Annual Reviews
Annu.Rev. Mic.,obiol. 1993.47:94543
Copyright©1993by AnnualReviewsInc. All rights reserved


Annu. Rev. Microbiol. 1993.47:945-964. Downloaded from
by University of Wisconsin - Madison on 10/19/05. For personal use only.

W. S. Reznikoff
,~)f Biochemistry,Collegeof Agriculturaland Life Sciences,University
of Wisconsin-Madison,

Madison, Wisconsin 53706

transposase, end sequences, gene regulation, cis-activity

The Reguhttion of Transposase and Inhibitor Protein Synthesis ............
The Transposase N-Terminal Sequence Is Critical for Sequence-Specific DNA
Binding and Other Transposase Activities
Recognition of Terminal 19-Base Pair Sequences ....................
pl Is a Cis-Active Transposase (and a Trans-Active Transposition Inhibitor) ....
Transposition MayBe Linked to Chromosome
Replication and~or Cell Division . .



The bacterial transposon Tn5 encodes two proteins, the transposase and a
related protein, the transposition inhibitor, whoserelative abundancedetermines, in part, the frequency of Tn5 transposition. The synthesis of these
proteins is programmed
by a complexset of genetic regulatory elements. The
host DNA
~aethylation function, dam,inhibits transposase promoter recognition and indirectly enhancesthe transposition inhibitor promoter.The inhibitor
lacks the N-terminal55 aminoacids of the transposase, suggesting that this
sequence plays a key role in the transposition process. Anintact N-terminal
sequence is required for the transposase’s recognition of the 19-bp end DNA
sequences.This is the first critical step in the transposition process. Transposase-end DNAinteraction is itself regulated by an intricate series of
reactions involving several host proteins: DnaA,Dam,and Fis. The transposase is a uniqueprotein in that it acts primarily in cis and inhibits its own
activity in trans. Modelsto explain these properties are described. Finally
circumstantial evidencesuggests that transposition occurs preferentially from
newly replicated DNAthat has yet to be partitioned to progenycells. This

Annual Reviews


timing of transposition is likely to havea selective advantagefor the host and
the transposable element.

Annu. Rev. Microbiol. 1993.47:945-964. Downloaded from
by University of Wisconsin - Madison on 10/19/05. For personal use only.

Transposition is a recombination process in which DNAsequences termed
transposable elements movefrom an original site on a DNAmolecule to a
newsite on the sameor on a different DNA
molecule. In addition, transposable
elements can cause, and are associated with, other types of genetic rearrangements such as deletions, inversions, and chromosomefusions. The genomes
of prokaryotic and eukaryotic organisms contain these elements. One could
consider themas an ancient genetic machineryfor causing genomicrearrangements and, therefore, for facilitating genomeevolution. In addition, their
associated biochemicalreactions are likely to be similar to other interesting
events involving the interaction of proteins and DNA.For these reasons
transposable elements are of considerable interest.
The transposable elements found in Escherichiacoli fall into three general
classes determined by their mechanismsof transposition. Transposons such
as Tn3 and "~ transpose through a two-step replicative mechanismin which
a cointegrate (fused replicon) structure is an intermediate. TransposonsTnlO
and Tn903 transpose through a conservative cut-and-paste mechanism.
BacteriophageMuand related viruses represent the third class of transposable
element. In these cases the transposition can occur through either of the above
two mechanisms,depending upon the proteins involved and the precise nature
of the DNAstrand cutting after an intermediate is formed between the
transposable element and the target DNAsequence. Tn5 is generally assumed
to transpose via a conservative mechanism(2); however,this presumptionhas
not been critically tested. The reader is encouragedto examinethe models
and evidence for these transposition mechanismsin the recent monographby
Berg & Howe(3).
The conservative and replicative mechanismsof transposition share many
basic characteristics. The transposable element encodestwo critical functions
required for the process--the end sequences and a protein termed the
transposase. The element is defined by the specific sequences at its end.
Transposition and related events removethe transposable element from its
original sequence context precisely at the ends of these sequences. Changes
in any base pair of these sequences typically reduces the frequency of or
abolishes transposition. The transposable element also encodesa protein called
the transposase. Thetransposase is a critical participant in manytransposition
functions including: specifically binding to the end sequences, bringing the
twoends together through a protein oligomerization process, cutting or nicking
the DNAadjacent to the end sequences, and inserting the transposable element
DNAinto a DNAtarget site.

and transposable element end sequences maybe a perfect .position process may give us insights as to how host DNA metabolismis organized and regulated.understanding their role in the tran. The terminal DNAsequences are at first glance simply an exampleof a target for protein binding. there are always surprising connections in biology. For personal use only.In addition to the role of host functions. Studyingthese events will help us understandother genetic processes. 1993. a quite rare. if the host functions that Tn5uses are organized in the cell in a unique sp~ttial fashion. Protein structure/function studies would seek answers to the questions about the organization of the peptide domains that perform these functions and how they interact. and the strategies. and regulating several steps in the transposition process. . Becausemanyof these host functions are ones involved in host DNA metabolism.annualreviews. example of. TN5 TRANSPOSITION 947 Host proteins also play critical roles in the transposition process such as facilitating the end-sequence binding of the transposase.47:945-964. However.Annual Reviews www. The mechanismsof the regulation vary remarkably among the manycases studied. studying Tn5transposition mayreveal that arrangement. highly regulated process. Rev. Finally.annualreviews. The transposase is a protein that performs multiple complexfunctions.their functionality is considerably more complexbecause they are often the target for more than one protein.tion mightenable the host cell to strike a balance betweeninsuring proliferation of the transposable element and insuring the cell’s owngenetic survival--the very process of transposition causes chromosome breakage and rearrangements. Workdone on other systems suggests that short DNA sequencescan dictate several extremely intricate reactions. are similar to ones found for other bacterial genetic systems.. Downloaded from arjournals. Transposition is. elucidating howtransposition is regulated in a given systemwill tell us how complex DNAmetabolizing processes might be controlled and give yet more examples of how gene expression can be by University of Wisconsin . Understanding how they use these functions will tell us muchabout the transposition process and about the functionality of the host functions themselves. nucleating the higher-order structure in whichthe ends are brought together. and the protein-end sequence interaction is associated with at least two events: protein binding and DNAstrand scission.. such compact complexity. Microbiol. By studying transposition we embarkon a largely unknown path into the cell’s metabolism. For instance. although some aspects are commonto all. Implicit in the above general description is the fact that several very interesting molecular events are involved in and regulate the transposition process.Madison on 10/19/05. in general. performingthe necessaryrepair or replication Annu. transposable elementencoded-functionsoften play a critical role in this regulation. perhaps not surprisingly. For instance. Such tight regul~. Transposable elements use host functions like biochemical parasites.

In cis it acts as a transposase. 948 REZN1KOFF Tn5 OE p3 Annu. Mylaboratory and several other investigators have been studying the bacterial transposable element Tn5as a model system. ’~ IS50R OE pl (Tnp) p2 (Inh) Figure1 Transposon Tn5.but the InhAUG is 55codonsdownstream fromthe TnpAUG. The possible function of this sequenceis an importanttopic of investigation. 19.annualreviews. A secondprotein [the inhibitor (Inh or p2)] is also encodedby IS50R(15. Interestingly. and . Rev. 19). ISSOL andIS50R. IS50Lcontainsan ochrecodonthat results in the synthesisof p3 andp4. and this review discusses our current understandingof this function. catalyzing the transposition of the Tn5 or IS50 sequences from which it was encoded (15.Annual Reviews www. Thus the regulation of these promoter activities becomesa crucial question.annualreviews. ISSORand IS50L(see Figure 1 for a schematic). which is thought to be the major meansof down-regulatingthis by University of Wisconsin .Tn5is a compositetransposonin whichgenesencodingthree antibioticresistanceproteinsare bracketed bytwoIS50elements. However. it is the host that regulates the relative activity of these twopromoters.IS50R encodes the transposase (Tnpor pl) anda second proteinthat inhibitstransposition (Inhor p2). apparently competing. nonfunctional analogues of TnpandInh. The paradoxof Tnpinhibiting the activity of other Tnpmoleculesis discussed in greater detail below. BothIS50 elements are delineatedby19-bpsequences. 1993. As discussed below. TnpandInh are translatedin the samereadingframe. IS50Ris a fully functional transposable element. The properties of Inh relative to the Tnpsuggest that the N-terminal 55 amino acids of Tnp play an important role in its activity. 33). 18. For personal use only. 18.Madison on 10/19/05. while IS50L contains an ochre codonthat results in the synthesis of inactive proteins (33).47:945-964. Inh’s only knownfunction is to inhibit Tn5 trans it primarily acts as an inhibitor of transposition (42). p4 IS50L IE kanr sttr bleo r IE ~ "~ ~. and the Tnphas twoopposingactivities. Inh is translated in the samereading frame as Tnpbut lacks the N-terminal 55 amino acids. Downloaded from arjournals. 43). IS50Rencodes the transposase (pl or Tnp) (15. promoters programTnp and Inh syntheses (21. The Tnp inhibitory activity is one meansby which Tn5 transposition is down-regulated. 45). The relative abundanceof Tnp and Inh plays a major role in determining the Tn5transposition frequency. Microbiol. separate. Tn5is an exampleof a composite transposon in which antibiotic resistance genes are flanked by two nearly identical insertion sequences. the insideend(IE)andthe outsideend(OE).

Whatsort of protein recognition reactions occur at the 19-bp terminal sequences? 4. Downloaded from arjournals. this chapter concentrates on areas of current and future research interest raised by the questions implied above. distinguisl~ting feature betweenIS50 and Tn5transposition is the choice of the transposable element by University of Wisconsin . 1993. a host function binds to the OEto enhance transposition while the host functions affecting the IE down-regulatetransposition. In addition.In addition. The translation initiation signals were designedsuch that they will only function a:~ part of a correctly initiated TnpmRNA (21. Douglas Berg (whose laboratory has contributed muchof what we know about Tn5) recently published an excellent review of Tn5(2). For personal use only. . As we shall see.Annual Reviews www. 37). Howmight transposition be linked to chromosome replication and/or cell division? The Regulation of Transposase and Inhibitor Synthesis Studies by Biek & Roth (4) first indicated that the frequency of Tn5 transposition was regulated by Tn5-encodedfunctions. which point to DNAreplication. have been suggested above. The specific topics covered are: 1. Tn5 sequences that were newlyintroducedinto a cell transposed at dramatically lower frequencies in a cell already containing Tn5as opposedto a cell lacking TnS. Twoof the relevant host functions (DnaAand Dam)also link the transposition process to DNA replication. this review is ntot a comprehensivetreatment of the subject. 34). Whatis the molecularbasis for the cis-active nature of the transposase? 5. Whatare the possible functions for the TnpN-terminal sequence? 3. TN5 TRANSPOSITION 949 the regulatory mechanismthat has evolved appears to link the occurrence of transposition to the DNAreplication process.Madison on 10/19/05. Tnp binds to both of these sequences during the transposition process. Rather. Microbiol. The strategy accomplishthis translation control maybe widespreadand is discussed below. Tn5 transposition utilizes two OEs. they are the sequencesrecognized by host functions.annualreviews. whereasIS50 transposition uses an OEand an IE sequence. Howare the syntheses of Tnp and Inh regulated? 2. Each IS50 is boundedby two unique 19-bp end sequences [termed outside (OE)and inside (IE) ends] that are critical for transposition (17. Someof these clues. Rev. Finally. we are just nowbeginning to obtain clues as to the possible relationship of Tn5transposition with overall cell processes. Therefore. and they likely perform other functions vital for Annu. Others will becomeobvious during the discussion of the lethal effect of overproducing Tnp.47:945-964. Tn5 has also evolved a mechanismfor preventing the spurious synthesis of Tnpby virtue of accidental placementwithin an active transcription unit (21).

and site-specific mutation studies prove. What then determines the abundanceof Tnp and Inh? The answers to this question are not only interesting for Annu.~ (Inh) TI-35 GATCT(~ATC LexA? Figure2 Controlling elements for Tn5/ISS0 TnpandInhsynthesis.Annual Reviews fivefold increase in T1 mRNA and a twofold decrease in T2 mRNA. see below) a consequence the Tn5-encodedInh protein. . probably competitive.The5’ endof IS50is defined bythe 19-bpOEsequence (see Figure4. TheAUGs for TripandInh are indicated(21). but the amountof cis-acting Tnp per Tn5 remains constant. Between the -35and. Moreover. Suchoverlapping promoters are found in other systems (e.g. the concentration of trans-acting Inh increases. An inspection of the DNAsequence indicates. promoters.47:945-964.annualreviews. Overlapping the TI . . Rev. as the copy numberof Tn5increases. For personal use only.Thepromoter for transposase synthesis(T1) initiates transcription66bpfromthe endof IS50(21). and this change in transposition seems to mirror (and presumablyis the consequence of) a four. The frequency of Tn5 transposition is 10-fold higher in damhosts.10regionare twoDam methylation sites that whenmethylated down regulateTnpmRNA synthesis(43).Madison on 10/19/05. that the relevant GATCdarn T1 AUG (Tnp) T2 OE -. Microbiol. Inh functions in trans to block transposition. they also reveal genetic regulatory motifs found in other systems. The opposite effect on promoter activity suggests that the promoters compete for RNApolymerase.annualreviews. below). TheT2(Inh) promoteroverlaps T1(21). As shownin Figure 2. This conclusion was deduced by a deletion analysis of in vivo promoter activities (21). Wenowknowthat this down-regulationis primarily (but not entirely. Downloaded from arjournals.. Thus the incoming Tn5 in the Biek & Roth experiment(4) encountereda preexisting pool of inhibiting Inh. the T2 transcript can only encodethe Inh while the T1 transcript encodes both proteins [but the Inh is translated inefficiently from the T1 message(37)]. 950 REZNIKOFF we (18) found that the Tn5 transposition frequency was constant regardless of the copy numberof Tn5(hence the transposition frequencyof an individual Tn5 decreased in the presence of additional Tn5s).org by University of Wisconsin . DNA(dam) methylation down-regulates the synthesis of the T1 (Tnp) transcript and appears to up-regulate the synthesis of the T2 transcript (43). see 30) and can programcontradictory functions.10regionsof is a weakLexA bindingsite (23).~. 1993. Thus by studying this Tn5 arrangement we will elucidate a more general regulation strategy.the frequency of transposition is in part set by the abundanceof Tnp and the ratio of Tnp to Inh. Tnp and Inh are expressed from overlapping.

Thisfigureis similarto one shown in Ref. Downloaded from arjournals.10 region of the T1 promoter. Moreover.Annual Reviews Annu. .Madison on 10/19/05.annualreviews. by University of Wisconsin . However. Microbiol. Thestart of the T1transcript(which will notforma secondary structure)andthe TnpAUG are indicated. 37). GUC C-~ U _~u U IJCCCGUUUUCCAGG G U AACUUCUGCUCUU 110 40 Figure 3 Secondary structurein read-through transcriptsblocksTnptranslation. 32).The specific relevance of damregulation of Tnp(and Inh) synthesis is that it should couple transposition to DNAreplication (about which more is discussed below) because newly replicated DNAis hemimethylated.47:945-964. Other transposon systems [in particular TnlO (31)] also display Dam methylation control of Tnp synthesis and of transposition. Also.these studies der~aonstrate in a quite precise mannera type of gene regulation displayed in many systems--chemical modification of specific DNAsequences to modulate protein binding. this regulation should stimulate transposition off of DNAthat is newly introduced into the cell. Tn5cou]td transpose into highly expressed genes. Rev. This mechanismreappears in the discussion of protein recognition of the IS50 IE sequence.:21.annualreviews.Transcripts that readthroughthe endof ISSOdonot expressTnpbecauseof a secondary structurethat occludes the Shine-Dalgarno sequence endandthe AUG (21.a set of experiments(21) has demonstratedthat read-through expression of transposase does not occur because such messagesdo not programthe translation of Tnp. For personal use only. Production of these transcripts lmight result in spurious transposase synthesis. thereby giving rise to read-through transcripts into the transposase gene. TN5 TRANSPOSITION 951 methylationsites overlap with the . whichis exactly the observedresult (27.

org by University of Wisconsin . Reznikoff. and (b) the sequence contexts of the Tn5 systems were different. presumably by binding to a weak LexAbinding site upstream of the T1 promoter (23) (see Figure 2).Many genetic regulatory proteins are enhanced in their DNA-bindingactivities through cooperative binding to a secondarysite (1). IS3 (39).Madison on 10/19/05. whethersuch a fortuitous nearby LexAbinding site could facilitate LexAbinding to its weakTn5 site. Jilk &W. M. For instance. The same mechanismappears to be shared by several other transposable elements [IS2 (13). 1993. As shownin Figure 3. suggesting that the N-terminal 55 aminoacids encodea domainof importance. unpublished results). Downloaded from arjournals. IS5 IS150 (38). and TnlO (5)] and maybe a general motif for Escherichia coli gene systems [for instance. IS4 (20). Our laboratory has found that LexAhas no significant control over transposase levels (40). the samepattern is seen for lac (36. 45). RNAsequences upstreamof the T1 transcript start site are necessaryfor the formationof this secondary structure. For personal use only. de la Cruz. Do other mechanismsexist that regulate transposase synthesis? Recent reports present conflicting evidence regarding the possible role of LexAin regulating transposase synthesis. but the Kuan Tessmanstudies were indirect (23). The Tnp.47:945-964. The former difference is a matter of technique. These studies differ in two respects: Weinreichet al performeddirect tests of a LexAeffect (40). Reznikoff. binding the lac repressor to its operator is thought to be stabilized through a bondto a secondary binding site. In vitro analyses of purified Tnp and purified Inh have shownthat at least one property missing in the Inh preparations is a DNAsequence-specific binding activity. while Kuan & Tessman postulate that LexArepresses transposase synthesis approximatelyfivefold. M.annualreviews. The TransposaseN-Terminal Sequence Is Critical for Sequence-Specific DNABinding and Other Transposase Activities The transposase and its inhibitor are identical in sequenceexcept that the Inh is missing 55 N-terminal amino acids (20).Annual Reviews www.annualreviews. This mechanismprevents read-through expression of Tn5 Tnp. Because Inh does not promote transposition. 11). and if so. A. Microbiol. Future studies might examine whether such a nearby LexAbinding site is present in the Kuan-Tessmansystem. submitted. Krebs & W. while the latter may suggest an interesting molecular phenomenon. R. but not in the one which we studied. T. 952 REZNIKOFF An RNAsecondary structure in these read-through RNAsoccludes the Tnp translation initiation signals (37). it is missingone or morecritical Tnpactivities (15-17. Recent studies have demonstrated that deletion of 11 . Wiegand. on the other Annu. Rev. binds to OE and IE end sequences specifically and shows the expected sensitivity to OEsequence mutations and Dammethylation of the IE (N. Weinreich. thereby forming a looped DNAstructure (10. 46) and gal (26)]. 33.

Weinreich & W. and yet the activity is cltearly there. Reznikoff. (Bottom)For the inside endsequence.Madison on 10/19/05. Reznikoff. D. Damsite methylation regulatesbothTnpbinding(43) and binding(41).-I |~-. 44).deletion experiment.TheDnaAbox is indicated for the outside endsequence (12. 17. Deletion of as little as three amino acids prevents this killing (M. Weinreich & W. unpublished results). A possible biological A i OE C 5’CTGACTC~~AGT (Dna A) 3’ C I E 5’CT GT CT C T T[~I’. Overproduction of the transposase (in the absence of transposition) kills host cells. unpublished results).Annual Reviews www. by University of Wisconsin . 41).l l@ll | t~m~.annualreviews. The11Achangegaverise to onedeletion in the secondclass. q?helower-caseletters indicate basepairs outsideof the required19-bpsequence. These experiments show that the N-terminal 55 amino acids are necessary for the sequence-specific DNA-bindingactivity and imply that the DNA-binding domain is in this region.the Damsites andthe overlapping Fis bindingsite are indicated(34. Determining the precise DNA-bindingdomain will be instructive because the Tn5 transposase does not contain a previously classified DNA-binding motif.annualreviews. D.. Downward-pointing arrowsdenotemutationsthat act like a deletion in the adjacent. Downloaded from arjournals.3’ (Fis) Figure4 (Top) Outsideandinside endsequences. Upward-pointing arrowsdenotemutationsthat constitutethe secondclass in the adjacentdeletionexperiment(16). For personal use only. Another surprising property requires the N-terminal three amino acids of Tnp. Microbiol. 1993. Rev. TheTnp/Inhtelrminationcodonis the complement to positions 12-14. .l¢ Annu. TN5 TRANSPOSITION 953 N-terminal arnino acids or introduction of 4 different N-terminal missense mutations destroys the DNA-binding activity of Tnp (M.

Jilk & W. Annu. Because .If the distal OEis deleted. Figure 4 displays the distribution of bp defects. T. de la Cruz. and almost all changes in these sequences greatly reduce or abolish the frequencyof transposition (24. Tn5 is delineated by two inverted OEsequences and IS50 by an OEand an IE sequence (see Figure 4) (17. Krebs & W. different portions of the OEsequencemayplay roles in different steps in the transposition process. For instance. However. somebase pairs could be recognized during the strand-cleavage reaction. All transposase-mediated genetic events (save one mentionedbelow) occur at the precise boundary the relevant 19-bp sequences. someof which are only partially defective in transposition and do not seem to impair transposase binding in vitro (16. transposase recognition of the OE may be muchmore complex than a simple binding reaction.47:945-964. These are most easily detected in situations in which the OEfar removed(distal) from the location of the adjacent deletion defective. For personal use only. Reznikoff. 34). unpublishedresults). Presumably the importanceof these sequenceslies in the fact that they are recognizedby proteins during the transposition process.annualreviews. submitted). Jilk W. Downloaded from least two proteins recognize the OEand three proteins recognize the IE. R.Reznikoff. 1993. Mutant sequencesof this class that have been tested for transposase binding in vitro bind transposase with a lower affinity than the wild-type OE(R.M. Recognition of Terminal 19-Base Pair Sequences The Tn5 and IS50 transposable elements are defined by the terminal 19-bp sequences. Twoclasses of adjacent deletions have been found. The first evidence that various OEsequencesdictate different steps in the transposition process came from a complicated set of in vivo observations (16). greatly reducing or blocking transposition. Wiegand. Tnpcan catalyze deletions adjacent to its site of insertion starting at the end of the OEsequence. the protein-DNA interactions occurring at the OEand IE sequencesclearly display a remarkable degree of complexity. The OEend is recognized by both the transposase and the host protein DnaA(17.Annual Reviews www. 954 REZNIKOFF significance for the cell killing will be describedin the section on chromosome replication below. These adjacent deletions are found in association with another subset of distal OEmutations. Microbiol.annualreviews. 29. 44). Tnprecognition of OEis fundamentalto the transposition process. unpublishedresults). then the adjacent deletions start immediately adjacent to the functional OE. and the nature of the class dependsuponthe precise changein the distal OE. Tnp binds specifically to OEsequences in vitro as judged by gel retardation assays (42. The secondclass of adjacent deletion is unusual in that this type starts one bp removedfrom the OE.Madison on 10/19/05. In addition. M. Rev. Althoughwe are not certain of all of the salient facts. A. SomeOEpoint mutations (which totally block transposition) also fall into this by University of Wisconsin . Reznikoff. A. 29). Weinreich.

14) Annu. and that a Tn5 derivative with a mutation in the OEDnaAbox did not appear to manifest sensitivity to the host dna genotype (29. it overlaps a binding site for a third protein. A possible explanation for someof the mutations that do not i~mpairtransposase bindingis that they are recognizedby a different protein. showingthat Tn5and IS50 transposition occurs at a reduced efficiency in dna hosts. The IE sequence and the OEsequence differ at out of 19 positions. some mutations that presumably alter transposase binding (SG. A closer examination of the results summarizedin Figure 4 suggests an additional level of complexity. Downloaded from arjournals. sugge. at different steps in the transposition reaction and/or by different proteins.Annual Reviews www.sting that these base pairs maybe recognized by both proteins. On the other hand. Possibly both DnaAand transposase recognize position 12. DnaA. Fis. Later experiments tested the relevance of this binding. we could hypothesizethat the positions that differ are not recognizedby Tnpeither because they do not play a critical protein-rec- . Rev.Thisfine structure is also seen in the binding of Tnp to the OE. 10T. At positions 2 and 12 different mutations are associated ’with different classes of adjacent deletion formation--perhapsin these two cases the samebase pair is recognizedin different waysat different steps in tl~te transposition process. Jilk &W. It contains recognition sites for two pro~teins (transposase and DamDNA methylase). A. For position 2. yet the transposase binds in vitro to an unmethylatedIE with an affinity resemblingits binding to the OE(R. 25). 1993.Madison on 10/19/05. and different base pair changes maydiscriminate differential]ty betweenthese two protein-recognition processes. Current experiments are examiningthe effect of DnaA transposase binding and are attempting to determinethe in vitro properties of DnaAbindling to various OEmutant sequences. Tnp might make different molecular contacts with the same base pair at different steps in the overall reaction. This result is quite surprising because it :~uggests considerable flexibility in the DNA recognition domain of Tnp. This observation was first made through footprinting studies of the OE(12). and also encodesthe termination codonfor the transposase (see Figure 4). 44). 11A) are within the DnaA box.Reznikoff. Thus. 44). Microbiol. The IE sequence is even more complicated. TN5 TRANSPOSITION 955 different subsets of OEpoint mutationsgive rise to different classes of adjacent by University of Wisconsin . someend-sequence base pairs are recognized in very complexways. Alternatively. This Tnp-IE binding result is consistent with the observed transposition preferences (9. SomeOEmutations disrupt Tnp binding while others do not. For personal use only. unpublished results). 3C) because the mutations are located outside of the DnaAbox as defined by sequence homology (12. flais implies a functional fine structure to the OE.However. A similar complexityfor end sequenceshas beensuggested for other transposable elements (7. DnaAal:~o recognizes the OEsequence.this explanation does not fit somecases (2A.

These studies gave quite different results depending upon whether a 19. This observation is consistent with direct DamDNAmethylation control over transposition reactions using the IE. The role of DamDNAmethylation regulation of IS50 transposition (utilizing the IE) is complicatedby the fact that a secondprotein (Fis) binds to a sequence overlapping the IE. it mayact to dampenIS50 transposition from newly replicated DNAduring the exponential phase. As noted below. but because Fis abundancevaries with the stage of bacterial growth. and this binding (which is also blocked by DamDNAmethylation) is thought to compete with transposase-IE binding.47:945-964. Jilk &W. DnaA). Rev. transcription initiation programmedby the T1 (transposase) promoter directly inhibited by DamDNAmethylation of sites overlapping the -10 region (43).Annual Reviews www.However. 43). Transposition of IS50 is sensitive to DamDNAmethylation because of the inhibition of two separate sequence-specific protein-DNAinteractions. Werecently showed that Dammethylation of IE DNAfragments reduces transposase binding in vitro (R. Wenowknowthat the 24-bp sequence (but not the 19-bp sequence)contains the overlappingFis-bindingsite. Somein vivo experiments designed to study the frequency of IS50 transposition actually used various recombinant DNAconstructs that have the same general structure as IS50 (OE-transposase gene-reporter gene-IE) but differ in the size of the IE sequence. unpublished experiments). . Downloaded from arjournals. Annu. As described above. 1993. whenit is most abundant. In vitro gel retardation and footprinting studies confirm that Fis binds to this sequenceand that it binds efficiently only to the unmethylatedsite (41).annualreviews. DNAmethylation regulation of transposition maysuggest a coupling between transposition and DNAreplication. Microbiol. However. DamDNAmethylation inhibits transposase binding to the IE. For personal use only. and that Fis acts to inhibit transposition in a Dam-dependentmanner(41).or a 24-bp sequence was used for IE. Verysimilar effects of DNA methylation that regulate TnlO/ISIOtransposition have also been observed (31).DamDNA methylation also inhibits IS50 (but not Tn5) transposition even when the transposase synthesis is programmedby a Dam-insensitivepromoter (tac).annualreviews.g. 956 REZNIKOFF ognition role [position 4 maybe an exampleof this (24)] or because they are recognized by someother protein (e. Thus we conclude that Tnp specifically recognizes two quite diverse sequences--another reflection of the complex substructures of IE and OE.the in vivo adjacent deletion studies suggest that mutations at someof these dissimilar positions do reduce Tnp binding directly (16).org by University of Wisconsin . This effect is presumably mediated through the Damsites within the IE (see Figure 4). 25). The 24-bp IE construct demonstrates muchlower transposition frequencies in a damhost (9.Madison on 10/19/05. indicating a direct effect of Dammethylation on IE recognition during transposition (24.Reznikoff. A. The physiological significance of this Fis effect is unclear.

chemicallystable in vivo (see 33). Microbiol. Wepropose that the nearly complete. The Tnp-Trip Oligomer ~ completion of translation incomplete tethered transposase free transposase ACTIVE ACTIVE free transposase [~~ INACTIVE Tnp-Inh Oligomer INACTIVE Figure 5 Transposase acts in cis. For personal use only. Once Tnpsynthesisis complete. by and large. 1993. Furthermore. Presumably.Madison on 10/19/05. Annu. This preferential cis activity could result from either a chemical or functional instability in the transposase. This observation has also been madefor the transposase proteins enc. the molecule favorsan inactiveconformation. Wehaveno information as to whether this influences transposition utilizing an IE or whetherTnpboundto the IE can influence the synthesis of the transposase. 18).annualreviews. pl Is a CJis-Active Transposase (and a Trans-Active Transposition Inhibitor) TheTn5transposase functions primarily in cis. and/or froma sequestration of the transposase during or shortly after synthesis. 19). andTnp-Tnp or Tnp-lnh oligomerization prior to end-sequence bindingwill alsoinactivateTnp. Downloaded from arjournals. but various studies have indicated that Tn5Tnpis. The Tn5 transposase functions pdmarly in cis (14.oded by other transposons such as TnlO and Tn903(8. by the release of the Tnp protein from the translation apparatus and by the oligomeriz~:tion of Tnp with monomersof Inh or Tnp.47:945-964. acting preferentially on OE-OE or OE-IEsequences located close to the site of transposase synthesis on the samerelicon (15. a low-abundanceactive conformation similar to the tethered protein and a high-abundanceinactive conformation. . the formation of Tnp-Tnpor Tnp-Inh oligomers probably occurs with the inactive conformation(or oligomerization inactivates the active conformation). 28). Tnp tethered to the translation apparatus can initiate the transposition by University of Wisconsin . tethered Tnp has a high affinity end-sequence binding activity and can initiate the transposition process prior to completionof its translation. The Tn903 transposase is knownto be chemically unstable (8).org/aronline TN5 TRANSPOSITION 957 Thetranslation termination codonfor the transposase (and its inhibitor) located within the IE at positions 14-12. Rev. Mycurrent hypothesis(pictured in Figure5) is that the Tn5Tnpis cis-active owingto protein conformationchanges that are regulated by two factors. Sucha propertywouldobviouslylead to cis but not trans activity. The released completed Tnp is proposed to have two conformations in equilibrium.Annual Reviews www.annualreviews.

Reznikoff.annualreviews.the completed Tnp is not completely inactive as Tnp does have some trans activity. However. submitted). Krebs &W. thereby blocking Tnp access to these sequences. M. For personal use Annu. unpublishedresults).org by University of Wisconsin . These mutant proteins may have altered the equilibrium between active and inactive conformationsof Tnp. Also suggesting that Tnp activity is regulated by oligomerization is the observation that Tnpitself inhibits transposition in trans. Alternatively. unpublished results). de la Cruz. 19. 958 REZNIKOFF longer a Tnp monomerexists without end-sequence binding the higher is the probability of oligomerization. thus this modelis not correct.Madison on 10/19/05. M. Weinreich. 18. but several of its predictions are clear. Wehave recently described Tnp mutants that increase the frequency of transposition both in cis and in trans (42). Evidencesuggests that Inh does oligomerize with Tnp and that the oligomers do have altered DNA-binding activity (N. T. Downloaded from arjournals. Wiegand. The MA56 mutant destroys the Inh start codon and introduces an alanine in place of methionine at codon 56 of Tnp. Althoughthe modeldescribed above and in Figure 5 is consistent with all . M. Mahnke&W. The proposed model includes aspects of sequestration (the incomplete tethered Tnpis held in close proximity to its gene) and functional instability (a conformational change facilitated by oligomerization).47:945-964. The evidence for this modelis quite circumstantial.Reznikoff.Annual Reviews www.Inh and other N-terminal deletions of Tnp cannot bind end-sequence DNAefficiently (N. Reznikoff. and the mutations are believed to have a conformational effect because the mutant Tnp proteins are more sensitive to proteolytic degradation (T. de la Cruz. This property of Tnpwas discovered during an in vivo analysis of a Tnp mutant that fails to makethe Inh protein (42). M. 45). This observation is consistent with the OE-bindingdomainresiding at the N terminus (see section 2 of this review) and suggests that this OEbinding domainis occluded in most of the intact Tnp. Krebs & W. However. This mutantis fully functional for transposition in cis but inhibits transposition in trans. Twosimple modelsexplain this inhibitory activity. Thus this property wouldalso lead to cis activity. Weinreich. Inh could form mixedoligomers with Tnp and alter Tnp activity. In vivo studies suggest that protein oligomerizationregulates Tnpactivity. Theprimary evidence supporting the notion that the tethered nascent protein maybind end sequences with an enhancedaffinity camefrom gel retardation experiments examining the binding of Tnp subfragments to OE DNA. T. submitted). Inh might bind to the end sequences forming inactive Inh-end sequence complexes. Wiegand. 1993. Wiegand&W.An in vitro synthesized Tnp subfragmentlacking 100 carboxyl terminal aminoacids binds specifically to OEDNAand does so with an affinity -10-fold higher than that of full-length Tnp(L. Reznikoff. Rev. The Inh protein is knownto inhibit transposase activity in trans (15.annualreviews. Microbiol.

Reznikoff. Downloaded from arjournals. unpublished results). Observations that suggest a functional link between transposition and chromosomepartition camefrom experiments analyzing the cellular consequences of transposase over least the link to chromosome partition can be bypassed. col~i mutants resistant to transposase overproductionkilling continue to divide whentransposase is overproduced. Onepossi- . 44). Weinreich. unpublishedresults). manyof the host functions participating in Tn5transposition are assumedto be componentsof a sequestered organelle involved in host-chromosome replication. unpublishedresults). As for the second. These results point to a possible link betweentransposition and chromosome-partitionfunction. Reznikoff.annualreviews. indicating a failure in partition-dependentseptation. Weinreich & W. The mutations mappedusing Hfr crosses reside at more than one TN5 TRANSPOSITION 959 the observations. D. For instance. Thesecorrelations are intriguing but do not showa mechanistic link. Rev. In particular the following host functions are knownto influence the frequency of Tn5transposition: Dam:a protein catalyzing postreplicative DNAmethylation. Cell death occurs in the absence of transposition as well as any other Tn5-encodedfunction (such as Inh or the end sequences) (M. In addition. This link is in additionto the preferential transposition of Tn5 or IS50 sequences off of newly replicated DNA(which results from damregulatory effects) and to the sharing of DnaAin both transposition and replication processes. Transposition May Be Linked to Chromosome Replication Somecircumstantial evidence might link Tn5 transposition to the process of chromosomereplication and partition.47:945-964. For personal use only. E. incomplete Tnp bind to OEDNAwith a high affinity? Does oligomerization favor the inactive Tnp conformation? Annu. fts genes encodefunctions required for septation. However. Weinreich &W.Annual Reviews www. Howis ~this coupling accomplished and what is the advantage of this arrangement? Wehope an analysis of the mutants mentioned above will suggest an answer to the first question. Microbiol. SulA:a protein that inhibits cell division. The dying cells form long multinuclear filaments. do the hypertransposing mutants alter the conformation of Tnp? Will tethered. transposition does occur in the mutants resistant to transposase overproduction (M. DnaA:an oriC-binding protein required for the initiation of DNAreplication (29. D. damcells have elevated transposition frequencies (43) by University of Wisconsin . 1993. D. direct experimentsare neededto test it. DeLong& Syvanen (6) reported that a small fraction transposase is associated with the inner membrane fraction upon cell lysis. sulA cells haveelevatedtransposition frequencies (35). dnaAcells have reduced transposition frequencies. The one that has been most closely examinedmapsnear thefts locus at 76 min (M.Madison on 10/19/05.

cleaving target DNAto give 9-bp 5’ overhangs. bringing the ends together (or inactivating the unbound Tnp) through oligomerization.47:945-964. a promoterfor Inh mRNA synthesis. One of the interesting aspects of Tn5molecular biology is the density of information encoded within a short sequence. e. Microbiol. the proposed coupling of Tn5 transposition to chromosome replication and partition might decrease the risk of genomic "suicide" occurring as a result of the conservative cut-and-paste transposition process. translation initiation signals for Tnp and Inh. cells that contain transposase. although cis-active sites mayalso be in this region [e. For instance.Thus in the first 259 bp. Thustransposition would be biased towards cells in which a copy of the parental genomeis maintained and can be inherited. Dammethylation sites regulating the Tnp promoter activity.a promoter for Tnp mRNAsynthesis. Conclusion The Tn5/IS50 system has been an amazingly productive object of experimental inquiry. Downloaded from arjournals. a Fis binding site of unknown function (41)]. This element encodes all of the Tn5 functions required for transposition and all of the Tn5 regulatory mechanisms. whichoverlapsthe IE.g. and sequences encoding the important N terminus of Tnp. The work to date has elucidated (and future work will continue to elucidate) the mechanismsby which transposition occurs and is regulated and will also continue to provide insights into important aspects of molecular biology. we find a complex Tnp-specific sequence. assuring that the ongoing chromosome replication wouldbe completedprior to cell division.Madison on 10/19/ Annu. 960 REZNIKOFF ble advantageto Tn5accruing from such an association is that a relationship between Tn5 and the same organelle might have evolved in order to insure that the organelle’s transposition apparatus wouldhave efficient access to these by University of Wisconsin . a DnaA-bindingsite. a possible LexAbinding site. Rev.g. cutting the DNAnext to the end sequences. transposase inhibits chromosomepartitioning. and inserting the .annualreviews. If. is involved with encodingTnpand Inh.annualreviews. Most of the rest of the sequenceup to the Fis site. For personal use only. Transposition is most likely to happensoon after DNA replication (whenthere are two copies of the donor DNAsequence) due to the influence of damDNA methylation on transposase synthesis and inside-end usage. starting at the OE. 1993. Alternatively. The IS50 sequence is 1533 bp in length. however. an imperfect symmetryelement that blocks the expression of read-through mRNA. then this might delay cell division in the very cells in whichtransposition is mostlikely to haveoccurred. I havenot discussed the bulk of the protein-coding sequencesin this review because the required Tnp(and Inh) structure-function studies have not been performed.Annual Reviews www. the Tnp presumably executes the following operations: binding of OEand IE.

DNAloop formation: role in generegulation and implications for DNA structure. R. TransposonTn5. M.Each of the functions requires particular Tnp domains of critical importance. Gene76:207-13 Eismann. and how many molecules are involved in synaptic complex formation? Finally. W.annualreviews.Proc. E.. 10. These and other questions will be the object of future experiments. 1989. The author is indebted to the many members of his laboratory who have . inverted USA84:8049-53 Derbyshire. 3.. 1993.K.A. Davi~. N. 195:949-52 .. D.. D. W. Sci. Hwang. the NSF (DMB-9020517). K. D. Syvanen. von Wilcken-Bergmann. Mol. Kleckner.te active form of the Tnp. See ~tef. Annu. K. K.. B. 1990. Natl. Proc.annualreviews. M.. by University of Wisconsin . Membrane association of the Tnpand Inh 7. W..47:945-964. Role of instability in the cis action of the insertion sequenceIS903transposase. J. 4. L.Sci. N. Moldave.. Berg. Howe.Cell 43:379-97 6.Washington..Cohn. Rev. 1990. 9.A. DC: Am.Annual Reviews www. Genetic analysis of the interaction of the insertion sequenceIS903transposasewith its terminal repeats. NewYork: Academic 2. and the editor of this volume for very helpful suggestions. Berg.. USA87:4048-52 Dodson. 1989.39:81-128. Roth.Madison on 10/19/05. 1990. Acad. Mobi.M. G.. Acad. Microbiol.R. In Progressin Nucleic Acid i~esearchandMolecularBiology. Grindley.. M. T. biochemical. Biek. Literature Cited 1.contributed their imagination and work. Downloaded from arjournals. D. Record. 1990. Even among the transposition operations for which we have substantial information.Specific destruction of the secondlac operator decreasesrepressionof the lac operon in Escherichiacoli fivefold. Muller-Hill. TN5 TRANSPOSITION 961 transposab]~e element into target DNA.. For instance.E. 1985. M. research efforts should be directed at determining if transposition is coupled to the cell-division cycle and how that linkage is accomplished. N. Soe. ACKNOWLEDGMENTS The research in the author’s laboratory has been supported by grants from the NIH (GM19670).Regulation of Tn5 transposition in Salmonella typhhnurium. Factorsaffectingtranspositionactivity of IS50 and Tn5ends. 1989. M. R. Where are they? Howare they organized in a three-dimensional structure? Understanding the molecular basis of Tnp cis activity should also reveal fascinating insights into this protein’s structure-function relationships. and the American Cancer Society (MV-554). 1987. pp.Natl. Jr. F. Bacteriol. F. D. 185-210 3. E. Microbiol.Tnl0protects itself at two levels fromfortuitous activation by e~:ternal promoters. E.Proc. and structural analyses. Natl.J. 8.B. D.x~ng. which will require detailed genetic. Berg. Acad. Simons. proteins of IS50R. Bellomy. much work is left to be done. Biol. For personal use only. M. 1987. ed. 172: 5516-19 Derbyshire.!e DNA. M. how does DnaA binding to the OE influence the Tnp-OE interaction? How do the different base pairs of the OEinteract with Tnp and at what steps of the transposition reaction does this occur? Howdo the different base pairs of the IE function with regard to Tnp activity? Howdoes Fis perturb the Tnp-IE interaction? What is t~. Kramer. Grindley. USA77:6047-51 5.

Johnson. 1987.. C. Transcriptional and translational sites M. by University of Wisconsin .annualreviews. W.. 84:9118-22 M. Nordmann. 147: 12.G. Sasakawa. nucleotide 25.. L. C.. S. Mol.-P. 105-13. Reznikoff. Dahiberg. 1988. 38. 1981.. lon-sulA regulatory function of IS50. Kleckner. right repeat proteins: control at the Donnelly. . J.. USA 80:7293-97 21. 177:64. Makris.. in the promotion and control of Tn5 R. Komberg. Ghosal. Tnl0 transposase 13. M.. Timmerman. Bacteriol. Roberts. 781-91 22. D.J. Biol. Cell 30:873-82 Cell 23:191-99 20. Acad. Krebs.. D.. 192: of Tn5 from ~. 35. Y. J. 1982. J. T. Schniz. Genet.. Kleekner. Reznikoff. R. gal operon proteins madeafter prophage Sci.. Hobom. 1988. C. Yoshikawa. 1986. R. J.. The dnaA protein complex 27.. C. E. Nucleic ends in vivo.. R. StrucRes. coli insertion element. Isberg. 1990. Escherichia coli in the lac operon. B. R. 1985. S. through transcripts. C. The control 1981. 1989. S. S. W.. Sequencesessential for transFritz.. Kleckner. M. Maquat. Acad.. O.. mechanismof repression at a distance Adhya. Mctransposon Tn5required for transposiClure. USA 15. Complexpromoters. A. W.-J. Cell 32:799-807 Acids Res. V. C. A. Merril.. 1981. Dual 26. 962 REZNIKOFF 11. C. DNAsequences at the ends of 31. K. Fuller. 142:879-84 tural analysis of insertion sequenceIS5. J. N.Annual Reviews www. Rezrfikoff. Burgess. 34. Aead. Rak.Madison on 10/19/05. Schwartz. In RNAP olymerase and the Regulation Cell 30:883-92 16.. New York: deletions. D. Kuhn. USA 85:2224-28 1988..I. Sommer.. Tillmann. J. sequence and phylogenetic relationships Orientation of IS50 Iransposase gene of a new E. Young. Reznikoff. Johnson. Johnson. M. Saedler. LexA mRNAfrom lacZ translation initiation protein of Escherichia coli represses mutants. R. Schnlz. S. Lazaar. Huisman. J. D. Sci. The position at the termini of ISS0. 1983. Gottesman. Carle. C. J. tion.. Mol. Acad. Biol. et al. S. Kim. Y. 1991. Commun. Jilk. 1982. A. I. E. M. 16:6789-99 5212-14 39. 170:889-94 Cell 38:889-900 28. N. Mol. Kinetics of Tn5 transposition. Bacteriol. J.. USA 85:8968-72 lambda induction. J. S.. 37. Proc. The functional differences in the of Tn5transposition in Escherichia coli inverted repeats of the Tn5are caused is mediated by protein from the right by a single base pair nonhomology.. Gralla. Biophys. Proc. C. 1983. and transposition. W. 211:427-45 expression of the Tn5transposase gene. IS150: distribution. Flashner. W. Bacteriol. Signon.. E. Kroger. M. Annu. level of the transposition reactions? E. Microbiol. 1993. Biol. Proc.. Bacteriol. Regulation of Tn5 by the 30.. C. adenine methylation.5-61 Gene 32:91-8 19.. P. Reznikoff. Nature 297:159-62 In vitro secondarystructure analysis of 23.. 173:6406-10 Translation initiation of ISSORread24. P. For personal use only. M. S.-J. Starlinger.S. Tu. E. Sci. K.. R.. 6:1111-22 29. P. Control of transposase and affects the efficiency of transposition inhibitor expression. Natl. P.. S. W. Biol. Implications of Tn5 associated adjacent Gross. E. R. R. Reznikoff. repeat. M. D. 1984. J. Bacteriol... W. S.H. R. W.. sequence of IS4. tive origin (oriC) and other DNA sites. M. V.annualreviews. R.. C. G. Mol. Proc.. R. eoli chromosomalreplieaTemporalcontrol of Tn5 transposition. . Hoopes.. L..-T. ed.. McCommas. W. S. C. Bertrand.Biochera. Downloaded from arjournals. Altman. Syvanen. F. C... 1979. Reznikoff. 1991.. 1984. Mol. W. N. Wickens. Rev. Krebs. D. S. L. D. Makfis.-P. H. Yin. 175:1264-71 Elsevier 17. P. W.. Identification of base pairs in the outside end of insertion sequence ISSO that L. Morisato. J. Klaer. Errada.. b221 ci857 Pam Oam to the chromosome.. S. 1987. W. H. Rothstein. J. Berg.. S. B. Uno. C. R. 181:169-75 Natl. J. W. Nature 304:280-82 ISI0 transposition is regulated by DNA 18. S. 221: Reznikoff. Record. O. 1982. Reznikoff. Funnell. C. E. L. Phadnis. L. pp.47:945-964. Kroger. Mutational 65-80 analysis of IS50 and Tn5 ends. EMBO are needed for IS50 and Tn5 transpod. Tessman. of Transcription.. Sasakawa. Gen. 171: Nucleic Acids Res. R. H. Nucleotide sequenceof the transacts preferentially on nearby transposon posable DNA-element IS2. 1983. Cell 43:117-30 1984. P. Way. Reznikoff. The role of the ISSORproteins 32. P. 1993. ~’. Makris. A. 198. 1987... 8:2101-9 sition. Reznikoff. Syvanen. S. 36. S.. Natl. Berg. E. 875-87 A. Natl. transposition. Rosetti. C. Mutational analysis of ISl0’s outside end. with the E. Kuan. B. R. Borchardt. 14. L. Jr. Reznikoff. H. Sci. 33. 1989..

Mol. TN5 TRANSPOSITION 1985. 169:4637-45 45. Induction of the SOSresponse in Escherichia coli inhibits Tn5and IS50 transposition.47:945-964. M. and Tn5 mutants. Characterization of two hypertranslx~sing Tn5mutants. X. S. Reznikoff. J. S.Annual Reviews www. Makris... M.. Biol. W. P. by University of Wisconsin . W. For personal use only. an essential host gene. C. M.L..annualreviews. 13:2127-39 Weinn. W. J... 172:355-62 .-P. Bacteriol. 199:35--46 44. Reznikoff. Yin. Wieg~md. W. 1988. Complete sequence of IS3. 173:6910-18 41.. Krebs. 1992. Reznikoff. W. D. Yin. W. M. Reznikoff. J. Bacteriol. W. Reznikoff. Bacteriol. W. p2 and the inhibition of Tn5transposition.’ich. S. The effect of dam methylation on Tn5 transposition. C. M. J. 1987. Annu. J.annualreviews. J. Molecular cloning and sequenceanalysis of trp-lac fusion deletions. Reznikoff. Mol. Microbiol.-P. 174:453037 42. Biol. Bacteriol. Munson. S.. Reznikoff. dnaA. J. T. Downloaded from arjournals. D. 174:1229-39 963 43. Rev. 1993. 1988. Nucleic Acids Res.-P. J.. C. Yin. S. Weinn. J. S. S.. 1984. C. Fis plays a role in Tn5 and IS50 transposition. 170:3008-15 46.Madison on 10/19/05. 1992. J. 40.qch. J. 1991. by University of Wisconsin . Downloaded from arjournals.Madison on 10/19/05. Microbiol. Rev. 1993. .47:945-964. For personal use only.annualreviews. by University of Wisconsin .Madison on 10/19/05.47:945-964.Annu. Downloaded from arjournals. .annualreviews. 1993. Rev. For personal use only.

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