Volume 6 NSumber 5 1979

Nucleic Acids Research

DNA sequences of the integration sites and inverted repeated structure of transposon Tn3

Tatsuo Takeya, Hisayuki Nomiyama, Jun Miyoshi, Kazunori Shimada and Yasuyuki Takagi Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan

Received 21 February 1979

ABSTRACT The nucleotide sequence of the "inverted repeat" structure of the transposon Tn3 was determined by the DNA sequencing procedure developed by Maxam and Gilbert(l). The sequence, 38 base pairs long, is as follows:

5'-GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG**(Tn3)

3'-CCCCAGACTGCGAGTCACCTTGCTTTTGAGTGCAATTC(T

-

The integration of Tn3 is associated with a directly repeated sequence of 5 nucleotides appearing at each end of Tn3. The two directly repeated sequences so far determined are not-the same. Furthermore, there is no homologous structure around the integration point of Tn3. INTRODUCTION It has recently been confirmed that several drug-resistance determinants are translocated from one replicon to another as discrete entities(2), and these translocatable elements have been named "transposons". Analysis of the mechanism of the translocation reaction is indispensable for solving the problem of the spread of drug resistance by plasmids. Information on the translocation system might also be valuable for understanding the mechanism of recombination between non-homologous DNAs. Tn3, a transposon that mediates ampicillin resistance(2,3, 4), is about 4,600 base pairs long(M.W. 3.2 x 106 daltons)(3,5) and encodes at least 3 proteins, including S-lactamase which hydrolyzes ampicillin(6,7). The frequency of its translocation ranges from 10 4 to 10 5 per recipient, depending on the system

used(8). Available evidence about the specificity of translocation of Tn3, in other words, the specificity of integration, so far suggests that there is no specific site for insertion(9,10). Therefore, the machinery for integration of Tn3 seems to be G Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

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Nucleic Acids Research different from that of bacteriophage lambda which is known for its site-specific integration, for example(ll). One essential feature of the structure of Tn3 DNA is the presence of a so called "inverted repeat" sequence at each end, and it has been suspected that this sequence may be essential for the translocation reaction(8). Since Tn3 has a rather simple structure and has been fairly well characterized, we determined the nucleotide sequence around its integration point and terminal region including the "inverted repeat" structure, to obtain information on the specificity of integration of Tn3. We found that the "inverted repeat" structure of Tn3 DNA comprises 38 base pairs and that there are no structures common to the regions containing the two integration points. We also found a directly repeated sequence of 5 nucleotide at each end of Tn3.

MATERIALS AND METHODS Plasmids: ColEl, pKY2289(previously named ColEl-amp2289 in ref.12), pKY96(ColEl-cosX-guaA in ref.13) and pKY2018(ColEl-cosX -guaA-Ap in ref.14) were used for DNA sequencing analysis(Fig.l). pKY2018 was isolated ir this laboratory by the transposition in vivo of Tn3 from Rldrd onto the in vitro constructed hybrid plasmid pKY96. pKY2289 was also isolated in this laboratory as a Tn3-induced mutant of ColEl lacking ability to produce the substance giving immunity to colicin E1(14). Tn3 present on pKY 2289 is derived from pKY2018. These plasmid DNAs were isolated from clear lysates containing the appropriate plasmids and purified further by isopycnic centrifugation in a CsCl/ethidium bromide gradient(15). Enzymes: All the enzymes used in this work were purified in this laboratory, except for bacterial alkaline phosphatase (from Worthington). Polynucleotide kinase was purified from T4infected E.coli B(17). The following restriction enzymes were purified by published methods: AluI(18), BamHI(19), EcoRI(20), (22),IgI(22), HphI(23), PstI(24) and TaI(25). Hae IE (21), HinfI was isolated essentially by the method used for isolation of HaeUlE, except that a G-150 Sephadex column was used for fur-

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Nucleic Acids Research a)

b)

EcoRI

Ec

RI

BamHI*/ oor

c)

Ad) R

A

BamHI

Tn3 ~~~~A AEcoRI EcoRI

Col El \E co

R

R~coI

CoEl c o El\ E gua

o RI

Figure 1. Plasmids used for DNA sequencing. a) ColEl, b) pKY2289, c) pKY96 and d) pKY2018.ori represents the replication origin of ColEl plasmid(16). The size of above figures is not proportionate to their molecular weights.

ther purification. PstI was also purified in the same way as HaeIEI. All reaction mixtures for restriction endonuclease digestion contained 10 mM Tris-HCl(pH 7.5), 7 mM MgCl2 and 7 mM mercaptoethanol(22), and that for EcoRI also contained 100 mM NaCl. Location of Tn3 on plasmid DNAs: The integration point of Tn3 was determined as follows. For location of Tn3 on pKY2289, ColEl DNA and pKY2289 DNA were digested with EcoRI and phosphorylated with 32P. The labeled DNAs were then treated with one of various restriction endonucleases and fractionated by polyacrylamide gel electrophoresis, to give two bands of material detectable by radioautography. The integration point of Tn3 was identified by assuming that if one of the two bands derived from ColEl DNA had the same mobility as that of pKY2289 DNA, while the other band had a different mobility, Tn3 was integrated into the latter fragment. The same procedure was employed for determining the location of Tn3 on pKY2018 DNA, using pKY96 DNA and 1833

Nucleic Acids Research pKY2018 DNA as materials. Isolation of DNA fragments and sequencing procedures: The appropriate DNA fragments were isolated from agarose gel by the method of Kamp, D. et al.(26). Dephosphorylation with alkaline phosphatase and phosphorylation with 32p using T4-polynucleotide 324 kinase and y- P-ATP were performed by the methods of Maxam and Gilbert(l). The labeled fragments at each end were then cleaved with the appropriate second restriction endonuclease and fractionated on polyacrylamide gel for sequence analysis. The sequencing procedure of Maxam and Gilbert(l) was used. For gel electrophoresis of chemically cleaved products, 10-20% of polyacrylamide gel(90 cm x 0.5 mm) was used. RESULTS Location of Tn3 in pKY2289 DNA and pKY2018 DNA: It has been reported that Tn3 may be located close to the EcoRI site of pKY2289 DNA and pKY2018 DNA(Fig.l, 12 and 14) and that the polarity of insertion of Tn3 is the same in the two plasmids. A more precise map of the integration point was constructed using various restriction endonucleases as described in the MATERIALS AND METHODS. The results obtained are summarized in Fig.2. AluI digests and TaI digests of ColEl DNA and pKY2289 DNA gave the same profiles on gel electrophoresis, whereas those of gaI, !phIe and HaellE digests gave different profiles(data not shown). These findings suggest that Tn3 is located between the first AluI site and first HaeI[ site from the EcoRI site of ColEl DNA. The same procedures were used to locate Tn3 in pKY2018 DNA. The restriction sites within Tn3 were confirmed by comparing them with those of pKY2289 DNA. Sequencing around the integration point in pKY2289 DNA: To examine the structure around the integration point and the "inverted repeat" sequence, we determined the nucleotide sequence around the juncture of Tn3 of pKY2289 DNA. For sequence analysis of the left-hand juncture, the DNA fragment covering the AluI site of pKY2289 DNA(Fig.2, Region 2) was isolated site and Hapi nucleotide its sequence was determined. For the right-hand and juncture, a HaeIE fragment consisting of 400 base pairs(Fig.2, Region 3) was recovered and analyzed. We also determined the

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Nucleic Acids Research Col El DNA: HphI EcoRI

I

_

A11I

Tay I

I

HaeM I

Ha¶pl

off

AluI

Region 1

TO3

pKY2289 DNA:

HphI EcoRI

TaI

Hggjfpm

Region

pKY2018 DNA:

AZuI TaqI

HaMHpqIN _

HapI[

AluI lphI

AluI

_

Haplm

HgaI

2

Region 3

Tn3

EcoRI HgaI HaeII

Hae f'htR7phfhhpll Region 4

Hae

Hp4I

HaeM EgaI

Region 5

200 base

pairs

Figure 2. Location of Tn3 in the plasmids and the restriction sites around the integration points and in the terminal region of Tn3. For details, see text. nucleotide sequence of the region between the AluI site and HaeflI site of ColEl DNA(Fig.2, Region 1). The results obtained are summarized in Fig.5. By comparison of the structures of regions 1, 2 and 3, we deduced that the inverted repeated structure was a sequence of 38 base pairs starting with GGGG. Another structural characteristic shown in Fig.5 is that integration of Tn3 is associated with a directly repeated sequence of 5 nucleotides(TATCA) appearing at each end of Tn3. Sequence analysis of pKY2018 DNA: To confirm the results obtained in pKY2289 DNA and to compare its structure around the integration point of Tn3 with that of a different derivative, 1835

Nucleic Acids Research

is.

_m C s3

S

.

These were deduced from left-side profile.

U-

U.S

'-3 6

~

.= .-

C.~~~~~~~~~~~~~~~~~~~~.

a;-a o

c i

a

a

as

CC

Figure 3. Autoradiogram of chemically cleaved products from Region 5 of Fig.2. Sequencing reactions, gel electrophoresis, and autoradiography were carried out by the methods of Maxam and Gilbert(l) as described in the MATERIALS AND METHODS.

we sequenced the fragment containing the integration point of Tn3 in pKY2018DNA.

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