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.

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

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

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

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

This mechanismreappears in the discussion of protein recognition of the IS50 IE sequence. whichis exactly the observedresult (27. Production of these transcripts lmight result in spurious transposase synthesis. However. Also. this regulation should stimulate transposition off of DNAthat is newly introduced into the cell. thereby giving rise to read-through transcripts into the transposase gene.annualreviews.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.Annual Reviews www. Downloaded from arjournals. 32). Rev.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.a set of experiments(21) has demonstratedthat read-through expression of transposase does not occur because such messagesdo not programthe translation of Tnp. Microbiol.Madison on 10/19/05. .:21. GUC C-~ U _~u U IJCCCGUUUUCCAGG G U AACUUCUGCUCUU 110 40 Figure 3 Secondary structurein read-through transcriptsblocksTnptranslation. 37). Other transposon systems [in particular TnlO (31)] also display Dam methylation control of Tnp synthesis and of by University of Wisconsin . Thestart of the T1transcript(which will notforma secondary structure)andthe TnpAUG are indicated. Moreover.annualreviews. Tn5cou]td transpose into highly expressed genes. For personal use only.47:945-964.10 region of the T1 promoter. Annu.Thisfigureis similarto one shown in Ref. TN5 TRANSPOSITION 951 methylationsites overlap with the .Transcripts that readthroughthe endof ISSOdonot expressTnpbecauseof a secondary structurethat occludes the Shine-Dalgarno sequence endandthe AUG (21.

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

TN5 TRANSPOSITION 953 N-terminal arnino acids or introduction of 4 different N-terminal missense mutations destroys the DNA-binding activity of Tnp (M.deletion experiment.l l@ll | Annu. q?helower-caseletters indicate basepairs outsideof the required19-bpsequence. Downloaded from arjournals.Annual Reviews www. unpublished results). For personal use only. D.annualreviews. 41).-I |~-. Determining the precise DNA-bindingdomain will be instructive because the Tn5 transposase does not contain a previously classified DNA-binding motif.the Damsites andthe overlapping Fis bindingsite are indicated(34. Downward-pointing arrowsdenotemutationsthat act like a deletion in the adjacent. Reznikoff. .l¢f. unpublished results).Madison on 10/19/05. 17. A possible biological A i OE C 5’CTGACTC~~AGT (Dna A) 3’ C I E 5’CT GT CT C T T[~I’. Deletion of as little as three amino acids prevents this killing (M. Microbiol. D. Another surprising property requires the N-terminal three amino acids of Tnp.annualreviews. 1993. by University of Wisconsin . and yet the activity is cltearly there. 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.TheDnaAbox is indicated for the outside endsequence (12.47:945-964. Weinreich & W. Weinreich & W. Upward-pointing arrowsdenotemutationsthat constitutethe secondclass in the adjacentdeletionexperiment(16). (Bottom)For the inside endsequence. 44). Damsite methylation regulatesbothTnpbinding(43) and binding(41). Overproduction of the transposase (in the absence of transposition) kills host cells.. The11Achangegaverise to onedeletion in the secondclass. Rev. 34. TheTnp/Inhtelrminationcodonis the complement to positions 12-14.3’ (Fis) Figure4 (Top) Outsideandinside endsequences.

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

10T. DnaA. we could hypothesizethat the positions that differ are not recognizedby Tnpeither because they do not play a critical protein-rec- . 14).org/aronline Annu. TN5 TRANSPOSITION 955 different subsets of OEpoint mutationsgive rise to different classes of adjacent deletions.Reznikoff. 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. This result is quite surprising because it :~uggests considerable flexibility in the DNA recognition domain of Tnp. The IE sequence is even more complicated. DnaAal:~o recognizes the OEsequence.Madison on 10/19/05. Downloaded from arjournals. it overlaps a binding site for a third protein. 3C) because the mutations are located outside of the DnaAbox as defined by sequence homology (12.sting that these base pairs maybe recognized by both by University of Wisconsin . Current experiments are examiningthe effect of DnaA transposase binding and are attempting to determinethe in vitro properties of DnaAbindling to various OEmutant sequences. sugge. Later experiments tested the relevance of this binding.this explanation does not fit somecases (2A. yet the transposase binds in vitro to an unmethylatedIE with an affinity resemblingits binding to the OE(R. It contains recognition sites for two pro~teins (transposase and DamDNA methylase). Jilk &W. 11A) are within the DnaA box. 44).47:945-964. A.Thisfine structure is also seen in the binding of Tnp to the OE. Possibly both DnaAand transposase recognize position 12. A similar complexityfor end sequenceshas beensuggested for other transposable elements (7.annualreviews. unpublished results). On the other hand. 44). and also encodesthe termination codonfor the transposase (see Figure 4). and that a Tn5 derivative with a mutation in the OEDnaAbox did not appear to manifest sensitivity to the host dna genotype (29. This observation was first made through footprinting studies of the OE(12). someend-sequence base pairs are recognized in very complexways. 1993. For personal use only. Alternatively. Microbiol. For position 2. A closer examination of the results summarizedin Figure 4 suggests an additional level of complexity. showingthat Tn5and IS50 transposition occurs at a reduced efficiency in dna hosts. The IE sequence and the OEsequence differ at out of 19 positions. Thus. Rev. SomeOEmutations disrupt Tnp binding while others do not. Fis. and different base pair changes maydiscriminate differential]ty betweenthese two protein-recognition processes. some mutations that presumably alter transposase binding (SG.annualreviews. Tnp might make different molecular contacts with the same base pair at different steps in the overall reaction. 25). A possible explanation for someof the mutations that do not i~mpairtransposase bindingis that they are recognizedby a different protein.Annual Reviews www. at different steps in the transposition reaction and/or by different proteins. This Tnp-IE binding result is consistent with the observed transposition preferences (9. flais implies a functional fine structure to the OE.However.

DnaA). As noted below. As described above. 25). Rev.DamDNA methylation also inhibits IS50 (but not Tn5) transposition even when the transposase synthesis is programmedby a Dam-insensitivepromoter (tac).annualreviews. Downloaded from arjournals. The physiological significance of this Fis effect is unclear.g. A. whenit is most abundant. it mayact to dampenIS50 transposition from newly replicated DNAduring the exponential phase. This effect is presumably mediated through the Damsites within the IE (see Figure 4).Madison on 10/19/05. but because Fis abundancevaries with the stage of bacterial growth. Thus we conclude that Tnp specifically recognizes two quite diverse sequences--another reflection of the complex substructures of IE and OE.47:945-964. . 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. indicating a direct effect of Dammethylation on IE recognition during transposition (24. The 24-bp IE construct demonstrates muchlower transposition frequencies in a damhost (9.However. 29. 43). 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. and that Fis acts to inhibit transposition in a Dam-dependentmanner(41). Jilk &W. Verysimilar effects of DNA methylation that regulate TnlO/ISIOtransposition have also been observed (31). In vitro gel retardation and footprinting studies confirm that Fis binds to this sequenceand that it binds efficiently only to the unmethylatedsite (41). Wenowknowthat the 24-bp sequence (but not the 19-bp sequence)contains the overlappingFis-bindingsite.Annual Reviews www.the in vivo adjacent deletion studies suggest that mutations at someof these dissimilar positions do reduce Tnp binding directly (16). 956 REZNIKOFF ognition role [position 4 maybe an exampleof this (24)] or because they are recognized by someother protein (e. Annu. transcription initiation programmedby the T1 (transposase) promoter directly inhibited by DamDNAmethylation of sites overlapping the -10 region (43). This observation is consistent with direct DamDNAmethylation control over transposition reactions using the IE.annualreviews. DamDNAmethylation inhibits transposase binding to the IE. DNAmethylation regulation of transposition maysuggest a coupling between transposition and DNAreplication. For personal use by University of Wisconsin . 1993. and this binding (which is also blocked by DamDNAmethylation) is thought to compete with transposase-IE binding.Reznikoff. Microbiol. unpublished experiments). These studies gave quite different results depending upon whether a 19. Werecently showed that Dammethylation of IE DNAfragments reduces transposase binding in vitro (R. Transposition of IS50 is sensitive to DamDNAmethylation because of the inhibition of two separate sequence-specific protein-DNAinteractions.or a 24-bp sequence was used for IE.

org by University of Wisconsin . the molecule favorsan inactiveconformation. a low-abundanceactive conformation similar to the tethered protein and a high-abundanceinactive conformation. This observation has also been madefor the transposase proteins enc. 1993. chemicallystable in vivo (see 33). Once Tnpsynthesisis complete. Microbiol. Wepropose that the nearly complete. the formation of Tnp-Tnpor Tnp-Inh oligomers probably occurs with the inactive conformation(or oligomerization inactivates the active conformation). For personal use only.Annual Reviews www. Downloaded from arjournals. Wehaveno information as to whether this influences transposition utilizing an IE or whetherTnpboundto the IE can influence the synthesis of the transposase. by the release of the Tnp protein from the translation apparatus and by the oligomeriz~:tion of Tnp with monomersof Inh or Tnp. The Tn5 transposase functions pdmarly in cis (14.47:945-964. 18). Sucha propertywouldobviouslylead to cis but not trans activity.Madison on 10/19/05. but various studies have indicated that Tn5Tnpis. The released completed Tnp is proposed to have two conformations in equilibrium. Tnp tethered to the translation apparatus can initiate the transposition reaction. 28).oded by other transposons such as TnlO and Tn903(8. pl Is a CJis-Active Transposase (and a Trans-Active Transposition Inhibitor) TheTn5transposase functions primarily in cis.annualreviews. and/or froma sequestration of the transposase during or shortly after synthesis. andTnp-Tnp or Tnp-lnh oligomerization prior to end-sequence bindingwill alsoinactivateTnp. 19). acting preferentially on OE-OE or OE-IEsequences located close to the site of transposase synthesis on the samerelicon ( TN5 TRANSPOSITION 957 Thetranslation termination codonfor the transposase (and its inhibitor) located within the IE at positions 14-12. by and large. . The Tn903 transposase is knownto be chemically unstable (8). Annu. Mycurrent hypothesis(pictured in Figure5) is that the Tn5Tnpis cis-active owingto protein conformationchanges that are regulated by two factors. Rev. tethered Tnp has a high affinity end-sequence binding activity and can initiate the transposition process prior to completionof its translation.annualreviews. 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. This preferential cis activity could result from either a chemical or functional instability in the transposase. Furthermore. Presumably.

18.Inh and other N-terminal deletions of Tnp cannot bind end-sequence DNAefficiently (N.Madison on 10/19/05. 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). Alternatively. The evidence for this modelis quite by University of Wisconsin . Inh might bind to the end sequences forming inactive Inh-end sequence complexes. However. The MA56 mutant destroys the Inh start codon and introduces an alanine in place of methionine at codon 56 of Tnp. T. T. Reznikoff. submitted). M. Krebs &W. M. 19. unpublishedresults). Evidencesuggests that Inh does oligomerize with Tnp and that the oligomers do have altered DNA-binding activity (N. M. Weinreich. Rev. Wehave recently described Tnp mutants that increase the frequency of transposition both in cis and in trans (42).annualreviews. 958 REZNIKOFF longer a Tnp monomerexists without end-sequence binding the higher is the probability of oligomerization. but several of its predictions are clear. Althoughthe modeldescribed above and in Figure 5 is consistent with all . and the mutations are believed to have a conformational effect because the mutant Tnp proteins are more sensitive to proteolytic degradation (T. unpublished results). Inh could form mixedoligomers with Tnp and alter Tnp activity. submitted). Weinreich. 1993. 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.47:945-964. These mutant proteins may have altered the equilibrium between active and inactive conformationsof Tnp. Reznikoff. Krebs & Annu. M. Mahnke&W.annualreviews. This mutantis fully functional for transposition in cis but inhibits transposition in trans. In vivo studies suggest that protein oligomerizationregulates Tnpactivity. Reznikoff. This property of Tnpwas discovered during an in vivo analysis of a Tnp mutant that fails to makethe Inh protein (42). Also suggesting that Tnp activity is regulated by oligomerization is the observation that Tnpitself inhibits transposition in trans.Annual Reviews www. Wiegand&W. de la Cruz. Twosimple modelsexplain this inhibitory activity.Reznikoff. The Inh protein is knownto inhibit transposase activity in trans (15.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. However.the completed Tnp is not completely inactive as Tnp does have some trans activity. For personal use only. thus this modelis not correct. 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. de la Cruz. thereby blocking Tnp access to these sequences. Thus this property wouldalso lead to cis activity. Wiegand. Downloaded from arjournals. Microbiol. Wiegand.

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

One of the interesting aspects of Tn5molecular biology is the density of information encoded within a short sequence. 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. assuring that the ongoing chromosome replication wouldbe completedprior to cell division. and inserting the . Most of the rest of the sequenceup to the Fis site. 1993. e.annualreviews. whichoverlapsthe Annu.a promoter for Tnp mRNAsynthesis. Downloaded from arjournals. Dammethylation sites regulating the Tnp promoter activity. starting at the OE. If. although cis-active sites mayalso be in this region [e. however. For instance. transposase inhibits chromosomepartitioning.Annual Reviews www.g. a Fis binding site of unknown function (41)]. we find a complex Tnp-specific sequence. a possible LexAbinding site. Thustransposition would be biased towards cells in which a copy of the parental genomeis maintained and can be inherited. cleaving target DNAto give 9-bp 5’ overhangs.47:945-964. the Tnp presumably executes the following operations: binding of OEand IE.g. and sequences encoding the important N terminus of Tnp. This element encodes all of the Tn5 functions required for transposition and all of the Tn5 regulatory mechanisms. The IS50 sequence is 1533 bp in length. Microbiol. 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 functions. a promoterfor Inh mRNA synthesis. bringing the ends together (or inactivating the unbound Tnp) through oligomerization. cutting the DNAnext to the end by University of Wisconsin . Alternatively. cells that contain transposase. 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. a DnaA-bindingsite. 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. is involved with encodingTnpand Inh. Rev.Madison on 10/19/05. 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. Conclusion The Tn5/IS50 system has been an amazingly productive object of experimental inquiry. 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.Thus in the first 259 bp. For personal use only. translation initiation signals for Tnp and Inh.

TransposonTn5.contributed their imagination and work. Factorsaffectingtranspositionactivity of IS50 and Tn5ends. See ~tef. Acad.Specific destruction of the secondlac operator decreasesrepressionof the lac operon in Escherichiacoli fivefold. 1990. Davi~.39:81-128. 1993.. Literature Cited 1. 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~. Berg. and how many molecules are involved in synaptic complex formation? Finally.Sci.Tnl0protects itself at two levels fromfortuitous activation by e~:ternal promoters. Berg. R. Sci. DC: Am.Cell 43:379-97 6.. Rev. Kleckner. M. E. Sci. M. Simons. 1989. 1985.Madison on 10/19/05. and the American Cancer Society (MV-554). These and other questions will be the object of future experiments. Natl.Each of the functions requires particular Tnp domains of critical importance. F.Natl. which will require detailed genetic. 1990. Soe.!e DNA. N. M. TN5 TRANSPOSITION 961 transposab]~e element into target DNA. M. Annu.R. USA77:6047-51 5..Proc... 1990. von Wilcken-Bergmann. Syvanen. B. Kramer.Annual Reviews www.J. Even among the transposition operations for which we have substantial information. For instance.. 1987.47:945-964. Acad. Microbiol.. E.K. Gene76:207-13 Eismann.J. W. K... D. Downloaded from arjournals. 1989.annualreviews. biochemical. Mobi.B. D. Bellomy..annualreviews. D..A.Proc. E. Howe. J. proteins of IS50R.. 1989. Proc. and structural analyses. In Progressin Nucleic Acid i~esearchandMolecularBiology.Cohn. For personal use only. Berg. Mol. Acad. D. Role of instability in the cis action of the insertion sequenceIS903transposase. DNAloop formation: role in generegulation and implications for DNA structure. 10. 195:949-52 . Biek. Genetic analysis of the interaction of the insertion sequenceIS903transposasewith its terminal repeats. M. 1990. research efforts should be directed at determining if transposition is coupled to the cell-division cycle and how that linkage is accomplished. Muller-Hill. Biol. Microbiol. Grindley. N. ed. 1987.Regulation of Tn5 transposition in Salmonella typhhnurium. W. D. the NSF (DMB-9020517). D. R. Grindley. G. Membrane association of the Tnpand Inh 7. pp. NewYork: Academic 2. 8.te active form of the Tnp. K. N. M. Del.Washington.. F. Moldave. much work is left to be done. and the editor of this volume for very helpful suggestions. inverted USA84:8049-53 Derbyshire.M.A. Hwang. Bacteriol. The author is indebted to the many members of his laboratory who have . 185-210 3. Natl. ACKNOWLEDGMENTS The research in the author’s laboratory has been supported by grants from the NIH (GM19670). Jr. K. Roth.x~ng. Record. USA87:4048-52 Dodson. 9. 172: 5516-19 Derbyshire. M. by University of Wisconsin .. 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. 3. W. 4.

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

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

Madison on 10/19/05.annualreviews. Microbiol. 1993. For personal use by University of Wisconsin . . Downloaded from arjournals.Annu.47:945-964. Rev.

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

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