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Transposable elements
Transposable elements David J. Finnegan U n i v e r s i t y of E d i n b u r g h , E d i n b u r g h , S c o t l a n d
Transposable elements comprise a major fraction of eukaryotic genomes. They are studied both because of their intrinsic biological interest and because they can be exploited as valuable research tools. Many interesting papers dealing with various aspects of the biology of these elements have been published during the past year and a number of new elements have been reported. Four areas in which particularly valuable contributions have been made are the mechanisms of transposition, the regulation of transposition, the use of transposable elements as research tools, and the biological function of transposable elements.
Current Opinion in Genetics and Development 1992, 2:861-867
Introduction Transposable elements are an important component of eukaryotic genomes, making up approximately 15% of the total DNA. They occur as families of dispersed repeat sequences scattered throughout the genome, with the number of copies varying from less than ten to several hundred thousand depending on the element and species concerned. They can be classified according to their structure and presumed mechanism of transposition (Fig. 1), and fall into two main classes: Class I elements that transpose by reverse transcription of an
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LTR
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RNA intermediate; and Class II elements that transpose directly from DNA to DNA. There are two groups of Class I elements. Class 1.1 elements resemble retroviruses in having long terminal repeats (LTRs) and open reading frames (ORFs) with the potential to code for polypeptides similar to the gag- and pol-encoded proteins. In this review, I shall refer to these as retrovirus-like elements. Class 1.2 elements also have ORFs similar to the retroviral gag and pol genes but have no terminal repeats and end with Arich sequences at the 3' end of their coding strands. Here, I shall refer to elements of this type as LINE-like elements
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Fig. 1. Schematic representation of the structure of eukaryotic transposable elements, (a) Class I elements are of two types, those that are retrovirus-like, Class 1.1, and those that are not, Class 1.2. All Class 1.1 elements have a gag-like I gag'l open reading frame (ORF), and an ORF that encodes a reverse transcriptase (RT). Some of them also have a third ORF of unknown function, indicated by (?). The long terminal repeats (LTRs) are also shown. Class 1.2 elements only have' gag" and RT ORFs. These elements have A-rich sequences (An) at their 3' ends. Within a family of such elements many copies may be truncated at their 5' ends, as indicated. (b) Class II elements have short terminal inverted repeats (arrowed}. They contain a gene encoding transposase, an enzyme that is required for their own transposition. This gene may have more than one exon (not shown here).
Abbreviations LTR--Iong terminal repeat; ORF--open reading frame.
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Genomes and evolution as the first exanlples to be detected were the man~malian LINE (long interspersed elements) or L1 elements. The SINEs, short interspersed elements, which are found in hundreds of thousands of copies in human and other mammalian genomes, also have an A-rich sequence at the 3' end of one strand. It has been shown that SINEs integrate at new sites by means of a transposition mechanisms, but must do so passively as the), do not encode proteins themselves. The Class II elements have inverted terminal repeats 10-200bp long, although one family of elements in Drosophila melanogaste,, the FB fanlily, has inverted repeats much longer than this. Elements of this type encode proteins, the transposases, that are required for transposition.
Mechanisms of transposition Transposition through an RNA intermediate Retroviral-like transposable elements are believed to transpose by a mechanism that is similar to the retroviral life-cycle and this has been directly established for Tyl elements of Saccharo*lo~ces cerevLqae [1]. Transcription of the RNA transposition intermediate initiates in one LTR and terminates in the other. This RNA is then packaged into virus-like particles that are constructed of proteins encoded by the gag- and pol-like ORFs [2-]. The RNA is subsequently reverse transcribed to yield linear double-strand DNA and integrated at new sites by element-encoded proteins. Studies in which an intron is introduced into a TI,I element show that it can be precisely removed during transposition, thus demonstrating that an RNA molecule acts as a transposition intermecliate. A similar observation has been made for the retroviruslike element copia of D. me&nogaster [3"]. In this case it appears that an intron nom~ally present in copia elements can be lost during transposition. LiNE-like elements are believed to transpose by a similar mechanism but with important differences. Transposition through an RNA intemlediate has been demonstrated for I elements of D. melanogaster [4,5] and mouse L1 elements [6.]. Again, this was shown by demonstrating that introns introduced into dlese elements can be removed precisely during transposition. The reverse transcriptase required for this event is presumably encoded by the pol-like ORF of these elements. Ivanov et al. [7"] have confirmed that jockey elements of D. melanogaster encode a reverse transcriptase by expressing it in E. coli, and a less direct but equally effective method has been used to demonstrate a similar activity associated with the second ORF of the CRE elements of Trypanosoma brucei [8"], human L1 elements [9"], and I elements of D. melanogaster (T Paterson et al., unpublished data). In these studies, the reverse transcriptase coding region of a TyI element, linked to an inducible promoter, is replaced by the equivalent region derived from the element in question, and virus-like particles are purified from yeast cells expressing the hybrid Tyl elements. In each case,
these particles were associated with reverse transcriptase activities distinguishable from that of wild-type Tyl particles. Before this could be done with a human L1 element a potentially functional element had to be identified. This was not as straightforward as it may sound as these elements are heterogeneous and no genomic clone had been identified that contained the two ORFs evident in the consensus L1 sequence. A potentially active element was identified when it hybridized to an oligonucleotide derived from a region of a recently transposed but truncated L1 element, and to oligonucleotides derived from the ends of the L1 consensus sequence [10-]. The RNA transposition intermediates of a LINE-like element must contain the entire sequence of the element, thus preventing it gradually decaying during successive rounds of transposition, and the promoters from which these RNAs are synthesized are presumably located within the elements themselves. The elements jock W [11], F [12-] and I ( C McLean et aL, unpublished data) of D. melanogaster have promoters that lie within their transcriptional unit and the sanle m W be true of all elements of this type. As LINE-like elements contain both gag and pol-like ORFs, their transposition intermediates are probably packaged in virus-like particles similar to those of retrovirus-like elements. Mouse embryonal carcinoma cells contain ribonucleoprotein particles associated with both plus-strand L1 RNA and polypeptides that react with the L1 gag-like gene-encoded product [13"]. This is consistent with the idea that these cells contain L1 virus-like particles. However, such particles have neither been purified nor shown to be involved in transposition. Similar results have been obtained using human embryonal carcinoma cells [14]. Two important questions about the transposition of LINE-like elements remmn to be answered. What are the primers for the reverse transcription of their RNA intermediates, and how are the products of reverse transcription integrated into the genome? LINE-like elements contain no obvious binding sites for primers of reverse transcription and are thought to use 3'OH groups generated at nicks in target DNA for this. DNA synthesis and integration thus occur simultaneously. Apart from elements inserted at specific sites in the genome there is no evidence that LINE-like elements code for nucleases involved in integration. The nicks used for integration may be produced by enzymes that are not directly involved in transposition. The precise mechanism of integration should be revealed by studies involving in vitro transposition systems. These systems will probably comprise virus-like particles containing transposition intermediates, assuming that such particles exist, and suitable target DNAs.
Transposition without an RNA intermediate Recently, a great deal has been learnt about the mechanism by which Class II elements transpose. For many years it was unclear whether this was a conservative or replicative process. Genetic studies of plant Class II el-
Transposable elements Finnegan
ements, the Ac and Spin elements of Zea mays and the Tam elements of Antirrhinum majus, suggest that they transpose conservatively. However, the situation is more complex for P elements of 19. melanogastem on the one hand, P transposase stimulates both excision and transposition suggesting a conservative mechanism, while on the other, transposition leads to an increase in the number of P elements, suggesting that it is replicative.
fled transposase. Transposition is scored by transfecting E.. coli with DNA from the assay mix and selecting cells that harbour the target plasmid plus the marked P element but not the donor DNA. Transposition is stimulated to modest but significant levels by the addition of transposase, and transposed copies of the marked P element integrate into target DNA generating 8 bp target site duplications just as they do in vivo. The only essential components in the reaction are Mg2+, GTP, tile transposase-fraction and a donor element with intact transposase-binding sites at both ends. There appears to be no requirement for an energy source or for DNA synthesis as transposition is stimulated by non-hydrolyzable analogues of GTP but not by dNTPs. Linear P-element DNA can act as a donor molecule in the absence of any flanking sequences provided that 3'OH groups are retained at each end. These data are consistent with Engels' proposal that transposition is a conservative excision/insertion process.
A solution to this paradox has been provided by the elegant genetic experiments of Engels et al. [15]. These indicated that P elements normally excise precisely from the genome and that the double-strand gaps created in the process are repaired using either sister chromatids or homologous chromosomes as templates (Fig. 2). If the template used has no P element at the original site, then the gap will be repaired to give the wild-type sequence and a precise excision. If the template contains a P element at the original site, as would be the case if the sister chromatid were used, then repair can introduce complete or deleted elements and this will be scored as either an incomplete excision or as no excision at all. The Tcl elements of Caenorhabdilis elegans have been shown to behave in a similar manner [16].
Is transposase the only protein required for transposition in this assay? Transposase itself does not bind the 31 bp terminal inverted repeats of P elements but all/9. melanogaster cells contain a protein that does. It is not yet known whether this protein is required for transposition and this will only become clear once the protein has been purified and added to the in vitro assay along with purified transposase.
Kaufman and Rio [17 ,°] have recently developed an in vitro system for studying P transposition. In this system, a donor plasmid containing a marked P element is mixed with a target plasmid in the presence of partially puff-
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Fig. 2. Repair of a double-strand gap left after Drosophila melanogaster P-element excision, with each line representing a single strand of DNA. Thin lines represent chromosomal DNA and thick lines a strand of P-element DNA. A dashed line indicates newly synthesized material, and the large crossovers show recombination. (a) Schematic representation of a site in the genome containing a P element. (b) The same site is shown with a double-strand gap generated by excision of the P element. (c) Details the sequential repair (see arrows) of the double-strand gap using as a template DNA without a P element. This could be DNA on the homologous chromosome. (d) Demonstrates the repair of the double-strand gap using as a template DNA with a P element at the original site. This could be a sister chromatid. (e) The double-strand gap is repaired by using as a template DNA that contains base changes (black dots). These could have been introduced in vitro and inserted at an ectopic site in the genome. For all cases (c-e), the sequential repair involves: (i), invasion of template DNA by the ends of the broken strands; (ii), DNA synthesis progressing from the 3'OH ends of the broken strands; and (iii), DNA synthesis completed and the gap repaired. Resolution of the structure seen in (ii) usually occurs without the recombination of outside markers.
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Genomesand evolution P elements can insert at many sites in the genome but not at random. Hot spots for insertion have been detected and a consensus 8 bp motif has been detected for target-site duplications. The sequence of the target site is not, however, the only constraint on insertion. Spradling el al. [18-] have recently sho~xaa that P elements preferentially transpose to sites a short distance from their original position, while a P element carrying regulator 3, sequences from the engrailedgene preferentially inserts near other genes that are expressed segmentally during embryogenesis [19o]. These observations could be explained if P-element-traaasposase complexes are found to be functional for only a short time after excision, thus needing to rapidly interact with target DNA for integration to take place. This would favour integration at sites in close proximity to the donor element either because they are both on the same DNA molecule or because they are linked by protein-DNA interactions.
The regulation of transposition There must be some constraints on the activity of transposable elements as uncontrolled transposition would be deleterious for any organism. P elements regulate their own actMty by a complex process that, in part, is the result of transcriptional repression from the P promoter, exerted by a polypeptide(s) related to transposase [20°%21,22.]. Transposition of retrovirus-like elements can also be regulated [23"]. Tyl transposition is regulated post-translationally [24,] by a mechanism that affects the levels of pol}q3eptides produced by the pol-like gene. Mumoons in RAD6 lead to a hundredfold increase in Tyl transposition [25]. These mutations affect one of the enzymes involved in protein ubiquitination and may well alter the stability of one of these Tyl-encoded pol~ge ptides. Over the years, several strains of D. me&nogaster have been reported as being genetically unstable with higher than usual levels of transpositon. Some of these strains carry claromosomal mutations that increase transposition of the elements involved [26"].
Transposable elements as experimental tools Cell ablation Transposable elements are valuable experinqental tools in many organisms. They can be used as insertional mutagens and as molecular tags with which to clone target genes. Transformation systems, based on P and hobo elements have been developed for Drosophila, and P elements carrying the E. coli lac Z gene have been used to detect enhancers stimulating gene expression in specific cell types. These studies have enabled the identification of regulatory elements that would have been difficult if not impossible to detect by other means. Some of these elements are active in small numbers of cells, the functions
of which may be unknown. In such cases it would be helpful to be able to observe the effect of controlled ablation of the cells. This can be done using P transfomlation vectors carrying genes coding for conditionally active cell autonomous toxins under fl~e control of regulatow sequences that stimulate their expression in the cells of interest {27"',28,,]. This system should prove to be particularly useful for studying tile functions of particular cells or groups of cells in complex tissues such as the nervous systenl.
Targeted alteration of genes in vivo Conclusions about the functions of regulator}, or coding sequences can only be made with confidence once the effects of changes in these sequences have been studied in otherwise wild-type genes. Exogenous DNA can replace endogenous sequences by recombination in some species but not in others, including D. melanogaster and C. elegans. P-element mediated transfom~ation can be used to introduce modified sequences at ectopic sites in the Drosophila genome but cannot replace wild-type sequences with DNA that has been modified i,7 vitro. This complicates many studies because effects on gene expression or protein function as the result of changes in sequence m W be masked by changes in gene expression mediated by flanking DNA at ectopic sites. Engels and his colleagues [29"] have overcome this problem by developing a method that allows specific base changes to be introduced into genes near sites of P-element insertion. They noted that ectopic copies of the white gene can serve as templates for repairing gaps resulting from excision of a P-element ,and argued that if repair synthesis were to extend some distance to either side of a gap, and if and the template differed in sequence from the original gene, then these differences would be transferred from one copy of the gene to the other (Fig. 2). Indeed this is the case and Gloor et aL [29"] have used the method to transfer up to 12 single base changes over a 2.8 kb region surrounding the site of a P-element insertion in white. TcI elements can be used in a similar way in C. elegans [30"]. This is similar to gene conversion associated with recombination and the average distance over which information is transferred from one copy of the gene to another is similar to the length of conversion tracts in meiotic recombination in D. melanogaster. Banga and Boyd [31"] have suggested that injected oligonucleotides can be used as templates for repair after P-element excision. This is an exciting possibility that should be investigated further as the data presented are not extensive and it is not clear whether transposase or even the oligonucleotide is required.
Do transposable elements serve useful functions? Transposable elements comprise a significant fraction of most eukaryotic genomes and yet transposition is dele-
Transposableelements Finnegan 865 terious under most circumstances. Why then are these elements maintained rather than being eliminated by selection? Transposition usually leads to an increase in the number of copies of an element in the genome so the answer could simply be that selective losses are balanced by transpositional gains. The alternative is that transposable elements are maintained by selection because they perform functions that are advantageous in the long-term. The most obvious function performed by transposable elements is to cause mutations. This may not appear beneficial but transposable elements cause some changes that are difficult to generate by other means [32] ,and the advantage accruing from this might be sufficient to maintain some elements. Another obvious effect of transposable elements is to increase the total mass of the genome. This may be of selective value particularly in plants in which there is a relationship between genome DNA content and generation time [33]. The LINE-like element, del2, of Lilium speciosum [34"] is present in about 250 000 copies (contributing nearly ten Z). melanogaster genome equivalents to this species) and m W be retained because it increases the genome mass. The ability of Class II elements to excise from the genome may also be advantageous in some circumstances. Many species are known to delete substantial anlounts of genomic DNA at specific times during development in a process known as genome diminution. In the ciliated protozoan Euplotes crassus, this is the result of the excision of sequences that appear to be Class II elements [35"]. This occurs during the formation of transcriptionally active macronuclei from transcriptionally silent micronuclei and ,night have evolved to minimize selection against insertions within genes. Indeed, some genes are known to be interrupted by such elements in micronuclear DNA but not in macronuclei. It is equally possible that it is excision of these elements that activates macronuclear genes, and dais may have been exploited by the organism as a means of regulating gene expression. A form of genome diminution is seen in polytene chromosomes of Drosophila where DNA in heterochromatic regions of the genome is under-represented. This has been assumed to be because of the differential replication of euchromatic and laeterochromatic sequences. Spradling el al., [18"] have pointed out that it could equally well be attributed to the excision of transposable elements concentrated in heterochromatin. Members of a family of putative transposable elements, the HeT-A elements, are associated with telomeres in 1). melanogaster [36",37"',38"]. These elements are located in centromeric heterochromatin and at telomeres but not in euchromatin. It is not clear whether they provide the function of the short repeats found at telomeres in other species - - thus far, no such repeats have been identified in 1)rosophila. The original HeT-A element was found on a fragment of DNA believed to derive from the tip of an X chromosome and other copies have been found at the broken ends of terminally deleted X chromosomes. These elements are similar to LINE-like transposable elements as there is a short run of A residues at the junction
between the 3' end of HeT-A DNA and X chromosome sequences. Furthermore, about 2.3 kb upstream of this there is a gag.like ORF similar to that in Drosophila LINE-like elements. No ORF that could encode a reverse transcriptase has yet been found but then nor has a complete element been sequenced. LINE-like elements are believed to reverse transcribe their RNA transposition intermediates by associating the A-rich sequence at the 3' end of the RNA with chromosomal DNA that is nicked in both strands. An element that could also do this at the ends of DNA molecules might eventually take over telomere functions. If HeT-A elements are LINE-like transposable elements, then their sequence organization is atypical but this is not surprising if the), have become adapted to perform a particular function within the genome.
Concluding remarks Nearly twenty years have passed since eukaryotic transposable elements were first identified at the molecular level. In that time, they have moved from being interesting peculiarities to being recognized as important components of all genomes. Much has been learnt about how they transpose and we have some understanding of how transposition is regulated. As a result, several elements have been tanaed and put to work in the laboratory. Mthough much remains to be learnt in all of these areas, the biggest gaps in our knowledge lie in understanding the significance of transposable elements for the structure and function of eukat3,otic genomes.
Acknowledgements I am grateful to H Biessmann, M-L Pardue, R Plasterk, S Ronsseray and A Spradling for discussing their unpublished data with me, and to G Bn,an for comments on this manuscript. My work is supported by research grants from the Medical Research Council and the Human Frontier Science Progranl.
References and recommended reading Papers of particular interest, published within tile annual period of review, have been highlighted as: , of special interest ,,. of oucstanding interest 1.
BOEKEJD: Transposable Elements in Saccharomyces cerevisiae. In Mobile DN,,t Edited by Berg DE, Howe MM. Washington DC: American Society for Microbiology; 1989:335-374.
2. •
BURNSNR, SAIBII. HR, WHITE NS, PARDON jF, TIMMINS PA, RICHARDSON,SMH, KINGSMAN SM, KINGSMAN AJ: Symmetry, Flexibility and Permeability in t h e Structure of Yeast Retrotransposon Virus-Like Particles. ~lqBOJ 1992, 11:1155-1164. The first detailed report of the structure of virus-like particles produced by a mmsposable element. The authors show that these particles are more variable in structure than those of retroviruses. 3. •
YOSHIOKIK, KANDA H, AKIBA H, ENOKI M, SHIBA T: I d e n tification of an Unusual Structure in t h e Drosophila
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Genomes and evolution melanogaster Transposable Element copi~ Evidence for copia Transposition T h r o u g h an RNA Intermediate. Gene 1991, 103:179-184. Direct evidence that a retrovirus-like element in Drosophila transposes by reverse transcription of an RNA intermediate. The data suggest that the RNA transposition intermediates of copia elements contain a sequence that favours packaging into vires-like particles. 4.
P.:';dJSSONA, FINNEGAN, DJ, BUCHETON A: Evidence for Retrotransposition of t h e I Factor, a LINE e l e m e n t of Drosophila melanogaster. Proc Natl Acad Sci USA 1992, 88:4907-4910.
5.
JENSEN S, HEIDMANN T: An Indicator Gene for Detection of Germline Retrotransposition in Transgenic Drosophila Demonstrates RNA-Mediated Transposition of the LINE 1 Element. ~%¢BOJ 1991, 10:1927-1991.
6. EVANS JP, PAIJ~IlTER RE): Retrotransposition of a Mouse L1 T Element. Pt~c Natl Acad Sci USA 1991, 88:8792-8795. he first direct evidence that mammalian LINE elements transpose through an RNA intermediate, as well as the first report of these elements transposing in an experimental situation. 7. .
IVANOVVA, MELNIKOVA, SIUNOV AV, FODOR I[, ILYIN YV: Authentic Reverse Transcriptase is Coded by jockey a Mobile Drosophila Element Related to Mammalian LINEs. EMBOJ 1991, 10:2489-2495. Direct evidence that the pol-like ORF of a Drosophila retrovims-like element encodes a reverse transcriptase. Most of the po/-like gene was expressed in E. coil and encoded a protein with reverse transcriptase but not RNAse H activity. GABRIELA, BOEKE JD: Reverse Transcriptase Encoded by a Retrotransposon from the Trypanosomatid Crythidia fasciculata. Proc Natl Acad Sci USA 1991, 88:9794-9798. Direct evidence that a LINE-like element encodes a reverse transcriptase. In this case, the reverse transcriptase coding region of a Tj,1 element is replaced by the equivalent region derived from a CRE1 element. The Tyl element used is transcribed from an inducible promoter, thus enabling high levels of expression. The resulting hybrid Tyl-CREviruslike particles are associated with a reverse transcriptase activity that is abolished when the CRE sequences are mutated. 8. •
9. ,
MATIilASSL, SCOTt AF, KAZAZIANHH, BOEKE JD, GABRIEL A: Reverse Transcriptase Encoded by a H u m a n Transposable Element. Science 1991, 254:1808-1810. Direct evidence for a reverse transcriptase encoded by a human L1 element. The method used and results obtained are similar to those described in [8*]. An epitope tag is used to detect the Ty1-L1 fusion polypeptide in the vires-like particles. 10. •
DOMBROSKI BA, MATI-IbVS SL, NANTt-bXKUMAR E, SCOTT AF, KAZAZI~'~HH: Isolation of an Active H u m a n Transposable Element. Science 1991, 254:1805-1807. Description of the cloning of a potentially active human L1 element. This appears to have been the donor of a truncated element found inserted within the Factor Vlli gene of a haemophiliac patient. 11.
MIZROKHm LJ, GEORGIEVA SG, ILYIN YV: jockey, a Mobile Drosophila Element Similar to Mammalian LINEs, is Transcribed from t h e Internal Promoter by RNA Polymerase II. Cell 1988, 54:685--691.
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MINCHIOTTIG, DI NOCERA PP: Convergent Transcription lnitiates from Oppositely Oriented Promoters within the 5' End Regions of Drosophila melanogaster F Elements. Mol Cell Biol 1991, 11:5171-5180. Describes an analysis of the promoter of the Drosophila LINE-like element E Sequences from the beginning of an F element are linked to a chloramphenicol acetyl transferase reporter gene and assayed in transfected cells. The results are complex but provide evidence for a promoter lying within the transcribed region and initiating transcription near the start of the element. A promoter that transcribes in the opposite direction is also observed. 13. •
MARTINSl2 Ribonucleoprotein Particles with LINE-1 RNA in Mouse Embryonal Carcinoma Cells. Mol Cell Biol 1991, 11:4804--4807.
A concise paper that details convincing evidence for there being ribonucleoprotein particles, which contain L1 RNA and proteins, in mouse embryonic carcinoma cells. 14.
DERAGON J-M, SINNETT D, LABUDA D: Reverse Transcriptase Activity from H u m a n Embryonal Carcinoma Cells NTera2D1. EMBO J 1990, 9:3363-3368.
15.
ENGELS XY.'R,JOHNSON-SCHLITZ DM, EGGIESTON XVB, SVED J: High-Frequency P Element Loss in Drosophila is Homolog Dependent. Cell 1990, 62:515-525.
16.
PLASTERKRH: The Origin of Footprints of the T c l Transposon of Caenorhabditis elegans. ~ I B O J 1991, 10:1919-1925.
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KAUFMANPD, RIO DC: P Element Transposition In Vitro Proceeds by a Cut-and-Paste Mechanism and Uses GTP as a Cofactor. Cell 1992, 69:27-39. Tile first description of an in vitro system for studying tile transposition of Class ll elements. Transposition requires added Mg2+ and GTP but not dNTPs. The donor P e l e m e n t must have intact transposase-binding sites at both ends but can be linear or circular. Linear molecules that end at the P termini can act as donors provided that they have 3'OH groups. This is a major step forward to understanding the mechanisms by which elements of this type transpose. 18. •.
SPRADUNGAC, KARI'EN G, GLASER R, ZH.M~G P: Evolutionary Conservation of Developmental Mechanisms: DNA Elimination in Drosophila. In EvolutionaoJ Conservation of De, velopmental Mechanismx Edited by Spradling A. New York: Wiley-Liss; 1992:39-53. This stimulating paper discusses the possibility that the elimination of DNA from chromosomes in somatic cells is not a rarity. The authors suggest that this p h e n o m e n o n may explain the under-representation of some sequences in polytene chromosomes, and that it may both contribute to position effect variegation and influence gene expression. These ideas are based in part on the authors' study of the behaviour of a mini-chromosome in D. melanogaster. 19. •
KASSmSJA, Non. E, VANSICKLE EP, ODENWALD \X/F, PERRIMON N: Altering t h e lnsertional Specificity of a Drosophila Transposable Element. Proc Natl Acad Sci USA 1992, 89:191 9-1923. Describes the behaviour of a P element carrying regulatory sequences from the Drosophila engrailedgene. The results indicate that the integration of P transfomlation vectors can be influenced by the sequences that they carry. This may help clarif3, the mechanism of target-site selection by transposable elements and factors that influence the organization of DNA sequences within nuclei. 20. RiO DC: Regulation of Drosophila P Element Transposition. •, Trends Genet 1991, 7:282-287. An excellent discussion of the regulation of P element transposition, which is subject to both tissue-specific and genetic controls. Transposition is restricted to the germ-line because a splicing event that is required for transposase production is inhibited in somatic cells. The factors that regulate transpositon in geml-cdls, as well as their inheritance, are less well understood. 21.
I..EMAITREB, COEN D: P Regulatory Products Repress in Vivo the P Promoter Activity in P-lacZ Fusion Genes. Proc Natl Acad Sci USA 1991, 88:4419--4423.
LE,VadTREB, RONSSERAYS, COEN D: P Cytotype Repression of the P Promoter is Exclusively Maternal in t h e Germline: A Model for P Cytotype. Genetics 1992, in press. Describes the use of P-lacZ fusion genes to investigate how Pelements are regulated. These experiments show that in strains carrying complete P elements lacZ expression is inhibited more strongly in germ cells than in somatic cells. The factors influencing expression in germ cells are inherited maternally, whereas those affecting somatic expression are not. The authors suggest an interesting model to explain these results. 22. •
23. CURCIOMJ, GARFINKELDJ: Regulation of Retrotransposition in •. Saccharomyces cerevisiae. Mol Microbiol 1991, 5:1823-1829 An excellent discussion of the factors influencing the activity of Ty elements in S. cerevisiae. This work shows that transposition is influenced by factors acting at many stages in the transposition cycle. 24. •
CURCmOMJ, GARFINKELDJ: Posttranslational Control of Tyl Retrotransposition O c c u r s at the Level of Protein Processing. Mol Cell Biol 1992, 12:2813-2825.
Transposable elements Finnegan 867 Describes experiments that indicate that Tyl transposition may be regulated by the rate at which primary translation products are processed and by the stability of the polypeptides derived from them. 25.
PICOLOGLOUS, BROWN N, LIEBMANSW: Mutations in RAD6, a Yeast Gene Encoding a Ubiquitin-Conjugating Enzyme, Stimulate Retrotransposition. Mol Cell Biol, 1990, 10:1017-1022.
KiM AI, BELYAEVA ES: Transposition of Mobile Elements g),psy (mdg4) and hobo in Germ-Line and Somatic Cells of a Genetically Unstable Mutator Strain of Drosophila melanogaster. Mol Gen Genet 1991, 229:437-444. Gives a detailed analysis of the behaviour of gipsy and 1~gbotransposable elements in the unstable strain MS of D. melanogaster.
following the excision of P elements. The data presented do not exclude the possibili W that oligonucleotides could be used to introduce base changes into chromosomal DNA even in the absence of P element excision. 32.
FINNEGAN DJ: Eukaryotic Transposable Elements G e n o m e Evolution. Trends Genet 1989, 5:103-107.
33.
BERNE'ITMD: Nuclear DNA C o n t e n t and Minimum Generation Time in Herbaceous Plants. Proc Roy Soc ser B 1972, 181:109--1335.
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27. ,,•
MOFFATKG, GOULD JH, SMITH HK, O'KANE CJ: Inducible Cell Abalation in Drosophila by Cold-Sensitive Ricin A Chain. Development 1992, 114:681~o87. Describes a potentially powerful method for killing specific cells at defined times during Drosophila development. This involves placing cold. .sensitive mutants of the ricin A-chain under the control of the P-element promoter in environments that restrict its expression to specific cell types. The activity of the toxin is then controlled by changing the ambient temperature. 28. ••
BELLEN HJ, D'EvEI.YN DA, HARVEY M, ELLEDGE SJ: lsolation of Temperature-Sensitive Diphtheria Toxins in Yeast and their Effects on Drosophila Cells. Det,elopment 1992, 114:787-796. A similar technique to that described in [27"']. Describes a method for inducible cell ablation using a temperature-sensitive mutant of the catalytic subunit of diphtheria toxin. 29.
GI~OR GB, NASSm NA, JOHNSON-SCHIJTZ DM, PRESTON CR, ENGELSWR: Targeted g e n e Replacement in Drosophila via P Element-induced Gap Repair. Science 1991, 253:1110-1117. Describes a novel method for targeting specific base changes in Drosophila genes at their normal chromosomal location. Previously, this has not been possible. The technique makes use of repair synthesis following excision of P elements from a target gene. • •
30. ••
PLASTERKRHA, GROENEN JTM: Targeted Alterations of the Caenorhabditis elegans G e n o m e by T r a n s g e n e Instructed DNA Double Stranded Break Repair Following Tc I Excision. ~9IBO • J 1992, 1 i:287-290. A demonstration that repair synthesis following Tel excision can be used to target base changes within the genome of C elegan.~ This has previously been described for Drosophila (see 129°• ] ). BANGASS, BOYD JB: Oligonucleotide-Directed Site-Specific Mutagenesis in Drosophila melanogaster. Proc Nail Acad Sci USA 1992, 89:1735-1739. Details experiments that indicate that oligonucleotides injected into Drosophila embryos may be used ms templates for repair .synthesis
and
34. .
LEETONPRJ, SMYTH DR: An Abundant LINE-like Element Amplitied in the G e n o m e of Lilium speciosum. Mol Gen Genet 1992, in press. A description of an unusually abundant LINE.like element in Lilium. 35. .
KiRKAUMF, JAHN CL: Tec2, a Second Transposon-Like Ele m e n t Demonstrating Developmentally Programmed Excision in Euplotes crassus. Mol Cell Biol 1991, 11:4751-4759. A description of a putative Class il element responsible for DNA rearrangements associated with the activation of micronuclear genes during formation of the macronucleus in a ciliated protozoan. 36.
BIESSMANNH, VALGEIRSDO'VI'IRV, I.OFSKWA, CIflN C, GINTHER B, LEVIS RW, PARDUE M-L: He'T-& a Transposable Element Specifically Involved in 'Healing' Broken C h r o m o s o m e Ends in Drosophila. Mol Cell Biol 1992, 12:3910-3918. A comparison of the sequences of four copies of a putative LINElike element, HeTA, that may be required for telomere function in D. melanogaster •
37. *,,
BIESSMANNH, CHAbIPION LE, O'HAIR M, IKENAGA K, KASRAVI B, MASON JM: Frequent Transpositions of Drosophila melanogaster HeT-A Transposable Elements to Receding C h r o m o s o m e Ends. EMBO J 1992, in press. A description both of the behaviour of HeTA elements located at the end of a broken X chromosome in D. melanogaster and of how they. change with time. These elements appear to insert at the broken end, thus preventing its gradual erosion. Anal)sis of the sequence of one of the elements found at the broken end strengthens the idea that this is a family of LINE-like elements. 38. ®
PARDUEM-k Do Some 'Parasitic' DNA Elements Earn an Honest Living? In The Dynamic Genome. Edited by Fedoroff N, Botstein D. Cold Spring Harbor: Cold Spring Harbor tabor-atory Press; 1992, in press. A personal account of the history of HeTA elements set in the context of McClintock's studies of chromosomal instabilities in maize.
31. •
DJ Finnegan, Institute of Molecular Biology, King's Buildings, Mayfield Road, Edinburgh University, Edinburgh EH9 3JR, Scotland.
Transposable elements comprise a major fraction of eukaryotic genomes. They are studied both because of their intrinsic biological interest and because they can be exploited as valuable research tools. Many interesting papers dealing with various aspects of the biology of these elements have been published during the past year and a number of new elements have been reported. Four areas in which particularly valuable contributions have been made are the mechanisms of transposition, the regulation of transposition, the use of transposable elements as research tools, and the biological function of transposable elements.
Current Opinion in Genetics and Development 1992, 2:861-867
Introduction Transposable elements are an important component of eukaryotic genomes, making up approximately 15% of the total DNA. They occur as families of dispersed repeat sequences scattered throughout the genome, with the number of copies varying from less than ten to several hundred thousand depending on the element and species concerned. They can be classified according to their structure and presumed mechanism of transposition (Fig. 1), and fall into two main classes: Class I elements that transpose by reverse transcription of an
(a}
LTR
"gag'
RNA intermediate; and Class II elements that transpose directly from DNA to DNA. There are two groups of Class I elements. Class 1.1 elements resemble retroviruses in having long terminal repeats (LTRs) and open reading frames (ORFs) with the potential to code for polypeptides similar to the gag- and pol-encoded proteins. In this review, I shall refer to these as retrovirus-like elements. Class 1.2 elements also have ORFs similar to the retroviral gag and pol genes but have no terminal repeats and end with Arich sequences at the 3' end of their coding strands. Here, I shall refer to elements of this type as LINE-like elements
RT
LTR
Class 1.1 'gag'
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An
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Fig. 1. Schematic representation of the structure of eukaryotic transposable elements, (a) Class I elements are of two types, those that are retrovirus-like, Class 1.1, and those that are not, Class 1.2. All Class 1.1 elements have a gag-like I gag'l open reading frame (ORF), and an ORF that encodes a reverse transcriptase (RT). Some of them also have a third ORF of unknown function, indicated by (?). The long terminal repeats (LTRs) are also shown. Class 1.2 elements only have' gag" and RT ORFs. These elements have A-rich sequences (An) at their 3' ends. Within a family of such elements many copies may be truncated at their 5' ends, as indicated. (b) Class II elements have short terminal inverted repeats (arrowed}. They contain a gene encoding transposase, an enzyme that is required for their own transposition. This gene may have more than one exon (not shown here).
Abbreviations LTR--Iong terminal repeat; ORF--open reading frame.
(~) Current Biology Ltd ISSN 0959-437X
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Genomes and evolution as the first exanlples to be detected were the man~malian LINE (long interspersed elements) or L1 elements. The SINEs, short interspersed elements, which are found in hundreds of thousands of copies in human and other mammalian genomes, also have an A-rich sequence at the 3' end of one strand. It has been shown that SINEs integrate at new sites by means of a transposition mechanisms, but must do so passively as the), do not encode proteins themselves. The Class II elements have inverted terminal repeats 10-200bp long, although one family of elements in Drosophila melanogaste,, the FB fanlily, has inverted repeats much longer than this. Elements of this type encode proteins, the transposases, that are required for transposition.
Mechanisms of transposition Transposition through an RNA intermediate Retroviral-like transposable elements are believed to transpose by a mechanism that is similar to the retroviral life-cycle and this has been directly established for Tyl elements of Saccharo*lo~ces cerevLqae [1]. Transcription of the RNA transposition intermediate initiates in one LTR and terminates in the other. This RNA is then packaged into virus-like particles that are constructed of proteins encoded by the gag- and pol-like ORFs [2-]. The RNA is subsequently reverse transcribed to yield linear double-strand DNA and integrated at new sites by element-encoded proteins. Studies in which an intron is introduced into a TI,I element show that it can be precisely removed during transposition, thus demonstrating that an RNA molecule acts as a transposition intermecliate. A similar observation has been made for the retroviruslike element copia of D. me&nogaster [3"]. In this case it appears that an intron nom~ally present in copia elements can be lost during transposition. LiNE-like elements are believed to transpose by a similar mechanism but with important differences. Transposition through an RNA intemlediate has been demonstrated for I elements of D. melanogaster [4,5] and mouse L1 elements [6.]. Again, this was shown by demonstrating that introns introduced into dlese elements can be removed precisely during transposition. The reverse transcriptase required for this event is presumably encoded by the pol-like ORF of these elements. Ivanov et al. [7"] have confirmed that jockey elements of D. melanogaster encode a reverse transcriptase by expressing it in E. coli, and a less direct but equally effective method has been used to demonstrate a similar activity associated with the second ORF of the CRE elements of Trypanosoma brucei [8"], human L1 elements [9"], and I elements of D. melanogaster (T Paterson et al., unpublished data). In these studies, the reverse transcriptase coding region of a TyI element, linked to an inducible promoter, is replaced by the equivalent region derived from the element in question, and virus-like particles are purified from yeast cells expressing the hybrid Tyl elements. In each case,
these particles were associated with reverse transcriptase activities distinguishable from that of wild-type Tyl particles. Before this could be done with a human L1 element a potentially functional element had to be identified. This was not as straightforward as it may sound as these elements are heterogeneous and no genomic clone had been identified that contained the two ORFs evident in the consensus L1 sequence. A potentially active element was identified when it hybridized to an oligonucleotide derived from a region of a recently transposed but truncated L1 element, and to oligonucleotides derived from the ends of the L1 consensus sequence [10-]. The RNA transposition intermediates of a LINE-like element must contain the entire sequence of the element, thus preventing it gradually decaying during successive rounds of transposition, and the promoters from which these RNAs are synthesized are presumably located within the elements themselves. The elements jock W [11], F [12-] and I ( C McLean et aL, unpublished data) of D. melanogaster have promoters that lie within their transcriptional unit and the sanle m W be true of all elements of this type. As LINE-like elements contain both gag and pol-like ORFs, their transposition intermediates are probably packaged in virus-like particles similar to those of retrovirus-like elements. Mouse embryonal carcinoma cells contain ribonucleoprotein particles associated with both plus-strand L1 RNA and polypeptides that react with the L1 gag-like gene-encoded product [13"]. This is consistent with the idea that these cells contain L1 virus-like particles. However, such particles have neither been purified nor shown to be involved in transposition. Similar results have been obtained using human embryonal carcinoma cells [14]. Two important questions about the transposition of LINE-like elements remmn to be answered. What are the primers for the reverse transcription of their RNA intermediates, and how are the products of reverse transcription integrated into the genome? LINE-like elements contain no obvious binding sites for primers of reverse transcription and are thought to use 3'OH groups generated at nicks in target DNA for this. DNA synthesis and integration thus occur simultaneously. Apart from elements inserted at specific sites in the genome there is no evidence that LINE-like elements code for nucleases involved in integration. The nicks used for integration may be produced by enzymes that are not directly involved in transposition. The precise mechanism of integration should be revealed by studies involving in vitro transposition systems. These systems will probably comprise virus-like particles containing transposition intermediates, assuming that such particles exist, and suitable target DNAs.
Transposition without an RNA intermediate Recently, a great deal has been learnt about the mechanism by which Class II elements transpose. For many years it was unclear whether this was a conservative or replicative process. Genetic studies of plant Class II el-
Transposable elements Finnegan
ements, the Ac and Spin elements of Zea mays and the Tam elements of Antirrhinum majus, suggest that they transpose conservatively. However, the situation is more complex for P elements of 19. melanogastem on the one hand, P transposase stimulates both excision and transposition suggesting a conservative mechanism, while on the other, transposition leads to an increase in the number of P elements, suggesting that it is replicative.
fled transposase. Transposition is scored by transfecting E.. coli with DNA from the assay mix and selecting cells that harbour the target plasmid plus the marked P element but not the donor DNA. Transposition is stimulated to modest but significant levels by the addition of transposase, and transposed copies of the marked P element integrate into target DNA generating 8 bp target site duplications just as they do in vivo. The only essential components in the reaction are Mg2+, GTP, tile transposase-fraction and a donor element with intact transposase-binding sites at both ends. There appears to be no requirement for an energy source or for DNA synthesis as transposition is stimulated by non-hydrolyzable analogues of GTP but not by dNTPs. Linear P-element DNA can act as a donor molecule in the absence of any flanking sequences provided that 3'OH groups are retained at each end. These data are consistent with Engels' proposal that transposition is a conservative excision/insertion process.
A solution to this paradox has been provided by the elegant genetic experiments of Engels et al. [15]. These indicated that P elements normally excise precisely from the genome and that the double-strand gaps created in the process are repaired using either sister chromatids or homologous chromosomes as templates (Fig. 2). If the template used has no P element at the original site, then the gap will be repaired to give the wild-type sequence and a precise excision. If the template contains a P element at the original site, as would be the case if the sister chromatid were used, then repair can introduce complete or deleted elements and this will be scored as either an incomplete excision or as no excision at all. The Tcl elements of Caenorhabdilis elegans have been shown to behave in a similar manner [16].
Is transposase the only protein required for transposition in this assay? Transposase itself does not bind the 31 bp terminal inverted repeats of P elements but all/9. melanogaster cells contain a protein that does. It is not yet known whether this protein is required for transposition and this will only become clear once the protein has been purified and added to the in vitro assay along with purified transposase.
Kaufman and Rio [17 ,°] have recently developed an in vitro system for studying P transposition. In this system, a donor plasmid containing a marked P element is mixed with a target plasmid in the presence of partially puff-
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Fig. 2. Repair of a double-strand gap left after Drosophila melanogaster P-element excision, with each line representing a single strand of DNA. Thin lines represent chromosomal DNA and thick lines a strand of P-element DNA. A dashed line indicates newly synthesized material, and the large crossovers show recombination. (a) Schematic representation of a site in the genome containing a P element. (b) The same site is shown with a double-strand gap generated by excision of the P element. (c) Details the sequential repair (see arrows) of the double-strand gap using as a template DNA without a P element. This could be DNA on the homologous chromosome. (d) Demonstrates the repair of the double-strand gap using as a template DNA with a P element at the original site. This could be a sister chromatid. (e) The double-strand gap is repaired by using as a template DNA that contains base changes (black dots). These could have been introduced in vitro and inserted at an ectopic site in the genome. For all cases (c-e), the sequential repair involves: (i), invasion of template DNA by the ends of the broken strands; (ii), DNA synthesis progressing from the 3'OH ends of the broken strands; and (iii), DNA synthesis completed and the gap repaired. Resolution of the structure seen in (ii) usually occurs without the recombination of outside markers.
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Genomesand evolution P elements can insert at many sites in the genome but not at random. Hot spots for insertion have been detected and a consensus 8 bp motif has been detected for target-site duplications. The sequence of the target site is not, however, the only constraint on insertion. Spradling el al. [18-] have recently sho~xaa that P elements preferentially transpose to sites a short distance from their original position, while a P element carrying regulator 3, sequences from the engrailedgene preferentially inserts near other genes that are expressed segmentally during embryogenesis [19o]. These observations could be explained if P-element-traaasposase complexes are found to be functional for only a short time after excision, thus needing to rapidly interact with target DNA for integration to take place. This would favour integration at sites in close proximity to the donor element either because they are both on the same DNA molecule or because they are linked by protein-DNA interactions.
The regulation of transposition There must be some constraints on the activity of transposable elements as uncontrolled transposition would be deleterious for any organism. P elements regulate their own actMty by a complex process that, in part, is the result of transcriptional repression from the P promoter, exerted by a polypeptide(s) related to transposase [20°%21,22.]. Transposition of retrovirus-like elements can also be regulated [23"]. Tyl transposition is regulated post-translationally [24,] by a mechanism that affects the levels of pol}q3eptides produced by the pol-like gene. Mumoons in RAD6 lead to a hundredfold increase in Tyl transposition [25]. These mutations affect one of the enzymes involved in protein ubiquitination and may well alter the stability of one of these Tyl-encoded pol~ge ptides. Over the years, several strains of D. me&nogaster have been reported as being genetically unstable with higher than usual levels of transpositon. Some of these strains carry claromosomal mutations that increase transposition of the elements involved [26"].
Transposable elements as experimental tools Cell ablation Transposable elements are valuable experinqental tools in many organisms. They can be used as insertional mutagens and as molecular tags with which to clone target genes. Transformation systems, based on P and hobo elements have been developed for Drosophila, and P elements carrying the E. coli lac Z gene have been used to detect enhancers stimulating gene expression in specific cell types. These studies have enabled the identification of regulatory elements that would have been difficult if not impossible to detect by other means. Some of these elements are active in small numbers of cells, the functions
of which may be unknown. In such cases it would be helpful to be able to observe the effect of controlled ablation of the cells. This can be done using P transfomlation vectors carrying genes coding for conditionally active cell autonomous toxins under fl~e control of regulatow sequences that stimulate their expression in the cells of interest {27"',28,,]. This system should prove to be particularly useful for studying tile functions of particular cells or groups of cells in complex tissues such as the nervous systenl.
Targeted alteration of genes in vivo Conclusions about the functions of regulator}, or coding sequences can only be made with confidence once the effects of changes in these sequences have been studied in otherwise wild-type genes. Exogenous DNA can replace endogenous sequences by recombination in some species but not in others, including D. melanogaster and C. elegans. P-element mediated transfom~ation can be used to introduce modified sequences at ectopic sites in the Drosophila genome but cannot replace wild-type sequences with DNA that has been modified i,7 vitro. This complicates many studies because effects on gene expression or protein function as the result of changes in sequence m W be masked by changes in gene expression mediated by flanking DNA at ectopic sites. Engels and his colleagues [29"] have overcome this problem by developing a method that allows specific base changes to be introduced into genes near sites of P-element insertion. They noted that ectopic copies of the white gene can serve as templates for repairing gaps resulting from excision of a P-element ,and argued that if repair synthesis were to extend some distance to either side of a gap, and if and the template differed in sequence from the original gene, then these differences would be transferred from one copy of the gene to the other (Fig. 2). Indeed this is the case and Gloor et aL [29"] have used the method to transfer up to 12 single base changes over a 2.8 kb region surrounding the site of a P-element insertion in white. TcI elements can be used in a similar way in C. elegans [30"]. This is similar to gene conversion associated with recombination and the average distance over which information is transferred from one copy of the gene to another is similar to the length of conversion tracts in meiotic recombination in D. melanogaster. Banga and Boyd [31"] have suggested that injected oligonucleotides can be used as templates for repair after P-element excision. This is an exciting possibility that should be investigated further as the data presented are not extensive and it is not clear whether transposase or even the oligonucleotide is required.
Do transposable elements serve useful functions? Transposable elements comprise a significant fraction of most eukaryotic genomes and yet transposition is dele-
Transposableelements Finnegan 865 terious under most circumstances. Why then are these elements maintained rather than being eliminated by selection? Transposition usually leads to an increase in the number of copies of an element in the genome so the answer could simply be that selective losses are balanced by transpositional gains. The alternative is that transposable elements are maintained by selection because they perform functions that are advantageous in the long-term. The most obvious function performed by transposable elements is to cause mutations. This may not appear beneficial but transposable elements cause some changes that are difficult to generate by other means [32] ,and the advantage accruing from this might be sufficient to maintain some elements. Another obvious effect of transposable elements is to increase the total mass of the genome. This may be of selective value particularly in plants in which there is a relationship between genome DNA content and generation time [33]. The LINE-like element, del2, of Lilium speciosum [34"] is present in about 250 000 copies (contributing nearly ten Z). melanogaster genome equivalents to this species) and m W be retained because it increases the genome mass. The ability of Class II elements to excise from the genome may also be advantageous in some circumstances. Many species are known to delete substantial anlounts of genomic DNA at specific times during development in a process known as genome diminution. In the ciliated protozoan Euplotes crassus, this is the result of the excision of sequences that appear to be Class II elements [35"]. This occurs during the formation of transcriptionally active macronuclei from transcriptionally silent micronuclei and ,night have evolved to minimize selection against insertions within genes. Indeed, some genes are known to be interrupted by such elements in micronuclear DNA but not in macronuclei. It is equally possible that it is excision of these elements that activates macronuclear genes, and dais may have been exploited by the organism as a means of regulating gene expression. A form of genome diminution is seen in polytene chromosomes of Drosophila where DNA in heterochromatic regions of the genome is under-represented. This has been assumed to be because of the differential replication of euchromatic and laeterochromatic sequences. Spradling el al., [18"] have pointed out that it could equally well be attributed to the excision of transposable elements concentrated in heterochromatin. Members of a family of putative transposable elements, the HeT-A elements, are associated with telomeres in 1). melanogaster [36",37"',38"]. These elements are located in centromeric heterochromatin and at telomeres but not in euchromatin. It is not clear whether they provide the function of the short repeats found at telomeres in other species - - thus far, no such repeats have been identified in 1)rosophila. The original HeT-A element was found on a fragment of DNA believed to derive from the tip of an X chromosome and other copies have been found at the broken ends of terminally deleted X chromosomes. These elements are similar to LINE-like transposable elements as there is a short run of A residues at the junction
between the 3' end of HeT-A DNA and X chromosome sequences. Furthermore, about 2.3 kb upstream of this there is a gag.like ORF similar to that in Drosophila LINE-like elements. No ORF that could encode a reverse transcriptase has yet been found but then nor has a complete element been sequenced. LINE-like elements are believed to reverse transcribe their RNA transposition intermediates by associating the A-rich sequence at the 3' end of the RNA with chromosomal DNA that is nicked in both strands. An element that could also do this at the ends of DNA molecules might eventually take over telomere functions. If HeT-A elements are LINE-like transposable elements, then their sequence organization is atypical but this is not surprising if the), have become adapted to perform a particular function within the genome.
Concluding remarks Nearly twenty years have passed since eukaryotic transposable elements were first identified at the molecular level. In that time, they have moved from being interesting peculiarities to being recognized as important components of all genomes. Much has been learnt about how they transpose and we have some understanding of how transposition is regulated. As a result, several elements have been tanaed and put to work in the laboratory. Mthough much remains to be learnt in all of these areas, the biggest gaps in our knowledge lie in understanding the significance of transposable elements for the structure and function of eukat3,otic genomes.
Acknowledgements I am grateful to H Biessmann, M-L Pardue, R Plasterk, S Ronsseray and A Spradling for discussing their unpublished data with me, and to G Bn,an for comments on this manuscript. My work is supported by research grants from the Medical Research Council and the Human Frontier Science Progranl.
References and recommended reading Papers of particular interest, published within tile annual period of review, have been highlighted as: , of special interest ,,. of oucstanding interest 1.
BOEKEJD: Transposable Elements in Saccharomyces cerevisiae. In Mobile DN,,t Edited by Berg DE, Howe MM. Washington DC: American Society for Microbiology; 1989:335-374.
2. •
BURNSNR, SAIBII. HR, WHITE NS, PARDON jF, TIMMINS PA, RICHARDSON,SMH, KINGSMAN SM, KINGSMAN AJ: Symmetry, Flexibility and Permeability in t h e Structure of Yeast Retrotransposon Virus-Like Particles. ~lqBOJ 1992, 11:1155-1164. The first detailed report of the structure of virus-like particles produced by a mmsposable element. The authors show that these particles are more variable in structure than those of retroviruses. 3. •
YOSHIOKIK, KANDA H, AKIBA H, ENOKI M, SHIBA T: I d e n tification of an Unusual Structure in t h e Drosophila
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Genomes and evolution melanogaster Transposable Element copi~ Evidence for copia Transposition T h r o u g h an RNA Intermediate. Gene 1991, 103:179-184. Direct evidence that a retrovirus-like element in Drosophila transposes by reverse transcription of an RNA intermediate. The data suggest that the RNA transposition intermediates of copia elements contain a sequence that favours packaging into vires-like particles. 4.
P.:';dJSSONA, FINNEGAN, DJ, BUCHETON A: Evidence for Retrotransposition of t h e I Factor, a LINE e l e m e n t of Drosophila melanogaster. Proc Natl Acad Sci USA 1992, 88:4907-4910.
5.
JENSEN S, HEIDMANN T: An Indicator Gene for Detection of Germline Retrotransposition in Transgenic Drosophila Demonstrates RNA-Mediated Transposition of the LINE 1 Element. ~%¢BOJ 1991, 10:1927-1991.
6. EVANS JP, PAIJ~IlTER RE): Retrotransposition of a Mouse L1 T Element. Pt~c Natl Acad Sci USA 1991, 88:8792-8795. he first direct evidence that mammalian LINE elements transpose through an RNA intermediate, as well as the first report of these elements transposing in an experimental situation. 7. .
IVANOVVA, MELNIKOVA, SIUNOV AV, FODOR I[, ILYIN YV: Authentic Reverse Transcriptase is Coded by jockey a Mobile Drosophila Element Related to Mammalian LINEs. EMBOJ 1991, 10:2489-2495. Direct evidence that the pol-like ORF of a Drosophila retrovims-like element encodes a reverse transcriptase. Most of the po/-like gene was expressed in E. coil and encoded a protein with reverse transcriptase but not RNAse H activity. GABRIELA, BOEKE JD: Reverse Transcriptase Encoded by a Retrotransposon from the Trypanosomatid Crythidia fasciculata. Proc Natl Acad Sci USA 1991, 88:9794-9798. Direct evidence that a LINE-like element encodes a reverse transcriptase. In this case, the reverse transcriptase coding region of a Tj,1 element is replaced by the equivalent region derived from a CRE1 element. The Tyl element used is transcribed from an inducible promoter, thus enabling high levels of expression. The resulting hybrid Tyl-CREviruslike particles are associated with a reverse transcriptase activity that is abolished when the CRE sequences are mutated. 8. •
9. ,
MATIilASSL, SCOTt AF, KAZAZIANHH, BOEKE JD, GABRIEL A: Reverse Transcriptase Encoded by a H u m a n Transposable Element. Science 1991, 254:1808-1810. Direct evidence for a reverse transcriptase encoded by a human L1 element. The method used and results obtained are similar to those described in [8*]. An epitope tag is used to detect the Ty1-L1 fusion polypeptide in the vires-like particles. 10. •
DOMBROSKI BA, MATI-IbVS SL, NANTt-bXKUMAR E, SCOTT AF, KAZAZI~'~HH: Isolation of an Active H u m a n Transposable Element. Science 1991, 254:1805-1807. Description of the cloning of a potentially active human L1 element. This appears to have been the donor of a truncated element found inserted within the Factor Vlli gene of a haemophiliac patient. 11.
MIZROKHm LJ, GEORGIEVA SG, ILYIN YV: jockey, a Mobile Drosophila Element Similar to Mammalian LINEs, is Transcribed from t h e Internal Promoter by RNA Polymerase II. Cell 1988, 54:685--691.
12. •
MINCHIOTTIG, DI NOCERA PP: Convergent Transcription lnitiates from Oppositely Oriented Promoters within the 5' End Regions of Drosophila melanogaster F Elements. Mol Cell Biol 1991, 11:5171-5180. Describes an analysis of the promoter of the Drosophila LINE-like element E Sequences from the beginning of an F element are linked to a chloramphenicol acetyl transferase reporter gene and assayed in transfected cells. The results are complex but provide evidence for a promoter lying within the transcribed region and initiating transcription near the start of the element. A promoter that transcribes in the opposite direction is also observed. 13. •
MARTINSl2 Ribonucleoprotein Particles with LINE-1 RNA in Mouse Embryonal Carcinoma Cells. Mol Cell Biol 1991, 11:4804--4807.
A concise paper that details convincing evidence for there being ribonucleoprotein particles, which contain L1 RNA and proteins, in mouse embryonic carcinoma cells. 14.
DERAGON J-M, SINNETT D, LABUDA D: Reverse Transcriptase Activity from H u m a n Embryonal Carcinoma Cells NTera2D1. EMBO J 1990, 9:3363-3368.
15.
ENGELS XY.'R,JOHNSON-SCHLITZ DM, EGGIESTON XVB, SVED J: High-Frequency P Element Loss in Drosophila is Homolog Dependent. Cell 1990, 62:515-525.
16.
PLASTERKRH: The Origin of Footprints of the T c l Transposon of Caenorhabditis elegans. ~ I B O J 1991, 10:1919-1925.
17. **
KAUFMANPD, RIO DC: P Element Transposition In Vitro Proceeds by a Cut-and-Paste Mechanism and Uses GTP as a Cofactor. Cell 1992, 69:27-39. Tile first description of an in vitro system for studying tile transposition of Class ll elements. Transposition requires added Mg2+ and GTP but not dNTPs. The donor P e l e m e n t must have intact transposase-binding sites at both ends but can be linear or circular. Linear molecules that end at the P termini can act as donors provided that they have 3'OH groups. This is a major step forward to understanding the mechanisms by which elements of this type transpose. 18. •.
SPRADUNGAC, KARI'EN G, GLASER R, ZH.M~G P: Evolutionary Conservation of Developmental Mechanisms: DNA Elimination in Drosophila. In EvolutionaoJ Conservation of De, velopmental Mechanismx Edited by Spradling A. New York: Wiley-Liss; 1992:39-53. This stimulating paper discusses the possibility that the elimination of DNA from chromosomes in somatic cells is not a rarity. The authors suggest that this p h e n o m e n o n may explain the under-representation of some sequences in polytene chromosomes, and that it may both contribute to position effect variegation and influence gene expression. These ideas are based in part on the authors' study of the behaviour of a mini-chromosome in D. melanogaster. 19. •
KASSmSJA, Non. E, VANSICKLE EP, ODENWALD \X/F, PERRIMON N: Altering t h e lnsertional Specificity of a Drosophila Transposable Element. Proc Natl Acad Sci USA 1992, 89:191 9-1923. Describes the behaviour of a P element carrying regulatory sequences from the Drosophila engrailedgene. The results indicate that the integration of P transfomlation vectors can be influenced by the sequences that they carry. This may help clarif3, the mechanism of target-site selection by transposable elements and factors that influence the organization of DNA sequences within nuclei. 20. RiO DC: Regulation of Drosophila P Element Transposition. •, Trends Genet 1991, 7:282-287. An excellent discussion of the regulation of P element transposition, which is subject to both tissue-specific and genetic controls. Transposition is restricted to the germ-line because a splicing event that is required for transposase production is inhibited in somatic cells. The factors that regulate transpositon in geml-cdls, as well as their inheritance, are less well understood. 21.
I..EMAITREB, COEN D: P Regulatory Products Repress in Vivo the P Promoter Activity in P-lacZ Fusion Genes. Proc Natl Acad Sci USA 1991, 88:4419--4423.
LE,VadTREB, RONSSERAYS, COEN D: P Cytotype Repression of the P Promoter is Exclusively Maternal in t h e Germline: A Model for P Cytotype. Genetics 1992, in press. Describes the use of P-lacZ fusion genes to investigate how Pelements are regulated. These experiments show that in strains carrying complete P elements lacZ expression is inhibited more strongly in germ cells than in somatic cells. The factors influencing expression in germ cells are inherited maternally, whereas those affecting somatic expression are not. The authors suggest an interesting model to explain these results. 22. •
23. CURCIOMJ, GARFINKELDJ: Regulation of Retrotransposition in •. Saccharomyces cerevisiae. Mol Microbiol 1991, 5:1823-1829 An excellent discussion of the factors influencing the activity of Ty elements in S. cerevisiae. This work shows that transposition is influenced by factors acting at many stages in the transposition cycle. 24. •
CURCmOMJ, GARFINKELDJ: Posttranslational Control of Tyl Retrotransposition O c c u r s at the Level of Protein Processing. Mol Cell Biol 1992, 12:2813-2825.
Transposable elements Finnegan 867 Describes experiments that indicate that Tyl transposition may be regulated by the rate at which primary translation products are processed and by the stability of the polypeptides derived from them. 25.
PICOLOGLOUS, BROWN N, LIEBMANSW: Mutations in RAD6, a Yeast Gene Encoding a Ubiquitin-Conjugating Enzyme, Stimulate Retrotransposition. Mol Cell Biol, 1990, 10:1017-1022.
KiM AI, BELYAEVA ES: Transposition of Mobile Elements g),psy (mdg4) and hobo in Germ-Line and Somatic Cells of a Genetically Unstable Mutator Strain of Drosophila melanogaster. Mol Gen Genet 1991, 229:437-444. Gives a detailed analysis of the behaviour of gipsy and 1~gbotransposable elements in the unstable strain MS of D. melanogaster.
following the excision of P elements. The data presented do not exclude the possibili W that oligonucleotides could be used to introduce base changes into chromosomal DNA even in the absence of P element excision. 32.
FINNEGAN DJ: Eukaryotic Transposable Elements G e n o m e Evolution. Trends Genet 1989, 5:103-107.
33.
BERNE'ITMD: Nuclear DNA C o n t e n t and Minimum Generation Time in Herbaceous Plants. Proc Roy Soc ser B 1972, 181:109--1335.
26. •
27. ,,•
MOFFATKG, GOULD JH, SMITH HK, O'KANE CJ: Inducible Cell Abalation in Drosophila by Cold-Sensitive Ricin A Chain. Development 1992, 114:681~o87. Describes a potentially powerful method for killing specific cells at defined times during Drosophila development. This involves placing cold. .sensitive mutants of the ricin A-chain under the control of the P-element promoter in environments that restrict its expression to specific cell types. The activity of the toxin is then controlled by changing the ambient temperature. 28. ••
BELLEN HJ, D'EvEI.YN DA, HARVEY M, ELLEDGE SJ: lsolation of Temperature-Sensitive Diphtheria Toxins in Yeast and their Effects on Drosophila Cells. Det,elopment 1992, 114:787-796. A similar technique to that described in [27"']. Describes a method for inducible cell ablation using a temperature-sensitive mutant of the catalytic subunit of diphtheria toxin. 29.
GI~OR GB, NASSm NA, JOHNSON-SCHIJTZ DM, PRESTON CR, ENGELSWR: Targeted g e n e Replacement in Drosophila via P Element-induced Gap Repair. Science 1991, 253:1110-1117. Describes a novel method for targeting specific base changes in Drosophila genes at their normal chromosomal location. Previously, this has not been possible. The technique makes use of repair synthesis following excision of P elements from a target gene. • •
30. ••
PLASTERKRHA, GROENEN JTM: Targeted Alterations of the Caenorhabditis elegans G e n o m e by T r a n s g e n e Instructed DNA Double Stranded Break Repair Following Tc I Excision. ~9IBO • J 1992, 1 i:287-290. A demonstration that repair synthesis following Tel excision can be used to target base changes within the genome of C elegan.~ This has previously been described for Drosophila (see 129°• ] ). BANGASS, BOYD JB: Oligonucleotide-Directed Site-Specific Mutagenesis in Drosophila melanogaster. Proc Nail Acad Sci USA 1992, 89:1735-1739. Details experiments that indicate that oligonucleotides injected into Drosophila embryos may be used ms templates for repair .synthesis
and
34. .
LEETONPRJ, SMYTH DR: An Abundant LINE-like Element Amplitied in the G e n o m e of Lilium speciosum. Mol Gen Genet 1992, in press. A description of an unusually abundant LINE.like element in Lilium. 35. .
KiRKAUMF, JAHN CL: Tec2, a Second Transposon-Like Ele m e n t Demonstrating Developmentally Programmed Excision in Euplotes crassus. Mol Cell Biol 1991, 11:4751-4759. A description of a putative Class il element responsible for DNA rearrangements associated with the activation of micronuclear genes during formation of the macronucleus in a ciliated protozoan. 36.
BIESSMANNH, VALGEIRSDO'VI'IRV, I.OFSKWA, CIflN C, GINTHER B, LEVIS RW, PARDUE M-L: He'T-& a Transposable Element Specifically Involved in 'Healing' Broken C h r o m o s o m e Ends in Drosophila. Mol Cell Biol 1992, 12:3910-3918. A comparison of the sequences of four copies of a putative LINElike element, HeTA, that may be required for telomere function in D. melanogaster •
37. *,,
BIESSMANNH, CHAbIPION LE, O'HAIR M, IKENAGA K, KASRAVI B, MASON JM: Frequent Transpositions of Drosophila melanogaster HeT-A Transposable Elements to Receding C h r o m o s o m e Ends. EMBO J 1992, in press. A description both of the behaviour of HeTA elements located at the end of a broken X chromosome in D. melanogaster and of how they. change with time. These elements appear to insert at the broken end, thus preventing its gradual erosion. Anal)sis of the sequence of one of the elements found at the broken end strengthens the idea that this is a family of LINE-like elements. 38. ®
PARDUEM-k Do Some 'Parasitic' DNA Elements Earn an Honest Living? In The Dynamic Genome. Edited by Fedoroff N, Botstein D. Cold Spring Harbor: Cold Spring Harbor tabor-atory Press; 1992, in press. A personal account of the history of HeTA elements set in the context of McClintock's studies of chromosomal instabilities in maize.
31. •
DJ Finnegan, Institute of Molecular Biology, King's Buildings, Mayfield Road, Edinburgh University, Edinburgh EH9 3JR, Scotland.