Introduction. In the 1950's, Barbara McClintock reshaped the ... ence of an autonomous element (McClintock, ..... Shapiro, and John E McDonald for helpful com-.
Genetica 86: 47-53, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.
Nonautonomous transposable elements in prokaryotes and eukaryotes D. L. Hartl 1, E. R. Lozovskaya 1 & J. G. Lawrence2 1 Department of Genetics, Washington University School of Medicine, 4566 Scott Avenue Box 8232, St. Louis, MO 63110, USA 2 Department of Biology, University of Utah, 205A Life Sciences Building, Salt Lake City, UT 84112, USA Received and accepted 10 March 1992
Abstract Defective (nonautonomous) copies of transposable elements are relatively common in the genomes of eukaryotes but less common in the genomes of prokaryotes. With regard to transposable elements that exist exclusively in the form of DNA (nonretroviral transposable elements), nonautonomous elements may play a role in the regulation of transposition. In prokaryotes, plasmid-mediated horizontal transmission probably imposes a selection against nonautonomous elements, since nonautonomous elements are incapable of mobilizing themselves. The lower relative frequency of nonautonomous elements in prokaryotes may also reflect the coupling of transcription and translation, which may bias toward the cis activation of transposition. The cis bias we suggest need not be absolute in order to militate against the long-term maintenance of prokaryotic elements unable to transpose on their own. Furthermore, any cis bias in transposition would also decrease the opportunity for trans repression of transposition by nonautonomous elements.
Introduction In the 1950's, Barbara McClintock reshaped the field of classical genetics by exploring the phenomenon of mobile genetic elements able to translocate within the genome of maize and induce chromosome rearrangements (McClintock, 1950; 1951; 1955; 1956a; 1956b). Because the mobile elements could also affect gene expression, McClintock called them controlling elements. She also distinguished autonomous copies of the elements, which were able to move on their own, from nonautonomous copies, which could move only in the presence of an autonomous element (McClintock, 1950). This distinction proved critical for understanding the genetic behavior of eukaryotic transposons. Transposable elements are found not only in maize but in most other eukaryotes. At the molecular level, the elements are particular DNA sequences, typically 103-104 base pairs in length, that have the ability to change position within and among replicons. The distinction between autono-
mous and nonautonomous elements can often be traced to differences in one or more proteins coded within the elements themselves. The coding regions of nonautonomous elements typically contain one or more nucleotide substitutions, large internal deletions, or, more rarely, internal rearrangements or even insertions of unrelated transposable elements, that render the elements unable to transpose on their own. However, many nonautonomous elements retain the sequences necessary for recognition by the transposase and other factors involved in transposition, and these elements can be mobilized by autonomous elements present in the same genome. Prokaryotic genomes also contain many distinct families of transposable elements (see Berg & Howe, 1989, reviews). While different from those in eukaryotes, there are often similar in structural features; for example, many prokaryotic transposable elements include terminal inverted repeats, and many elements encode their transposase and]or regulatory proteins within their own sequence. However, in prokaryotes, defective, nonautonomous
48 copies of transposable elements are apparently less common than in eukaryotes (Lawrence et al., 1992). The reasons for the lower relative frequency of nonautonomous elements in prokaryotes have not been thoroughly explored. There may be several levels of explanation, and the mechanisms may be complex and not uniform for all classes of transposable elements. This paper is concerned with transposable elements that exist exclusively in the form of DNA, and hence retroviruses and other elements that translocate via an RNA intermediate are excluded. We will also focus on nonretroviral transposable elements that terminate in inverted repeats and that code for their own transposase protein, for example, the transposable elements P and mariner in Drosophila and the insertion sequences (IS) in Escherichia coli. For these classes of nonretroviral transposable elements, some considerations that are relevant to the persistence of nonautonomous elements are as follows: 1) Nonautonomous copies of transposable elements may serve an important function in regulating transposition, particularly in eukaryotes (Rio, 1991). On the other hand, regulatory effects of altered or truncated transposase proteins are not restricted to eukaryotes. For example, the insertion sequence IS50R, which is part of the bacterial transposon Tn5, codes for both a transposase and an inhibitor of transposition produced from a second translational start site within the transposase mRNA (Isberg et aL, 1982; Johnson et al., 1982). 2) An important difference between the transmission of prokaryotic and eukaryotic nomadic elements is that bacterial plasmids mediate the horizontal transfer of prokaryotic elements among strains, apparently at a high rate (Sawyer et al., 1987). Mathematical models of transposition that incorporate plasmid transmission give a very satisfactory account of the distribution of numbers of IS elements among strains (Sawyer et al., 1987), and they also explain why unrelated elements have a statistically significant tendency to occur together (Hartl & Sawyer, 1988). Although the models do not explicity include provision for functional and nonfunctional elements, it seems likely that plasmid transmission would impose a selection for functional elements. The reasoning is illustrated by the following example. For a functional element to be transferred from the chromosome of one bacte-
rial strain into that of another by means of plasmidmediated transfer, the element must first transpose from the chromosome of the donor strain into a plasmid, then the plasmid must be transferred into a recipient strain, and then the element must transpose from the plasmid into the chromosome of the recipient strain. For a non-autonomous element, this chain of events is complicated further by the requirement for both the donor strain and the recipient strain to contain functional elements that mobilize the nonautonomous element in trans. The additional requirement suggests that frequent plasmidmediated horizontal transfer must promote the dissemination and persistence of functional transposable elements more so than nonautonomous elements. 3) There is also a fundamental difference in molecular biology between prokaryotes and eukaryotes because, in prokaryotes, transcription and translation are coupled processes. This coupling may very well increase the opportunity for immediate cis activation of transposition of the particular element that has been transcribed and translated, relative to trans activation of elements elsewhere in the genome, and thus may help explain the relatively fewer nonautonomous transposable elements found in prokaryotes. Further development of this idea is the main theme of this paper. We do not suggest that cis activation is obligatory, only that there is a bias toward cis activation, since even a slight bias might be sufficient to select against the long-term maintenance of elements that cannot transpose on their own. At the same time, the coupling of transcription and translation also decreases the opportunity for trans repression of transposition by nonautonomous elements. Coupled transcription and translation may therefore have dramatic effects on the population biology and evolution of transposable elements.
Transposition in eukaryotes The classical case of autonomous and nonautonomous transposable elements is the Ac-Ds system in maize. McClintock identified Dissociation (Ds) as the site of chromosome breakage, although the breakage was not spontaneous but required the presence of the Activator (Ac) element. At the molecular level, the autonomous Ac element is a 4600
49 bp DNA sequence that terminates in 11 bp inverted repeat sequences (Pohlman et al., 1984). In contrast, the Ds elements comprise a heterogeneous assembly of DNA sequences, some obviously deleted versions of Ac and others highly divergent, but all containing the characteristic inverted repeats that are required for mobilization of the elements (Doting et al., 1984; Sutton et al., 1984). It is now clear that families of transposable elements in eukaryotes frequently include copies that are nonautonomous. Indeed, even in maize, McClintock showed that there are families of transposable elements other than As/Ds that include autonomous and nonautonomous copies, for example, Spm (Suppressor-Mutator) (see Fedoroff, 1989). Among the most extensively studied organisms in this regard is Drosophila melanogaster, the genome of which contains the P element along with about 50 other transposon families (Ashburner, 1989). The autonomous P element is 2,907 bp but also occurs in nonautonomous forms, many of which contain deletions (Bingham et al., 1982; Spradling & Rubin, 1982; see Engels 1989 for review). One particular nonautonomous element, called the KP element, accounts for over half of the P element copies in some natural populations (Black et al., 1987). The KP element contains a deletion of 1753 bp (eliminating nucleotide positions 808-2560 in the complete P element), but it retains the 31 bp terminal inverted repeats and the 11 bp subterminal inverted repeats about 125 bp from each end. The KP element codes for a 207 amino acid protein that might act to repress transposition (Black et al., 1987; Rio, 1991), perhaps by a mechanism similar to the repression effects of a 576 amino acid protein (the 66 kDa protein) produced from somatic transcripts of the complete P element (Misra & Rio, 1990; Rio, 1991). The genome of D. melanogaster also includes nonautonomous copies of the transposable element hobo (Streck et al., 1986), and that of D. mauritiana includes nonautonomous copies of mariner. In the case of mariner, some of the nonautonomous forms result from nucleotide substitutions rather than deletions (Maruyama & Hartl, 1991a). Because transcription and translation are uncoupled in eukaryotes, all transposition of nonretroviral elements in eukaryotes results from trans activation. Indeed, it is this feature of eukaryotic biology that made it possible to use transposable elements
as vectors for germline transformation in Drosophila (Spradling & Rubin, 1982). Differences in mobilization of particular copies of transposable elements must generally reflect differences in the binding of transposase and other relevant proteins or in the accessibility of the element as a function of its position in the genome (e.g., in heterochromatin versus euchromatin, or in A+T rich isochores versus G+C rich isochores).
Transposition in prokaryotes Analysis of certain unstable mutations in bacteria led to the discovery of transposable elements three decades after their discovery in eukaryotes (Jordan et al., 1968; Shapiro, 1969; Michaelis et al., 1969; Hirsch et al., 1972). The class of bacterial transposable elements called insertion sequences (IS) is comparable to the eukaryotic transposons such as P and mariner in being relatively short (up to several thousand bp), flanked by inverted repeats, and not transposing via an RNA intermediate (in contrast with retroviral elements such as Tyl in yeast and copia in Drosophila, as well as LINE elements in mammals). (IS elements are reviewed in Galas & Chandler, 1989). Interestingly, in bacteria, a distinction between autonomous and nonautonomous elements has never been necessary. For example, insertion mutations in bacterial genes typically contain transposable elements that are functional. On the other hand, a few copies of bacterial transposable elements that are at least potentially nonautonomous have been reported (Reinitz et al., 1989). The situation with regard to transposable elements in archeobacteria is still unclear, but the data suggest that mobile elements in these prokaryotes resemble their bacterial counterparts (Charlebois & Doolittle, 1989). Transposable elements in the Archaea have been discovered as insertions in gas vacuole genes and bactetiorhodopsin (Bop) genes (Weidinger et al., 1979; Pfeifer et al., 1981). An autonomous transposable element has been identified as the insertion in some of the Bop mutations in Halobacterium (Simsek et al., 1982). On the other hand, putative nonautonomous elements do occur, as evidenced by the 1384 bp element ISH26, for which a 1705 bp variant containing a small internal duplication has been described (Ebert et al., 1987).
50 Regardless of whether a particular prokaryotic transposable element transposes in a conservative or replicative manner, its transposase protein is of fundamental importance. Transposase proteins are known to bind to the inverted repeats of many transposable elements (Morisato et al., 1983; Derbyshire et al., 1987; Nakayama et al., 1987; Wiater & Grindley, 1987; New et al., 1988; Leung et al., 1989; Stadler et al., 1990), where it is assumed that the protein promotes DNA cleavage initiating transposition. The coupling of transcription and translation makes it possible for the transposase proteins to operate preferentially on the inverted repeats of the element from which they were produced. Chandler et al., (1980) have shown that the IS1 transposase has an affinity for inverted repeats that are nearby, and the transposases of a number of IS elements have been shown to be primarily cis-acting (Grindley & Joyce, 1981; Machida et al., 1982; Morisato et al., 1983). Since the N-terminal domains of several transposases bind specifically to the ends of the transposable element (Stadler et al., 1990; Zerbib et al., 1987), it has been suggested that the cis-action of transposases may also play a role in the regulation of transposition (Lawrence & Hartl, 1991). On the other hand, there is no doubt that trans activation of transposition can also occur at appreciable rates, for example, in Mu (Pato, 1989), Tn3 (Sherratt, 1989) and Tn5 (Berg, 1989). Nevertheless, a mechanism of transposition in which the transposase is even somewhat biased toward cis-activation would tend to favor the transposition of elements that are intact and fully functional. Copies that are defective would undergo transposition only if they were in close physical proximity to functional elements, as in the case of IS50L in the bacterial transposon Tn5 (Berg, 1989), or when transposase acted in trans. Hence, nonautonomous elements would be less likely to be propagated by transposition than in eukaryotes. However, they would still be liable to deletion from the genome by processes unrelated to transposition. Furthermore, elements less able to transpose onto bacterial plasraids would forfeit the principal mechanism for becoming disseminated among strains (Sawyer et al., 1987; Hartl & Sawyer, 1988), making it even more difficult for longterm persistence in the population. The cis-biased model of transposition predicts that nonfunctional copies of transposable ele-
ments should be less common in prokaryotes than in eukaryotes. What are the data? Most studies of bacterial transposable elements do not address the issue. One relevant observation is that defective copies of IS/ were not found among the multiple copies found in the laboratory strain Escherichia coli K12 (Umeda & Ohtsubo, 1991). The most extensive data are those of Lawrence et al. (1992), who carried out DNA sequence analysis of 11 independent copies of IS/ (including both major isoforms), 12 copies of IS3, and 12 copies of IS30, among natural isolates of Escherichia coli and related enteric bacteria. The relevant result is that fewer than 5% of the elements examined contained any mutation that was likely to eliminate transposase function (Lawrence et al., 1992), and each of these was found only once. Furthermore, none of the potentially nonfunctional elements exhibited large deletions. These results contrast with those in eukaryotes, in which 50% or more of the copies of many nonretroviral transposable elements may contain large deletions (Black et al., 1987; Engels, 1989; Maruyama & Hartl, 1991a),
Population models of transposable elements Models for the population dynamics of transposable elements typically include parameters for rate of transposition, regulation of transposition, excision, and natural selection (reviews in Charlesworth & Langley, 1989; Ajioka & Hartl, 1989). The prokaryotic models implicity assume that all elements are functional and therefore that cis or trans activation is immaterial. However, models incorporating preferential cis activation and a parameter for rate of generation of nonfunctional elements would be of considerable interest. Although plasmid transmission itself might tend to favor the dissemination of autonomous elements, for reasons outlined in the introduction, this effect would be enhanced by a cis bias in transposition. The average persistence and equilibrium frequency of nonautonomous elements would certainly decrease as the bias toward cis activation increased, but an explicit population model would show the quantitative relationships between the various parameters. For nonretroviral transposable elements in eukaryotes, it seems reasonable to suppose that
51 nonautonomous elements are equally likely to be mobilized as autonomous elements, assuming that they retain all the sequences necessary for efficient transposase recognition and binding. Therefore, nonautonomous elements will be propagated along with autonomous elements because of trans activation of transposition, and from generation to generation, the relative numbers of autonomous and nonautonomous elements will change according to a stochastic process. Hence, there is a finite probability of evolving to a state in which all of the elements present in a population are nonautonomous. This state is an absorbing barrier (i.e., is irreversible), since transposition can no longer occur. Since fixation of nonautonomous elements is a permanent state, but fixation of autonomous elements is not, the ultimate disappearance of autonomous elements is inevitable in a finite population. This implication is not affected by possible regulatory effects of nonautonomous elements, which, indeed, may accelerate the rate of fixation. Stochastic loss is suggested with regard to the P element in D. guanche, the genome of which contains 20-50 tandem copies of a defective P element capable of coding for a protein highly similar to the 66 kDa repressor protein, but no active P elements or transposase-coding sequences (Miller et al., 1991). This situation also occurs in the closely related species D. subobscura and D. madeirensis. The case for stochastic loss of mariner elements in species of the D. melanogaster species subgroup is also very strong (review in Capy et aL, 1992, see also article by Capy et al. in this volume). The element occurs in five of the eight species in the subgroup, and DNA sequence comparisons strongly imply that it was present in the ancestor of the subgroup (Maruyama & Hartl, 1991a). The element was lost independently at least twice (in the lineage leading to D. erecta/D, orena and that leading to D. melanogaster). Furthermore, D. sechellia contains two mariner elements at fixed positions in the genome; one is certainly defective because it has three deletions and is missing much of the 3' end, while the other appears to have, at most, a low level of activity (Capy et al., 1991). In addition, in D. teissieri, about 80% of the mariner elements have a deletion of 716 base pairs spanning positions 544-1260, but the inverted repeats remain intact (Maruyama et al., 1991). The susceptibility of eukaryotic transposable ele-
ments to stochastic loss, through the fixation of nonautonomous copies, contrasts with the situation in prokaryotes, where preferential cis activation favors autonomous transposable elements and thus should promote long term persistence in bacterial species. For example, homologues of the E. coli element IS3 occur in species of the genus Serratia, and molecular comparisons suggest that the element was present in common ancestor of these species (Lawrence et al., 1992), the divergence of which is estimated at 250-300 million years (Ochman & Wilson, 1987). In eukaryotes, apparently, the only countervailing force against ultimate loss is the possibility of horizontal transmission of autonomous elements across the reproductive barriers between species. While horizontal transmission is well documented, the rate at which it occurs appears to be quite low (Daniels et al., 1990; Mamyama & Hartl, 1991b). Nevertheless, when autonomous elements are first introduced into naive genomes, they have the opportunity to multiply before accumulating significant numbers of nonautonomous mutants. Even if the accumulation and ultimate fixation of nonautonomous elements limits the persistence of a transposable element in any particular eukaryotic species, the likelihood of complete extinction of the element is offset by the occurrence of horizontal transmission on a time scale that may be roughly comparable to the rate of speciation (Maruyama & Hartl, 1991a, 1991b).
Acknowledgements We are very grateful to Howard Ochman, James A. Shapiro, and John E McDonald for helpful comments and suggestions on the manuscript. This work was supported by NIH grants GM40322 and GM33741.
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