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.

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

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

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

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

Thestart of the T1transcript(which will notforma secondary structure)andthe TnpAUG are indicated. Downloaded from arjournals.a set of experiments(21) has demonstratedthat read-through expression of transposase does not occur because such messagesdo not programthe translation of Tnp. However. Tn5cou]td transpose into highly expressed by University of Wisconsin .Transcripts that readthroughthe endof ISSOdonot expressTnpbecauseof a secondary structurethat occludes the Shine-Dalgarno sequence endandthe AUG (21.Annual Reviews www. Production of these transcripts lmight result in spurious transposase synthesis.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.annualreviews. Moreover. GUC C-~ U _~u U IJCCCGUUUUCCAGG G U AACUUCUGCUCUU 110 40 Figure 3 Secondary structurein read-through transcriptsblocksTnptranslation. This mechanismreappears in the discussion of protein recognition of the IS50 IE sequence.annualreviews.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. Microbiol.Madison on 10/19/05. TN5 TRANSPOSITION 951 methylationsites overlap with the . Also. 32). thereby giving rise to read-through transcripts into the transposase gene. this regulation should stimulate transposition off of DNAthat is newly introduced into the cell. Rev.10 region of the T1 promoter. whichis exactly the observedresult (27. For personal use only. 1993.Thisfigureis similarto one shown in Ref. . 37).

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

l¢ by University of Wisconsin ..Annual Reviews www. . TheTnp/Inhtelrminationcodonis the complement to positions 12-14. Overproduction of the transposase (in the absence of transposition) kills host cells. Downloaded from arjournals. The11Achangegaverise to onedeletion in the secondclass. Weinreich & W. D. Weinreich & W. (Bottom)For the inside endsequence. and yet the activity is cltearly there.the Damsites andthe overlapping Fis bindingsite are indicated(34. 17. D.annualreviews.Madison on 10/19/05.deletion experiment. Microbiol. For personal use only.-I |~-.l l@ll | t~m~. 1993. q?helower-caseletters indicate basepairs outsideof the required19-bpsequence. 34. 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. A possible biological A i OE C 5’CTGACTC~~AGT (Dna A) 3’ C I E 5’CT GT CT C T T[~I’. 44).3’ (Fis) Figure4 (Top) Outsideandinside endsequences. Rev. Downward-pointing arrowsdenotemutationsthat act like a deletion in the adjacent. Deletion of as little as three amino acids prevents this killing ( Annu. Reznikoff.TheDnaAbox is indicated for the outside endsequence (12. Damsite methylation regulatesbothTnpbinding(43) and binding(41). unpublished results). 41). unpublished results). Determining the precise DNA-bindingdomain will be instructive because the Tn5 transposase does not contain a previously classified DNA-binding motif. Reznikoff.annualreviews. Upward-pointing arrowsdenotemutationsthat constitutethe secondclass in the adjacentdeletionexperiment(16). Another surprising property requires the N-terminal three amino acids of Tnp. TN5 TRANSPOSITION 953 N-terminal arnino acids or introduction of 4 different N-terminal missense mutations destroys the DNA-binding activity of Tnp (M.47:945-964.

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

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

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

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

Inh could form mixedoligomers with Tnp and alter Tnp activity. In vivo studies suggest that protein oligomerizationregulates Tnpactivity. The evidence for this modelis quite circumstantial. Microbiol. unpublished results). M. Krebs &W. 958 REZNIKOFF longer a Tnp monomerexists without end-sequence binding the higher is the probability of oligomerization. Twosimple modelsexplain this inhibitory activity.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. by University of Wisconsin .the completed Tnp is not completely inactive as Tnp does have some trans activity.annualreviews. 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.Inh and other N-terminal deletions of Tnp cannot bind end-sequence DNAefficiently (N. de la Cruz. The MA56 mutant destroys the Inh start codon and introduces an alanine in place of methionine at codon 56 of Tnp. M. submitted). submitted). but several of its predictions are clear. Reznikoff. Wehave recently described Tnp mutants that increase the frequency of transposition both in cis and in trans (42). 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. Also suggesting that Tnp activity is regulated by oligomerization is the observation that Tnpitself inhibits transposition in trans.Annual Reviews www. 19. 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). Evidencesuggests that Inh does oligomerize with Tnp and that the oligomers do have altered DNA-binding activity (N. Thus this property wouldalso lead to cis activity. thereby blocking Tnp access to these sequences. Wiegand. These mutant proteins may have altered the equilibrium between active and inactive conformationsof Tnp. However.annualreviews. 18. T. 45). This property of Tnpwas discovered during an in vivo analysis of a Tnp mutant that fails to makethe Inh protein (42). Althoughthe modeldescribed above and in Figure 5 is consistent with all .Madison on 10/19/05. Mahnke&W. unpublishedresults). Weinreich. de la Cruz. However. For personal use only. 1993. Wiegand.47:945-964. thus this modelis not correct. Annu. Wiegand&W. Inh might bind to the end sequences forming inactive Inh-end sequence complexes. The Inh protein is knownto inhibit transposase activity in trans (15. This mutantis fully functional for transposition in cis but inhibits transposition in trans. Reznikoff. Rev. Downloaded from arjournals. Weinreich. Reznikoff. and the mutations are believed to have a conformational effect because the mutant Tnp proteins are more sensitive to proteolytic degradation (T. T. Alternatively.

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

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

Factorsaffectingtranspositionactivity of IS50 and Tn5ends. ACKNOWLEDGMENTS The research in the author’s laboratory has been supported by grants from the NIH (GM19670). 1990.. M.. Mol.K. 1987. 1985. E. which will require detailed genetic.annualreviews.J. 1987. The author is indebted to the many members of his laboratory who have . Davi~. biochemical. much work is left to be done. Sci. Berg. inverted USA84:8049-53 Derbyshire. For instance. Acad. USA87:4048-52 Dodson.te active form of the Tnp... D. Literature Cited 1. W. For personal use only. W.39:81-128.J.Cell 43:379-97 6..A.Proc. 195:949-52 . D. Jr. D. K. 1993. TransposonTn5. 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. Berg. M. Bellomy. Kleckner. Moldave.Annual Reviews www. J. the NSF (DMB-9020517).B.. Del. M.. 9.Cohn. 10.R. Berg. T. Downloaded from arjournals. Role of instability in the cis action of the insertion sequenceIS903transposase. and the editor of this volume for very helpful suggestions. Muller-Hill. research efforts should be directed at determining if transposition is coupled to the cell-division cycle and how that linkage is accomplished. Biol. N. G. Gene76:207-13 Eismann. Grindley. ed.Regulation of Tn5 transposition in Salmonella typhhnurium. Acad. TN5 TRANSPOSITION 961 transposab]~e element into target DNA.Natl. Bacteriol. Biek. Proc.. Microbiol. M. Acad.Madison on 10/19/05. DC: Am. These and other questions will be the object of future experiments. Roth.. D. 1989. N. N. K. F. Simons.A. M. Mobi.47:945-964. pp. Genetic analysis of the interaction of the insertion sequenceIS903transposasewith its terminal repeats. 1989. 3.Specific destruction of the secondlac operator decreasesrepressionof the lac operon in Escherichiacoli fivefold. 1989. Natl. L. See ~tef. Annu. and the American Cancer Society (MV-554). F.contributed their imagination and work. Membrane association of the Tnpand Inh 7. Syvanen. Rev.... and how many molecules are involved in synaptic complex formation? Finally.x~ng. Soe. USA77:6047-51 5. Howe. Hwang. D. Natl.M. In Progressin Nucleic Acid i~esearchandMolecularBiology. K. Microbiol. DNAloop formation: role in generegulation and implications for DNA structure..!e DNA. 1990. von Wilcken-Bergmann. Grindley. E. 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~. B. 8.Each of the functions requires particular Tnp domains of critical importance. M. and structural by University of Wisconsin . 4. D. M. Kramer.Tnl0protects itself at two levels fromfortuitous activation by e~:ternal promoters. 185-210 3.annualreviews. R. Even among the transposition operations for which we have substantial information. R.Proc. W. Sci. Record. NewYork: Academic 2.Sci. 172: 5516-19 Derbyshire.Washington.. proteins of IS50R. E.E. 1990.

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

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

org by University of Wisconsin . For personal use only. Downloaded from arjournals. 1993.Madison on 10/19/05.annualreviews.47:945-964. .Annu. Rev. Microbiol. by University of Wisconsin . Microbiol.annualreviews. For personal use only. Downloaded from arjournals. 1993.Madison on 10/19/05.47:945-964. . Rev.

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