IS50-mediated inverse transposition

J. Mol. Riol. (1982) 159, 257-271

ISSO-Mediated Inverse Transposition Discrimination

Between the Two Ends of an IS Element

of Microbiology and Immunology and of Genetics University School of Medicine. Saint Louis, MO. 63110, IJ.S.A.

Departmwts

Washington

(Receiwd

19 February

1982, and in ree*ised form

7 April

1982)

The tclrmini of the kanamycin resistance transposon Tn5 consist of a pair of nearI> identical insertion sequences named IS501, and 1S;iOR arranged in inverted orientation. The ends of these IS50 elemrrits consist of an extremely short (8 of 9 base-pairs) inverted repeat. Assays of ISSO-mediated gene transposition from pRR322 : : Tn5 and related bacterial plasmids to bacteriophage h show that the two ends of IS50 are not equivalent as sites for transposase recognition. Pairs of outsid? (0) ends of IS50 are found to be used up to three orders of magnitude more efficiently than pairs of inside (I) ends, but only slightly more efficiently than one 0 end and one I end. These data suggest that the eight base-pair inverted repeats at t,he termini of IS50 comprise only a partial transposase recognition site, and that sequences at the 0 end beyond the common eight base-pairs are needed for efficient transposase recognition.

1. Introduction Movement of transposable elements to new non-homologous sites in a genome is believed t,o require element-specific transposase proteins which recognize distinctive DXA sequences at the ends of each element. The simple IS (insertion sequence) elements generally encode their own transposases and contain no genes unrelated t.o their own t.ransposition, while transposons, which are more complex. contain auxiliary genes whose functions are unrelated to the transposition process (e.g. the transposable antibiotic resistance determinants frequently found in bacterial R factor plasmids). Many transposons are composites in which functional TS elements bracket the auxiliary genes as direct or inverted repetitions (Tn,ii(Kan’), TnS(Cam’). and TnlO(Tet’)); others contain no, or just one. complete IS element (e.g. TnS(Amp’), TnSOl(Mer’). TnlY21(Tet’) (see Berg & Berg, 1981 : (‘alas 8: Miller. 1980; Campbell et al., 1979)). The 5.7 kb$ long kanamycin resistance element Tn5 (Berg et al., 1975; Berg. 1977) is representative of the composite transposons. Tn5 contains nearly identical 1.5 kb long t,erminal inverted repeat arms (Auerswald et al., 1980) which have recently

P57

258

(’ SASAli.4~VA

ANI)

I)

K. H&R(:

been shown to be transposable (Berg et ~2.. 1980~,1982: Hirschel & Herg, 1982: Isberg & Syvanen, 1981): they are unrelated to IS elements present in the chromosome of Escherichia coli Kl2 (Berg Nr Drummond, 1978), and have been named (IS50R (right) and IS5UL (left) (Berg it nl., 198Ou). IS50R cont,ains a functional transposase gene, ISSOL, an ochre allele of this gent (Rothstein rt trl.. 1980aJ). The termini of IS50 consist of hyphenated eight of nine base-pair inverted repeats (Berg et al.. unpublished results), which are shorter than the invert’ed repeats at the termini of ot’her known I8 elements. In initial tests of whether the Tn5 arms are IS elements. we had attempted to detect transposition involving pairs of inside (I) ends of Tn5 arms (inverse transposition; Chandler et al., 1979). Infection of E. coli with the Att. - phage A6221 rer : : Tn5 ~1857 at 3O”C, which permits the establishment of immunit,y. resulted in Kan’ transductants at, frequenc+s of about IO -‘, Most of these transductant’s WX~I‘V not lysogenic for A. having resulted from transposition of ‘l’n:i to the E. ~1; chromosome (Berg. 1977). Stable A lysogens wfarv found af a frequency of about 10Y5. and each of 50 tested was Kanr (Berg. unpublished results). This failure to detect, inverse transposition of an TS50-h-IS50 segment to give A-lysogcnic Kan’ transductants was consistent with the possibilities that the inside end of one of the Tn5 putative IS elements was inactive, or that the 40,000 base-pair h genome used in these experiments was too large to be transposed efficiently. The demonstrat)ions that each of the Tn5 inverted repeats is t,ransposable by itself (BPrg ut ~1.. 198Oa.1982: Isberg & Syvanen, 1981: Hirschel 6-z Berg. 19X2), and that blocks of’ DXA as large as t,he A genome could move when bracketed by Tn5 elements (Berg c’+ whether invrrsr Stamberg. 1979; Guarent,e rt al.. 1980). led us to rr-examine transposit,ion involving pairs of I ends can be found with l’n.5. Our result,s demonstrate that pairs of I ends can fun&ion t,oget,her. although they are used VW? inefficiently relative to a pair of 0 ends, and that an 0 and an 1 end func%ion almost as efficiently as a pair of 0 ends. Models for transposase action suggest,rd by thesc~ findings are presented.

2. Materials and Methods I’lasmids and E. co/i K I:! bac%erial strains used arc’ Ii&cad in ‘I’abl~~ 1 ‘l’n,jdii I 1 pro\~ided 1)~ 31. How% and reft~rrcd to hrrwfter a.s ‘I’rt,jLt. is ahout. 4000 base%-pairs long (illstrad of 5.7 kh of TnS-WT) : the segment, adjacent to lH5UR including t,he Tn5 unique Smal. Srtll a.nd BornHI sites (see ,Jorgensen et al.. 1979) is deleted in TnSd and only a - 0.9 kb piece of I>,hu‘A which contains kan’ remains between ISSOL and IS.50K. Phagr Crud- is hOam h51<5hcil9 intam redAl irw&JcI’” Sam7: it contains about 9.5 kb less I)N4 than Ai (Hirsch4 k Kerg. 1982).

Hactrria \veIp routinely gro\vn in LN broth (I ‘A0 (w/v) NY%aminr. 0..3?. (w/v) I)iti:o yrast extract, O.SO, (V./V) Na(‘l), or on LX agar (LN broth solidified with l,.SFo (w/v) I)ifco 12acto agar). N plates for h plaque format,ion contain l’?C, N% aminr. O..i’?,, Na(“l atld I4% Kavt,o agar. Wht~rr nrcessary. kanamyvin, ampicillin and tc~t,rac~yc*linrwert~ uwd at ~ot~cc~utr;ltiotls of 50. 2.3) a.4 123 pg/ml. rrsJwctiut~ly. I’lasmid I)NA was rxt,ra.ctcd 1)~thv I
DISC’RIMINATION

BETWEES

IS50

ENDS

259

TABLE 1 Plasmids

and bacterial

strains Reference or source

Genot)pe

Designation A. I’lasmids pBR333

-2-I

pBR(:lo

6 kb: Tn&l transposed to pHR333

kb (Hachman et rrl.. 1078)

pBRGIIR

10 kb: prrrious1.v called pBR322 : : Tn.? Hip/v (Hcrg Pt 01.. 198Oa)

pHRGI6

IWB kb : see Fig. 4 (This work)

pHR(Gli

lO?i kb: see Fig. 4 (This work)

pM HS

55 kb (Holivar

el rrl.. 1977)

I)HI 14

F- AtrpEF, Q-1 sup/C+

Egnrr $ Herg (1981)

I)HIIIR

.SUpF+

,I. Crlis strain .I(%4

I)BlT,O4

F- AproB-lnc AtrpE.5 supli:+

Herp rt 01. (I 980h)

l)Bl64X

F- AtrpES AproB-lar WCA I rp.sL

Egner & Herg (1981)

1)Hl86:3-2

F- At+“:, AproB-lnc supE+ recil I rpsL @red-) (pBRG 11R (dimer))

Transformation dinw

I)HIXZ

F - Afrph’:, AproB-lac supB+ rw.4 1 rpsL (hrrd - )

DB1648 (hrrd-)

I)HIHi.?-2

F- AtrpE:, AproB-lnc sup&’ rrr.4 1 rp,sL (bred-) (pBRG10 (dimrr))

Transformation dimvf

of DBl873

with

pBRGl0

I)HIX9I

Hirsrhel & Berg (198’1) F - AtrpiC.5 h&l Asrl-wc.4306 of DHl873 Fm AtryE’ AproH-lar supE+ wc.1 1 Transformation monomer rpsL (hwd-) (pBRG16 (monomer))

with

pBR(:I 6

l)Hl908-I I)Hl!W-1

xupE+

Fm AtrpE8 AproR-lac supE+ rec.4 1 Transformation rpsl, (hrc&) (pBRG17 (monomer)) monomer

of DB1873 with pBR(:I I K

of DB1873 with

pBR(:17

The isolation of the 10 kb pBR322 :: Tn.j plasmid pBRGl1 R (formerly called pBR322 : : ‘l’n%Ni/!v) has hrrn described (Herg at ~1.. 19SOn; Hirschel & Herg. 1982). To generate the small (6 kb) plasmid pHR(:lO. we selected for transposition of 4 kb Tn5A to the 2 kh Amp’ plasmid pBR333 (a delrtion derivative of pBR322) by extracting plasmid DNA from a strain rarrying pBR333 and a chromoaotnal insertion of T&P. and transforming competent recipient cells to an Ampr Kan’ phenotype. Plasmid pBR(+lB was constructed as follows: (I) inverse transposition of h from h : : Tn5 vd- to pMB9 was selected by inducing a A : : Tn5 red- lysogen of strain DB1648 carrying pMB9. and identifying h Trt’ transducing phage which were Kan”: (2) rec.4 - cells carrying Amp’ ban’ pBRGl0 were transduced to Tet’ at 3O’C by this phage stock: (3) derivatives of these trnnsductants able to grow at 41’C were selected to enrich for t,hose which had undergone recombination between IS.50 elementas and lost h phage sequences (Fig. 4); (4) plasmid DNA extracted from a pool of t,hese clones was used to transform strain DB1648 to an Amp’ Tet’ phenotype. and an Amp’ Tet’ plasmid of the expected 1Wi kb size was saved and named pBR(:16. ‘l’hv reciprocal plasmid, pBRG17. \vas constructed in an analogous fashion : (1) inverse transposition from h : : Tn:i r-6 to pBR333; (2) transduction of rec.-l + rells carrying a pMB9 : : Tn5 plasmid with the resulting h Amp’ Kan” phage, and selection of 9mpr Tet’ transductant,s at 30°C; (3) enrichment for sulwlc~nc~sin which rrcomt)inat,ion between IS:W elements had occurred by selection of Amp’ and Tet’ at 41 (‘: (4) plasmid DNA extra&on. transformation. and identification of a l@S kb Amp’ Tet’ plasmid. The strwtures of pHRG16 and pBRG1i shown in Fig. 4 wtw verified by digestion with ShoI. which cleaves IS.50 4fG base-pairs from its 0 end. but dws not rleavr the 55 kb pMB9 or 2.1 kb pBR333 segments. Shol digestion generated 3.1 kb (amp’) and i.,5 kh (W) fragments from pBRGl6, and 4.2 kb (nw,$) and 6.3 kh (fc/‘) fragment,s from pBRG1 i.

2fN

(‘. SASAKA\V;\

ASI)

I).

R

I
esspntiall~ as dcscrihed 1)~ Brown of 01. (1959). If?r’As LVP~Cdig&ed H it.h rc~xtrictic),l endonuclt~asc~s obtained from Ne\v England Biolahs and from Krt,hrsda R~~sc~arch Laboratories according to suppliers’ inst,rurtions. and rlectrophort~sed in horizont.al acetatrbuffered 0.7% (u-/v) agarosr gels (Berg P/ 01.. 1980b). l’hage induction and transductions ~vc~r’e carried out as described hy l?gner Hr Berg (1981) and Hirsrhel & Berg (1982). using spa&c* modifications indicated in thr legends to Tahlrs 2 and Z. (c) Sfmir/s cc~rryiny puw popu/ntio~c.s oj dinwric plusrnids The populations of pHR322-related plasrnid I)NAs in WC.I + WIIS art’ hetrrogrnrous a11d generally consist of monomt+r, dimtlric and higher oligomt~ric specks. uhrreas in rr~:I - WIIS thr plasmid population is uniform (Hedhrook & Ausuhel. 1979: Hirschcal & Btq. l!M.Z). Thus. to construct, clones carr?;ing stable populations of dimeric forms of pHR($lO and pHR(:I 1Ii. rrcd - strain DHl648 was transformed with thtx hetwogwlcv~us plasmid DNA populations from WCA + ~11s. and individual transformant VIOIWS\vf’rf‘ srrcbcxncldhy plasmid caxt.ra.c+on and c~lt~ctrophorr~sisto identify those which contained thts I2 kt) pKR(: IO or 20 kh plZK(; I I I< dimeric plasmids. Ilsually ahout :i’$, of W-.-I - vIo~ws transformed nith pHR(: IO. or pKR(: I I plasmids from WC.-I+ cells contained uniform populat,ions of dimeric plasmids.

3. Results (a) Expurimrntal

plan

The finding that each of the Tn5 arms is individually transposable (Berg et&., 1980a: Hirschelk Berg, 1982) made it evident that the inside (I) end ofeach IS50 arm can join to new target. sequences. at, least in comhinat,ion with an outside (0) end. In the present work, we decided to search for MS&mediated inrcrsc transposition involving a pair of I ends under conditions in which we could also detect the usage of one 0 and one I end, or a pair of 0 ends. We analyzed the products of transposition from the pBR322 : : TnS-related plasmids pBRGl0 and pBRGl1R to Ared- in rncA - E. coli. As detailed in Figures 1 and 2. when dimericforms of these plasmids are the donors, use of a pair of 0 ends results in h Kanr Amps phage ; use of a pair of I ends results in h Amp’ Kan’ phage : and use of the 0 end of one IS50 element and the I end of another results in a h Amp’ Kan’ phage whieh contains three, rather than two, copies of TS.50. In preliminary analyses of h Kanr phage generated from plasmid pBRGl0 in DBI873, about 40!/’ of those formed using a dimeric: plasmid were Amp’ the monomeric cointegrates, whereas only about, 3 y& formed in strains carrying plasmid were Amp’ cointegrates. The conservative (non-replicative) model for Tn5 transposition (Berg, 1977) predicts the formation of h-pBR322 : : Tn5 cointegrates by direct transposition from dimers, but not from monomers, whereas some of the replicative transposition models (Bukhari. 1981) predict cointegrates from either monomer or dimer donors. Hence, to make the outcome of studies of the 0 and I ends of IS50 independent of the mechanism of Tn5 and IS50 transposition we decided to use dimeric forms of pBRGl0 and pBRGllR as donors in transposition. (b) Direct tra.nnposition To detect (Fig. 2. left)

from dimrric

plasmid

donors

transposition from dimeric pBRGl1 R (Fig. 1. left) and pBRGl0 plasmids, we induced phage development in r~crlhrpd- lysogen

l)IS(‘RIMINATIOS

RETWEEN

261

IS.50 ENDS Products

Tronspos~tion

*

Target i

0 L km ( d ,(-

R

_-__sk+2+l _--_---.-

amp feet

____-------,p

Ll ‘, A __-. ,’

FIG:. I. Products of transposition from the dimerie pKR322 :. Tn.5 plasmid pHRC I I R to phape A. Left. pBRGI IR donor plasmid and phage X target. Right,: (a) simple direct transposition using Tn5 outside (0) ends: (b) simple inverse transposition using IS50 inside (I) ends; (c) transposition of a segment, containing 3 ISSO elements mediated by 0 and I ends of 2 TSSOR elements; (d) transposition of a segment containing 3 IS50 elements mediated by 0 and I ends of 2 ISOL elements. Zig-zag lines. IS50 elemenm: broken line, h target: R and L. ISOR and ISSOL. respectively: 0 and I. IS50 0 and I ends. respectively : restriction sites (shown only in pBR322 and Tn5 sequences): Bm, RrrmHI : Bg, RglII. Distanres between RnmHI and Rg!II sites are indicated in kb. Classes (a). (c) and (d) are obtained when A/rot/’ phape are selected. and classes (I)), (c) and (d) when hemp’ I&’ are selected. dim&r

DB1873 carrying t.hese plasmids and selected A Kan’ and h Amp’ transducing phage. Table 2 shows that the majority of A Kanr transducing phage do not carry the plasmid ampI marker, and thus have resulted from simple direct transposition involving t,he 0 ends of two ISdO elements (cf. Fig. l(a)).

(c) Inverse

transposition

frown dimrric

plasmid

donors

Amp’ transductants from these lysates were screened to identify those which were KanS and thus likely to carry hPplasmid cointegrates formed by inverse transposition using pairs of 1 ends (Figs l(b) and 2(a)): only about 1‘40 kb) plasmid. These plasmid DNAs were digested with BgIII, which cleaves pBRGl0 and pBRGllR at two sites near the 1 end of each IS50 element and cleaves h once. Digestion of the cointegrates from pBRGl0 donors generated a 5.1 kb fragment consisting of pBR333 plus most of each IS50 element (Figs 2(a) and 3(a)); similarly, BglII digestion of /\ Amp’ KanS DNAs from the pBRGl1 R donor generated a 7.5 kb fragment consisting of pBR322 plus most of each IS50 element (Figs l(b) and 3(b)). The - 1 kb and 2.5 kb BgbII fragments which consist of the central regions of Tn5d and Tn5 in pBRGl0 and pBRG1 IR were missing. We conclude that these h Amp’ Kar? cointegrate plasmids resulted from ISSO-mediated inverse transposition. Tests using nine other plasmids, similar in st.ructure to pBRG1 I R, also resulted in low frequencies ( - 1%) of inverse

262

(‘. SASAKAWA

ANI)

I). IC. HE:R(: Y!eld

Products I s.. ..-.

(d)i

I

\----.-

I : 3/710 x.-. :

L5.12 --------.----------------.,~-

(01;

,-

0 amp 0

OampO c

3.1 --I

IkI

OompO

2.9----13.1-f

I h -‘I

O/60

-. .-.----.-------,~~.-,

PIG:. 2. h Amp’ transducing phages generated by transposition from a dimeric pBKUl0 plasmid to a phage A target. Left: dimeric pBRGl0 donor plasmid and phage h target. Right: (a) simple inverse transposition mediated by the I ends of a pair of IS50 elements: (b) simple direct transposition mediated by the 0 ends of a pair of IS50 elements of the complex transposon TnSd-pBR333-T&d. a segment containing 4 IS50 elements: (c) transposition of a segment containing 3 IS50 rlement~s mediated by 0 and I ends of 2 ISM elements; (d) inverse transposition of a complex segment containing 4 ISSO elements and mediated by the I ends of a pair of IS50 elements (not detected in the sample of 60 A Aml? Kan’ cointegrates tested). Zig-zag lines. IS.50 elements: broken lines, sequences of h phage targtat : 0 and I, IS50 0 and I ends: k and camp, resistances to kanamycin and ampicillin. respectively. The sizes in kb of diagnostic fragments generated by RglII digestion ((a)) and by Xhol digestion ((b) to (d)). are indicated.

h Karr’ transducing phagr in lysatrs prrparrd by thermal induction of h Iysogrnic strains I)HlXW:! and Dl3187R-4. which contain dimeric plasmids. were selected 1,~ infection of DB114 at a multiplicity of 02 phage/cell. and selection of Kan’ transductants at 31 C. Hecause t,he vast majority of Ampr phape ~rrr also Kan’ (column -5). the frequencies of Amp’ phage were calculated directly from thr Amp’ fraction of Kan’ phagr (columns 1 and 2). The determination of h Kan’ phage from pBR1ZIO and pBR(:l I K was bawd on 14 and 3 separate phage lysatw. respectivelv. The fraction of Amp’ phage that was Kan’ (column 3) was based on tests of Amp’ transdurtants &orn Iysatrs of 35 subclones of DB1875-2, and 33 subclones of DB1863-2 (20 to 21 amp’ transductants per Iynate). So mow than ow Kan” Amp’ transductant was found in each set of 20 to 21 transductants. The direct transposit,ion frequency (column 3) is calculated using the data in columns 1 and 2. and the inverse transposition frequency (column H) is calculated using the data from c~~lumr~s1 and 5. t Direct transposition. $ Inverse transposition.

DISCRIMINATION

BETWEEN

IS50

ENDS

263

12345

1234567

(a)

6

78

(b)

FIN:. 3. &arose gel analysis of Bglll digests of h Ampr Kan’ plasmid l)SAs resulting from inwrst transposition from (a) pBRG10 and (h) pBRGI1R. (a) BglII-digested h Amp’ Kan” I);h;As (lanes 1 to 5) and pHR(:IO 1)SA (lane ti): law 7. HindIlLdigested A+ l)NA size standard. (b) HglII-digested A Amp’ Kan’ 1)S.h (laws I to 6) and pBR(:I 1 R DSA (lane 7): Iant’ 8. HhdITI-digested X+ USA size standard.

pBRGI6

km,

(b)

y”‘r 1)

-0

I

,pEIRGl7

Flc:. 4. Construction and structures of pBR(: I6 and pHR(:I5 plasmids. These composite plasmids contain Amp’ plasmid pBR333 joined to Tet’ plasmid pMRY by inverted repeats of IS50 elements. pBRGl6 was generated by homologous recombination between pKRGI0 and a h--pMH9 phage ; pBR(:li was generated by homologous recombination b&veen a pMB9 : : Tn.5 plasmid and a LpBR333 phage (see the legend to Table 1). Arrows point to Xhr~l sites present in IS50 (4X5 base-pairs from 0 twls). and absent. from pMB9 and pBR333 components.

transposition (Lowe, Sasakawa &, Berg. unpublished results). Thus, pairs of I ends of IS50 elements can function together to mediate inverse transposition, but at, a frequency which is low relative to ot’her ISSO-mediated transposition events. 1

(d) Inverse tmnsposition

from plasmidP3 lacking

thf Tn.5 central region

To determine if inefficient usage of a pair of I ends is a property of TS50 elements themselves, or due to some property of the Tn5 central region. we generated the composite Amp’ Tet’ plasmids pBRG16 and pBRG17, in which Amp’ and Tet’ replicons are joined by inverted repeats of IS50 (Fig. 4). Because inverse transposition products from the dimeric plasmids used above were so much rarer than cointegrates formed using one 0 end and one I end (see section (e). below), we analyzed transposition products formed in DB1873 carrying pBRGl6 and pBRG17 plasmids as monomers. Inverse transposition product,s (h Amp’ Tet” phage from pBRG16 and /\ Tetr Amp” phagc from pBRG17) were obtained 103-fold less frequently than the reciprocal direct t,ransposition products (Table 3). Shol restriction endonucleasc digest,ion (Fig. ;‘,) of representative X Amp’ anti A Tetr phage generated the predicted fragments. We conclude that inefficient, use of pairs of I ends is a property inherent to TS50. and is not due to sequences in the central region of Tn5.

I)IS(‘KlMISATIOS

HETWEES

IS50

ENDS

26.5

TAIII,E 3 Inverse arid dirrct

transposition

frequencies

unaffected

‘I’rwrsposition donr,r

h Tet’ Amp’

Frequency h Amp’ Tet’

pI3Kt:lti pBR(:li

4.1 x 10-V 3.5 x 10-s:

4.7 x 10-91 4.4 x 10-V

by the Tn5 central reyion Ratio inverse/dirr~ct transposition I.1 x 10-s I .3 x IO - 3

The selection for h transducing phage was carried out as described in the legend t,o Table 2. except that the multiplicity of infection of strain DB1891 was 1 phage/cell. Each of 60 Tet’ transductants from I)Kl9(WI Iysates tested was Amp’ and thus resulted from direct transposition. Similarly. each of I60 Arn$ transduct.ants from DBI909-I Iysates tested was TetS. and thus also resulted from direct transposition. The inverse transposition frequenries given in the Table are corrected for a high frequently of Amp’ Tet’ transductants among Amp’ from pBR(:IS (56 of 60 Amp’ were also Tct’). and am~mg Tct’ from pKRGl7 (66 of 76 Tet’ were also Amp’). t Direct transposition. : Inverse transposition.

(e) Transposition

involving

one 0 crnd one I end

of h Amp’ Kan’ plasmids, which formed the largest group of transposition products from dimeric pBRGl0 and pBRGl1R donors. were used to assess t,he abilit,y of an I end of one IS50 element and an 0 end of another to act in t.ransposit.ion. XhoI cleaves pBRGl0 only within its IS50 components and generates a 2.9 kb fragment, containing kan,r and IS50 I ends and a 3.1 kb fragment containing amp’ and IS.50 0 ends. Consequently. XhoI digest,ion can be used to identify cointegrates formed using pairs of 0 ends. an 0 end and an I end, or pairs of 1 ends. based on the numbers of 2.9 kb and 3-l kb fragments per genome (Fig. 2). Of 60 cointegrates from pBRGl0 dimers a,nalyzed by XhoI digestion as in Figure 6. 34 yielded a 2 : 1 ratio of the 2.9 kb to 3.1 kb fragment: these contained complete TnSd elements flanking pBR333 sequences, and resulted from insertion mediated by a pair of 0 ends. Twent.y-six of 60 yielded a 1 : 1 ratio of 2-9 kb to 31 kb fragments: these resulted from transposition mediated by one 0 and one I end. We conclude that, an I end in combination with an 0 end can be used almost as efficiently in transposition as a pair of 0 ends. None of the 60 cointegrates yielded the 1 : 2 ratio of 2.9 kb to 3.1 kb fragments expected of inverse transposition (see Fig. 6). This supports the conclusion of experiments in sections (c) and (d). above. that pairs of 1 ends a.re used only rarely in transposition. \Ve also analyzed the h Amp’ Kan’ coint,egrates due to transposition from dimerir pBRBl1R. Because BamHl cleaves asymmetrically both within the Tn5 central region and in pBR322 (Fig. 1, left), digestion of cointegrates formed using directly repeated ISSOR elements generates a 5.9 kb ISSOL-containing fragment regardless of the position or orientation of the insert in the h genome (Fig. 1(c)). Digestion of cointegrates formed using directly repeated ISWL elements generates a 4.2 kb ISSUR-containing fragment (Fig. l(d)). Of the .S6h Amp’ Kan’genomes analyzed in this way, 27 resulted from IS5OR and 29 resulted from IS5UL-mediated .4nalyses

266

C. SAGAKAWA

1

2

3 4

AND

5 6

I).

E.

BER(:

7 8 9 10 11 12

(a) -.

r-------

tet

0

0

ori

‘I

\\ .___-- ----- - --------------------------,

I I

- - - - - -,

L7.5-

:

I

r------------

\ .___

amp L

-------

-----

-----

or/

+x-/’ I

e--

------___, \

34-J _-___

-----

----

-+

-__--A

1

-’ I

(b) FIG:. 5. l’roducts of transposition from monomeric pBK(: I6 and pHH(: Ii plasmids to phagr h. (a) Agarow gel electrophoresis of X&-digested h Tet’ Amp’ (lanes 2 to 7). h Amp’ Tet” (lanes 8 to 11) cointegrat,e plasmids from pBRGl6 and pBRGl6 control (lane 12). Lane I. HindIII-digested h’ DNA. (b) Distinctive 75 kh and 3.1 kh XhoI fragments in h Tet! Amp” and h Amp’ Tet’ cointegrate plasmids reflect the use of a pair of 0 ends (direct transposition). and a pair of I ends (inverse transposition). respectively. (c) Agarose gel rlwtrophoresis of Xhol -digested h Tet’ Amp5 (lanes 2 to 4) and h Amp’ ‘I’&” (laws 5 to 7) cwintegrate plasmids generated hy transposition from pKR(G Ii. and pBRG17 control (lane 8). Lane 1, Hi,ldIII-digested h+ IJS.4. (d) Distinctiw 4.2 kh and ti.4 kl) Shol fragments in h 4mp’ ‘I’& and h Tet’ Amp” cointegrate plasmids reflect the use of a pair of 0 ends. and a pair of I ends. respectively.

transposition (see Fig. 7 for representative digests). In none of the 56 cointegrates did BamHI digestion generate both the 5-9 and the 4.2 kb fragments expected of cointegrates in which direct repeats of complete TnS elements bracketed pBR322 sequences. (Since cointegrates analogous to these were recovered from the smaller pBRGl0 plasmid (Figs 2(b) and 6). we presume t’hat they probably were formed intracellularly and that the resulting 56 kb cointegrate phage genome was too large to package efficiently (cf. Berg, 1974).) B ecause the h Amp’ Kanr cointegrate phage

DISCRIMINATION

BETWEEN

I850

6

,-----------: 1 \ .-------------------------

,---s-m ; I \

0

I

olnp ori -

78

0

------------.

\

4.2- ------

ori

tet -6.4

.__-----------

267

ENDS

-- -,‘A ----

I

x

------_

\

----

---------------,/L

----

\ ’ -1

x

I ’ d’

(d) FIG:. 5(v). (cl).

comprise a major fraction of those obtained when either h Amp’ or h Kan’ phage are selected, it is clear that an I end of either ISSOR or lS5OL, in combination with an 0 end, functions well in transposition from the pBRGllR dimeric plasmid. In previous studies we had found a 20 : 1 ratio in the use of pairs of ISSOR verms pairs of ISSOL elements in the formation of cointegrates from pBRGllL (Berg et n2., 1981 ), a plasmid which differs from pBRGl1 R used here only in the orientation of Tn5 in pBR322. Possibilities of an important influence of the position or orientation of Tn5 in the control of transposition are suggested by comparing that) 20 : 1 ratio with the 27 : 29 ratio in the current experiments, and we are intent on determining how this control is exerted.

(‘. SASAKALV.4

1

2

3

4

ASI)

I). E. l
5

6

7

8

9

4. Discussion The results present,ed here extend our earlier conclusion (Berg et al.. 1980a.1982 : Hirschel & Berg, 1982) that each of the Tn5 1.5 kb terminal inverted repeat arms is an IS element (IS50). and that pairs of IS50 elements mediate the transposition 01 any Dh’A segment between them. Our present data show that pairs of 0 ends of IS50 are about three orders of magnitude more efficient in transposition than pairs of/ ends, even though a pair of 0 ends is only slightly more efficient than an 0 and an I end. Analyses using plasmids which contained inverted repeats of ISBO. but none of the Tn5 central region, showed that the inefficiency of I end usage is inherent in the DNA sequence of IS50 itself, and is not due to inhibitory sites in t.he Tn5 central

I)IS(‘RIMINATION

3

BETWEEN

4

5

8

7

8

9

IS50

ESI)S

lo

11 12

269

plasmids of BnmHI digests of h Amp’ Trt’ Kan’ cointegratr i. Agaro se gel elertrophoresis indicative of from dimeric pRRGIIR to phage k The 59 kb fr agmwt ng from t ransposition of transposition is evidmt in lanes 2. 3. 5. 6. 7 and 9. The 4.2 kl) fi ragment indicative -rnrdiated RntnHIis evident in laws I. 4. 8 and IO. Laws I1 and I 12 umtaiu m tadia t,rd transposition A+ I)SA st,andards. rrspwtivrly. cad pHR(:I 1 R DNA and HindTI-digested

region. Isberg & Syvanen (1981) have also concluded that the IS50 I ends are used inefficiently. Thr termini of IS50 consist of a hyphenated eight of nine base-pair inverted repeat (Berg et al.. 1982), which is smaller than that found at the termini of any other characterized IS element. Thus, the rarity of inverse transposition might conceivably reflect fundamentally different roles for 0 and I ends. For example. in rolling circle (replicative) models in which transposition initiates at one end, and t,erminat’es at the other end of an element (Bukhari, 1981). 0 ends might be used primaril,v for initiation, and I ends for termination. The very large proportion of cointegrates obtained when inverse transposition was selected in strains carrying primarily monomeric pBR’G16 and pBRG17 plasmids (see the legend to Table 3) is consistent with this interpretation. However, because of data suggesting that direct transposit,ion of Tn5 may in fact be conservative (Results, section (a): and unpublished results), we currently prefer the alternative explanation that the eight base-pairs common to both ends represent only a minimal recognition site, and that transposase most efficiently acts on a sequence at the IS50 0 end which ext’ends beyond the eight base-pairs common to both ends. Two sequences near the IS50 0 end can now be pinpointed as possible caomponents of an extended strong recognition sit,e for an ISBO-encoded transposasr: (1) base-pairs 56 to 61, which are a direct repeat of base-pairs 1 to 6 (Auerswald et al., 1980) may comprise a site at which transposase protein could bind to IS50 and repress transposase gene expression (Johnson & Reznikoff, 1981) :

270

(‘. SASAK.AW.4

AXl1) I)

E:. lSEK(;

(2) base-pairs 15 to 26 of IS50 resemble sequences often found at sites of ‘l’n5 insertion (Bossi & Ciampi, 1981). Tn5 is known to insert non-randomly (Shaw Xr Berg, 1979; Berg et al., 198ob; Miller et al., 1980), and it is possible that sites in target DNAs favored for transposable element insertion are chosen by virtue of their similarity to the sequences in the element recognized by the transposase protein. In contrast to what we find analyzing IS50, the two ends of the IS10 inverted repeat arms of tetracycline resistance transposon TnlO appear to be used with similar efficiencies (Foster et al., 1981). The occurrence of ISIO-mediated direct and inverse

transposition

at

about

the

same

frequency

implies

that

complete

transposase recognition sites may be present at both ends of IWO. The efficient use by Tn5 of one 0 and one I end, relative to a pair of I ends. suggests to us that transposase binding to an element may involve two steps. In one step, efficient binding depends strongly on the complete recognition sequence found at the 0 end, Binding to a second end is less sensitive to the difference between strong (0) and weak (I) binding sites. Because sequences resembling the IS50 I end should occur by chance at about 100 sites in bacterial chromosomes, single copies of IS50 present near one of these of bacterial genes bounded on one sites should be able to mediate the transposition side by IS50 which supplies 0, and at the other end by a sequence which resembles in transposase-DNL4 recognition may provide an the IS50 I end. Such flexibility important source of new transposable elements in natural populations.

We are grateful to 1,. McDivitt and R. Ramabhadran for supA technical assistancr. to I)rs C. %I. Berg and S. Brenner for stimulating discussions. to I.. (“are for hospitality during the preliminap experiments. and to fil. Howe for the gift of Tn547711. This work was supported by Public Health Service research grant, 5 ROl AI 14267 (to D.B.). American Cancer Society Institutional Grant IN-X, and Public Health Service international Research Fellowship 1 FO,i TWO2949 (to (1.8.). Preliminary experiments were supported by a grant from the Fonds National Suisse de la Recherche Scientifiqur to 1,. (“art).

RFFFRFNVFci 1 A 1 Auerswald. E.. I,udaig, 107--I 13. Bachman. I(.. Betlach.

(:. & Schaller. Jl.. Bayer.

1,

H. (1980). Cold ,Sprir~y H&or H. & Tanofsky.

Sytt/y.

Qwr,ct. Rio/. 45.

S. (1978). (‘old Spritrg Hrxrhor S!lnrp.

Qwrnt. Biol. 43. 69-76. Bedbrook, J. R. &. Ausubel. F. M. (1979). Crll. 9. 707 716. Berg. C. M. & Berg. D. E. (1981). In &1icroDiology. I!#31 (Rchlrssinger. I).. ~1.). pp. IOi 116. ASM Publications. Washington. I).(‘. Berg. D. E. (1974). .J. Mol. Biol. 86. 5!#-68. I’loart~ids rind E'pisomrs (Hu khari, Berg, D. E. (1977). In I).Y,J Ir~swtion Elements, A. I.. Shapiro. J. A. & Adhya. S. L.. rds), pp. 205-212. (‘old Spring Harbor Press. (‘old Sprmg Harbor. Berg. I). E. & Drummond. 31. (1978). J. Bactrriol. 136. 41!)+%22. Berg. I). E. ;G Stamberg. J. (1979). Grnutics, 91, si. Berg. D. E.. Davies. J.. Allet. H. & Rochaix. J-1). (1!)75). I’roc. .\ir/. .-Irnd. SC;.. 7T.LS..-l. 72. 3628-3632.

DISCRIMINATION

BETWEEN

1650 ENDS

271

Berg, D. E., Egner, C., Hirschel, B. J., Howard, J.. Johnsrud, L., Jorgensen, R. A. & Tlsty. T. D. (1980a). Cold Spring Harbor Symp. Quant. Biol. 45, 115123. Berg. D. E.. Weiss, A. & Crossland, L. (19806). J. Bucteriol. 142, 439-446. Berg, D. E., Sasakawa, C.. Hirschel, B. J., Johnsrud, L.. McDivitt, L., Egner, C. & Ramabhadran, R. (1981). In Molecular Biology, Pathogenicity and Ecology of Bacterial PlaswGds (Levy, S. B.. Clowes. R. C. & Koenig, E. I,., eds), pp. 381-390, Plenum Press. New York. Berg, D. E., Johnsrud, L., McDivitt, L., Ramabhadran, R. & Hirschel. B. J. (1982). Proc. Sat. Acad.

Sci.,

U.S.A.

79, 3632-3635.

Birnboim. H. C. & Daly, J, (1979). ,I’ucl. dcids Rrs. 7, 1513-1523. Bolivar, F., Rodriguez, R. L.. Greene. I’. J., Betlach. M. C.. Heyneker, H. L.. Bayer, H. W., Cross, J. H. & Falkow. S. (1977). Gen,e. 2, 95-113. Bossi, L. & Ciampi, M. S. (1981). Mol. Uen. Gen,et. 183, 4OfS408. Brown, M. G. M.. Weston, A.. Saunders. J. R. & Humphrey, (:. 0. (1979). FEMS Microbial. Letters, 5, 219-222.

Bukhari. A. I. (1981). Trends B&hem. Sci. 6. 56-66. Calos, M. P. & Miller, J. H. (1980). c)ell, 20, 579-595. Campbell. A.. Berg, D. E.. Botstein. D.. Lederberg, E. M.. Novick, R. P., Starlinger. P. & Szybalski. W. (1979). Gene. 5. 197-206. Chandler. M., Roulet’. E.. Silver, I,.. Boy de la Tour. E. & Care. I,. (1979). Mol. (Zen. (&net. 173, 23-30.

Egner c’. & Berg. D. E. (1981). Z'roc. Alid. dead. Sci.. U.S.=l 78. 459-463. Foster. T. J.. Davis. M. A.. Roberts. D. E.. Takeshita. K. & Kleckner. N. (1981). Cell. 23. 201-213. (iuarente. L. P.. Isberg, R. R.. Syvanen. M. & Silhavy. T. J. (1980). J. Mol. Biol. 141, 23S 248. Hirschel. B. J. & Berg, D. E. (1982). J. Mol. Biol. 155. 105120. Isberg. It. R. & Syvanen, M. (1981). J. Mol. Biol. 156, 15-32. Johnson. R. C. 8 Reznikoff. W. S. (1981). Sucl. dcids Res. 9, 1873-1883. Jorgensen. R. A., Rothstein, S. J. & Reznikoff, W. 8. (1979). Mol. Nen. Genet. 177. 56-72. Miller. J. H., Calos. M. P.. Galas. I).. Hofer. M., Buchel. D. E. & Muller-Hill, B. (1980). J. Mol.

Biol.

144. l-18.

Rothstein. S.. Jorgensen, It., Pestle. K. & Reznikoff. W. (1980a). Cell. 19, 795-805. Rothstein. S. J.. Jorgensen. R. A.. Yin. J. C. l’.. Yong-di. Z.. Johnson. R. & Reznikoff, W. S. (198ob). Cold Spring Harbor Symp. f&ant. Rio!. 45, 99--10.5. Shaw. K. ,J. & Berg. C. M. (1979). (:enPtics. 92. 741-747. Edited

by J. H.

Afiller