The Structure of R1<i>drd19</i>: a Revised Physical Map of the Plasmid

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Reference

The Structure of R1 drd19 : a Revised Physical Map of the Plasmid

CLERGET, MICHEL OLIVIER, CHANDLER, Michael, CARO, Lucien

Abstract

We have analyzed derivatives of the plasmid R1drd19 carrying the transposon Tn10 by electron microscopy following denaturation and renaturation of the molecules, and by digestion with various restriction enzymes, gel electrophoresis and Southern blotting. We show: 1) that the published restriction map of R1drd19 is inconsistent with our results. We present a modified map which is consistent with our data. 2) that R1drd19 carries a single resident copy of the element IS10 which is normally associated with Tn10 as an inverted repeat, and 3) that R1drd19 carries three copies of the insertion element IS1 in the resistance determinant region.

CLERGET, MICHEL OLIVIER, CHANDLER, Michael, CARO, Lucien. The Structure of R1 drd19 : a Revised Physical Map of the Plasmid. Molecular and General Genetics , 1981, vol. 181, no. 2, p. 183-191

DOI : 10.1007/BF00268425 PMID : 6268938

Available at:

http://archive-ouverte.unige.ch/unige:150492

Disclaimer: layout of this document may differ from the published version.

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The Structure of Rldrdl9: A Revised Physical Map of the Plasmid

Michel Clerget, Michael Chandler, and Lucien Caro

D6partement de Biologic mol6culalre, Unlversit6 de Gen~ve, 30, Qual Ernest Ansermet, CH-1211 Geneve 4, Switzerland

Summary. We have analyzed derivatives of the plasmid Rldrdl9 carrying the transposon TnlO by electron microscopy following denaturation and renaturation of the molecules, and by digestion with various restriction enzymes, gel electrophoresls and South- ern blotting. We show:

1) that the published restriction map of Rldrd19 is inconsistent with our results. We present a modified map which is consistent with our data.

2) that Rldrd19 carries a single resident copy of the element ISIO which is normally associated with TnlO as an inverted repeat, and

3) that Rldrd19 carries three copies of the insertion element IS/ in the resistance determinant region.

Introduction

The plasmid Rldrd19 (Meynell and Datta 1967) is closely related to the multiple antibiotic resistance plasmids R6 and R100.1.

It has considerable homology with these plasmids (Sharp et al 1973) and is composed, in a similar way~ of a cluster of drug resistance genes (r-determinant or r-det) flanked by two directly repeated IS/elements (Hu et al. 1975: Ptashne and Cohen 19751 and of a region, the RTF, which carries genes responsible for autonomous replication and conjugal transfer. Like R6 and R 100.1, its structure and replication properties have been studied extensively in recent years. Although closely related, the three plasmids are not identical. Heteroduplex molecules formed between Rldrd19 and R100.1 (Sharp et al. 1973 and Fig 7a) show three major differences in structure~ One is associated with the insertion of the tetracychne resistance transposon TnlO into the RTF region of RI00.1 ; two are located in the r-determinant.

The two latter occur at the extremity proximal to the origin of vegetative replication and involve the insertion into Rldrdl9 of the ampicillin resistance transposon Tn3 and of a 9.6 kilobase (kb) segment carrying resistance to kanamycin (Kmr). (Kopecko et al. 19761.

A detailed restriction map of Rldrdl9 has recently been pub- hshed (Blohm and Goebel 19781. While analyzing isolates of Rldrdl9 into which we had inserted the Tc r transposon Tnl0, we have observed several discrepancies in this map. In the work reported here we analyze three independant TnlO insertion deriv- atives of Rldrdl9 by electron microscopy, by digestion with restriction enzymes, and by Southern transfers using IS/ and TnlO specific probes. We present a modified restriction map Offprint request to" M, Clerget

which is consistent with the data obtained and also with the bulk of the data of Blohm and Goebel (1978).

Rldrdl9 gives rise fairly' frequently to variants which have lost either Km r or all the other r-det associated resistance genes (Meynell and Cooke 1969; Kopecko and Cohen 1975; Mohn et al. 1979). We show by hybridization of an IS1 specific probe to Southern transfers of digested plasmid DNA that Rldrdl9 carries a third copy of IS/ within the Km r region of the r-det.

This additional copy is probably implicated in the formation of the Rldrd19 deletion derivatives. The results of similar transfers hybridized with a TnlO specific probe, together with the results obtained by electron microscopy, suggest that Rldrd19 also carries a resident copy of IS/0,

In sum, these findings provide a clearer picture of the structure of Rldrd19 and provide a basis for an explanation of the relative instability of the various drug resistance markers of this plasmid. The presence of IS10 homologous material at a position of Rldrd19 distinct from the position of TnlO on R100.1 and R6 supports the idea that ISIO can behave as an insertion element.

Materials and Methods

Bactemal Strains and Bactettophuges. The bacterial strains employed are described in Table 1 Bacteriophages ,{:'IS/ ()~r14=)ocI857cII :IS1N53N7, Brachet et al 1970) and )L :Tnl0 (2ci857. Tnl0) (Jor- gensen et al. 19791 have been described

Media. Bacterial cultures were grown In L broth (Lennox 19501 supple- mented with 10 pg/ml thymine or in M9 (Adams 19591 supplemented with 0.4% glucose, 0.4% casamino acids (Dmfco) and 10 Ixg/ml thymine

Antibiotic resistances were tested on L agar supplemented with the following concentration of antibiotics, streptomycin (Sm) 20 lag/ml, chloramphemcol (Cm) 20 ~tg/ml, tetracycline (Tc) 25 pg/ml, kanamycln (Km/ 20 lig/ml, and ampicillin (Ap) 25 pg/ml

lsolatlon o/Rldrdl9 TnlO Dern,atwes LC798, lysogenlc for 2 .TnlO and carrying Rldrd19 was crossed with LC80I in liquid culture at 33 ° C overnight. The culture was concentrated tenfold by centrifuga- tion and plated Selection was made for cotransfer of tetracycline resistance (Tc r) and Rldrdl9 associated Cm r.

DNA Isolauon. PlasmId DNA was isolated from cleared lysates (Clew- ell and Helinski 19691 of stationary phase cultures as described pre- viously (Chandler et al. 19771 The preparation of bacteriophage DNA has also been described (Bird et al. 1972, Louarn et al 19741.

Electron Microscopy Formation of internal (homo-)duplex and heteroduplex molecules was performed as described by Davis et al (19711 0026-8925/81/0181/0183/$01.80

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Table 1. Bacterial strains Strain Genotype

LC771 thy leu (2c1857S7: :Tnl0) LC798 LC771/R l drd19

LC801 leu purE trp his metA ilv argG proC thy ara lac xyl mtl gal strA T6 r 2 r

Preparations were spread on a water hypophase, d~X174 viral and RFII molecules were used as internal length standards for single and double stranded DNA respectively (Sanger et al. 1977). Length measurements were made from photographs, using a Numonics digitizer (Numonics Corp.).

Restriction Endonuclease Analysis of Plasmid DNA. Digestion of plasmid DNA by HindIII, EeoRI and SalI enzymes was performed using standard conditions (Chandler et al. 1977). Digested DNA preparations were subjected to electrophoresis on 0.6% or 1.0% agarose slab gels. DNA fragments were purified from gels by removing agarose strips carrying specific fragments and subjecting these strips to electrophoresis in a tube gel. The DNA was collected in a dialysis bag placed over the lower end. The samples were then dialyzed against 10 2MTris, 10 3MEDTA(pHS.0).

Southern Transfer, Hybridization and Autoradiography. DNA fragments from agarose slab gels were denatured and transferred to nitrocellulose filters according to the technique Southern (1975). The filter was briefly washed with 2xSSC (SCC is 0.15M NaC1, 0.015M Na-citrate pH 7.0), air dried and baked under vacuum at 80 ° C for 2 h. Various probe DNAs were nick-translated using the method of Manniatis et al.

(1975). The reaction was terminated by the addition of 3 ml 50%

formamide, 2 × SSC; 30 p,g of unlabelled, sonicated calf thymus DNA was added and the DNA was heated to 90 ° C for 10 rain before use.

Filters from Southern transfer experiments were hybridized overmght with 3 x 105-1 x 106 cpm of 32P-labelled probe DNA in a final volume of 15 ml 2xSSC, 50% formamide at 42 ° C. They were then washed for 1 h at room temperature in 2 x SSC, 50% formamide, for a further 1 h in 2 x SSC. air dried, and autoradiographed using X-Omat (Kodak) X-ray film.

Results

Isolation and Characterization

of Rl." : TnlO Derivatives: a Resident ISIO on Rldrdl9

Rldrdl9::Tnl0 derivative plasmids were isolated by selection for cotransfer of Tc r and Cm ~ from a strain lysogenic for 2 : : TnlO and carrying the plasmid Rldrdl9. The observed frequency of cells having received both resistances compared to those having received Cm r alone was found to be 10-7-10 -s. This value is in accord with measurements of the frequency of TnlO transposition made by others under similar conditions (Foster 1977).

In order to confirm the presence of TnlO in three putative insertion derivatives (Rldrdl9-.-Tnl0#ll, 67 and 72), plasmid DNA was denatured, rapidly renatured, and observed using the electron microscope. Under these conditions TnlO forms a double stranded snap-back, due to renaturation of the inverted ISIO elements (Sharp et al. 1973), and a characteristic single- strand loop. Each derivative plasmid exhibits this structure (Fig. 1 a). In addition, each insertion derivative shows an alternative structure in which the length of the double-strand stem is identical to that of ISIO (1.45 kb) but in which the size of the TnlO loop is larger and that of the plasmid smaller than expected. The relative size of the loops is characteristic for each individual insertion derivative. Measurements of the two derivat-

~ 3

^O.

620

b

C

73 0

Fig. 1. a Drawing of Rldrd19: :Tnl0¢/ll showing the typical stem and loop structure of TnlO following denaturation and renaturation of the molecules (snap-back). b The alternative stem and loop structure observed for Rldrd19: :Tnl0#l 1. Duplex formation has occurred between one of the inverted repeat elements (IS/0) of TnlO and a resident ISIO. Measurements were made on four molecules, e The alternative stem and loop structure observed observed for Rldrd19: :Tn10~/72.

Measurements were made on seven molecules

Table 2. Rldrd19 restriction fragments (kb)

EcoRI HindIII SalI EcoRI EcoRI + HindlII + SalI

A 17.5 63.5 23.0 17.5 14.0

B 17.5 17.5 21.4 17.5 10.5

C 12.8 4.4 15.0 12.8 9.5

D 9.5 3.0 15.0 9.5 8.7

E 7.7 2.9 8.8 7.7 7.7

F 6.5 2.7 8.0 4.2 5.0

G 5.3 0 35 3.0 3.0 4.3

H 4.2 2.8 4.2

I 2.8 2.8 3.7

J 2.6 2.6 3.0

K 2.0 2.55 2.95

L 1.95 2.05 2.8

M 1.4 1.8 2.7

N 1.3 1.6 2.6

O 0.9 1.4 2.1

P 0.3 1.3 2.0

Q 0.25 1.05 1.9

R 09 14

S 1.4

T 1.3

Fragment sizes were determined from the known sizes of EcoRI generated fragments of R100.1 (Chandler et al. 1977; Tanaka et al. 1976) run in the same gel. The size of the smaller fragments was determined by electrophoresis in a 7.5% acrylamide gel using pBR322 cut by MspI as a reference (Sutcliffe 1978)

ives Tnl0#11 and Tn10#72 are shown in Fig. 1 b and c, respectively.

This observation suggests the presence of a resident copy of the ISIO element on the parent plasmid Rldrd19. The alternative structure would then be due to hybridization between the resident ISIO and one of the two copies of ISIO associated with

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Fig. 2. a 1% agarose gel of EcoRI digested Rldrdl9. Tn10/J72 (channel 1): Rldrdl9 (channel 2) and R100 1 (channel 3). b A schematic representation of the gel shown in a) indicating the position of 14 of the i7 EcoRI fragments (Blohm and Goebel I978) of Rldrd19 (channel 2) and showing the presence of an additional fragment in the insemon derivative Rldrdl9" :Tn10/~72 (channel 1) TnlO insertion in this case has occurred in EcoRI fragment B (Fig. 4b). The change m size of the parental fragment cannot be detected on this gel. Channel 3 shows the EcoRI fragment pattern of R100.1 (Tanaka et al. 1976). e Autoradiograph of a Southern transfer of the gel shown in a following hybridization with 32p labelled )~: :Tnl0 DNA

the TnlO transposon. The loop size would depend on the position of the inserted TnlO

To confirm this interpretation, DNA of Rldrdl9 and of the /:/72 insertion derivative was digested with EeoRI, transferred to a nitrocellulose filter by the technique of Southern (1975) following electrophoresis on a 1% agarose gel, and probed for TnlO homologous sequences by hybridization with 32p-labelled 2::Tnl0 DNA. The results of this experiment are shown in Fig. 2. EcoRI generates 17 fragments in Rldrdl9 (Blohm and Goebel 1978; Table 2). The largest of these are shown in Fig. 2 channel 2a and schematically in channel 2b. TnlO has one EcoRI cutting site (Jorgensen et al. 1979); insertion of TnlO will, therefore generate two EcoRI fragments at the expense of the fragment into which insertion has occurred. Derivanve//72 exhibits three fragments having homology with )~::Tnl0 (Fig. 2, channel 1 c).

Two fragments show a strong homology, presumably because they carry part of the Tc loop in addition to ISIO. The third band exhibits a less strong response. This fragment, EeoRI C,

also shows homology in the parent plasmid Rldrdl9 (Fig. 2, channel 2c). EeoRI digested R100.1 included as a control carries two bands with homology to TnlO (Fig. 2, channel 3c, Chandler et al. 1979a). We cannot rule out the possibility that some additional TnlO material is associated with the IslO element present in Rldrdl9.

Further Restriction Enzyme Anal3.sis of the hTsertion Derwatives. Modificatton of the Published Restriction Map of Rldrdl9

Single and double digestions using the restriction enzymes EcoRI, HindIII, and SalI were used to determine the position of the TnlO insertion in the three derivative plasmids by comparison with similar digestions of the parent plasmid. A list of the fragments generated by these enzymes in the parent plasmid can be found an Table 2. Since TnlO carries one site for EcoRI and three sites for HmdIII (Jorgensen et al. 1979), insertion of

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Eco R I

1 2 3 4

A+B . . . .

C ~ 11.5~" 11.2~ m C)~

T m J~

m m

m n

Hind f

1 2 3 4

A . . . .

B ~ ~ D

12.5~

9 D 6.8 m

5m

C ~ C+4.6 D C +4.6~ E+~.6 D

Soil

1 2 3 4

A~ >A~.. > A.=,.

B~ 18.5~

[+D . . . .

F . . . .

G~ D m

EcoRI + Hinder

1 2 3 4-

A+B~ ~

[ ~ ~

II D

D D J 0 ~

6.t,D

F . . . .

3.8~ 3.6~ 3.8~

G . . . .

H+I n J . . . .

Fig. 3. Composite diagram showing EcoRI, HtndIII, SalI and EcoRI + HmdIII fragment patterns of R ldrdl9 (channel 1); Rldrdl9::TnlO#11 (channe12);Rldrd19::TnlO//67(channe13)andRldrd19::TnlO#72(channe14) The smallest fragments generated by these enzymes are not included

%n10~7 2

L K

D C

\

-.P

0

f

J

Fig. 4. a Restriction map of the plasmid R ldrdl9 taken from Blohm and Goebel (1978). b Restriction map of R ldrdl9 modified according to the data presented in this communication showing the location of the resident insertion elements ISIO and IS/and the location of the TnlO insertions in derivatives #11,//67 and #72

TnlO will introduce additional site for these enzymes. The results of the various digestions are shown schematacally in Fig. 3.

The Rldrdl9::Tnl0¢/11 derivative is missing EcoRI fragment D but exhibits two additional fragments having molecular lengths of 7.1 and 11.5 kb (Fig. 3, channel 2). It also carries two additional HindIII fragments of 4.6 and 5.0 kb one of which (4.6 kb) represents the interior region of TnlO (Chandler et al.

1979a). This fragment has the same size as fragment HindIIIC thereby giving rise to a double band. There is no apparent change or loss of the parental HindIII fragments. The fragment EcoRI-HindIII D disappears and two fragments, of 3.8 and

11 kb, can be seen Finally, Rldrdl9::Tn10¢/11 has lost SalI fragment B and gained a SaII fragment larger than SalI A.

The Tnl0 transposon is, therefore, located on the fragments EcoRI D, HindIII-EcoRI D, and SalI B.

Rldrd19::TnlO#67 has likewise lost EcoRI fragment D and carries two additional fragments, of 11.2 and 7.4 kb (channel 3). HindIII fragment B ~s missing and two additional fragments, of 9 and 12.5 kb, appear as well as the 4.6 kb TnlO fragment.

The fragment EcoRI-HindIII D disappears, giving rise to two additional fragments of 3.6 and 10 kb. Again, as m the previous derivative, the fragment SalI B is absent and an additional frag-

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Fig. 5. Digestion of isolated Sill fragments of the derivative Rldrd19. Tnl0~/11. EcoRi+ SaII digestion of the parent plasmld Rldrd19 (channel 1) EcoRI digestion products of fragment SalI B: .TnlO (channel 2): SalI A (channel 3), SalI C+D (channel 4), SaII E (channel 5): SalI F (channel 6) and SalI G (channel 7l Minor bands are due to cross-contamination of the fragments during isolation and to partial digestion products. These are shown as dotted lines in the drawing

ment having a molecular length greater than SalI A appears.

The TnlO transposon is, therefore, located in EcoRl D, HindIII B, EcoRI-HindIII D and SalI B.

Similar arguments lead to the positioning of TnlO in the third derivative /(72 on SolI fragment E (Fig. 3, channel 4).

The experiment shown in Fig. 2, in which we determined the EcoRI fragments of this derivative which carry TnlO homology, demonstrate that TnlO is inserted in EcoR! A or B (see above).

A comparison of the results obtained with Rldrdl9::Tnl0 derivatives//11 and /~67 with the published restriction map of the parent plasmid (Blohm and Goebel 1978 and Fig 4a) reveals a major contradiction. According to this map, insertions into EcoRI fragment D should appear in SalI fragment A. Our results demonstrate that TnlO insertions into EcoRI D appear, instead, in Sa[I B. In order to resolve this anomaly, we have isolated the individual SalI fragments, as described in Materials and Methods, and digested them with EcoRI. For this purpose, we have employed the TnlO insertion derivative ¢/11 Since TnlO

does not carry a SalI site (Jorgensen et al. 1979), the TnlO Inser- tion into SalI B provides a greater separation of SalI A and B during electrophoresis.

The sizes of the fragments of Rldrdl9 generated by EcoRI together with SalI are shown m Table 2. The fragment EcoRI- SalI C is not present in the TnlO insertion derivative /411 but gives rise to two fragments of 7,1 and 11.5 kb. The results of digestion of the SalI fragments with EcoRI are shown in Fig. 5.

The minor bands seen in this figure are due to cross-contamination of the fragments during isolation and to partial digestion products.

Channel 1 shows an EcoRI-SalI digest of the parent plasmid Rldrd19 It can be seen that fragment Sall B (channel 2) gives rise to EcoRI-SalI fragments H, N, Q and R or S. The two additional fragments of 7.1 and 11.5 kb resulting from insertion of TnlO, with its unique EcoRI site, into EcoRI-SaII C can also be observed. Fragment SalI A (channel 3) gives rise to EcoRI-SalI fragments A, G, L. and T. Since SalI fragments

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Fig. 6a d. Location of ISI element on Rldrdl9. a and b Restriction fragment pattern of Rldrdl9 digested with EcoRI + SalI (channel 1); SalI (channel 2) and EcoRI (channel 3). e Autoradlograph of a Southern transfer of the gel shown in a following hybridization wath 32p-labeled 2.:IS1. d SalI digestion of Rldrd19::TnlOf~67 (channels 1 and 2) and autoradiograph of a Southern transfer of the gel following hybridization with 32P-labelled 2::IS1 (channel 3).

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Fig. 7. a Electron mlcrograph of a heteroduplex molecule formed between R100.1 and Rldrdl9 in the region of the r-determinant. It shows three single strand insertion loops: the Km r region (1) and Tn3 (2) associated with Rldrd19 and a third of unknown function associated with R100.1 (3) b Diagram of the region showing lengths in kllobases. Measurements were made on nine molecules c Alignment of the restriction maps of R100.1 and Rldrd19 +EcoRI sites; " HindIII sites: v SalI site. (Chandler et al. 1977 ; Tanaka et al. 1976, Iida 1980:

Blohm and Goebel 1978: this work)

C and D are not separable under the conditions we use, we have digested a mixture of the two fragments. The results are shown in channel 4. These fragments give rise to EcoRI-SalI fragments B, E, I, M. O, P and R or S. It will be noted that none of the three sets of fragments are found to be contiguous on the published map (Fig. 4a) The results of digestion of the remaining SalI fragments (channels 5, 6, 7) are consistent with the published map.

In order to clarify these inconsistencies we propose the following modifications to the Rldrdl9 map: reversal of SalI A and B, a change in the order of the EcoRI fragments E, M, A, the corresponding EcoRI-SalI fragments A, I, R, E and EcoRI-HindIII fragments A, O, E, and an inversion of the EcoRI-SalI segment H-Q-G. These modifications are shown in Fig. 4b. Using this modified map and the restriction enzyme data (Fig. 3) we have located the three TnlO insertions #11, 67 and 72. These are shown in Fig. 4b. For brevity we do not present the arguments which lead to the assignment of these positions. They are available on request.

Location of the IS1 Elements. a Third Copy o/ IS1

The revised map presented in Fig. 4b is consistent with the observation of Blohm and Goebel that an RTF derivative of Rldrd19 loses the r-det EcoRI fragments F, J, O, D, H, L, G, P, Q, N, and I and the EcoRI-SalI fragments F, S, N, C, G, Q, H, T, and L. If ISis are involved in r-det excision (Chandler et al.

1977) this leads to the predicltion that one of the two IS/ elements is located on EcoRI fragment A, SalI A and EcoRI-SalI A and not, as is implicit in the map of Blohm and Goebei, on EcoRI E, SalI B and EcoRI-SalI E.

To determine the position of the IS/ elements, we have employed 32p labelled nick-tranlated 2r14 (2: :IS/) DNA as a probe in hybridizations with Southern transfers of Rldrdl9 following digestion with EcoRI, SalI and EcoRI-SalI. The results are shown in Fig. 6. In case, fragments having the size of EcoRI A and F (channel 3)~ SalI A and F (channel 2) and EcoRI-SalI A and F (channel 1) have IS/ homology. This is in agreement with the modified map It should be noted here that neither

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190

EcoRI A and B nor SalI A and B are resolved on this gel.

A surprising result from this experiment is the observation that EcoRI D (channel 3) and EcoRI-SalI C (channel 1) also have IS1 homology. This suggests the presence of a third copy of IS1 on Rldrdl9. A similar result is found with the insertion derivative Rldrd19: :Tn10#67. This derivative carries the TnlO insertion in SaII fragment B (Fig. 4b). We have used this plasmid to obtain a greater separation of SalI A and B in order to determine on which SalI fragment the third copy of IS1 occurs.

The results of this experiment are shown in Fig. 6d. Both SalI A and SalI B : :Tnl0, in addition to SalI F, carry IS1 homologous materials. This demonstrates that the third copy of IS1 is located in SalIB. These data are included in the modified map (Fig. 4b).

Comparison of the Physical Map of RlO0.1 wtth Rldrd19 We have compared the physical map of R100.1 with Rldrd19 by analysis, in the electron microscope, of heteroduplex molecules formed between the two plasmids. These structures have been used to correlate the restriction maps of both plasmids.

A photograph of a heteroduplex in the region of the r-det is shown in Fig. 7a and schematically in Fig. 7b. The structures were oriented with respect to the TnlO transposon. The structure of the entire heteroduplex molecule (data not shown) is in good agreement with those of Sharp et al. (1973) except for the large insertion loop which codes for Km r. The alignment of restriction maps, shown in Fig. 7c was deduced from a knowledge of the position of IS/b, with respect to the various restriction enzyme sites (Chandler et al. 1977). It will be noted that the sequences of R100.1 and Rldrd19 in this region are remarkably similar.

The differences are found only in the three regions in homology; the Km insertion, Tn3, and a small insertion of 1.45 kb which appears in R100.1.

Discussion

The results of an analysis of three derivatives of Rldrd19 into which the transposon TnlO had inserted cannot be explained using the current detailed restriction map of the plasmid (Fig. 4 a). They can be explained however if several modifications are made to this map (Fig. 4b). These modifications are supported by two lines of evidence: 1) the results of redigestion by EcoRI of purified SalI restriction fragments, 2) the assignment of IS/ elements to individual restriction fragments. It should be noted that we have not attempted to verify the entire map presented by Blohm and Goebel (1978).

The presence of TnlO homologous sequences on Rldrdl9 was unexpected since the plasmid does not specify Tc r. The absence of characteristic TnlO snap-back structures in the parent plasmid (data not presented) indicates that only part of TnlO is carried by Rldrd19. Snap-back structures observed in the case of the three TnlO insertion derivatives exibit a 1.45 kb double strand stem (Fig. 1) indicating that Rldrd19 carries at least a complete copy of IS10. Although we have not analysed the homology between TnlO and Rldrd19 in detail, it seems unlikely that the plasmid carries much more than a single ISIO element.

We infer this from the results of hybridizations between Rldrdl9::Tn/0~/72 and 2::TnI0 (Fig. 2). It will be noted that two of the three TnlO homologous fragments, those which carry the two portions of the inserted transposon, show intense bands on the autoradiograph. The third band, specific to the parent plasmid is significantly less intense. Further indirect evidence comes from an analysis of heteroduplexes formed between R100.1 and Rldrd19 (Sharp et al. 1973, our unpublished data). The

data shown in Fig. 2 locates the ISIO element on fragment EcoRI C, on the Rldrdl9 map (Fig. 4b). We observe a small insertion loop of 1.39 kb at approximately 31 kb from ISlb towards TnlO (data not shown). This lies within EcoRI fragment C. We suggest that the insertion is a single copy of ISIO. Since its relative position in Rldrd19 is distinct from the position of TnlO in R100.1 and R6 (Sharp et al. 1973). it is unlikely that it was generated by deletion of the TnlO transposon. The observation lends support to the notion that ISIO can behave as an independent transposable element.

The results of experiments designed to locate the IS1 elements on Rldrd19 (Fig. 6) demonstrated the presence of three fragments with IS1 homology in contrast to the two copies present in R100.1 In the case of R100.1, the r-det is flanked by two directly repeated copies of IS1 (Hu et al. 1975; Ptashne and Cohen 1975). These IS/ elements are involved in r-det excision (Chandler et al. 1977) and tandem duplication (Rownd and Mickel 1971). Rldrd19 frequently gives rise to variants which have lost all the r-det specified resistances except for Km r (e.g.

Rldrdl6: Meynell and Datta 1967), variants which have lost only Km r (e.g. pSC50:Kopecko and Cohen 1975), and variants which have lost all resistances (RTF, Cohen and Miller 1970).

This behavior, by analogy with R100.1, is likely to occur via recombination between the IS1 elements. The independent segre- gation of Km r with respect to the other drug resistances could be explained then by the presence of a third directly repeated copy of IS1 within the r-det separating Km r from the other resistance determinants.

Inspection of the heteroduplex map (Fig. 7a, b) shows that the third IS1 element of Rldrd19 must occur within one of the two insertion loops in this plasmid since Rldrd19 is homologous to R100.1 over the remaining r-det regions. Furthermore, since the smaller of these insertions has been identified as Tn3 (Sharp et al. 1973) which carries no IS/ homology (data not presented), the IS1 element must be inserted in the 9.6 kb loop.

This interpretation is in agreement with our observations that an IS1 element is located m the EcoRI D and SalI B fragments as well as at the extremities of the r-det, (Fig. 6). In the map shown in Fig. 7c we have located the third IS1 element at the right hand extremity of the 9.6 kb insertion in fragment EcoRI- D. This location is based on the results of experiments in which the 10.4 kb (9.6 kb+ISlb) segment has been transposed to a 2 receptor replicon (Clerget et al. 1980). It should be noted that some supporting evidence that all three copies of IS/in Rldrd19 occur in the same orientation comes from our inability to form snap-back internal heteroduplexes with a double stranded IS/

stem in these 2::Km r derivatives (Clerget et al. 1980) or in the heteroduplexes formed between R100.1 and Rldrdl9 (Fig. 7a).

Such structures are easily observed in molecules of similar dimen- sions which carry inverted copies of IS1 (Chandler et al. 1979b).

Acknowledgements. We would like to thank G. Churchward, H. Krisch and P. Prentkl for invaluable discussions during the course of this work, and E. Gallay for the electron microscopy. This work was supported by grant No. 3.591.0.79 from the Swiss National Science Foundation.

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Communicated by J. Schell Received July 28 / November 15, 1980

Note Added in Proof

In Fig 7c we have omitted a SalI restriction site which is located in EcoRI fragment F of Rldrdlg.