation is conditioned by an insert of the modulator element (Mp) (now recognized as Ac) in the P locus (Emerson, 1914; Brink and Nilan, 1952). When a second Mp element is present, the phenotype changes. The new phenotype has a distinctly fine type of variegation as opposed to the coarse type of variegation with the original mutable pericarp locus. Coincident with this change in phenotype, the excision of the M p leads to a colored phenotype. In crosses where medium pericarp variegation (the c o m e type) was observed, there were twin sectors of colored and fine type. This suggested that the fine and the red co-twin were the produce of one event. Numerous twin sectors were found. When it was analyzed, it was shown that the red sector lacked the Mp and the fine sector had the extra Mp identified as rrMp (transposed Mp). This would indicate that a loss of Mp at one position (coarse type) yielding a red full-colored phenotype meant a gain of the element at another position, resulting in two phenotypes as twins (Greenblatt and Brink, 1962, 1963; Greenblatt, 1966, 1968, 1974). The most important feature is that the various rrMp arising from transposition from the P-RR-Mp had their origin from the P locus (because the red phenotype appeared), indicating that transposition did occur from the P locus. There was another surprise. From one transposition, two trMp arose, and when tested via linkage studies, both indicated that they were at homologous positions on two different chromosomes. This became a useful and revealing method concerning the transposition mechanism (Greenblatt, 1984). McClintock’s studies were reconfirmed by the Greenblatt and Brink studies, and transposition was now established. An example is presented to illustrate the transposition of En from a f -m(papu). The a f -m(papu) allele is an autonomously mutable allele inserted in the second exon of the A 1 locus. From several studies it became clear that the En was located at the locus because of the correlative transmission of al-m(papu) and En in testcrosses (Fig. 12). Here, most of the non-Af (colored) kernels are variegated, indicating that mutability follows the a f -m(papu) allele. This was verified in both a self and a backcross population. The few colorless kernels arising in the backcross and in the self populations are disturbing. A test of these colorless exceptions is followed in Fig. 12B, which will determine the nature of the newly arisen colorless kernels. In this cross, the colorless kernels are crossed to a reporter allele, the a l - m f allele (a reporter allele “reports” the presence of En). This is useful because a l -mf is closely linked to sh2, and one can assume that when the shrunken phenotype is observed, sh2 is following the a l - m f allele. The product of this cross is variegated, which indicates that the newly arisen colorless kernels do carry En, and that these changed

104

PETER A. PETERSON Bl-rn(a@g) X A Z a 1-m(papu) A1 4

A

F1

x

a 1-m 1(papu)

4 112 colored:l/2 variegated: few colorless

48 4 B colorless

3 colored: 1variegated:few colorless = al-ml?)Shl x 9-m-1 sh alsh2 a-m-7 sh

4

a1-mlnrJShZ

C

m

alsh2

x

a 1-m lsh2 variegated

4

m

alsh2

112 colorless:l/2 colored sh:few variegated sh

D

I

I

I

I

I

el-m(nr) Sh2 f

f

En

I

MmBmmHmmHWMmmmmmmmm

f

X

mmmmmmmmmmmmmmmmmmm f

I

al-ml

I

I

sh2

I

Figure 12. The genetic determination of a transposition event at the A/ gene. At B, the newly originated colorless round kernels originating from al-mlpapu) (A) are an unknown but are proven to be a/m(nr) (nonresponding) (contain an En-see variegated at C ) derivatives from the excision of En. In D, the linkage of the newly excised En to the alm(nr) allele is shown and the variegated shrunken kernels arise by a crossover at X that links the En to the a l - m l reporter allele. (al-mpapu. variegated; A / , colored.) f,continuation of gene sequence.

types are defective derivatives that no longer respond to En. That they do not respond indicates that defectives (excision repair difficulties-see Section 111, F3) do arise and are not responsive. But in this case, they do carry En and therefore are considered to be nonresponding a1 -m(nr) derivatives (Fig. 12C). This could be concluded from the cross indicated in Fig. 12B. When the cross is made as is shown in Fig. 12C, there is an indication that the En transposed from the initial a1 -m(papu) allele is close to its original site. This could be concluded because most of the derivative round (Sh2) colorless kernels from the cross in Fig. 12C are colorless, indicating that the En is in a repulsion phase with reference to the al-ml(sh2) reporter allele. The progeny resulting from the cross indicates the consequences of the transposition of En. In Fig. 12D a diagrarnmatic chromosome is shown to graphically indicate the events that occur in Fig. 12C. It would take a crossover (X)to link the a1 -ml reporter allele with the

TRANSPOSABLE ELEMENTS IN MAIZE VARIABILITY

10s

En that yields the few variegated shrunken kernels in the progeny of the cross. Thus, the transposition event would be established. More extensive data on En transposition were given by Nowick and Peterson (1981). The genetic studies demand that the transposable element be excised cleanly from the originating site. The basis for this is that the transposable element at the new site has all the fidelity of its original composition. Otherwise, the En functions would not be expressed. Thus, the mechanism required for this excision of the element must be precise and definitive in cutting out the element. As indicated previously, the mutable pericarp studies were definitive in describing the transposition mechanism. In later studies by Greenblatt (1984), a model was shown that illustrates the mechanism. In brief, the excision event takes place and the excision of the element occurs from a replicated segment. The new site for this transposing element is an unreplicated chromosome segment. Since it is copied before replication, this indicates that after replication we now have two elements where we had only one transpose. This is illustrated in Fig. 13A (Dash and Peterson, 1993). Such a model was aided and abetted by the availability of the twin sectors that yielded the two events in the same position. Thus, it could be established that from one transposition event, the two phenotypes appeared, namely, the colored and the fine type. When it was finally determined that the colored lack the M p and the fine pattern had two Mp's, it was obvious that one event causing the loss of M p yielded two rrMp's. The original site in the M p case was the P locus. And the P locus could be screened for M p to determine that the M p was no longer at the P locus. This was shown molecularly in later studies by Chen et al. (1987) and by Athma et al. (1992). It could be confirmed by showing that the donor site no longer had the element and that the element now was at a new position. What was also shown is that the new positions were close to the donor site. This indicated that the transposition occurred to nearby sites, which is a favorable feature for attempts to target genes. With this possibility, one could place the target close to the element or rather the element close to the target to maximize targeting efficiency for a gene. Constructs are possible to enhance the process (Dash and Peterson, 1989; Chang and Peterson, 1993). Further details on this are shown in several reviews (e.g., Peterson, 1987).

2. Key Features Leading to Variability:Alteration in Genes There are two components to the feature that contributes to gene changes and subsequent variability induction by transposable elements. Recall that the insertion of a transposable element in a host site induces a target-site duplication. In essence, nucleotides are added to the genome. Each system has its own signature with reference to the number of nucleotides added. The least number of nucleotides in a target-site duplication is the 3 bp contributed by the EnlSpm system. The Ac and the Uq elements contribute eight nucleotides to the target-

106

PETER A. PETERSON

IIIIIIII111IIIIIIIIIIIIIIIIIIIIIIIIIIIII; I' II 11111111111111111111llllllllllllllll

B

1. A transposase generates staggered nicks

m

=C I I

1. A transposase generates staggered nicks

5 &&3 5 - - 3

--5

t

1

< 3

5

t

32. Hypothetical intermediates Integration of a "clean" element

2. Hypothetical intermediates

I

3 Possible excision products

I

or

,

I

-

-

I I

I

3 Figure 13. (A) Transposition of En. Like Pvv, E n excises and yields two chromosome strands with an En. (B) 1. Excision of a plant transposable element. The wavy line represents sequences of the termini of an inverted repeat. The arrows indicate the staggered nicks. 2. Exonuclease degradation is indicated by fine dotted arrows; polymerase action is illustrated by bold dashed lines. It is here that the errors occur leading to altered sequences as shown as possible excision products in 3. (C) Integration of a transposable element. Arrows indicate the staggered nicks. D) Same symbols as in B, illustrating the possible origin of one excision event. The nicking occurs as in B. but the element has not been released. The boxed-in sequences (e.g., GTT) represent TSDs. Thin, dashed half-circles represent the extent of exonuclease degradation. Polymerase action is illustrated by a thick. dashed line. The resulting sequences are different from the original. (After Saedler and Nevers, 1985, with permission.)

TRANSPOSABLE ELEMENTS IN MAIZE VARIABILITY

107

site duplication. The Mu element contributes upward of 200 bp. Thus in each insertion event, the nucleotide sequence in the gene at the site where the insertion takes place increases by that number of nucleotides-3, 8, or many more. At some time in the growth of the tissue, the excision of the element takes place. The excision process is not precise. It will be shown that in over 90% of the excisions of an element, the host site is different than it was before insertion. As the transposase acts to cut out the element (recall that the cut in the element is very precise since the new site of insertion has a complete, functional element that includes the 13-bp motif of the En system or 1 1-bp TIR motif in the Ac system), errors are made in the replication and ligation of the host site. The problem lies with the whole sequence of the host site. If the gene locus was left as it was before element insertion, the duplication would not be present. However, in most events the excision process is not precise, and this should be described. First, consider that the excision process involves a protein-DNA complex (Fig. 14B)in which the endonuclease enzymes, during the binding process, cut out the element leading to excision. As hypothesized by the studies of Frey et al. A

lxxxl -Target

site duplication TSD

11111111111111111111lllllllllllllllllllllll111111111111111111

B

GTC AA

ATA ATA AAC GCG

Figure 14. (A) Parts of a transposable element are identified. The xxx of the TSD (3 bp) and TIR (13 bp) are generalizations of nucleotide sequences. Specific bp numbers are given in Table 1. The functional part (of En) is shown in Fig. 6. (B)Diagram of the DNA-protein complex illustrating the TPNA binding and the TPND cutting event. This protein complex is described in Frey e t a / . (1990b). (After Peterson, 1986a. with permission.)

108

PETER A. PETERSON

(1990a,b), the transposase brings the termini of the element together (Figs. 5 and 14A), allowing the enzymes to commence excision. This is initiated by staggered nicks at the ends of the target-site duplication. As a consequence of these nicks, the 5' ends of the target-site duplication and the 5' end of the TIR are converted into complementary single-stranded fringes after repair synthesis (Fig. 13B). This is the reverse of the integration process. In integration, the mobile element also includes the same enzymatic complex (Fig. 14B) that has an affinity for the ends of the specific mobile element. The insertion event is accompanied by staggered nicks at the target site (Figs. 13B and C). The distance between the staggered nicks characterizes the TSD of each of the individual elements (see Table I). As the cut ends spread apart, the gaps are filled by repair synthesis, resulting in a duplication of the target site (Fig. 13C). It is possible that the templates are switching during repair synthesis (Fig. 13D). During the excision process, there is template switching combined with exonuclease degradation of the single-stranded nucleotides as shown in Fig. 13B. This template change leads to the alteration in the fidelity of the original sequence and the resulting replication of the template can lead to an excision product that is apparent. It is clear from these events that altered nucleotide sequences result from the excision process. Because these altered nucleotide sequences affect the reading frame of the gene, the alteration in the template will change the coding, resulting in altered proteins.

3. Footprints and Altered Products of Excision

,

If the excision process merely cut out the element leaving the target-site duplication intact, an examination of the nucleotide sequence of genes would uncover many instances of short duplicated segments such as ACT ACT in the En/ Spm system, or TACTAGGC TACTAGGC in the Ac system. The appearance of a series of these duplications would cause some interest. The role of transposable elements in contributing these duplicated segments would explain their presence. Yet the excision process is not wholly precise, and the footprints would be readily evident. If one makes a determined effort to isolate revertants and examines the junction site where the element left, changes in the sequence are found. This was done in several studies. In the first study, revertants were isolated from the Adhl locus (Sachs et al., 1983). Sachs et af. (1983) described the flanking sequences of the site of the Ds excision of four revertants and showed that although the duplicated sequences were retained, changes occurred at the junction of the duplicated segments (Fig. 15). In two revertants, RVI and RV2, the nucleotides show strand inversion. In two others, RV3 and RV4, the sequences showed 2-bp deletions in addition to the strand inversion. What surprised these investigators was that the qualitative level of gene action was not affected despite the addition of nucleo-

109

TRANSPOSABLE ELEMENTS IN MAIZE VARIABILITY

.

4

Element, gene and revertant

5' of element

Ds1 in Adhl-Fn335 TSD

S'GGGACTGA

RVl

GGGACTCTCGGACTGA

3' of element

<<

GGGACTGA 3'

References

Sachs e t ai.. 1 9 8 3

Dennis el a/.. 1 9 8 6

-1Sh-mti233 revertant C

I

CTTGTCC

Wildtype (Wxl

GTC AAG.TT

Spm-18 in Wx TSD

gtc aaG TT

I

I
Pohlman e t a/.. 1 9 8 4 .TTGTCCC

Weck er al.. 1 9 8 4

C AAC GCG

SchwarzSommer e t a/., 1985

G TTc aac gcg germinal revertants

wx- 1

gtc aaG TTA

. TTc aac gcg

wx-2

gtc aaG T.

CG TTc aac gcg

gtc aaG T.

ACG TTc aac gcg

arc aaG T T

G TTc aac Qca

wildtype *I AA chanaes

+ leu + ser

Somatic exc iions a1 in leaf DNA

I frameshift + leu

Figure 15. A compilation of revertants originating from different elements and at different loci identified. Boldface represents altered nucleotides; dots indicate missing nucleotides; capital letters represent the TSDs of different elements; lowercase letters refer to host sequences.

110

PETER A. PETERSON

tides relative to the progenitor sequence. In a later study with the same locus, Dennis et af. (1986) reported similar effects of excision, such as nucleotide substitutions (RV10, RV6) and nucleotide losses (RV31, RV43, RV46). There were 1-, 2-, or 3-bp alterations in the duplicated segments. It is noted that the assays used for determining the effects of protein differences might not have been adequate to detect slight differences. Schwarz-Sommer et al. (1985) found that most of the events were imprecise excisions leading to gene alterations at the junction of the excised IldSpm elements from the wx-m8 allele. They uncovered 16 revertants or revertant sectors and only 2 were of the precise sequences of wild type. The other 14 included assorted changes. Some were frameshift and, therefore, nonfunctional alleles. Others added amino acids to the proteins (leucine and serine) and others deleted amino acids. Excision of the Mu1 element generates deletions (Taylor and Walbot, 1985) with the excisions having 34-bp deletions or 5-bp additions (Britt and Walbot, 1991). Similar changes were reported in Antirrhinum with the excision of TAM1 (Martin et af., 1989; Sommer et al., 1988). Imprecise excisions are the key to the genetic variability question. Excisions out of gene sequences are a rich source of changes that have consequences on gene function and expression.

G. EFFECTSON GENEEXPRESSION 1. Phenotypic Changes It has been demonstrated that the footprints left by transposable element excisions lead to altered gene sequences, and it is appropriate to examine the kinds of changes that have been recognized at the phenotypic level. Some of the changes uncovered are novel and have not previously been seen. This is not unexpected because the excision event yields random kinds of changes. Phenotypes that have been uncovered include a wide range of color phenotypes of the aleurone color alleles, Wx-starch types that can be distinguishable by Ikl staining, and effects on the alcohol dehydrogenase gene. Changes have also been realized in the unstable Delphinium alleles originally described by Demerec (1935) and described molecularly with the Tam series in Antirrhinum (Sommer et al., 1988). Rhoades (1936, 1938) uncovered 29 full-colored mutations induced by Dt at the a1 -dr allele, and two different types could be distinguished among these. Of the 29, 27 were the standard A1 type, which was confirmed by the red-colored pericarp and would be considered identical to the original A1 allele. This is surprising because most result in different phenotypes. There is a bias, however,

TRANSPOSABLE ELEMENTS IN MAIZE VARIABILITY

111

because full-colored types were uncovered and others may not have been recognized. Further, definitive assays, such as sensitive enzyme tests, are possible with the now established A1 enzyme (Reddy et al., 1987). The remaining two exceptions of the 29 were interesting in their phenotype and what they showed. One of them gave a recessive brown pericarp color. That is, in the heterozygote Al-srlAl-br, the pericarp was red, but in the homozygote Al-brlAl-br, the pericarp was brown. This recessive brown pericarp of Al-br is in contrast to the dominant red of the A1 allele and dominant brown pericarp allele of Al-b. This particular colored derivative showing a recessive brown phenotype was a novel, previously undescribed allele, and it was designated Al-br. The second exception of the 29 full-colored exceptions was identified as A1 -rb; this was also a new allele. This allele produces a reddish-brown pericarp color in conjunction with the P allele, which is dominant to the brown of A1 but recessive to the red of A l . There was a nonfull color pale aleurone allele that was designated a1 -br. This gave a recessive brown pericarp color instead of the dominant brown produced by the previously isolated a1 -p allele originally described by Emerson and Anderson (1932). This range of phenotypes shown by the excisions out of al-dt illustrate the diverse expressions that can be uncovered with the Dt transposon. If the 27 fullcolored alleles were further examined in other kinds of tests, possibly other kinds of differences in phenotype would have been shown. They were never subjected to molecular analysis, and they could have been perfect excisions, which is possible because the a1 -dt studies have not included molecular analyses to any great extent. In McClintock’s last studies with the cl-ml and the cl-m2 alleles, numerous kinds of expressions from the mutability at those alleles were observed. What was readily seen was a wide range of quantitative expressions associated with pigmentation in the aleurone. These intermediate types were not full-color types and they were clearly distinguishable. The induced changes of the a1 -m allele yielded a large number of variable phenotypic types (Peterson, 1959, 1961). One can say that the Wx revertants from wx-ml were overproducers (McClintock, 1949). These examples illustrate a graded expression arising from these various alleles with transposon inserts that were transactivated by their regulatory elements. Again, the sequence changes caused several different forms, which provides a rich source of variability. 2. Enzyme and Molecular Changes

The first studies to examine possible enzymatic changes were with the Adh-1 gene. This was possible because this is one of the first genes isolated and available for biochemical analysis. Sachs et al. (1983) found reduced levels of mRNA

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PETER A. PETERSON

in Northern blots of the Ds-induced Adh-1 -Fn 335 allele when compared with either the progenitor allele or the revertant. These authors speculated that these mutants reduced the mRNA levels because of the position of the insert near the start of the transcription or because the processing of the dehydrogenase message may be affected by the insert leading to an inefficiency of the normal transcript. The analysis reported by Sachs et al. (1983) was supported by the Osterman and Schwartz (1981) studies. They showed that the enzyme produced by this allele was more thermolabile than that produced with the progenitor allele. The modified protein from this allele must be a consequence of the effect of the Ds insert (the 1.5- kb Ds insert near the 5' of the Adh-1 gene). Other studies of the Adh-1 gene showed that variation occurred when the particular tissues or pollen were subjected to diverse tests. Osterman and Schwartz (1981) demonstrated that the derivative alleles are definitely altered. Alternative forms are also seen in the starch studies with the Wx locus in maize. With the wx-rn allele in the presence and absence of En, differences can be seen at the mRNA level (Gierl et al., 1985). In the section on footprints, the various isolated wx derivatives with altered amino acid content were described (Schwarz-Sommer er al., 1985). These wx derivatives were never subjected to tests at the biochemical level, but one can readily see that such changes are possible and could be realized phenotypically. Two products after Ds excision at the wx locus were found to have a range of enzyme activities (Wessler et al., 1986). These changes showed lower levels of enzyme activity, indicating changes in amino acid content of the protein. Numerous changes have been isolated from transposon changes in the anthocyanin color series in maize. The case with Dt at the A1 locus has been discussed. Another example is the regulatory gene C1. In the absence of C l action, color is absent in the maize kernel. The structural genes require the CI product that binds to structural gene promoters, thus activating transcription leading to corn kernel anthocyanin coloration. A number of alleles of the C1 locus have been described (Paz-Ares et al., 1987, 1990; Goff er al., 1991). One allele, Cl-S, conditions a more abundant message (mRNA), and it expresses a stronger potency against the effect of the inhibitor C1-I. This is obvious from the observation that the Cl-S allele is less inhibited by the standard Cl-1. How then does the C1-S differ from the C l ? When one examines Box I1 of the promoter (Fig. 16), a GCA sequence is changed to a TAG sequence. The possibility that this alteration is caused by a transposon is noted by a typical 5-bp transposon-induced footprint in the vicinity of this 3-bp change in Box I1 (Fig. 16). This change in sequence at the C l locus promoter results in the overexpression of the C1 allele. Interestingly, this same sequence in Box I1 of the promoter is also present in the C1-I (standard) allele. This overexpression is very likely the basis for the dominance of C1-1 in the heterozygote C1-IICl. The reason for this

TRANSPOSABLE ELEMENTS IN MAIZE VARIABILITY

........ .....

R BOX II

113

..... ..... .....

Cl lTfAGG TGCAG TGCAGG C1-I......lTfAGG lTAGG TGCAGG C1-S lTfAGG lTAGG TGCAGG

Figure 16. Box I1 in the promoter of the CI locus of three different alleles.

observation is that Cl-1 competes favorably with the C1 product for activation of transcripts of the structural genes in competitively binding to promoters of structural genes. The phenotype is therefore dominant colorless in the heterozygote, indicating the dominance of the Cl-1 allele. Because the truncated C1-I protein lacks a basic domain, transcription cannot proceed in the continuation of the transcript of the structural gene. Yet the transactivation site, either at A1 or at C2, is occupied and, therefore, excludes the C1 product from proper transcript activation. So, how does a transposon explain these differences? It is very likely, in view of the associated footprints, that a transposon-inducedchange caused the 3-bp promoter change that results in a more productive transcript. Further, the change occurred in a promoter of a regulatory gene that is at the beginning of the coloration development process. And this anthocyanin regulator and the downstream structural genes represent a model of the hierarchy of gene activity leading to most gene products, especially those governing quantitative traits. Another case of transposon-induced alteration in the promoter region was reported by Sommer ef al. (1988). These investigators found four solid-color revertants from the highly variegated niv-53 ::Taml mutant in which the Tam1 transposable element is integrated in the promoter region of the chalcone synthase gene. In four derived mutant lines, the Taml element was deleted together with the flanking nucleotides of the chalcone synthase promoter. In one case, the TATA box of the chalcone gene was removed, resulting in an extremely low expression of the gene, and causing gene transcription to initiate at a new position. Among the revertants, the amounts of chalcone synthase mRNA varied (with 100 being the wild type) from 107 to 2. Two of the mutants niv-531 (107) and niv-164 (103) were overexpressive in terms of the wild type. The other four were 2, 25, 35, and 26 (percentage chalcone synthase mRNA) for niv-5311, niv-5312, niv-5313, and niv-5314, respectively. Not only were the effects of the promoter change seen, but the analysis allowed Sommer el al. (1988) to explore the promoter parts necessary for proper promoter activity. The analysis of the Cl gene provides further information following a computer analysis of protein efficiency. Analysis of the various transposons randomly inserted at C l indicates that a broad diversity of available allelic possibilities can be generated. These are generated by transposable element excision products. In a study of a number of sites of the C l gene that had inserts, Franken ef al. (1992), identified sites with effects on protein performance. At these sites, each

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PETER A. PETERSON

excision illustrated the kind of phenotypic effect that could be generated by this excision. This probably affected the three-dimensional formation of proteins and, consequently, their functional activation and efficiency (Branden and Tooze, 1991). It is evident in this brief survey of transposons that the effects of the excisions of transposons from genes could be on proteins and on the potency of expression of promoters. Though only two promoters were analyzed, it is clear that when other transposons are examined, similar changes will be seen.

H. PRESENCE IN THE MAIZEGENOME In view of the demonstration of transposon effects in the genome, could they be prevalent enough to cause these changes? First, it was emphasized that their presence in the genome could only be observed if there is a reporter allele to register its presence. This was tested by crossing a number of the reporter alleles to various cycles of the Iowa stiff stalk synthetic (BSSS) of maize. The results of these tests made evident the pervasiveness of the U q elements throughout various cycles of the improved BSSS populations (Peterson, 1986a; Peterson and Salamini, 1986; Cormack et al., 1988). Because 15 of the lines used to formulate BSSS were recovered (Stucker and Hallauer, 1992), these were examined, and la159 was found to have the Uq element (Cormack et al., 1988). The Uq element has persisted in BSSS during the course of improving this maize population. U q has been maintained since the initiation of this breeding population because BSSS is a closed population, meaning that no new germplasm has been introduced. Lamkey et al. (1991) examined this material for active Uq in 5 of the 11 completed cycles that have been of reciprocal recurrent selection [BSSS(R)(1I)]. In a parallel study, these same authors examined 10 of the 13 completed cycles of the half-sib and S2 progeny selection [BSSS(S)(l3)] for active Uq. Uq increased from 19% (BSSS) to 91% [BSSS(S)(l3)] at a linear rate after 13 cycles of half-sib and S2 progeny recurrent selection. This increase occurred without any cognizance of Uq because primary emphasis was on the yield performance of the advancing populations. This was not the case for the other population. Active Uq decreased from 15% (BSSS) to 0% [BSSS(R)(Cl l)] after 11 cycles of reciprocal recurrent selection. The extinction of the Uq element occurred between the fifth and sixth cycles of selection. Lamkey et al. (1991) concluded that random genetic drift played a key role in the increase and decrease of Uq in the two populations. A selective advantage of a region linked to Uq was also considered, and such a region, if it existed, would be a “bonanza” for plant breeders. How active is a transposon such as Uq in a breeding population? Uq is a

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modified form of a nonactive Ac (Pisabarro er al., 1991). Whereas Ac can excise all Ds’s, Uq is limited to action only on Dsl (Caldwell and Peterson, 1992). When a reporter allele is crossed to various maize lines, spontaneous activation is observed in the form of sectors (Pan and Peterson, 1988). That these are quiescent U q that are spontaneously activated was verified by the isolation of spontaneous germinal originated Uq’s (Pan and Peterson, 1991a). In a further examination, these Uq’s were found to be clustered in a linkage group (Pan and Peterson, 1991b). One of these clustered Uq’s was later confirmed to be an Ac (P. A. Peterson, unpublished). Whether this Ac was a change from a previous Uq is not known, but given the relation between the two, this is probable. The evidence is plentiful that the Uq element is pervasive. Mrh is also pervasive in a number of maize breeding lines, even more so than Uq (Peterson and Salamini, 1986; Peterson. 1988). One significant feature relative to transposable element activity is that observations are confined to elements that have been genetically identified. Uq escaped detection until Sprague and McKinney (1966) began experiments with virus infection of maize that later led to the genetic isolation and identification of Uq (Friedemann and Peterson, 1982). The Mrh element that is also pervasive was not isolated and identified until 1982 (Rhoades and Dempsey, 1982). Other elements, whether they are or are not transposing, is not known, yet they are likely present in the genome. This includes the Cin elements that are present in a high frequency and are highly heterogeneous (Shepherd et al., 1982, 1984). Transposable element inserts are also prevalent in the maize progenitor, teosinte. Although their transposition activity has not been recorded in teosinte, these elements do have the characteristics of transposable elements (Blumberg et al., 1990; Shepherd et al., 1983).

I. NEOTENY AND REALIGNMENT OF RESOURCES FOR MORE EFFICIENT NETWORKS The plant breeder is well aware of the complexity of the properties associated with yield in plants. This should not be a surprise. From the perusal of any textbook on plant biochemistry (e.g., Goodwin and Mercer, 1988), it becomes evident that there are many processes involved in plant metabolism and growth. Further, pathways are often in parallel, leading from common substrates. This would suggest that a breeder is selecting the most efficient pathway that meets the needs of the program. In a discussion on quantitative inheritance, Peterson (1992) suggested that a plant breeder is selecting different “cassettes” and that these cassettes are most efficiently used as a unit by the plant. Though tropical and temperate maize have an overlap in metabolic usage, the “fine-tuning” of

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PETER A. PETERSON

each of the maize lines is constrained by the cassettes that best support the growth potential of individual plant types. There is a further point. There must be a certain amount of “downgrading” in plant breeding improvement. The large tassel and robust leaves are not needed in most maize-growing environments. Genes must be bridled or reduced in activity in this downgrading process. Although nucleotide alterations induced by transposons can lead to improved protein performance, many of the changes lead to null alleles. In the wx-m8 case reported by Schwarz-Sommer el al. (1985), most of the derivative products following excision were altered or null products. By this process, altered pathways could develop that lead to more effective “networking” of limited resources. This should lead to a better performance among the selected lines of a breeding population.

J. SUMMARY OF EFFECTS Conclusive proof of the role of transposable elements in the induction of significant variability could come from the isolation of genes known to advance the performance of populations. Because these quantitatively important genes are evasive, this form of proof is remote. Yet all genes probably contribute to performance although conclusive proof on this statement is not readily forthcoming. Given that genes are proven to be altered by transposons, it is likely that the contribution of transposons to altered genes leading to altered proteins and, subsequently, variability is a clear possibility. As more genes are uncovered and maize research gets closer to isolating quantitative trait loci (Lander and Bodstein, 1989), it is hopeful that a clearer picture will emerge on the role of transposons in maize populations.

DEFINITIONS OF TERMS AND SYMBOLS Controlling elements Controlling element allele Controlling element systems Controlled allele

A collective term for transposable elements associated with mutable alleles that affect gene activity. An allele with an inserted controlling element at the locus. Includes two units, receptor and regulatory elements, that express a specific interaction (see Fig. 4C). An allele under the control of a controlling element system; includes an inserted receptor or regulatory element.

TRANSPOSABLE ELEMENTS IN MAIZE VARIABILITY

Receptor elements (rcp) Regulatory elements (Rg)

DS Z 1-102

a2-ml a-ml

a' a-m(r)

117

Elements such as dt, Ds,and I that suppress gene activity when in cis position to the locus. Elements such as Dt, Ac, F a , and En that alter or excise receptor elements so that the locus under control becomes functional. Dissociation; a receptor element. In combination with Ac, some states result in chromosome breakage at the site of Ds (McClintock, 1951). Inhibitor; a receptor element of the En system (Peterson, 1960). The insert, specific to the al-m(r) allele. A receptor allele at A2. Without En/Spm, it is full colored; with En/Spm, it is colorless with sectors of color from an inactivating En. (al-ml 5719 contains a 789-bp insert, located between the last two nucleotides of the second exon of the A1 gene. The gene expression of this allele is hypothesized to result from the presence of an intron donor that if used in mRNA splicing, permits formation of a modified exon.) This allele is a derivative of al-ml 6078. It is a recessive allele of A1 and responds to En. In the absence of En, the aleurone is pigmented pale; in its presence, colored spots are produced on a colorless background (Peterson, 1965). A neutral nonresponding allele. al-ml 5719; recessive allele of A and responds to En. In the absence of En, the aleurone is colorless; in its presence, colored spots are produced on a colorless background expressing the change of a1 to A 1 (Peterson, 1961).

Ac

Activator; a regulatory element (that triggers changes at

En

Enhancer; a regulatory element (that triggers changes a t UdSpm). Suppressor function of En/Spm. Can turn on or turn off activity of controlled loci; can be assayed only with certain responding alleles. Mutator function of En/Spm resulting in spots on kernels.

S

M

Ds).

118

Med

FCU

MP

Pr Pr

P”

PETER A. PETERSON

Mediator; required along with En to activate the c2-rn881058Y allele. Associated with a three-element system (Muszynski and Peterson, 1990). Factor Cuna; a regulatory element of the Feu system (Gonella and Peterson, 1977). Modulator; a transposable element that can behave as a regulatory element with Ds (Brink and Nilan, 1952). Purple aleurone with all color genes. Red aleurone with all color genes. The mutable pericarp locus. Associate with Mp in a cis condition. Transposability of Mp is expressed as changes from colorless to colored pericarp (Brink and Nilan, 1952).

wx844:En1 wx W X

wx wx

En at the wx locus. This allele gives purple starch with KI staining. The phenotype of this allele. This allele gives red or brown starch with KI staining. The phenotype of this allele.

REFERENCES Athma, P., Grotewold, E., and Peterson, T. (1992). Insertional mutagenesis of the maize P gene by intragenic transposition of Ac. Generics 131, 199-209. Avery, 0. T., Macleod, M., and McCarty, M. (1944). Studies on the chemical nature of the substance inducing transformation of pneumococcal types; Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type 111. J . Exp. Med. 79, 137-158. Beadle, G. W. (1932). A gene for sticky chromosomes in Zea mays. Z . Indukr. Absramm. Vererbungsl. 63, 195-217. Bhattacharyya, M. K.. Smith, A. M., Ellis, T. H. N., Hedley, C., and Martin, C. (1990). The wrinkled-seed character of pea described by Mendel is caused by a transposon-like insertion in a gene encoding starch-branchingenzyme. Cell (Cambridge, Mass. j 60, 115- 122. Blumberg vel Spalve, J., Schwarz-Sommer, Zs., Saedler, H., and Peterson, P. A. (1990). The Cin2 and the Cin3 insertion elements of Zea mays ssp. parviglumis might serve as markers to monitor the evolution of Zea mays. Maydica 35, 151- 156. Branden, C., and Tooze, J. (1991). Introduction to protein structure. In “Molecular Biology in Three Dimensions,” p. 302. Garland, New York. Bridges, C. B. (1936). The bar ‘gene’ a duplication. Science 83,210-21 1. Brink, R. A,, and Nilan, R. A. (1952). The relation between light variegated and medium variegated pericarp in maize. Generics 37, 519-544.

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Britt, A. B., and Wdlbot, V. (1991). Germinal and somatic products of Mu1 excision from the Bronze-] gene of Zea mays. Mol. Gen. Genet. 227, 267-276. Caldwell, E. E. 0.. and Peterson, P. A. (1992). The Ac and U9 transposable element systems in maize: Interactions among components. Genetics 131, 723-731. Castleberry, R. M., Crum. C. W., and Krull, C. F. (1984). Genetic yield improvement of U.S. maize cultivars under varying fertility and climatic environments. Crop Sci. 24, 33-36. Chang, R. Y . , and Peterson, P. A. (1993). Chromosome labeling with transposable elements in maize. Accepted for publication. Chen. J., Greenblatt. 1. M., and Dellaporta, S. L. (1987). Transposition of Ac from the P locus of maize into unreplicated chromosomal sites. Genetics 117, 109- 116. Chomet, P., Lisch, D., Hardeman, K. J., Chandler, V. L., and Freeling, M. (1991). Identification of a regulatory transposon that controls the mutator transposable element system in maize. Generics 129, 261 -270. Cormack, J. €3.. Cox, D. F., and Peterson, P. A. (1988). Presence of the transposable element 179 in maize breeding material. Crop Sci. 28, 941 -944. Darwin, C. (1868). “The Variation of Animals and Plants under Domestication,” Vol. I . John Murray, London. Dash, S., and Peterson. P. A. (1989). Chromosome constructs for transposon tagging of desirable genes in different parts of the maize genome. Maydica 34, 247-261. Dash, S.. and Peterson. P. A. (1993). Frequent loss of the En transposable element following excision and its relation to chromosome replication. Genetics (submitted for publication). Demerec, M. (1935). Unstable genes. Bot. Rev. 78,233-248. Demerec. M. (1946). Annual report of the Director of the Department of Genetics. Year BookCarnegie Inst. Washington 45, 139- 192. Demerec, M. (1947). Annual report of the Director of the Department of Genetics. Year BookCartiegie Inst. Washington 46, 123- 170. Dennis, E. S . , Gerlach. W. L., and Peacock, W. J. (1986). Excision of the Ds controlling element from the Adhl gene of maize. Maydica 31,47-57. Duvick, D. N. (1977). Genetic rates of gain in hybrid maize yields during the past 40 years. Maydica 22, 187-196. Duvick, D. N. (1984). Progress in conventional plant breeding In “Gene Manipulation in Plant Breeding” (J. P. Gustafson, ed.), pp. 17-31. Plenum, New York. Duvick, D. N. (1986). Genetic contributions to yield gains of U.S. hybrid maize, 1930 to 1980. In “Genetic Contributions to Yield Gains of Five Major Crop Plants” (W. Fehr, ed.), CSSA Spec. Publ. No. 7, pp. 15-47. Crop Sci. Soc.Am., Madison, WI. Duvick. D. N. (1991). Genetic contributions to advances in yield of US. maize. Maydica 37, 69-79. El-Lakanyu, M. A., and Russell, W. A. (1971). Relationship of maize characters with yield in testcrosses of inbreds at different plant densities. Crop Sci. 11,698-701. Emerson, R. A. (1914). The inheritance of a recurring somatic variation in variegated ears of maize. Am. Nat. 48,87- 115. Emerson, R. A. (1918). A fifth pair of factors, Aa, for aleurone colour in maize and its relation to the Cc and Rr pairs. Mem.-N.Y. Agric. Exp. Stan. (Irhaca) 16, 225-289. Emerson, R. A. (1929).The frequency of somatic mutation in variegated pericarp of maize. Genetics 14,488-51 1 . Emerson, R. A., and Anderson, E. G.(1932). The A series of allelomorphs in relation to pigmentation in maize. Genetics 17, 503-509. Fedoroff, N., Wessler. S . , and Shure, M. (1983). Isolation of the transposable maize controlling elements Ac and Ds. Cell (Cambridge. Mass.) 3S, 235-242.

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