A PRODUCT OF THE TN5 TRANSPOSASE GENE INHIBITS TRANSPOSITION

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Copyright 0 1983 by the Genetics Society of America

A PRODUCT OF THE TN5 TRANSPOSASE GENE INHIBITS

TRANSPOSITION

JOHN B. LOWE' AND DOUGLAS E. BERG

Departments of Microbiology ond Immunology and of Genetics, Woshington University Medical School, St. Louis, Missouri 63120

Manuscript received November 5, 1982 Revised copy accepted December 16,1982

ABSTRACT

The bacterial transposon Tn5 possesses a regulatory mechanism that allows it to move with higher efficiency when it is first introduced into a cell than after it is established. Tn5 is a composite transposable element containing inverted repeats of two nearly identical elements, ISSOR, which encodes the transposase protein necessary for Tn5 movement, and IS50L which contains an ochre mutant allele of the transposase gene. Data presented here show that Tn5 transposition is inhibited about 50-fold in cells of Escherichia coli which already carry IS50R in the multicopy plasmid pBR322. If the cells contain a plasmid carrying either IS5OL instead of ISSOR, or derivatives of ISSOR in which the transposase gene has been mutated, little if any inhibition of Tn5 transposition is found. Although inhibition had previously been hypothesized to require interaction between the products of IS50L and IS50R, our results show that IS5OR alone is sufficient to mediate inhibition and suggest that the inhibitor is a product of the transposase gene itself.

HE movement of transposable elements to new sites in a genome is mediated

T

by element-specific transposase proteins and does not require the extensive

DNA

sequence homology essential for classical recombination. Each of the

prokaryotic elements tested to date has evolved mechanisms of transposition

that increase its copy number relative to other genomic sequences, and several

of the transposable elements indigenous to

E. coli are present in five to ten

copies per genome (for reviews,

CALOS

and

MILLER

1980;

STARLINGER

1980;

KLECKNER

1981;

SAPIENZA

and

DOOLITTLE

1980).

Transposition can cause mutations, alter the expression of genes near inser-

tion sites and lead to a variety of genome rearrangements. Unrestrained increase

in the number of copies of any

DNA

sequence is also likely to be harmful.

Consequently, it is probable that selection has favored elements that transpose

at higher frequencies when first introduced into a cell than after they are

established. Much as prophage immunity blocks lysogenization by temperate

phages, such regulation should benefit resident transposable elements directly

by inhibiting the proliferation of homologous and potentially competing ele-

ments introduced into the occupied cell. Regulated transposition has been

demonstrated using several different prokaryotic elements:

$3

and Tn3 (Amp')

(2)

606

J.

B.

LOWE AND D. E. BERG

(CHOU et

al.

1979; GILL, HEFFRON

and FALKOW

1979; GRINDLEY

et

al.

1982),

Tn5(Kan") (BIEK and ROTH 1980a,b), TnZO(Tet') (BECK, MOVED

and INGRAHAM

1980) and bacteriophages

X

and Mu (HERSKOWITZ

and HAGEN

1980;

BUKHARI

1976).

In principle, transposition could be regulated by

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repression of transposase

synthesis, as is found with Tn3 and y8, and phages

X

and Mu, (2) binding of an

inhibitor to the transposase protein, or

(3)

binding of an inhibitor to

DNA

sequences that transposase must recognize when it mediates transposition.

Transposon Tn5, unlike Tn3,

y8,

X

and Mu, has only one gene whose product(s)

is involved with transposition and may, therefore, regulate its own transposition

differently. Tn5 is a composite element containing terminal repeats of insertion

sequences named IS50R and IS50L (BERG

et

al.

1980,1982a;

ISBERG

and SYVANEN

1981). IS5OR encodes transposase, whereas IS50L, which differs from IS50R at

a single site, contains an ochre mutant allele of the transposase gene

(ROTHSTEIN

et

al.

1980; ROTHSTEIN

and REZNIKOFF

1981; AUERSWALD,

LUDWIG

and SCHALLER

1980). The transposase gene occupies nearly the full length of IS50R, and two

proteins which are 421 and 461 amino acids long are translated from staggered

in-phase translation initiation sites in the transposase message

(ROTHSTEIN

et

al.

1980;

ROTHSTEIN

and

REZNIKOFF

1981). There is no other long open reading

frame likely to encode a separate repressor (AUERSWALD,

LUDWIG and SCHALLER

1980), and the central region of Tn5 between its IS50 elements does not encode

functions involved in transposition

(BERG et

al.

1982a; SASAKAWA

and

BERG

1982).

It has been proposed (BIEK and

ROTH

1980a,b) that inhibition requires the

presence of IS50R, and also of IS50L which encodes a pair of truncated proteins

26 amino acids shorter than those of IS50R. The results presented here and in

a preliminary report

(BERG

et

al.

1982b) show that IS50R alone inhibits Tn5

transposition and indicate that inhibition is mediated by a product of the

transposase gene.

MATERIALS AND METHODS

Phage and bacterial strains: Phage X::Tn5 is a derivative of Ab221 rex::Tn5 cI857 (BERG 1977)

containing the mutant alleles Oom29 Pam80 and was obtained from T. SILHAVY. The bZ21 deletion makes the phage unable to undergo integrase-promoted insertion into the bacterial chromosome, and the 0 and P alleles make it unable to replicate autonomously in nonsuppressing (sup-) E. coli. Xbbnin is Ab515 b519 xisam6 c1857 nin5 Sam? Xred- is

X

b.515 b519 intam29 redAI5 imm2lc" Sam7 (BERG et al. 1982a; HIRSCHEL and BERG 1982). These phages integrate and excise in sup+ bacteria, and the prophages can be induced by thermal inactivation of their thermolabile repressors; the genomes of Xbbnin and of X red- are about 7.5 and 9.5 kilobases (kb), respectively, smaller than that of A-wild type, and, thus, are suitable for detecting the transposition of elements such as Tn5-410

(7.7 kb) as well as Tn5 (5.7 kb) (see Figure 1).

All bacterial strains are derived from E. coli K-12. DB1873 is F- recAl AproBlac AtrpE5 supE' (ked-) (SASAKAWA and BERG 1982). DB1977 is F- gal rpsL sup- recA-srl::Tn10(Tetr)A306, generated by transduction of strain 594 (CAMPBELL 1961) with P1 grown on E. coli carrying the TnlO-linked recA deletion of JCl0289 (CSONKA and CLARK 1979). DB114 is F- AtrpE5 supE' hfl-1 and can be efficiently lysogenized by X after infection at low multiplicity ((1 phage/cell) (EGNER and BERG 1981).

Microbial procedures: LN broth (BERG, WEISS and CROSSLAND 1980) and minimal medium E

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INHIBITOR OF T N 5 TRANSPOSITION 607

IS501

IS50R

Tn5-WT

0

tnp'

I

kan'

I

t n p +

0 I

S50A

H

_Y

I

S50A

**- * * X $ $ -**

0

t r p f +

0 T n 5 - 4 1 0

FIGURE 1.-Maps of Tn5 elements. Tn5 is 5.7 kb long, is not homologous to sequences in the chromosome of E. coli K-12 (BERG and DRUMMOND 1978). and is a composite element containing terminal inverted repeats of the 1.5-kb transposable elements IS5OR and ISSOL (thickened lines) (BERG et al. 1982a; ISBERG and SYVANEN 1981). The transposase gene, whose product is necessary for Tn5 and for IS50 movement, is within IS50R. It is transcribed inward from a promoter about 49 bp from IS50Rs outside end (ROTHSTEIN et al. 1980; JOHNSON and REZNIKOFF 1981). The message has two in-phase initiation sites 120 bases apart. Translation of these two proteins is terminated by a UGA codon at position 1521 (14 bases from IS50s inside end) (ROTHSTEIN et al. 1980). ISSOL differs from IS50R by a single base pair at position 1443 and contains an ochre mutant allele of the transposase gene (ROTHSTEIN et al. 1980; ROTHSTEIN and REZNIKOFF 1981; AUERSWALD, LUDWIG and SCHALLER 1980). Tn5-410 was generated by replacement of Tn5-wild type's central HindIII fragment with a 5.3-kb fragment containing a trpE+ gene; the internal 340 bp of each IS50 element is missing in Tn5-410 (indicated by the symbol

A),

and hence, Tn5-410 cannot transpose unless complemented by the transposase encoded by another element (MEYER, BOCH and SHARPIRO 1979). Sites recognized by restriction endonucleases HindIII, BglII, BclI and BamHI are indicated as H, Bg, Bc and Bm, respectively. The positions of the HindIII, BglII and BclI sites in base pairs from the outside ends of IS50 are 1196, 1516, and 1521, respectively (AUERSWALD, LUDWIG and SCHALLER 1980; COLLINS, VOLCKAERT and NEVERS 1982).

extracted by a n alkaline sodium dodecyl sulfate lysis method (BIRNBOIM and DOLY 1979). Restriction endonucleases and T4 DNA ligase were used in accordance with instructions of the suppliers (New England Biolabs and Bethesda Research Laboratories). Transformation of E. coli with plasmid DNA and electrophoresis of plasmid DNA in horizontal agarose slab gels were carried out according to standard procedures (BERG, WEBS and CROSSLAND 1980 HIRSCHEL and BERG 1982). General condi- tions for phage X growth have been described (EGNER and BERG 1981; SASAKAWA and BERG 1982).

Transposons: The structures of the kanamycin resistance transposon Tn5 (BERG et al. 1975) and its transposition deficient trpE+ substitution derivative Tn5-410 (MEYER, BOCH and SHAPIRO 1979) are diagrammed in Figure 1.

Plasmids: The salient segments of the plasmids constructed to localize regions necessary for inhibition of transposition are diagrammed and described in Figure 2. The construction and characterization of a hoterodimeric plasmid for cis-complementation analysis of transposase func- tion is diagrammed and described in Figure 3.

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608 J. B. LOWE AND D. E. BERG

P H R H S

-

b

<

pBRG551

H H

r

0

pBRG2

H R H

+

-T

--

pBRG552

H H

0 ISSOR I konr I ISSOL 0

ti

r

Hi.& Bm I Bc

II

Bg L H Bm

I 1

5

0

r

Bm Bm

0

B C Bm

0

BQ Bm

- .

.

H H Bg Bc Bm Bc BgH Bm S

0

Y

r

Bc Bm

0

89 Bm

pBRG68R

pBRG553

pBRG557

p BR G 554

pBRG66L

pBRG556

their deletion derivatives. FIGURE Z.-Maps of segments of pBR322::IS50, pBR322::Tn5 and

Thickened lines indicate IS50 sequences, 0 and I denote the outside and inside ends of IS50, respectively, and the open boxes represent the extent of the deletions generated in vitro. P, S, R, H, Bm, Bc and Bg indicate sites recognized by restriction endonucleases PstI (in pBR322), SdI, EcoRI, HindIII, BamHI, BclI and BglII, respectively. Only the segments extending between the PstI and

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IJ'HIBITOR OF

TN5

TRANSPOSITlON

609

RESULTS

The Kan' colonies formed after

E. coli is infected with a X::Tn5 phage defective

in both replication and integration result from transposition of Tn5 from the

A

vector to the host genome

(BERG

1977). The presence of Tn5 inhibits the

transposition of a second Tn5 element in the same cell (BIEK

and

ROTH

1980a,b).

To identify segments of Tn5 that inhibit transposition we constructed a set of

derivatives of the recA- strain DB1977, each harboring one of the series of

pBR322-derived plasmids containing portions of Tn5 (Figure 2), and measured

the efficiency of formation of Kan' transductants after infection by A::Tn5.

Because elements such as Tn3 (Amp') transpose to plasmids preferentially

(GETSCHMER

and COHEN

1977) we carried out tests to determine whether

pBR322 also served as a trap for

Tn5

transposition from

A. Plasmid DNA was

extracted from pools of >10,000 Kan' transductant colonies generated after

h::Tn5 infection of strain DB1977 harboring pBR322

or

the pBR322::IS50R

plasmids pBRGl

or

pBRG2, and plasmid DNA was extracted and used to

transform plasmid-free DB1504. In each of the three cases only about 1

of

lo4

Amp' transformants also carried the Kan' marker of Tn5. These results indicate

that Tn5 does not transpose preferentially to pBR322 plasmids.

Tests of inhibition of transposition of Tn5 from A::Tn5, using pBRGl and

pBRG2 which carry

IS50R

but no other sequences from Tn5, showed that IS50R

alone caused an approximately 40-fold inhibition in Tn5 transposition (Table

1,

lines 2 and 3). In contrast, derivatives of pBRGl and of pBRG2 lacking comple-

mentary one-fifth and four-fifth segments of

IS5OR

(pBRG551 and pBRG552,

respectively; Figure

2)

did not inhibit Tn5 transposition (Table

1,

lines

6

and 7).

To assess whether a product of the transposase gene of IS50R is responsible

for inhibition, deletion derivatives of pBR322::Tn5 plasmids were generated

in

vitro,

Plasmid pBRG557, generated

by

ligation of the BclI site near the inside

(I)

end of IS50R and the BamHI site in pBR322, (Figure 2) should encode a normal

length transposase (Figure 4), although it is missing sequences at IS50Rs I end.

Like intact ISSOR, this truncated IS50 element inhibits transposition of Tn5 from

an infecting

A::Tn5

phage; a complementation test showed that this fusion does

indeed encode

a

transposase that can promote Tn5-410 movement (Table 1, line

Since we did not have a pBR322::IS50L pIasmid similar

tu

the IS50R plasmids

pBRGl and pBRG2, we generated pBRG558, a fusion of the BclI site of IS50L to

the BamHI site pBR322. This plasmid neither inhibited Tn5 transposition nor

complemented Tn5-410, (Table

1,

line

8).

Its failure to make a functional

transposase reflects the ochre allele in IS50L,

26 codons before the 3'-terminus

5).

were generated from pBRG68R using double digestion with BamHI and either BclI (pBRG557) or

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610

J ,

B.

LOWE AND D. E. BERG

pBRG559

(7)

INHIBITOR OF

TN5

TRANSPOSITION

611

TABLE 1

Identification of sequences in Tn5 that affect Tn5 transposition

_____ _______ _ _ _ _ _ _ ~ ~~

Complementa- Tn5 transposition Transposition to X tion of Tn5-410 Plasmid" Salient characteristic from k T n 5 (X

lo-')*

(x (X 10-6)d

1. pBR322 2. pBRGl 3. pBRG2 4. pBRG553 5. pBRG557 6. pBRG551 7. pBRG552 8. pBRG558 9. pBRG554 10. pBRG556

no Tn5 sequences 1.5 (f0.85) IS50R 0.04 (f0.04) IS5OR 0.04 (20.05) IS5OR 0.02 (f0.009) IS5OR ABclI 0.02 (fO.009)

IS50L ABclI 1.1 (f0.04) IS5OR AHindIII 1.5 ( f l . 1 ) M O R AHindIII 2.0 (f0.6)

IS50 R ABgl I1 0.83 (f0.3) ISSOL ABglII 1.2 (f0.3)

0.018 (fO.O1) 0.90 (f0.09) 1.1 (fO.O1) 1.6 (f0.70) 0.018 (f0.003) 0.017 (f0.005) 0.026 (zkO.005) 0.025 (k0.004) 0.016 (f0.004) 0.023 lf0.001)

<0.05 nd' nd 5.9 (f7.1) 3.3 (f2.5)

nd nd 0.002 0.05 0.001

a The plasmids used are diagrammed in Figure 2.

'

Inhibition of the transposition of Tn5 from a X::Tn5 phage was determined a s follows. Cells of strain DB1977 carrying appropriate plasmids were grown overnight in broth containing 0.2% maltose and 20 mM MgSOa, infected at a multiplicity of <1 phage/cell, diluted 20-fold, and grown 5 hr at 30' before plating on LN kanamycin agar to select Kan' transductants.

To assay the transposition proficiency of IS50 elements, a measure of transposase synthesis and intactness of the outside (0) and inside (I) ends if IS50, plasmids were transformed into recA+ X lysogen DB104, phage development was induced, and Mmp'-transducing phage selected after infection of strain DB114. The formation of Mmp'-transducing phage relies on the frequent formation of oligomeric forms of the plasmids by homologous recombination in recA+ cells (BEDBROOK and AUSUBEL 1976; BERG 1983) and the transposition of a segment of the oligomer consisting of one set of pBR322 vector sequences bracketed by direct repeats of IS50 elements. The 0 end of one copy of IS50 and the I end of a second copy of IS50 are found at the junctions with

A target DNAs (BERG et al. 1982a; SASAKAWA and BERG 1982; BERG 1983).

T o assay the ability of IS50 elements to synthesize a functional transposase independent of the intactness of their ends, plasmids carrying them were fused with a pBR322::Tn5-410 (Trp+) plasmid to form heterodimers (as diagrammed in Figure 3), the heterodimers were introduced into the recA-

h red- lysogen DB1873, phage development was induced and the frequency of hTrp+-transducing phage was determined. The frequencies of complemented Tn5-410 transposition reported in lines 1 and 8-10 are averages of pools of between four and 16 transductant colonies in a total of four separate assays. Standard deviations were not calculated.

of the transposase gene (ROTHSTEIN

et

al.

1980; ROTHSTEIN

and REZNIKOFF

1981).

Its failure to inhibit transposition indicates that this control is mediated by a

product of the transposase gene.

Plasmid pBRG554, generated by ligation of the BglII site in IS50R (just 5 bp

away from the BclI site used to make pBRG557) and the BamHI site in pBR322,

should encode a protein containing the amino acids of transposase joined to an

formed by crossing over in the region of greatest homology (as depicted here in the middle frame). Other heterodimer plasmids indicated in Table 1 were generated in a similar fashion. Symbolism: thin lines, pBR322 sequences; thickened lines, IS50 sequences; stippled regions, the trp+ segment of Tn5-410; hatched lines, sequences of Tn5 internal to IS50 elements; 0 and I, outside a n d inside ends of IS50 A, 338-bp deletions of I end segments of IS50; R, H and Bm, sites recognized by restriction endonucleases EcoRI, HindIII and BamHI, respectively. The distances between HindIII sites are indicated. Top, Parental plasmids pBRG553 and pBRG556. Middle, Pairing configuration of the parental plasmids in the 3-kb region of greatest homology. Bottom, Heterodimer plasmid pBRG559, generated by a single reciprocal crossover between pBRG553 and pBRG566 when paired in the region of greatest homology.

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612 I

S50

J. B. LOWE AND D. E. BERG

Bgl

II

B c l I

I

end

1534

t

A T C A A G A T C T G A T C A A G A G A C A G

I I

iie /us ile STOP

371 Bum HI 388

C

t

T G T

G G A T C C

i

T

C T A C G C C

.)

pBR322

IS50 pBR322

1512 Bci I/&m HI 388

4 +

t

pBRG557

A T C A A G A T C T G A T C C T C

T

A C G C C

I ile I y s ;le STOP

I

S50

1 5 12 BO/ n/som HI

pBR322 625

t

- *+

+

pBRG554

A T

C

A A

G

1

A T C C T C T A C G C C

(77codonr)

T C C

T

A A

/ r

ile fys /le leu tyr ala ser STOP

FIGURE 4.-Deletions of the I end of ISSOR. Line 1, DNA sequence near the I end of IS50 and the predicted carboxyterminal amino acid sequence of transposase. Line 2, DNA sequence near the BamHI site in pBR322. Line 3, DNA sequence of the fusion plasmid pBRG557 in the vicinity of the BclI/BamHI fusion and a portion of the predicted amino acid sequence of transposase. Line 4, DNA

sequence of the fusion plasmid pBRG554 in the vicinity of the BglII/BomHI fusion and a portion of the predicted amino acid sequence of the fusion peptide. Nucleotide positions in IS50 are taken from AUERSWALD. LUDWIG and SCHALLER (1980) and COLLINS, VOLCKAERT and NEVERS (1982); those in pBR322 are from SUTCLIFPE (1978).

additional segment of

81 amino acids encoded by pBR322 (Figure

4).

This fusion

plasmid failed to inhibit Tn5 transposition, failed to complement Tn5-420

transposition (Table

1,

line

9)

and, thus, provided additional evidence that a

product of the transposase gene controls transposition. The comparable

BglII/

BamHI

fusion involving

IS50L

(pBRG556) was also defective both in inhibition

of

Tn5

transposition and in complementation of Tn5-420 (Table

1,

line

10).

From these experiments

we

conclude that Tn5’s ability to inhibit its own

transposition results from the action of a product of the transposase gene of

I550R, and that it does not require the presence of ISSOL.

DISCUSSION

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INHIBITOR OF

TN5

TRANSPOSITION

613 certain derivatives of a chromosomal Tn5 element which had been selected for

relief of the transcriptional polarity characteristic of wild-type Tn5, they also

postulated that synthesis of the inhibitor required both of Tn5’s inverted

repeats--IS5OR, which encodes transposase, and IS50L, which contains an

ochre mutant allele of transposase

(BIEK

and

ROTH

1980a).

We used a set of related pBR322::Tn5-derived plasmids to investigate the

genetic basis of transposition inhibition. Our finding that plasmids with no Tn5

sequences other than IS50R (pBRG1 and pBRG2) inhibit Tn5 transposition

shows that the inhibitor is encoded by the ISSOR arm of Tn5. The other arm,

IS50L, does not participate in the inhibition of transposition (Table

1).

Thus, the

polarity relief mutants generated by BIEK and

ROTH

must have been more

complex than they had assumed. IS50R contains one long open reading frame.

A pair of in-phase sites for the initiation of translation results in a pair of

proteins 461 and 421 amino acids long, and the longer protein is known to be

essential for transposition

(ROTHSTEIN

et

al.

1980;

ROTHSTEIN

and

REZNIKOFF

1981;

JOHNSON

and

REZNIKOFF

1981). The fusion plasmid pBRG557, which

encodes a normal transposase, inhibits transposition even though the inside (I)

end of IS50R is missing, and thus the element cannot itself transpose. The

comparable IS50L fusion (pBRG558) does not inhibit transposition, nor does the

IS50R plasmid pBRG554, which encodes a complete transposase protein inac-

tivated by the addition of a pBR322-encoded peptide.

The results presented show that the only Tn5-encoded requirement for

inhibition of transposition is a product of the functional transposase gene in

IS50R. Although our results do not distinguish among a number of possible

inhibition mechanisms, two reports which present complementary results have

appeared since this work was submitted

(JOHNSON,

YIN and

REZNIKOFF

1982;

ISBERG,

LAZAAR

and SYVANEN

1982). They show that the shorter (421 amino

acid) ISSOR-encoded protein is the inhibitor of transposition, and that inhibition

does not operate through repression of transposase gene transcription. Several

classes of explanations of inhibition remain to be tested: Since supercoiling of

potential target molecules is important for efficient transposition (ISBERG

and

SYVANEN

1982), if the inhibitor possesses a generalized nicking-closing activity

analogous to that of phage

h

integrase

(KIKUCHI

and

NASH

1979), inhibition

might result from induced changes in the conformation of potential target

DNAs. Alternatively, the inhibitor might operate by binding to the functional

transposase

or

by binding to its recognition sites at the termini of IS50.

We are grateful to DR. C. M. BERG for critical readings of the manuscript. This work was supported by United States Public Health Research grants 5 R01 A114267 and 1 RO1 A118980 to D. B., American Cancer Society Institutional grant IN-36 to Washington University and American Cancer Society Postdoctoral Fellowship P.F-2089 to J. L.

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L.

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L.

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412.

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G. VOLCKAERT and P. NEVERS, 1982 Precise and nearly-precise excision of the sym- metrical inverted repeats of Tn5; common features of recA-independent deletion events in Escherichia coli. Gene 19: 139-146.

CHOU, J., P. G. LEMAUX, M. J. CASADABAN and S. N. COHEN, 1979 Transposition protein of Tn3: identification and characterization of an essential repressor-controlled gene product. Nature

CSONKA, L. N. and A. J. CLARK, 1979 Deletions generated by the transposon TnlO in the srl recA

EGNER, C. and D. E. BERG, 1981 Excision of transposon Tn5 requires the inverted repeats, but not

GILL, R. E., F. HEFFRON and S. FALKOW, 1979 Identification of the protein encoded by the

GRINDLEY, N. D. F., M. R. LAUTH, R. G. WELLS, R. J. WITYK, J. J. SALVO and R. R. REED, Transposon-mediated site specific recombination: identification of three binding sites

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region of the Escherichia coli K-12 chromosome. Genetics 93: 321-343.

the transposase functions of Tn5. Proc. Natl. Acad. Sci. USA 78: 459-463.

transposable element Tn3 which is required for its transposition. Nature 2 8 2 797-801.

1982

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INHIBITOR OF T N 5 TRANSPOSITION

615

HERSKOWITZ, I. and D. HAGEN, 1980 The lysis-lysogeny decision of phage A: explicit programming

HIRSCHEL, B.

1.

and D. E. BERG, 1982 A derivative of Tn5 with direct terminal repeats can

ISBERG, R. R., A. L. LAZAAR and M. SYVANEN, 1982 Regulation of Tn5 by the right-repeat proteins:

ISBERG, R. R. and M. SYVANEN, 1981 Replicon fusions promoted by the inverted repeats of Tn5:

ISBERG, R. R. and M. SYVANEN, 1982 DNA gyrase is a host factor for transposition of Tn5. Cell 3 0

JOHNSON, R. C. and W. S. REZNIKOFF, 1981 Localization of the Tn5 transposase promoter using the

JOHNSON, R. C., J. C. P. YIN and W. S. REZNIKOFF, 1982 Control of Tn5 transposition in Escherichia

KIKUCHI, Y . and H. A. NASH, 1979 Nicking-closing activity associated with bacteriophage A int

KLECKNER, N., 1981 Transposable elements in prokaryotes. Annu. Rev. Genet. 1 5 341-404.

KRETSCHMER, P. J. and S. N. COHEN, 1977 Selected translocation of plasmid genes: frequency and regional specificity of translocation of the Tn3 element. J. Bacteriol. 130: 888-899.

MEYER, R., G. BOCH and J. A. SHAPIRO, 1979 Transposition of DNA inserted into deletions of the Tn5 kanamycin resistance element. Mol. Gen. Genet. 171: 7-13.

ROBERTS, R.

J.,

1981 Restriction and modification enzymes and their recognition sequences. Nucleic Acids Res. 9: r75-196.

ROTHSTEIN, S . J., R. A. JORGENSEN, K. POSTLE and W. S. REZNIKOFF, 1980 The inverted repeats of Tn5 are functionally different. Cell 1 9 795-805.

ROTHSTEIN, S. J. and W. S. REZNIKOFF, 1981 The functional differences in the inverted repeats of Tn5 are caused by single base pair nonhomology. Cell 23: 191-199.

SAPIENZA, C. and W. F. DOOLITTLE, 1980 Genes are things you have whether you want them or not. Cold Spring Harbor Symp. Quant. Biol. 45: 177-182.

SASAKAWA, C. and D. E. BERG, 1982 IS50 mediated inverse transposition: discrimination between the two ends of an IS element. J. Mol. Biol. 159: 259-271.

SASAKAWA, C., J. B. LOWE, L. MCDIVITT and D. E. BERG, 1982 Control of T n 5 transposition in E.

coli. Proc. Natl. Acad. Sci. USA. 7 9 7450-7454.

STARLINGER, P., 1980

SUTCLIFFE, G., 1978 Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Nucleic

VOGEL, H. J. and