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10 Transposons and Genome Evolution in Plants NINA FEDOROFF Although it is known today that transposons comprise a signifi- cant fraction of the genomes of many organisms, they eluded discovery through the first half century of genetic analysis and even once discovered, their ubiquity and abundance were not recognized for some time. This genetic invisibility of transposons focuses attention on the mechanisms that control not only trans- position, but illegitimate recombination. The thesis is developed that the mechanisms that control transposition are a reflection of the more general capacity of eukaryotic organisms to detect, mark, and retain duplicated DNA through repressive chromatin structures. T he 50 years that have elapsed since the publication of Stebbinsâ Variation and Evolution in Plants have seen extraordinary changes in our understanding of how genomes are structured and how they change in evolution. The bookâs publication date roughly coincides with the first reports by Barbara McClintock that there are genetic The Pennsylvania State University, University Park, PA 16802 This paper was presented at the National Academy of Sciences colloquium âVariation and Evolution in Plants and Microorganisms: Toward a New Synthesis 50 Years After Stebbins,â held January 27â29, 2000, at the Arnold and Mabel Beckman Center in Irvine, CA. Abbreviation: LTR, long terminal repeat. 167
168 / Nina Fedoroff elements capable of transposing to different chromosomal locations in maize plants (McClintock, 1945). The book contains a brief mention of Marcus Rhoadesâ observation that a standard recessive a1 allele of a gene in the anthocyanin biosynthetic pathway can become unstable and revert at a high frequency to the dominant A1 allele in a background containing a dominant Dt (âdottedâ) allele (Stebbins Jr., 1950). But transposable ele- ments were not yet common fare, nor was it known that Dt is a transposon. Today we know that transposons constitute a large fractionâeven a majorityâof the DNA in some species of plants and animals, among them mice, humans, and such agriculturally important plants as corn and wheat. Given what we now know about genome organization, it is para- doxical that the discovery of transposable elements lagged so far behind the discovery of the basic laws of genetic transmission. And it is equally curious that even when they were discovered, acceptance of their general- ity and recognition of their ubiquity came so slowly. It is perhaps an understatement to say that McClintockâs early communications describ- ing transposition were not widely hailed for their explanatory power. Indeed, McClintock commented in the introduction to her collected pa- pers that the response to her first effort in 1950 to communicate her dis- covery of transposition in â. . . a journal with wide readership . . . ,â specifically the Proceedings of the National Academy of Sciences, convinced her that â. . . the presented thesis, and evidence for it, could not be ac- cepted by the majority of geneticists or by other biologistsâ (McClintock, 1987). By contrast, the explanatory power of Watson and Crickâs 1953 Nature paper on the structure and mode of replication of nucleic acids was recognized immediately (Watson and Crick, 1953). An informative parallel is provided by the contrast between the imme- diate recognition of the importance of Darwinâs theory of evolution and the long delay between Mendelâs articulation of the laws of heredity and their wide acceptance in evolutionary thinking (Carlson, 1966). It can be speculated that this was because Darwinâs theory provided immediate explanations in the realm of the perceptible, whereas the hereditary mechanisms under- lying variation were obscure. Variation, in Darwinâs view, was continuous. Geneticists sharing his view formed the âbiometrical school,â devoted to the statistical analysis of inheritance. It was not at all clear how the simple rules derived by Mendel for the hereditary behavior of âdifferentiating charactersâ bore on the problem of evolution (Carlson, 1966). The relevance of discontinuous variation or the production of âsports,â as morphological mutations were called, was even less obvious, because the biometric ap- proach treated offspring as statistical combinations of parental traits. Thus the idea that the study of mutations was central to understanding evolution was close to unimaginable a century ago.
Transposons and Genome Evolution in Plants / 169 Equally unimaginable at mid-20th century was the idea that trans- posable elements are essential to understanding chromosome structure and evolution, much less organismal evolution. The efforts of Bateson and other geneticists had firmly established Mendelian âlawsâ as the cen- tral paradigm of genetics and the identification and mapping of genetic âlociâ through the study of mutant alleles was proceeding apace. Because genetic mapping is predicated on the invariance of recombination fre- quencies, there was plentiful evidence that genes have fixed chromo- somal locations. Written at this time, Stebbinsâ book in general and in particular his third chapter, titled âThe Basis of Individual Variation,â clearly acknowledges the existence of many chromosomal differences among organisms in a population, including duplications, inversions, translocations, and deletions. At the same time, the book reflects the pre- vailing view that these â. . . are not the materials that selection uses to fashion the diverse kinds of organisms which are the products of evolu- tionâ (Stebbins, Jr., 1950). Instead, Stebbins concludes that the majority of evolutionarily important changes in physiology and morphology are at- tributable to classical genetic âpointâ mutations. Another half century has elapsed and the geneticistâs âblack box,â sprung open, spills nucleotide sequences at an ever accelerating pace. Our computers sift through genomes in search of genes, knee-deep in transposons. How could we not have seen them before? The answer is as straightforward as it is mysterious and worthy of consideration: they are invisible to the geneticist. Well, almost invisible. And of course it depends on the geneticist. THE DISCOVERY OF TRANSPOSITION The study of unstable mutations that cause variegation dates back to De Vries, who formulated the concept of âever-sporting varietiesâ and eventually came to the conclusion that these types of mutations do not obey Mendelâs rules (de Vries, 1905). The first person to make sub- stantial sense of their inheritance was the maize geneticist Emerson, who analyzed a variegating allele of the maize P locus during the first decades of this century (Emerson, 1914, 1917, 1929). His first paper on the subject opens with the statement that variegation â. . . is distin- guished from other color patterns by its incorrigible irregularityâ (Emer- son, 1914). What follows is a brilliant analysis of âfreak earsâ containing large sectors in which the unstable P allele has either further mutated or reverted. Emerson was able to capture the behavior of unstable muta- tions in the Mendelian paradigm by postulating that variegation com- menced with the temporary association of some type of inhibitor with a
170 / Nina Fedoroff locus required for pigmentation. Emersonâs suggestion was that normal pigmentation was restored upon loss of the inhibitor. Several prominent geneticists, among them Correns and Goldschmidt, dismissed unstable mutations as a special category of âdiseased genesâ (Goldschmidt, 1938; Fedoroff, 1998). It was their view that little could be learned from the study of such mutations that was relevant to the study of conventional genes. But the drosophilist Demerec and the maize ge- neticist Rhoades shared Emersonâs view that there was no difference in principle between stable and unstable mutations. Indeed, the Rhoades mutation cited in Stebbinâs volume illustrates the important point that instability is conditional. Rhoadesâ experiments had revealed that a stan- dard recessive allele of the maize A1 locus, isolated decades earlier and in wide use as a stable null allele, could become unstable in a different genetic background. The key ingredient of the destabilizing background was the presence of the Dt locus, which caused reversion of the a1 allele to wild type both somatically and germinally (Rhoades, 1936, 1938). In the late 1930s, McClintock had begun to work with broken chro- mosomes and by the early 1940s she had devised a method for producing deletion mutations commencing with parental plants, each of which con- tributed a broken chromosome 9 lacking a terminal segment and the telomere. Searching for mutants in the progeny of such crosses, she ob- served a high frequency of variegating mutants of all kinds (McClintock, 1946). She noted that although reports of the appearance of new mutable genes were relatively rare in the maize literature, she already had isolated 14 new cases of such instability and observed more. She chose to follow the behavior of a locus, which she called Dissociation (Ds), for its propen- sity to cause the dissociation of the short arm of chromosome 9 at a posi- tion close to the centromere, although she soon appreciated that chromo- some dissociation required the presence of another unlinked locus, which she designated the Activator (Ac) locus (McClintock, 1946, 1947). By 1948, she had gained sufficient confidence that the Ds locus moves to report: âIt is now known that the Ds locus may change its position in the chromosomeâ (McClintock, 1948; Fedoroff, 1998). The relationship between the chromosome-breaking Ds locus and variegation emerged as McClintock analyzed the progeny of a new variegating mutation of the C locus required for kernel pigmentation. She carried out an extraordinary series of painstakingly detailed cytological and genetic experiments on this new mutation, c-m1, whose instability was conditional and depended on the presence of the Ac locus (McClintock, 1948, 1949; Fedoroff, 1998). She showed that the origin of the unstable mutation coincided with the transposition of Ds from its original position near the centromere on chro- mosome 9 to a new site at the C locus and that when it reverted to a stable wild-type allele, Ds disappeared from the locus. Having established that
Transposons and Genome Evolution in Plants / 171 Ds could transpose into and out of the C locus germinally, she inferred that somatic variegation reflects the frequent transposition of Ds during development. Transposition explained both Emersonâs and Rhoadesâ ear- lier observations. McClintock and Rhoades were good friends, of course, and it is evident from their correspondence that McClintock immediately saw the parallels between the behavior of the c-m1 mutation and Rhoadesâ a1 mutation (Lee Kass, personal communication). The Ac and Ds elements are transposition-competent and transposi- tion-defective members of a single transposon family. In the ensuing years, McClintock identified and studied a second transposon family, called Suppressor-mutator (Spm) (McClintock, 1951, 1954). Her studies on these element families were purely genetic, and she was able to make extraordinary progress in understanding the transposition mechanism because she studied the interactions between a single transposition-com- petent element and one or a small number of genes with insertions of cognate transposition-defective elements (Fedoroff, 1989). Two points about this early history of transposition merit emphasis. First, the active elements were denumerable and manageable as genetic entities, despite their propensity to move. Second, the number of different transposon families and family members uncovered genetically was (and still is) small. Hence the genetic impact of transposable elements was limited. McClintock recognized that the high frequency of new variegating muta- tions in her cultures was linked to the genetic perturbations associated with the presence of broken chromosomes (McClintock, 1946, 1978). Her inference, extraordinarily prescient, was that transposons are regular in- habitants of the genome, but genetically silent. PLANT TRANSPOSONS IN THE AGE OF GENOMICS With the cloning of the maize transposons, first the Ac element in my laboratory and later the cognate En and Spm elements in Heinz Saedlerâs and my laboratories, the picture began to change (Fedoroff et al., 1983; Pereira et al., 1985; Masson et al., 1987). To begin with, it became obvious immediately that the maize genome contains more copies of a given trans- poson than there are genetically identifiable elements. Although most of these sequences are not complete transposons, there are nonetheless more complete transposons than can be perceived genetically (Fedoroff et al., 1984). Importantly, it was clear almost immediately that a genetically active transposon could be distinguished from one that was genetically silent by its methylation pattern (Fedoroff et al., 1984; Banks and Fedoroff, 1989). Both of these observations bear on the genetic visibility of transposons. As maize genes and genome segments began to be cloned and se- quenced, the discovery of new transposons accelerated. Although the
172 / Nina Fedoroff transposons that McClintock identified and studied were DNA trans- posons, both gypsy-like and copia-like retrotransposons were soon identi- fied in the maize genome and subsequently in many other plant genomes (Shepherd et al., 1984; Flavell, 1992; Purugganan and Wessler, 1994; White et al., 1994; Suoniemi et al., 1997, 1998). It has also become evident that non-long terminal repeat (LTR) retrotransposons are abundant in maize, as well as other plant genomes (Schwarz-Sommer et al., 1987; Noma et al., 1999). Many additional maize transposon families have been identified through their sequence organization and their presence in or near genes (Spell et al., 1988; Bureau and Wessler, 1992, 1994a, b; Wessler et al., 1995; Bureau et al., 1996). We now know that transposons and retrotransposons comprise half or more of the maize genome (Bennetzen et al., 1998). WHAT DO TRANSPOSONS DO? Commencing with McClintockâs elegant analyses of transposon-asso- ciated chromosomal rearrangements and extending into the literature of today, the range of transposon-associated genetic changes has continued to expand (McClintock, 1951). Insertion of plant transposons, like almost all known transposons, is accompanied by the duplication of a short flank- ing sequence of a few base pairs (Schwarz-Sommer et al., 1985a). Plant transposons excise imprecisely, generally leaving part of the duplication at the former insertion site (Schwarz-Sommer et al., 1985a). The conse- quences of insertion and excision of a transposon therefore depend on the location within the coding sequence and excision of an insertion from an exon commonly results in either an altered gene product or a frame-shift mutation. Transposon insertions can alter transcription and transcript processing, and there are cases in which transposons are processed out of transcripts by virtue of the presence of splice donor and acceptor se- quences (Kim et al., 1987; Wessler, 1989; Giroux et al., 1994). Transposons also can promote the movement of large segments of DNA either by transposition or by illegitimate recombination (Courage-Tebbe et al., 1983; Schwartz et al., 1998). THE PARADOX One might think that given their abundance, transposable elements would rapidly randomize genome order. Yet the results of a decade of comparative plant genome studies has revealed that gene order is sur- prisingly conserved between species. Close relationships among genomes have been demonstrated in crop plants belonging to the Solanaceae, and the Graminae, between Brassica crops and Arabidopsis, among several le- gumes, and others (Tanksley et al., 1992; Gale and Devos, 1998; Lager-
Transposons and Genome Evolution in Plants / 173 crantz, 1998). The synteny among the genomes of economically important cereal grasses is so extensive that they are now represented by concentric circular maps (Gale and Devos, 1998). There are rearrangements, but a relatively small number of major inversions and transpositions is required to harmonize the present day maps. Such maps, of course, are crude representations of the genome, and rearrangements can emerge as the level of resolution increases (Tanksley et al., 1988, 1992; Tikhonov et al., 1999). The frequency of rearrangements also can differ markedly and there is evidence that rearrangements are more prevalent just after poly- ploidization (Song et al., 1995; Gale and Devos, 1998). Even within a con- servative lineage, however, some gene families are more heterogeneous in composition and map distribution than others (Leister et al., 1998). SYNTENY AND DIVERGENCE What are the useful generalizations? First, synteny can extend down to a very fine level, but it is far from perfect. A detailed sequence compari- son of the small region around the maize and sorghum Adh1 loci reveals a surprising amount of change in a constant framework (Tikhonov et al., 1999). The sorghum and maize genomes are 750 and 2,500 Mbp, respec- tively. The Adh1 gene sequences are highly conserved, and complete se- quencing revealed that there were seven and 10 additional genes in the homologous regions of maize and sorghum, respectively. The region of homology extends over about 65 kb of the sorghum genome, but occupies more than 200 kb in the maize genome. The gene order and orientation are conserved, although three of the genes found in the sorghum Adh1 region are not in the maize Adh1 region. The genes are located elsewhere in the maize genome, suggesting that they transposed away from the Adh1 region (Tikhonov et al., 1999). Although homology is confined largely to genes, there are also homologous intergenic regions. There are simple sequence repeats and small transposons, called MITES as a group, scattered throughout this region in both sorghum and maize. MITES are found primarily between genes, but several are in introns. The small MITE transposons are found neither in exons nor in retrotransposons. There are three non-LTR retrotransposons in the maize Adh1 region and none in the sorghum Adh1 region (Tikhonov et al., 1999). The major difference between the maize and sorghum Adh regions is the presence of very large continuous blocks of retrotransposons in maize that are not present in sorghum. Although most blocks are between genes, one appears to be inside a gene sequence. They are present in many, but not all intergenic regions. There is a relatively long stretch of almost 40 kb containing four genes in maize and seven genes in sorghum, which con- tains no retrotransposon blocks in maize and in which there is about 10 kb
174 / Nina Fedoroff of extensive homology, some genic and some intergenic. Thus synteny extends down to a relatively fine level and includes both genic and inter- genic sequences. PLANT GENOMES EXPAND A second generalization is that plant genomes grow. Genome sizes among flowering plants vary dramatically over almost 3 orders of magni- tude, from the roughly 130 Mbp Arabidopsis genome to the 110,000 Mbp Fritillaria assyriaca genome (Bennett et al., 1982). Genome size variation greatly exceeds estimates of differences in gene numbers (Bennetzen and Freeling, 1997). This, of course, is the celebrated C-value paradox (Tho- mas, 1971). Plant genomes expand by several mechanisms, including polyploidization, transposition, and duplication. Thus, for example, a fine- scale comparison of the Arabidopsis thaliana and Brassica nigra genomes reveals that the Brassica genome contains a triplication of the much smaller Arabidopsis genome, as well as chromosome fusions and rearrangements (Lagercrantz, 1998). There is evidence that the maize genome is a segmen- tal allotetraploid (White and Doebley, 1998). It is estimated that up to 70% of flowering plants have polyploidy in their lineages (Leitch and Bennett, 1997). Thus replication of whole genomes or parts of genomes is a com- mon and important theme in plant genome evolution. TRANSPOSITION Transposition is also a major cause of plant genome expansion. To begin with, transposition generates DNA. Retrotransposition results from transcription of genomic retrotransposons, followed by insertion of reverse transcripts into the genome at new sites (Howe and Berg, 1989). Plant transposons generate additional copies of themselves by virtue of excising from only one of two newly replicated sister chroma- tids and reinserting into as yet unreplicated sites (Fedoroff, 1989). Ab- sent countering forces, genome expansion is an inevitable consequence of the properties of transposable elements. The accumulation of retro- transposon blocks between genes is a major factor in the size difference between the maize genome and those of its smaller relatives (SanMiguel et al., 1996, 1998). Retrotransposon blocks occupy 74% of the recently sequenced 240-kb maize Adh region (Tikhonov et al., 1999). These blocks contain 23 members of 11 different retrotransposon families, primarily as complete retrotransposons, but also occasionally as solo LTRs (Tikho- nov et al., 1999). Within these blocks, retrotransposons are commonly nested by insertion of retrotransposons into each other (SanMiguel et al., 1996, 1998).
Transposons and Genome Evolution in Plants / 175 What is perhaps most surprising about the maize retrotransposon blocks that have been characterized is that they grow quite slowly. The transposi- tion mechanism assures that retrotransposon ends are almost always identi- cal when an element inserts, hence the divergence between the LTRs of a single element reflects the age of the insertion. Bennetzen and his colleagues found that the sequence difference between the LTRs of a given element is almost invariably less than the sequence difference between the LTRs of the element into which it is inserted. Using these differences to order and date the insertions, they inferred that all of the insertions have occurred within roughly the last 5 million years, well after the divergence of maize and sor- ghum (SanMiguel et al., 1998). Importantly, no retrotransposons have been found in the corresponding Adh1 flanking sequence in sorghum (SanMiguel et al., 1998; Tikhonov et al., 1999). This raises the possibility that retrotrans- poson activity may differ between closely related lineages. AMPLIFICATION AND REARRANGEMENT New copies of transposons and retrotransposons provide new sites of homology for unequal crossing over. Evidence that transposable elements are central to the evolutionary restructuring of genomes has accumulated in every organism for which sufficient sequence data exist. Exceptionally detailed examples of the role of transposition, retrotransposition, amplifi- cation, and transposon-mediated rearrangements in the evolution of a contemporary chromosome are provided by recent studies on the human Y chromosome (Saxena et al., 1996; Schwartz et al., 1998; Lahn and Page, 1999a, b). Although the level of resolution is not yet sufficient in many cases to determine the molecular history of each duplication, it is evident that many, if not a majority of plant genes belong to gene families ranging in size from a few members to hundreds (Michelmore and Meyers, 1998; Riechmann and Meyerowitz, 1998; Martienssen and Irish, 1999; Rabino- wicz et al., 1999). R genes, for example, comprise a superfamily of similar myc-homologous, helixâloopâhelix transcriptional activators of genes in anthocyanin biosynthesis (Ludwig et al., 1989; Perrot and Cone, 1989; Consonni et al., 1993). Detailed analysis of the R-r complex, a well-studied member of the R superfamily, reveals a history of transposon-catalyzed rearrangement and duplication (Walker et al., 1995). There also may be other genetic mechanisms that drive genome expan- sion. A recent analysis of the behavior of maize chromosomal knobs reveals that the pattern of segregation under the influence of a âmeiotic driveâ locus of as yet unknown function results in the preferential transmission of chromosomes with larger knobs over chromosomes with smaller knobs (Buckler et al., 1999). Maize knobs are blocks of similar short tandemly repeated sequences, ranging from as few as 100 copies to as many as 25,000
176 / Nina Fedoroff per site (Ananiev et al., 1998a). Their structure and dispersed occurrence further suggest that they are transposable (Ananiev et al., 1998a, b; Buckler et al., 1999). The combination of transposability and preferential transmis- sion of chromosomes with expanded knobs thus provides an additional mechanism for genome expansion. GENOME CONTRACTION Are there genetic mechanisms that contract genomes? Careful analy- sis of the relative deletion frequency and length in drosophilid non-LTR retrotransposons supports the inference that there are more deletions per point mutation in Drosophila than in mammals and that the average dele- tion size is almost eight times larger (Petrov and Hartl, 1998). Thus mecha- nisms that contract genomes by preferential deletion may exist, as well. Bennetzen and Kellogg have argued that despite ample evidence for the operation of mechanisms that expand genomes in plants, there is little evidence that plant genomes contract (Bennetzen and Kellogg, 1997). The maize intergenic regions that have been analyzed, for example, comprise predominantly intact retrotransposons, rather than solo LTRs, which can arise by unequal crossovers between the repeats at retrotransposon ends and are common in other genomes (Bennetzen and Kellogg, 1997). How- ever, it also is known that both the Ac and Spm transposons of maize frequently give rise to internally deleted elements, and Ac ends are very much more abundant in the maize genome than are full-length elements, suggesting deletional decay of transposon sequences (Fedoroff et al., 1983, 1984; Schwarz-Sommer et al., 1985b; Masson et al., 1987). So it would not be surprising to find mechanisms that preferentially eliminate sequences. And indeed, preferential loss of nonredundant sequences early after poly- ploidization has been detected in wheat (Feldman et al., 1997). CONTROLLING TRANSCRIPTION, RECOMBINATION, AND TRANSPOSITION Despite our growing awareness of the abundance of plant transpos- able elements and the role they have played in shaping contemporary chromosome organization, the fact is they eluded discovery for the first half century of intensive genetic analysis. Thus what is perhaps the most striking observation about transposable elements is not their instability, but precisely the opposite: their stability. Not only are insertion muta- tions in genes infrequent, but retrotransposition events are so widely sepa- rated that the time interval between insertions in a particular region of the genome can be counted in hundreds of thousands to millions of years (SanMiguel et al., 1998). Chromosomes containing many hundreds of
Transposons and Genome Evolution in Plants / 177 thousands of transposable elements are as stable as chromosomes con- taining few. By what means are such sequences prevented from transpos- ing, recombining, deleting, and rearranging? The transposon problem can be viewed as one aspect of a larger prob- lem in genome evolution: why does duplicated DNA persist? Duplica- tions are a by-product of the properties of the DNA replication and re- combination machinery. Short stretches of homology suffice to give rise to duplications by slippage during replication, homology-dependent un- equal crossing-over, and double-strand breakage/repair (Gorbunova and Levy, 1997; Liang et al., 1998). But duplications are problematical. Once a duplication exists, the mechanisms that generated it also permit unequal crossing over between identical repeats (Anderson and Roth, 1977; Perel- son and Bell, 1977; Koch, 1979). Prokaryotes readily duplicate genetic material, but do not retain duplications (Perelson and Bell, 1977; Romero and Palacios, 1997). Thus the ability of genomes to expand by duplication is predicated on their ability to sequester homologous sequences from the cellâs recombination machinery and retain them, which may necessitate the invention of mechanisms to recognize and differentially mark dupli- cations. Some lower eukaryotes, including Neurospora crassa (Selker and Garrett, 1988; Selker, 1997) and Ascobolus immersus (Rossignol and Fau- geron, 1994, 1995), have the capacity to recognize and mark duplicated sequences by methylating them. Sequence methylation silences transcrip- tion, enhances the mutability of the duplicated sequence, and inhibits recombination (Selker, 1997; Maloisel and Rossignol, 1998). Some years ago, Adrian Bird pointed out that there are two evolu- tionary discontinuities in the average number of genes per genome (Bird, 1995). The first is an increase between prokaryotes and eukaryotes and the second is between invertebrates and vertebrates. He suggests that with a given cellular organization there may be an upper limit on the tolerable gene numbers imposed by the imprecision of the biochemical mechanisms controlling gene expression. He suggested that the transcrip- tional ânoise reductionâ mechanisms that arose at the prokaryote/eu- karyote boundary were the nuclear envelope, chromatin, and separation of the transcriptional and translational machinery, as well as RNA pro- cessing, capping, and polyadenylation to discriminate authentic from spu- rious transcripts. He proposed that genome-wide DNA methylation is the novel ânoise reductionâ mechanism that has permitted the additional quantal leap in gene numbers characteristic of vertebrates. HOMOLOGY-DEPENDENT GENE SILENCING The results of both classical and contemporary studies on the silenc- ing of redundant gene copies in plants suggests that both methylation
178 / Nina Fedoroff and other epigenetic mechanisms reflect a much more fundamental ability to recognize and regulate gene dosage (Kooter et al., 1999). Mc- Clintock understood that transposable elements exist in a genetically intact, but cryptic form in the genome and she carried out genetic analy- ses of Spm transposons undergoing epigenetic changes in their ability to transpose (McClintock, 1962). We later found that the genetically inac- tive Spm transposons are methylated in critical regulatory sequences (Banks and Fedoroff, 1989). It also has been reported that the large intergenic retrotransposon blocks in maize are extensively methylated (Bennetzen et al., 1994). The discovery that the introduction of a transgene can lead to the transcriptional silencing and methylation of both the introduced gene and its endogenous homolog brought gene silencing mechanisms un- der intense study (Park et al., 1996; Kooter et al., 1999). Genes can be silenced both transcriptionally and posttranscriptionally consequent on the introduction of additional copies. Posttranscriptional silencing appears to be caused by RNA destabilization, whereas transcriptional gene silencing involves DNA methylation (Vaucheret et al., 1998; Kooter et al., 1999). There is also some evidence that posttranscrip- tional silencing triggers DNA methylation (Wassenegger et al., 1994). The results of recent studies on the classical epigenetic phenomenon of R locus paramutation in maize have revealed that local endoredu- plication of a chromosomal segment both triggers silencing and can render the endoreduplicated locus capable of silencing an active allele of the gene on a homolog (Kermicle et al., 1995). Similar observations have been made with transgenes, as well as endogenous gene duplica- tions at different chromosomal locations in tobacco and Arabidopsis (Matzke et al., 1994; Luff et al., 1999). A connection between gene silencing and chromatin structure has come from the analysis of mutants altered in methylation and in tran- scriptional gene silencing (Jeddeloh et al., 1998; Kooter et al., 1999). Both approaches have identified alleles of the ddm1 locus, which encodes a protein with homology to known chromatin remodeling proteins. This suggests that the repressive mechanisms of DNA methylation and chro- matin structure are linked in plants, as they are in animal cells (Ng et al., 1999; Wade et al., 1999). Evidence also is accumulating that double- stranded RNA mediates gene silencing, both in plants and in a variety of other organisms (Waterhouse et al., 1998; Fire, 1999). Analyses of mutants altered in posttranscriptional gene silencing in Neurospora have identified an RNA-dependent RNA polymerase, as well as a RecQ helicase-like pro- tein, homologs of which are known to be involved in DNA repair and recombination (Cogoni and Macino, 1999a, b).
Transposons and Genome Evolution in Plants / 179 THE ORIGIN OF TRANSPOSONS AND METHYLATION Although it is popular to assert that transposons are genomic âpara- sitesâ and that DNA methylation evolved to control them, I suggest that the evidence supports neither notion (Yoder et al., 1997). The idea that transposons as parasitic, selfish DNA comes from a couple of essays writ- ten two decades ago, one by Doolittle and Sapienza (1980) and one by Orgel and Crick (1980). These essays sought rightly to free us from the then prevalent notion that genome structure is optimized by phenotypic selection. But the persistence of the moniker âselfish DNAâ has become an impediment to further understanding of the origin, historical contribu- tion, and contemporary role of transposons in chromosome structure. Transposons may be an inevitable by-product of the evolution of sequence-specific endonucleases. Complete transposons have been shown to arise from a single cleavage site and an endonuclease gene (Morita et al., 1999). Although the successful constitution of a transposon from the recognition sequences used in Ig gene rearrangement and the RAG1 and RAG2 proteins was interpreted as evidence that the V(D)J recombination system evolved from an ancient mobile DNA element, the fact is that the critical components of a transposon and a site-specific rearrangement sys- tem are the same (Hiom et al., 1998). Thus questions about the origin of certain kinds of transposons may devolve to questions about the asso- ciation of sequence-specific DNA binding domains with endonuclease domains. Although the majority of methylated sequences in a genome can be transposable elements, the view that DNA methylation evolved to control transposons seems implausible in the light of evidence that duplications of any kind trigger methylation in organisms that methylate DNA (Yoder et al., 1997; Garrick et al., 1998; Selker, 1999). And organisms that do not methylate DNA also have mechanisms for detecting duplications and sequestering repeats (Pirrotta, 1997; Sherman and Pillus, 1997; Henikoff, 1998). Genome expansion by duplication is predicated on preventing ille- gitimate recombination between duplicated sequences. Although differ- ent eukaryotic lineages appear to have invented different mechanisms, what is common to repeat-induced silencing in all eukaryotes is the stable packaging of DNA into ârepressiveâ chromatin. It may be that the evolu- tion of mechanisms that recognize, mark, and sequester duplications into repressive chromatin structures, among which some involve DNA me- thylation, were the prerequisites for expansion of genomes by endore- duplication at all scales. The additional benefit of such ârepressiveâ mechanisms in minimizing spurious transcription could be secondary sequelae. Because sequence duplication is inherent in transposition, the ability to recognize and repress duplications would serve to minimize
180 / Nina Fedoroff both the activity and the adverse impacts of transposons, rendering them genetically invisible and favoring their gradual accumulation. An important and as yet underappreciated property of compacted, inactive genomic regions is their ability to impose their organization on adjacent, as well as nonadjacent, active regions, often in a homology- dependent manner. This is evidenced in position effect variegation in Drosophila, an organism that does not methylate its DNA, as well as in plant paramutation, which involves DNA methylation (Kermicle et al., 1995; Henikoff, 1998). What has been learned recently from analyzing gene silencing and paramutation suggests that it does not take many tandem duplications to trigger the formation of a compacted, silenced region. A silenced region then may become a âsinkâ for insertions within it, as well as a silencer for homologous sequences located adjacent to it or elsewhere in the genome (Henikoff, 1998; Jakowitsch et al., 1999). CONCLUSIONS The key to understanding the prevalence of transposons in contem- porary genomes, as well as their genetic invisibility, therefore may lie not in transposons themselves, but in the much more fundamental capacity of eukaryotic organisms to recognize and sequester duplications. Whether transposons, retrotransposons, and other repetitive elements accumulate extensively in a given evolutionary lineage may depend on several fac- tors, among them the efficiency of repressive mechanisms and the rate at which the sequences undergo mutational and deletional decay. For ex- ample, methylation of C residues enhances the mutability of CG base pairs, hence methylation accelerates the divergence rate of newly arising duplications. This happens in an extreme form in Neurospora, in which many methylated CGs are mutated in the span of a single generation, and at more measured rates in plants and mammals, in which the mutability can be detected by virtue of a marked deficiency of the base pairs and triplets that are normally methylated (Selker, 1990; SanMiguel et al., 1998; Wang et al., 1998). The burgeoning analyses of genomes also makes it evident that re- pressive mechanisms are imperfect. However slowly, genomes are inexo- rably restructured by transposition and rearrangements arising from ec- topic interactions between dispersed transposons. Thus there is little remaining doubt that transposons are central to genome evolution. What is less clear is the relationship between genome restructuring and mor- phological change. We know that the magnitude of the morphological differences between species does not necessarily reflect the magnitude of the genetic or chromosomal differences between them. It recently has become evident, for example, that the marked morphological and devel-
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