A novel type of transposon generated by insertion element Is102 present in a psc101 derivative

Cell, Vol. 30, 29-36, August 1982, Copyright 0

1982

by MIT

A Novel Type of Transposon Generated by Insertion Element IS102 Present in a pSCIO1 Derivative Yasunori Machida, Chiyoko Machida and Eiichi Ohtsubo Department of Microbiology School of Medicine State University of New York at Stony Brook Stony Brook, New York 11794

Summary We describe a novel type of transposon in the tetracycline resistance plasmid pYM103, a derivative of pSClO1 carrying a single copy of an insertion element IS102. The new transposons we found were identified as DNA segments, approximately 6 kb (Tn1021) and 10 kb (Tn1022) in length, able to mediate the cointegration of pYM103 with plasmid Col El. The resulting cointegrate contains either of these pYM103 segments duplicated in a direct orientation at the junctions of the parent plasmids. A direct duplication of a g bp sequence at the target site in Col El is found at the junctions for cointegration. Both transposons have IS102 at one end and also contain different lengths of the pYM103 DNA adjacent to IS102, including the tetracycline resistance gene. Each transposon contains terminal inverted repeats of a short nucleotide sequence. These results and the fact that IS102 can itself mediate plasmid cointegration, giving rise to a duplication of a g bp target sequence, indicate that IS102 is responsible for generation of Tn1021 and Tn1022. They are quite different from the common IS-associated transposons, which are always flanked by two copies of an IS element, and may be similar to transposons such as those of the Tn3 family and phage Mu.

ever, have no flanking IS element, but contain short terminal inverted repeat sequences (see reviews by Calos and Miller, 1980; Kleckner, 1981). These latter Tn elements encode a protein or proteins required for their transposition. IS and Tn elements can promote various kinds of genomic rearrangements. One of these is cointegration of two different plasmid genomes such that a plasmid carrying an element integrates into the second plasmid. During the cointegration event, the element in the cointegrate is duplicated in a direct orientation at the two junctions of the parental plasmid sequences, and a nucleotide sequence at the target site on the recipient plasmid is duplicated in a direct orientation at the cointegration junctions (Ohtsubo et al., 1980a, 1980b; McCormick et al., 1981). Plasmid cointegration has been very useful for functional analysis of an element as well as for identification of new IS or Tn elements (Grindley and Joyce, 1981; Ohtsubo et al., 1981; McCormick et al., 1981; Machida et al., 1982). We report that the IS element IS102 (1056 bp in length), a natural component of plasmid pSClO1 (Ohtsubo et al., 1980b; Bernardi and Bernardi, 1981), can generate a new type of Tn element carrying the tetracycline resistance region of pSC101. These elements were observed as DNA segments that mediated plasmid cointegration. Unlike the known IS-associated Tn elements, these new Tn elements were not flanked by two copies of IS102, but contained only a single copy of IS1 02 at one end and variable lengths of DNA, including the tetracycline resistance region that is adjacent to IS1 02 in pSC101. The observation of the Tn elements generated by IS102 suggests a mechanism by which the Tn elements with no flanking IS elements, such as the Tn3 family and phage Mu, could have evolved.

Introduction Results An insertion sequence (IS) element is a defined segment of DNA that translocates itself into other DNA sequences. In the past decade, several kinds of IS elements have been reported (see reviews by Calos and Miller, 1980; Kleckner, 1981). Each IS element contains short terminal inverted repeat sequences characteristic of the element. These inverted repeats are thought to be recognition sites for a protein or proteins specified by each IS element to promote its translocation. One group of transposable elements, or transposons (Tn), is known to be associated with IS elements. They encode genes for resistance to antibiotics, such as chloramphenicol, tetracycline and kanamycin, and for enterotoxin production. These genes are always flanked by two copies of an IS element, which are primarily responsible for transposition of the composite transposons. Other types of Tn elements, such as the ampicillin resistance transposon Tn3, its related transposons and phage Mu, how-

Isolation of pCM Plasmids Formed between pYM103 and Col El The plasmid pYMlO3 (10.2 kb) carries a single copy of IS102 and a mutant of another insertion element, ISl, named IS1-3 (Figure 1). Plasmid pYMlO3 was derived primarily from the tetracycline resistance plasmid pHS1, a temperature-sensitive replication mutant of pSClO1, which was inserted by the mutant IS1 -3 with a deletion of 20 bp within the wild-type IS1 sequence by a series of genetic manipulations (Machida et al., 1982). We have previously demonstrated that in pYM103 both IS102 and IS1 -3 can mediate cointegration with plasmid Col El (6.7 kb) (Ohtsubo et al., 1981; Machida et al., 1982). The resulting cointegrates contain a duplication of either IS102 or IS1 -3 at the cointegration junctions in a direct orientation, and thus are of two types, as shown in Figure 1. These cointegrates can be readily detected in cells

Cell 30

A

B abcde

abcde

kb

27 23 17.9 kb

10.2

kb

10.2 6.7

6.7

Figure 2. Agarose (0.6%) Gel Showing Closed and Open DNA of the Cointegrate Plasmids as well as Their Parents

Figure 1. Structures of pYMlO3 mids Formed between Them

and Col El and Coin&grate

Plas-

The filled boxes in pYM103 represent 15102, and the open boxes represent IS1 -3. The ends of IS1 02 are indicated by 7 and 0 to orient its sequence. The solid and dashed lines with arrows along the pYM103 circular sequence show the duplicated segments present in pCM plasmids. In pCM1, a pYMlO3 sequence of approximately 6 kb (the solid line) is duplicated at junctions between pYMlO3 and Col El. In pCM2, a pYMlO3 sequence of approximately 10 kb (the dashed line) is duplicated at the cointegration junctions. The open and filled rectangular boxes on the Col El sequence show the sites of 9 bp sequences which are duplicated during formation of pCM1 and pCM2, respectively. Cleavage sites for the restriction endonucleases are shown by the short solid and dotted lines. Since the integration site of pYMlO3 into Col El to form the IS1 02- or IS1 -3-mediated cointegrates is unique for each cointegrate, the cleavage sites on the Col El region in these cointegrates are shown by the dotted lines. ori: origin of replication. Tc’: tetracycline resistance gene.

harboring pYMlO3 and Col El by selecting the tetracycline resistance of pYM103 and the character of temperature-resistant DNA replication of Col El. The cointegrates are formed at a frequency of about 1 O-9 per division cycle. During examination of a number of cointegrates, we found one rare plasmid of extremely large size, about 23 kb in length; much larger than the IS1 02- or IS1 -3mediated cointegrates, which were about 17.9 kb and 17.6 kb, respectively. We were able to isolate two other large plasmids from recA+ and recA - cells carrying pYM103 and Col El. We named these three plasmids pCM1, pCM2 and pCM3 and subjected them to extensive structural analysis. The PCM plasmids were found among 35 independent cointegrates examined. Thus, considering the frequency of cointegration mediated by IS1 02 and IS1 -3, the frequency of generation of pCM plasmids can be estimated to be

Circular

(A) (Lane a) Parental plasmid Col El; (lane b) parental plasmid pYMlO3; (lane c) pCM1 DNA, and (lane d) pCM2 DNA, prepared by the crude lysis method. Common faint bands are contaminating chromosomal DNA. (Lane e) An IS102-mediated cointegrate shown for comparison of size. The position of closed circular DNA of each plasmid is shown by the arrows, and their molecular lengths are given in kilobase pairs. (13) Lanes a, b and e are same as those in (A). Lanes c and d show purified pCM1 and pCM2 DNAs, respectively. The arrows indicate the position of closed circular DNA for each plasmid. The thick arrow shows the closed circular DNA position of the segregant of pCM2 (see text).

lo-” to 10-l’ per division cycle. Figure 2A shows the gel electrophoresis pattern of pCM1 DNAs, isolated from recA- hosts, and of pCM2 DNAs, isolated from recA + hosts. Lengths were approximately 23 kb for pCM1 and 27 kb for pCM2 (see the following restriction endonuclease cleavage analysis); pCM3, also isolated from a recA+ host, was identical in length with pCM2 (not shown in Figure 2). Physical Structures of the pCM Plasmids Because of the complexity of the structures of the pCM plasmids, we will first describe their general structural characteristics and then present the experimental results used to support these descriptions. All of the pCM plasmids contain the entire sequences of the two parental plasmids, pYM103 and Col El, and a duplication of a large segment of pYMlO3. As shown in Figure 1, the duplication in pCM1 is approximately 6 kb in size, extending from one end (called the Bend) of IS1 02 to a site within the ISl-3 sequence. This duplication includes the entire IS102 sequence, the tetracycline resistance region and the cleavage sites for restriction endonucleases Sst II, Eco RI and Bst Eli within pYM103. It is present at the junctions between pYM103 and Col El in a direct orientation. Similarly, pCM2 contains a direct duplication of an approximately 10 kb sequence of pYMlO3. This duplicated sequence also begins at the 6’ end of IS1 02, but ends at a different site on pYMlO3. It includes the entire IS1 02 sequence, the tetracycline resistance region, the replication origin of pYM103 and the re-

&Novel

Type of Transposon

striction cleavage sites for Sst II, Eco RI and Bst Eli within pYMlO3 (Figure 1). The recombination site on Col El used to form pCM2 is different from that used to form pCM1. The third pCM plasmid, pCM3, contains a direct duplication of the exact same segment duplicated in pCM2 (not shown in Figure 1). The target site on Col El used to form pCM3, however, is different from that used to form pCM2. Cleavage Analysis Plasmid pYMlO3 has a single cleavage site for each of the restriction endonucleases, Sst II, Eco RI and Bst Eli; Col El has a single cleavage site for Sst II and Eco RI, but two cleavage sites for Bst Eli (Figure 1). We analyzed the pCM plasmids using these three restriction enzymes. Figure 3 shows the gel electrophoresis pattern of the digests of pCM1 and pCM2 as well as their parents. Digestion of both pCM plasmids with Sst II or Eco RI produced three fragments, whereas digestion with Bst Eli produced four fragments. These results indicate that pCM1 and pCM2 are not simple recombinants between pYM103 and Col El, since each pCM plasmid contains one extra cleavage site for each restriction enzyme. An important note is that in digests of pCM DNA with each enzyme, one fragment was identical with pYM103 (10.2 kb). However, the Sst II and Eco RI digests of each pCM plasmid did not contain an intact Col El fragment (6.7 kb), while the Bst Eli digest contained one (1.2 kb) of the two Col El fragments but not the other (5.5 kb). Instead, each digest contained two new fragments characteristic of each pCM plasmid. These results suggest that pCM1 or pCM2 contains a direct duplication of a sequence of pYMl03, which includes the Sst II, Eco RI and Bst Eli cutting sites, at the junctions with a unique target site on Col El for each pCM plasmid. Thus digestion with each restriction enzyme produces a fragment identical with intact pYMlO3 and two new smaller fragments, instead of a Col El fragment. To support this putative structure, we examined the pCM1 and pCM2 plasmids as well as their parents by double digestion with Sst II and Bst Ell. As shown in Figure 3, the digest of each pCM plasmid contained

a b cd

abed

abed

six fragments. As expected from the structures deduced above, two fragments (5.6 and 4.6 kb) generated from the parent pYM103 were also found in the digest of each pCM plasmid. The 5.6 kb fragment in each pCM digest was relatively brighter than that in pYM103 digests (Figure 3). This can be explained by the existence of a duplicated pYMlO3 segment, which contains the sequence including the Sst II and Bst Eli cleavage sites, resulting in the production of a double amount of the 5.6 kb Sst II-Bst Eli fragment. The other fragments of the pCM1 digest included two Col El fragments, 2.4 kb and 1.2 kb in length, but did not include a third fragment of 3.1 kb. We assume that the 3.1 kb Col El fragment contains the target site for the integration of pYM103 and therefore becomes a part of the other two pCM fragments, which do not correspond to any fragments in the digests of the parental pYM103 and Col El plasmids (Figure 3). Similarly, the pCM2 digest did not contain one Col El fragment 2.4 kb long, but contained two new fragments instead (Figure 3). This indicates that the Col El fragment absent from pCM2 contains the target site for integration of pYM103, and that the two new fragments must contain parts of the pYM 103 and Col El sequences. In the double digest of the third pCM3, we obtained results very similar to those from pCM2, except that the two new fragments which appeared were slightly different in length from those from pCM2 (data not shown). From the length analysis of restriction fragments, we calculated molecular lengths of approximately 23 kb for pCM1, 27 kb for pCM2 and 27 kb for pCM3, which are longer than the sum of the lengths of their parents, 16.9 kb. Thus the lengths of the duplicated segments must be approximately 6 kb in pCM1, 10 kb in pCM2 and 10 kb in pCM3. These include the Sst II-Bst Eli 5.6 kb fragment which is known to contain the tetracyline resistance region and the Eco RI site as well (Figure 1). Nucleotide Sequence Analysis We determined the nucleotide sequences of the restriction fragments containing the junctions between pYMlO3 and Col El sequences in each pCM plasmid Figure 3. Agarose (0.7%) Gel Electrophoresis of Restriction Endonuclease Digests of Col El, pCM1, pCM2 and pYMlO3

abed

kb 10.2 + 6.7~

kb -5.6 ~4.6 - 3.1 -2.4 -

Sstll

EcoRI

BsfEII

SstIl + BstEIl

1.2

(Lane a) Col El ; (lane b) pCM1; (lane c) pCM2; (lane d) pYMlO3. Restriction endonucleases used are indicated below each panel. Sst II, Eco RI and Bst Eli cleave pYMlO3 DNA at only one site, generating a 10.2 kb fragment. Col El DNA has single cleavage sites for Sst II and Eco RI, generating a 6.7 kb fragment, while Bst HI cuts Col El DNA at two sites, producing 5.5 kb fragment and 1.2 kb fragment. Double digestion of Col El with Bst Eli and Sst II produces 3.1 kb, 2.4 kb and 1.2 kb fragments. The position of each fragment is shown by the arrow with the molecular length in kilobase pairs.

as well as those of the Col El fragment containing the target site for integration of pYM103. These restriction fragments were readily identified by comparing the digestion pattern of each pCM plasmid with that of the parental plasmids after cleavage with different restriction enzymes. Figures 4 and 5 show the restriction fragments sequenced by the method of Maxam and Gilbert (1977) and the critical parts of the nucleotide sequences. Figure 6 shows examples of autoradiograms from polyacrylamide gels used to determine the nucleotide sequences of fragments containing the

cointegration junctions in pCM1. The results in Figure 4 showed pCM1 to have the following structural features. At one junction between the pYM 103 and Col El sequences, the sequence of Col El (labeled with B) was connected to a sequence in pYM103. The sequence was found to be the 0 end of the insertion element IS1 02 present in pYM103. At the other junction, the sequence of Col El (labeled with A) was also joined to a pYM103 sequence inside the mutant IS1 3. The 9 bp sequence at the Col El target site for cointegration was duplicated in a direct orientation at EcoRI

ECORI I

IS102 e end in pVM103 fISlO2

T&71

6 end in pYM103 L&III

GCTTTGTTGAATAAATCGAAC---GGCC CGRAACAACTTATTTAGCTTG---CCGG ThQI

/

1 “CXIII

\

6 in CalEl

F-A

Thai

in ColEl

J

Thd

DdeI

\

Eco RI

A in Col~i

Eco RI

SUU3A

I

ori ‘ori

ori

Figure 4. Critical between pYMlO3 on Col El

Part of the Nucleotide Sequences of the Junctions and Col El in pCM1 as well as the Target Sequence

The dotted circles on Col El and pCM1 plasmids denote the regions whose sequences were determined by the restriction fragments indicated inside of the plasmids. The small arrows in the dotted circles represent the direction of nucleotide sequencing. The Col El sequences labeled A and B are underlined below the nucleotide sequences. The rectangular boxes in the dotted circles and in the nucleotide sequences show the 9 bp target sequence on Col El that is duplicated in a direct orientation at junctions. The solid lines along the pCM1 circle represent the duplicated pYM103 segment.

Figure 5. Critical between pYMlO3 on Col El

Part of the Nucleotide Sequences of the Junctions and Col El in pCM2 as well as the Target Sequence

Strategy used to determine the junction or target sequences is shown in the dotted circles on the two plasmids. The Col El sequences labeled A and B are underlined below the nucleotide sequences. The rectangular boxes in the dotted circles and in the nucleotide sequences show the 9 bp target sequence on Col El. The dashed arrows along the pCM2 circle represent the direct repetition of the pYMlO3 segment.

A Novel Type of Transposon 33

the two junctions between the pYM103 and Col El sequences in pCM1 (see boxed sequences in Figure 4). These results suggest that the segment of pYMlO3 DNA, as represented in Figure 7, has been involved in cointegration with Cot El to form pCM1. The results from sequencing pCM2, as summarized in Figure 5, showed that at one junction the Col El sequence (labeled with B) was also joined with the B end of IS1 02 in pYM103, but at the other junction the

Col El sequence (labeled with A) was joined with a pYMlO3 sequence different from that seen in pCM1. This pYM103 sequence is within the essential replication region of pYM103, most of which has been sequenced in this laboratory (unpublished results). We also observed a duplication of a 9 bp target sequence of Col El different from that of pCM1 (see boxed sequences in Figure 5). The results suggest that the segment of pYM 103, as represented in Figure 7, has been involved in cointegration with Col El to form pCM2. The sequencing results of pCM3 showed that it was formed by cointegration mediated by the same pYM103 segment as that seen in pCM2 (data not shown). We also observed that a 9 bp sequence at the target site on Col El was duplicated at the cointegration junctions in a direct orientation. The duplicated sequence was CCAG GGTC

CAAGCAAGC GTTCGTTCG

TAAA ATTT,

which was 55 bp away from the Col El target site used to form pCM2. These results demonstrate that one end of the duplicated segments of pYM103 is the 6’ end of IS102, but the other end is variable. As shown in Figure 7, each of these segments contained inverted repeat sequenes at its ends, although they were very short (see boxed regions in Figure 7). The common sequence among these inverted sequences in the two segments was 5’- RGCNTTGNNN -3’. Figure 6. Sequence Ladders pYMlO3 and Cal El in pCMl

Showing

Two

Junctions

between

The restriction fragments containing these junctions are shown in Figure 4. These sequences were determined by the procedure described by Maxam and Gilbert (1977). The thick letters represent the nucleotide sequences actually shown in the autoradiogram, and the thin letters show the sequences of the complementary strands. (a) 8% polyacrylamide gel showing nucleotide sequence of one junction between Cal El and pYMlO3. The Cal El sequence is connected to the sequence at position 242 in IS1 (Ohtsubo and Ohtsubo, 1978). (b) 20% polyacrylamide gel showing the other junction sequence. The Cal El sequence is connected to the Bend of IS1 02 (Ohtsubo et al., 1980b). The sequence in the boxes represent the 9 bp repetition at the target site on Cal El.

Stability of pCM Plasmids Because each pCM plasmid contains a direct duplication of a large DNA segment, recA-directed homologous recombination at the duplicated region within the pCM sequence may resolve it into two segment plasmids, one identical with a parent pYMlO3 and the other a Col El derivative containing the duplicated segment of pYM103. As shown in Figure 2A, DNA preparations of pCM1 from a recA- host and pCM2 from a recA+ host do not seem to contain any of the above segregants. However, when a large amount of

ISI-

Ori /

I--.-

6-kb

DNA

SEGMENT

Figure

7. Schematic

2:

Representation

/'

1 I 1

I

-. 2. ATAAAT&A---------------G&CCG TATTTAGCT:---------------CCAACGGGC

of the Linearized

pYMIO3

Genome

The locations of the 6 kb segment duplicated in pCM1 and of the 10 kb segment duplicated in pCM2 are shown, as well as the nuclaotfda sequences at both ends of each segment. The sequences in open boxes show the inverted repeats found at ends of each segment. The solid lines above or below the sequences show that they are common among four ends.

Cell 34

such DNA was subjected to gel electrophoresis, we found that the pCM2 DNA preparation from recA+ contained a small proportion of DNA molecules approximately 17 kb in length, as shown in Figure 2B. The pCM1 DNA preparation from a recA- host did not contain any such small plasmid DNA (Figure 28). The small plasmid DNA molecules seen in the pCM2 preparation are similar in size to the Col El derivative containing the duplicated 10 kb segment in pCM2namely, 6.7 + 10 = 16.7 kb-suggesting that this is one of the segregants formed by the homologous recombination within pCM2. Similarly, the pCM3 DNA preparation isolated from a recA+ host also contained a small amount of the plasmid DNA molecules identical in size with those seen in the pCM2 preparation (data not shown), suggesting that pCM3 also had resolved. We did not observe the other possible segregant plasmid identical with pYMlO3 in any of the pCM DNA preparations. This is probably because the plasmid DNAs were extracted from cells grown at 37°C a restrictive temperature for replication of pYMlO3. Discussion IS and Tn elements mediate cointegration between two plasmids, giving rise to a direct duplication of the element that mediated the event. We have demonstrated that the pCM plasmids formed between pYMlO3 and Col El are cointegrate plasmids containing a direct duplication of either a 6 kb or a 10 kb DNA segment which carries the tetracycline resistance region of pYMl03. Therefore, these segments must be the tetracycline resistance transposons. We named the 6 kb transposon Tn1021 and the 10 kb transposon Tn1022. In contrast to most transposons, which are flanked by two copies of an IS element, TnlO21 and Tn1022 contain only a single copy of IS1 02 at one end, but a sequence of variable length which was originally adjacent to IS1 02 at the other end. It is possible that Tn1022 is a transposon that has come from one plasmid by transposition into another plasmid to give plasmid pSClO1 (and thus its derivatives, pHS1 and pYM103), and that Tn1022 mediated cointegration to form two different cointegrates, pCM2 and pCM3, as shown in Results. However, it is clear that TnlO21 did not exist in the original plasmid pSClO1 and its temperature-sensitive replication mutant pHS1, because one end of TnlO21 was within the IS1 -3 sequence, which was added to pHS1 to create pYMlO3 by a series of genetic manipulations (Machida et al., 1982; see also Experimental Procedures). This suggests that TnlO21 and Tn1022 are not transposons that have come from other genomes, but could be spontaneously generated within the pYMlO3 sequence at a low frequency. Our results also showed that TnlO21 and Tn1022 both generated a 9 bp sequence at the target site

during formation of the pCM plasmids. We have previously shown that cointegration mediated by IS102 alone results in generation of a duplication of a 9 bp sequence at the target site (Ohtsubo et al., 1980b). This fact, together with the result showing that IS1 02 appeared at one end of Tn1021 and Tn1022, indicates that IS102 is responsible for the spontaneous generation of new transposons. This type of genetic rearrangement by a single IS element has been unknown and illustrates a novel pathway for the introduction of new genetic material onto the same or different genomes. It has been reported that IS102 may encode a protein involved in IS1 02-mediated plasmid cointegration (Bernardi and Bernardi, 1981; Machida et al., 19821, and that IS1 02 has 18 bp inverted repeats at its ends which may be recognized by the putative IS1 02-encoded protein (Ohtsubo et al., 1980b; Machida et al., 1982). The results presented here show that two new transposons also contain short terminal inverted repeat sequences, much shorter than those in ISlO2. The limited length of the inverted sequences might reflect the frequency of generation of the pCM plasmids, which is lower than that of cointegration mediated by IS102. As described in Results, there is a common terminal inverted repeat sequence, RGCNTTG, in Tnl021 and Tn1022. This sequence could be the minimal sequence required for recognition by the IS1 02 protein and could appear randomly approximately once per 2000 bp in a genome. It would be likely that whenever the sequence RGCNTTG and its adjacent sequence make a better match with the terminal inverted repeat sequence of ISlO2, such a sequence can contribute to the generation of a transposon containing a single copy of IS1 02. Tn elements such as the Tn3 family and phage Mu are not flanked by two copies of an IS element, but contain short terminal inverted repeat sequences (see review by Kleckner, 1981). These transposons, however, encode a protein that probably binds to the terminal inverted repeats to promote their own transposition (Heffron et al., 1977; Faelen et al., 1978). It is intriguing to speculate that such transposons could be generated during evolution as transposons in which only a single copy of an IS element was present, as demonstrated in this paper. Although the new transposons may at first have very low transposition frequencies because of the limited homology of the inverted repeat sequences, they may become more efficient by creating more homologous inverted repeat sequences through base substitutions in the terminal sequences. Furthermore, during evolution, the other terminal inverted repeat sequence of the IS element, now located in the middle of the new transposon, could be altered with no drastic changes in the sequence encoding the protein responsible for transposition.

A Novel Type of Transposon 35

Experimental

Procedures

Bacteria and Plasmids Escherichia coli K12 strains used were JE5507 and JE5519, which were previously described (Ohtsubo et al., 1980a). JE5519 is a RCA derivative of JE5.507. The plasmid pYMlO3 was derived from the plasmid pHS1, which was inserted by a single copy of an insertion element IS1 followed by mutagenizing inside the IS1 (Machida et al., 1982). Plasmid pHSl is a temperature-sensitive replication mutant of pSClO1 (Hashimoto and Sekiguchi. 1976). The plasmid Col El, which belongs to a different incompatibility group from pSClO1, was used for the analysis of cointegration with pYMlO3. The E. coli strains harboring pYMlO3 and Col El were described previously (Machida et al, 1982). Restriction Endonucleases Eco RI was purchased from Miles Laboratories, and Mbo II was obtained from New England BioLabs. Sst II, Bst Eli, Hae Ill, Tha I, Alu I, Hinf I, Pst I, Sau 961, Sau 3A and Dde I were obtained from Bethesda Research Laboratories. These enzymes were used as recommended by the laboratory from which they were obtained. Other Materials Bacterial alkaline phosphatase was purchased from Worthington, and T4 polynucleotide kinase from P-L Biochemical Corporation: Y-~*PATP was obtained from ICN Chemical and occasionally was prepared by the method of Schendel and Wells (1973). Reagents purchased for sequencing were dimethyl sulfate (Aldrich Chemical Company), hydrazine (Eastman) and piperidine (Fisher Scientific Company). Autoradiography was performed by exposure to x-ray film (SB5, Kodak) with an intensifying screen (Blue Max or Du Pant) at -70°C. Isolation of Cells Carrying Cointegrate Plasmids JE5507 or JE5519 cells harboring pYMlO3 and Col El were grown in L broth at 25°C overnight. Of a 1 :l O6 dilution 0.1 ml was inoculated into test tubes containing 5 ml L broth, and cells were grown at 25°C for 36-48 hr. Of each culture 0.1 ml was plated on agar plates containing 20 pg/ml tetracycline for JE5507 or 10 pg/ml tetracycline for JE5519. and incubated at 42°C for 24 hr. We collected 35 colonies from 35 independent cultures, and then analyzed them for size and structure of cointegrated plasmid DNA as prepared by the crude lysis method described below.

each plasmid by ethidium bromide-cesium chloride equilibrium centrifugation as described by Ohtsubo et al. (1978). After removal of ethidium bromide with cesium-chloride-saturated isopropanol, DNA was dialyzed against 10 mM Tris-HCI (pH 8.0) and 0.1 mM EDTA. Gel Electrophoresis Native duplex DNA fragments were separated by gel electrophoresis on 0.7% agarose (13 by 15 cm) or 6% or 8% polyacrylamide slab gels (acrylamide:bisacrylamide, 2O:l). DNA bands were visualized by staining with ethidium bromide (0.4 pg/ml) under ultraviolet light. For DNA sequencing, 8% and 20% polyacrylamide gels (13 by 40 by 0.04 cm) containing 100 mM Tris-borate (pH 8.3), 2 mM EDTA and 7 M urea were used. Determination of Nucleotide Sequence DNA fragments generated by restriction endonuclease digestion were treated with 5 to 10 units/ml of bacterial alkaline phosphatase in IO mM Tris-HCI (pH 8.0). 1 mM MgCI? and 0.1 mM EDTA. After treatment with phenol and extraction with ether, the DNA was precipitated with ethanol. The precipitated DNA was suspended in 10 mM Tris-HCI (pH 8.0) and 0.1 mM EDTA and then phosphorylated with 0.2 to 1 PM y32P-ATP and 66 units/ml T4 polynucleotide kinase in 50 mM Tris-HCI (pH 8.0), 10 mM MgC12 and 5 mM dithiothreitol. “P-labeled fragments were occasionally obtained by strand separation with gel electrophoresis or cleaved with another restriction endonuclease (Maxam and Gilbert, 1980). The nucleotide sequence of the “P-labeled fragment was determined as described by Maxam and Gilbert (1977). Acknowledgments We thank H. Ohtsubo. K. Armstrong and M. McCormick for critical reading of the manuscript. We are also grateful to D. Davison for help with manuscript preparation, S. Donaldson for typing the manuscript and J. Demian for the photography. This work was supported by grants to E. 0 and H. 0 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

April 6, 1982

References Rapid Detection of a Plasmid by the Crude Lysis Method Content and length of plasmid DNA within a clone were examined by the crude lysis method as follows. A 0.5 ml aliquot of an overnight culture of cells harboring a plasmid was mixed with an equal volume of 75% ethanol, 2% phenol, 20 mM Tris-HCI (pH 8.0) and 10 mM EDTA. The cells were collected by centrifugation in an Eppendorf centrifuge for 3 min and resuspended in 100 pl of 20 mM Tris-HCI (pH 8.0) and 1 mM EDTA. Of 25% sodium lauryl sulfate 4 ~1 was added, and the suspension was incubated at 37°C for 5 min. After centrifugation for 15 min, 10 to 20 pi of the supernatant was mixed with 5 pl of 0.05% bromophenol blue and 50% glycerol and was run on a 0.7% agarose gel. Plasmid DNA was visualized by staining with 0.4 pg/ml of ethidium bromide. For restriction endonuclease digestion or transformation of plasmid DNA isolated in this way, the above supernatant (100 pl) was chilled in ice and mixed with 100 pl of 0.1 M NaOH. After 2 min in ice, 100 ~1 of 3 M sodium acetate buffer (pH 4.5) was mixed in well, and the mixture was chilled for 30 min in ice. White precipitate was removed by centrifugation in an Eppendorf centrifuge for 5 min. Plasmid DNA in the supernatant was precipitated with ethanol and rinsed with chilled ethanol. After drying, the precipitated DNA was resuspended in 10 mM Tris-HCI (pH 8.0) and 0.1 mM EDTA. When necessary, the sample was treated with RNAase I (50 to 100 sg/ml) at 37°C for 30 min before the alkaline treatment. Purification Covalently

of Plasmid DNA Molecules closed circular DNA was isolated

from

cells

harboring

Bernardi. A. and Bernardi, element present in pSClO1. Calos, M. P. and Miller, 20, 579-595.

F. (1981). Complete sequence Nucl. Acids Res., 9, 2905-2911. J. H. (1980).

Faelen, M., Olivier, H. and Toussaint, Mu-l early functions in Mu-mediated Nature 271, 580-582.

Transposable

of an IS

elements.

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