Tn903 induces inverted duplications in the chromosome of bacteriophage lambda

J. Mol. Biol. (1980) 139, 1-17

Tn903 Induces Inverted Duplications in the Chromosome of Bacteriophage Lambda MICHAEL SYVANEN Department

of Microbiology and Molecular Harvard Medical School Boston, Mass. 02115, U.S.A.

(Received 27 November 1978, and in revised form

Genetics

4 September 1979)

Specialized transducing strains of bacteriophage lambda have been isolated that carry the transposable kanamycin resistance element, Tn903. Tn903 carries an inverted duplication of 1130 base-pairs flanking the kanamycin resistance gene. Often, when h: :Tn903 particles are infected into bacterial cells, the lambda chromosome is rearranged into a defective lambda plasmid which replicates with the bacterial cell. The formation of the defective plasmids (called Tn903/\dv) is most likely induced by the Tn903 insertion itself. This follows from the fact that the novel DNA sequence found in these plasmids, with respect to the ancestral is always adjacent to the Tn903 element. Physical chromoXTn903 chromosome, somal mapping of these plasmids shows that they contain large inverted duplications of lambda sequences situated about the Tn903 insertion. The formation of the Tn903hdv plasmids from the ancestral XTn903 is not dependent on the recombination functions provided through the phage red gene or the host recA gene.

1. Introduction Antibiotic resistance genes found on bacterial resistance factors are known to spontaneously undergo genetic transposition. A simple demonstration of this property can be carried out by growing a temperate bacteriophage on a cell carrying an R factor, followed by isolating a phage that has incorporated drug resistance genes into the phage chromosome (Kleckner et al., 1975; Berg et al., 1975; Gottesman & Rosner, 1975). In addition to the property of transposition, these elements frequently cause other genetic rearrangements of the chromosomes in which they are located. For example, elements coding for a kanamycin resistance and a tetracycline resistance, called Tn5 and TnlO, respectively, can cause deletion mutations (Botstein & Kleckner, 1977; Berg, 1977) and TnlO induces chromosomal inversions (Kleckner et al., 1979). This paper describes a genetic rearrangement that is induced by Tn903, a kanamycin/ neomycin resistance transposon from the R factor R-6 (Ohtsubo & Ohtsubo, 1977). Tn903 consists of a unique internal region of 900 base-pairs flanked by a duplicated, but inverted, sequence of 1130 base-pairs. The unique region contains the structural gene for kanamycin resistance (Armstrong et al., 1977) and the duplicated sequence, called 78, is of unknown function. Tn903 has been shown to transpose from R-6 into a variety of phage chromosomes, such as fd (Nomura et al., 1978), A (Berg et al., 1978;

0022-2836/80/130001-17

$02.00/O

0 1980 Academic

Press Inc. (London)

Ltd.

2

M.

SYVANEK

Young et al., 1979) and P-4, Mu, and P-l (unpublished results). On the other hand, transposition of Tn903 into the Escherichia coli chromosome or the sex factor F occurs at barely detectable frequencies (Berg et al., 1978; Young et al., 1979). During efforts to monitor transposition of Tn903 from a phage lambda donor. we saw that this element, rather than transposing, induced a rearrangement of the bacteriophage chromosome. The rearrangement was an extensive inverted duplication of the phage sequences. In general, the event leading to an inverted duplication within a chromosome would be lethal. These could be detected with phage lambda because the rearrangement converted lambda with Tn903 into a defective lambda plasmid. Defective lambda plasmids (Xdv) were described originally by Matsubara & Kaiser (1968). The hdv plasmids carry, at minimum, the lambda replication genes 0 and P. a control gene cro and the rightward promoter, Pr (Matsubara, 1975; Berg, 1974). E. coli that carry these plasmids are partially resistant to infection by either wild-type lambda or by vir mutants of lambda: this distinguishes a Xdv-carrying strain from a lambda lysogen, since in a lysogen Xvir will grow, whereas lambda itself cannot. This paper presents evidence showing that Tn903 causes the genetic rearrangement that gives rise to the defective plasmids.

2. Materials and Methods (a) Bacterial

and phage strains

E. coli K-12 strains W3110 or W3101 recA 13 are used as the recipients for the kanamycin resistance Tn903. Phage titers are det,ermined by plat.ing the phage on Ymel (contains swpF). Table 1 lists the lambda strains used in t,his study. Bacteria, are normally grown in TYE medium, a broth with Tryptone (O.5e/0), yeast. ext,ract (0.1 “4) and NaCl (0.524). Phage are stored in 20 mM-Tris (pH S), 20 mM-Mg&. (b) Isolation

of kanamycin-resistan,t

strains

carrying

defective plasmids

A total of lo7 of the hTn903 isolate was added to 0.1 ml of growing W3110 at. 5 x 10’ cells/ml. After allowing the phape to adsorb for 5 min, 1 ml of broth was added and the culture was incubated for 30 min at 37”C, witlr aeration. Then 0.5 ml of the infect,ed TABLE

1

Phage strains Phage designation hpkl0 hpk8 Xpk35 Xpk3 hdk6 Xpk21 hpk3 redXTnlO

(:omplete

genotype

hb519 Tn903 ~1857 nin5 Sam7 Xb51Q Tn903 (0,705) ~I857 nin5 Snm7 hb515 b519 ~1857 TnQ03 (0.797) nin5 Sum7 hb519 cl857 Tn903 (0.923) Snm7 Xb519 ~I857 win5 ,Stcm7 Rz : : Tn903 (0.952) hb519 b515 ~I857 nin5 Sam7 Tn903 (0.993) hb221 rednm270 ~1857 Tn903 (0.923) Sam7 A6515 b519 ~1857 nin5 Snm7 with TnlO

The number in parentheses following !Fn903 gives the location of the insertion in fractional X lengths. In X$10, Tn903 is located in the b region between the b519 deletion and the attachment site. In ATnlO the site of insertion was not determined. Map positions were determined by measuring electron micrographs of heteroduplex molecules (data not shown; Young et (II., 1979).

Tn903

INDUCES

INVERTED

DUPLICATIONS

IN

X

3

bacteria was spread onto TYE agar plates containing kanamycin sulfate at 40 pg/ml. After 24 h at 42”C, single colonies that appeared were carried through 2 successive single colony isolations on kanamycin-containing medium. Approximately half of the original clones failed to give normal growth upon restreaking. The st.ability of t,he kanamycin resistance phenotype was examined for each isolate. This was done by inoculating cells from a kanamycin-resistant strain into liquid TYE medium, growing the bacteria to saturation, then plating out for single colonies on TYE agar and testing these isolated colonies for kanamycin resistance.

(c) Marker

rexue

and complementation

of h genes

The presence of lambda genes in kanR bacteria was determined by marker rescue tests. A solution of tester phage, with an amber mutation in the given gene was placed on the surface of a TYE agar plate spread with 0.1 ml of a liquid overnight culture of the strain to be tested mixed into 2.5 ml of a soft agar overlay. The plates contained 40 pg kanamycin sulfate/ml. For each tester phage a 10.~1 drop of 3 dilutions, log, lo7 and IO5 plaqueforming units/ml, was added to the plate. 4 multi-replicator prong was used to transfer 20 drops at a time. If a wild-type copy of the gene is present in the bacteria, a clearing zone will appear under the lo9 spot and single plaques will be seen under the lo7 drop. A positive complementation is indicated if either a zone of clearing or numerous single plaques can be seen under the lo5 drop. The phage used in these tests were: Ximm434cAam32, Ximm434cts Kam24, himm434 Nam53, Ximm21 Pam3, XQam73Qam501, and Ximm434 Ram5 (d) The test for

Xcro gene

The presence of the hero product in a given bacteria was tested by plating lambda for single plaques onto a lawn of the bacteria and examining the plaque morphology. For the parental W3110, h+ will make relatively large turbid plaques, whereas if the cro product is present, either no single plaque or very small clear plaques result. In a second test for the presence of cro product, the ability of lambda to form lysogens in the particular strain was measured. The phage used to test for lysogens is hcI857 TnlO. This allows the format,ion of lysogens to be measured directly by the appearance of tetracycline-resistant clones at 30°C which do not grow at 42°C. In this test 2 x lo6 XTnlO phage were added to 5 x lo* bacteria. The mix was then grown in 1 ml of broth for 30 min at 3O”C, and l/10 of this was spread onto TYE agar plates containing 20 pg tetracycline/ml, and, for kanamycinresistant strains 40 pg kanamycin sulfate/ml. The plates were incubated overnight at 3O’C. For W3110 and derivatives without cro product, about 100 tetracycline-resistant clones appeared after 24 11. For the strains that scored posit,ive for cro function, no tetracycline-resistant clone appeared after 24 h. After 2 or 3 days at 3O”C, however, a few hundred tiny colonies would often appear. But these would grow at 42°C.

(e) Preparation

of DNA

To purify plasmid DNA, the protocol described by Sharp et al. (1972) was followed exactly. In this procedure, whole cell DNA is denatured in alkali and then rapidly renatured. Small circular DNA will renature, while chromosomal DNA remains singlestranded and can be removed with nitrocellulose. After the supercoiled DNA was purified on CsCl/ethidium bromide centrifugal equilibrium gradients, it was dialyzed into 0.1 M-KCl, 0.02 M-Tris.HCl (pH SO), O-5 mM-EDTA. To prepare the plasmids for electron microscopic examination, the DNA was first nicked in order to relax the super coils. This was done by reacting the DNA with EcoRI endonuclease in the presence of ethidium bromide. This treatment causes single strand nicks at r1 restriction sites (Feunteun et al., 1976). After nicking, EDTA was added to 10 mM and ethidium bromide was removed by extraction with n- butanol saturated with a 0.1 M-NaCl.

4

M.

SYVANEN

The plasmid DNA, where indicated, was cut with EcoRI endonuclease, and with XhoI endonuclease (both purchased from New England BioLabs). Each enzyme was reacted with the plasmid DNA for 30 min at 37°C in the buffer recommended by BioLabs. (f) Electron microscopy To examine for inverted duplications in plasmid sequences, the DNA that had been nicked was denatured and quickly renatured by the following procedure : to 25 ~1 of DNA at 8 pg/ml, which had been nicked or cut with one of the endonucleases, was added 25 ~1 of 0.2 nl-NaOH,

20 mM-EDTA.

Then

5 ~1 of 1.8 >I-Tris.HCl

(pH

7.3) was

added.

Immediately

25 ~1 of this is added t.o a solution to give a final concn of 40% formamide, 0.1 mg cytochrome/ml, 0,2 M-Tris.HCl (pH 8.5), 10 mM-EDTA in 100 ~1. The DNA was examined in the electron microscope using the 40% formamide spreading technique described by Davis et al. (1971).

(g) Agarose gel electrophoresis After being digested by EcoRI endonucleases the plasmid DNA was analyzed by vert,ical 0.7% agarose slab gel electrophoresis using Tris-borate buffer (Sharp et al., 1973). The molecular weights of the various EcoRI restriction fragments were determined by measuring the distance each fragment migrated into the gel, and comparing this distance with a standard of known molecular weight. The standard was h DNA digested with EcoRI endonuclease. The log of the known molecular weight for each fragment was plotted against the distance that the fragment migrated through the gel. A best curve was fitted through the points and used as the standard for determining the molecular weights of the unknown DNA samples.

3. Results Tn903 is a transposable element that carries a gene for kanamycin resistance. The defective lambda plasmids (Tn903hdv) carrying Tn903 were found after infecting drug-sensitive E. coli with phage h : : Tn903 and then by selecting kanamycin-resistant bacteria. Under the conditions used here lambda is unable to establish itself as a lysogen. Table 2 gives the frequency of kanamycin-resistant bacteria following such TABLE 2 Isolation XTn903 used as donor pkl0 W pk35 pk3 dk6 pk21 No phage

of kanamycin-resistant Location of Tn903 in donor

b,

region 0.705 0.797 0.923 0.952 0.991

bacteria from hTn903 Frequency kanR bacteria 1.5 x 10-e 3 X 10-4 5 % 10-S 1.2 x 10-J 3x10-5 1.5x 10-b < 10-10

Fraction Stable 7/g e/12 o/10 o/11 o/12 o/7

The frequency of kanamycin-resistant (kanR) bacteria is the ratio between the number of kanR bacteria appearing on a plate per number of bacteria infected with the indicated hTn903 phage (see Materials and Methods). Each bacteria was infected by one phage. When bacteria were multiply infected, the yield of kanR bacteria was reduced (not shown). Stability of the kanamycin resistance phenotype was tested in selected clones as described in Materials and Methods. All selections for kanamycin resistance were done at 42°C to prevent lysogenization (see the text).

Tn903

INDUCES

INVERTED

DUPLICATIONS

IN

h

5

infections by different lambda-Tn903 insertions. As is indicated, Xpk35, hpk3, hdk6 and /\pk21 give rise to kanamycin-resistant bacteria but the resistance is unstable. In these four cases if the bacteria are grown for 12 generations in the absence of kanamycin, from 20% to 95% of the bacteria become kanamycin-sensitive. Genetic traits that are this unstable suggest that a plasmid is involved. On the other hand, hpk8 and hpkl0 yield some stable kanamycin-resistant strains where, presumably, Tn903 becomes inserted into the bacterial chromosome. The formation of the unstable kanamycin-resistant strains is not dependent on general recombination functions. This is shown in Table 3, where the yield of kanamycin-resistant strains at 42°C is given when the bred - mutation is crossed into hpk3 and the recA- mutation is introduced into the bacterial recipient. These kanamycinresistant strains are also highly unstable for Tn903. TABLE 3 Isolation

from XTn903 in infections

of kanamycin-resistance

recombination-dejcient Recipient

DOnOT ATn903

bacteria

W pk3red pk3 pk3red The same protocol strain is W3101recA13.

as is given

-

in Table

Frequency bacteria

kanR ( x 105)

ret +

27

ret +

10

recA

14

rectl

5

2 was followed.

(a) The kanamycin-resistant

The ret + strain

derivatives

contain

is W3110

and the recA

h genes

The fact that kanamcyin resistance in many of the isolates given in Table 2 is unstable suggested that Tn903 in these cells is carried on a plasmid. If this is so, then certain lambda genes would be present in these strains, especially genes 0 and P, which are required for plasmid replication. Therefore, the presence of the lambda genes were tested for by marker rescue, using a set of heteroimmune lambda hybrids with amber mutations in different lambda genes (Table 4). As is shown, most of the kanamycin-resistant strains have gene P. All of the strains that are highly unstable carry gene P. It is not shown, but the strains that are P+ by marker rescue also complement P- phage. The unstable strains lose their kanamycin resistance and their lambda genes simultaneously. This was shown by picking one kanamycin-sensitive segregant each from strains derived from pk8, pk35, pk3 and pk21, and showing that they also had lost their lambda genes (data not shown). These data suggest that Tn903 is associated with a hdv-like plasmid. Some qualification should be made about calling the plasmids Xdv, since according to previous studies Xdv plasmids express the cro protein, and that an active cro product. is required

M. SYVANEN TABLE 4 Marker Ancestral hTn903

pkl0

Site of Tn903 in ancestral phage ; between h genes : K+N

(left of kil)

pk8

pk35

K+N

(right

of kil)

CT0 + P

rexue tests for X genes

Number of kanR recipients

to have h genes: A

7 2

-. +

4

-I-

1

+

1

-

2 3 4 3

10

K

‘iiT

cm

-

.-

+ i +

-

CT

-

+ -

+ + i.-t

+ t+ +

-

-

pk3

QSR

10

-

3

-

pk21

R-CA

10 3 1 1

-. -

pk3 red-

Q+R

14

.--

P

--. + + + + + +

+ + -

+ + U

+ + + ii +

-L-

+

+ +

Q

R

+ + + iF + .+t i-+ -t +

The recipient host for hTn903 is W3110 when XpklO, pk8, pk35, pk3 and pk21 were used as the donor (see Table 2). The recipient is W3101 recA13 when hpk3 red- is used as the donor (see Table 3). The presence of A genes A, K, N, P, Q or R was determined by marker rescue. The presence of the cm product was tested by 2 phenotypes that this protein confers upon bacteria. One is the restriction of X growth, the other the inability of h to lysogenize bacteria that’ express em (see text and Materials and Methods). The site of each insertion with respect to the various h genes is given for convenience (see Fig. 1 and Table 1 for a more precise location).

for both the establishment and maintenance of /\dv (Matsubara, 1975). It was therefore somewhat unexpected to find that Xpk35 gave rise to hdv-carrying strains that do not express the cro + phenotype. The cro + phenotype has two manifestations : one is that lambda will make a small clear plaque when grown on a cro+ strain, and the second is that lambda is unable to lysogenize such a strain. By both of these criteria, the recipient strains carrying the lambda plasmids whose ancestral phage is ;\pk35 are cro-. I would like to pursue this point in more detail before considering the chromosomal rearrangements that give rise to the Tn903Xdv plasmids. A problem is that the plasmids whose ancestral phage are hpk35 are apparently missing the rightward promoter (pR) from which genes 0 and P are normally transcribed (see Fig. 1). A possible solution is that the lambda late promoter, pR’, becomes juxtaposed during the original chromosomal rearrangement such that it can now permit transcription of genes 0 and P. If so, then the Q product, a positive controlling element for transcription from pR’, should be present. Therefore, the presence of different lambda gene products

Tn903

INDUCES

INVERTED

DUPLICATIONS

IN

7

A

was examined by eomplementation tests in the Tn903Xdv-carrying strains. Table 5 gives the results of these complementation tests. The strains listed in this Table were selected from the larger group given in Table 4. As can be seen, the CTO- hdv149carrying strains will complement ~Qam73Qam501. This result lends support to the possibility that the transcription of 0 and P is controlled by the Q protein from the late promoter pR’. The pattern of complementation shown for MS149 in Table 5 was also seen for six other independent isolates of W3110 Tn903Adv plasmids whose ancestral phage is Xpk35 (data not shown). 62

FIG. 1. The A chromosome. Lambda has a linear genetic map, but during vegetative growth the two ends close to give the (:OR site. The genes referred to in this paper are shown on t,he outside of the circle, the bars indicate t.ho sites of the various Tn903 insertions and the circles indicate the EcoRI cutting &es. The sit? of the pkl0 insertion is only known approximately. The relative order of the late promoter, pR’, and the pk3 insertion is unknown. The direction of transcription from the 3 promoters is indicated by the 3 arrows.

TABLE

Complementation

Rnctcri:tl

SY146 SY149 SY150 SY151 SY152 SYl53 SY214

strains

5

and marker rescue of h genes in selected kanamycin-resistant strains

Ancest.ral

hTn903

pk8 pk35 pk3 pk3 tlk6 pk21 pk3 red -

Positive complementation of the gene product + and no gene by -. The U indicates uncertainty spotting various dilutions of tester phage on each and Methods. The bacterial strains were selected strains derive from W3110 except for SY214 which

-

.-.

4

ii -

-

-

-. -

++ A+

~

At+J-

-

-t+r

U

+A ++ ++ ++ Sf

+ ++ + + + i+

-

4-t -u + -t -

is denoted by + +, positive marker rescue by (see the text). The tests were performed by of the indicated bacteria as listed in Materials from the larger group shown in Table 4. The derives from W3101 recA13.

8

RI.

SYVANEN

(b) Tn903 is involved in the formdon

of the A plaswkls

There are two lines of evidence to indicate that Tn903 is involved in forming the lambda plasmids. One argument is based on the frequency of their formation. It can be seen in Table 2 that hpk35, /\pk3, hdk6 and hpk21 give rise to kanamycin-resistant, clones (all of which carry lambda plasmids) at frequencies between 3 x 10e5 and 3~ 10m4. On the other hand, lambda by itself gives rise to hdv plasmids at a lower frequency, 10m6 to 1W7 (Matsubara, 1975). Tt seems likely that’ Tn903 is causing this increased frequency. The second argument is based on the distribution of genes found with each Tn903hdv. The discontinuit’y in X gcncs during Tn903Adv formation is always adjacent’ to the Tn903 in the ancestral phage. This is apparent, if one compares the distribution of X genes in the strains shown in Table 4 with the genetic map in Figure 1, which gives the site for each of the Tn903 insert’ions in the ancest,ral phagc. From the comparison, three rules can be stated which summarize the data in Table 4. Keep in mind that when we originally isolated t,hese strains we selected for kanamycin resistance (hence Tn903), and cell growth at’ 42°C. (1) The strains never carry the region of the lambda chromosome that includes the entire leftward operon under N cont’rol bet)ween the b, region and the 0.797 coordinates of hpk35. This is probably due to the presence of the kil gene. which is controlled by the N gene and is found in this region (Greer, 1975). (2) dll genes between the Tn903 insertion and t,he replication region, 0: P and ori, are included. (3) Genes immediately distal to Tn903 wit’h respect to the replication region are also excluded from the plasmid. The simplest explanation for why these rules are followed is that Tn903 causes an excision of a section of the lambda chromosome and connects a site adjacent to Tn903 to some second site on lambda. Thus t’he novel DNA sequence, not found on the ancestral phage, should be present on the plasmid adjacent to the kanamycin resistance element. The second site is not of any specilic sequence but must be consistent’ with the three rules above. A more complicated explanation that relies ent’irely on via Tn903 selection of the various Tn903hdv plasmids, rather than by induction followed by selection, can be formulated. In order to decide between these possibilities. I determined the physical structure of the plasmids from selected strains.

(c) Physical

properties

of four

T’n903Xdv

plasmids

Plasmid DNA was isolated from the strains SY150, SY151, SY153 and SY214 (Table 5). This proved the existence of plasmids, as judged by the appearance of the appropriate satellite band of DNA in a CsCl/ethidium bromide sedimentation equilibrium density gradient of whole cell DNA. First let me summarize the results of the physical analysis of the Tn903hdv plasmids. Each plasmid contains the complete Tn903 sequence and certain lambda sequences that are duplicated and inverted about Tn903 in the form - - 0 P - - Tn903 --P 0 --. This structure was demonstrated from an electron micrographic analysis of plasmid DNA which has been cut, denatured and renatured. The property of an inverted duplicated DNA structure is that each DNA strand contains intrastrand

Tn903

INDUCES

INVERTED

DUPLICATIONS

IN

A

9

homology. Thus, during rapid renaturation of this DNA, regions within each strand can reanneal (or “snap back”) within themselves to give the characteristic snap back duplex stems. If unique DNA separat,es the duplicated regions, then the overall structure: will appear as a linear duplex with a single st,rand loop. Examples of such electron micrographs are shown in Figure 2. Plasmid DNA from strain SY214 is shown here. Pictures of DNA that n-as first’ nicked on one strand to allow separation of the two strands when denatured in alkali, are shown in Figure 2(a) and (b). In (a) two molecules having a long double-stranded region with t,wo single strand loops at each terminus are evident. They have the appearance of bar bells. This was t.he initial evidence suggesting that these molecules contain large inverted duplications. The fact t,hat there is a single-strand loop at each end shows that the inverted duplication is separated by two unique regions. Since both + strands and - strands should form stem and loop structures, we would expect t’hat a loop from a -+ bar bell structure would have its homologous sequences on a loop from a - bar bell structure and that such a pair could stick together at, these homologous loops. Such a structure was found a,nd is shown in Figure 2(b). Annealing of two closed loops cannot be perfect. since there are no free ends to permit release of torsional st’rain, hence the tangled appearance in Figure 2(b). In order to determine which sequences are duplicated and their orientation and relationship to t’he unduplicated regions, the plasmid DNA was cut with the two different restriction endonucleases. EcoRI and Xhol, which cut lambda and Tn903 at known sites. Based on the marker rescue tests, we know that the plasmid in SY2 14 carries the EcoRI site at 0.806. In addition, Tn903 has no EcoRI site. Therefore, if the region at, 0.806 is duplicated, two unique fragments would be produced by digestion of the SY214 plasmid by EcoRI. This occurs. I first digested this plasmid with EcoRI and did agarose gel electrophoresis upon the digested DNA, which gave two discrete bands on the gel. The larger band represented a DNA size that is 30% to 4091, of full-lengbh h DNA and the other one that is 9.8% of whole lambda (data in Table 8). When this EcoRI digest of Xdv214 was denatured, reannealed and examined in t’he electron microscope, two different snap back structures resulted. Examples of these are shown in Figure 2(c) and (d). The two structures seen have a large loop and a short stem and a long stem and a small loop. The two added together create the ba.r bells seen in Figure Z(a) and (b) (measurements are given in Table 6). I also cut /\dv214 with XhoI, which has a known cutting site within the unique region of Tn903. but does not cut the X DNA. When an XhoI digest of Xdv214 DNA was self-annealed and examined, a snap back structure resulted that had a singlestrand loop at one end only, as can be seen in Figure 2(e). The small loop seen on the bar bells disappears, showing that this sequence is the unique region of Tn903. As would be expect’ed, the length of the stem shown in Figure 2(e) is the same length as the st’em shown in (a) (see Table 6). Li large number of micrographs like those of Figure 2(a), (c), (d) and (e) were made, the different distances measured. and the average values of those distances calculated (Table 6). Phage PM-2 DNA, which is 0.213 times the size of lambda, was used as the size st,andard to normalize the various measurements. There is one salient measurement in Table 6 that establishes an important feat’ure of the struct’ure of hdv214. That, is. the distance of the longer of the two stems

(a)

c2

(b)

FIG. 2. Electron micrographs of hdv snap-backs. Purified plasmid DNA from SY214 (see Table 5) was denatured in alkali, rapidly renatured and prepared for elec~on m~roscopy and examined for self-annealed structures. Before denaturation, the plasmid DXA was either ((a) and (b)) nicked by means of EcoRI endonuclease digestion in digested with &oRI endonuclease or (e) digested with XhoI endonuclease. Table 6 the presence of ethidium bromide; ((c) and (d)) completely gives the measurements made on a larger number of these structures. In the interpretive drawing the solid line designates double-stranded DNA and the broken line designates single-stranded DNA.

C..,.’ 2

Tn903

INDUCES

INVERTED

DUPLICATIONS

IN

h

11

TABLE 6

Dimensions of Adu214 and its inverted duplication hdv214 Whole Melt

Distance

DNA

and reanneal

Stem

after

Each value double-stranded 15%. t Examples $ Examples $ Examples

digestion

of A

Loop(s)

0.169 0.149 0.026 0.178

Nickingt EcoRI digestionS XhoI

as a fraction 0.400

circles

$

0.018 0.019 0.044 0.043

and 0.038

represents the average of 10 to 17 measurements. The standard deviation for the stems is 5% to 7% of the average, and that for the single-stranded loops is about shown shown shown

in Fig. in Fig. in Fig.

2(a). 2(c) and 2(e).

((1).

produced after EcoRI digestion is equal in length to the distance between the 0306 EcoRI cutting site and the unique region of Tn903 found in Apk3, the ancestral phage that gave rise to hdv214 (see Table 5). This means that hdv214 contains an inverted duplication of the lambda sequences beginning at the stems of the Tn903 in Xpk3. Thus, Tn903 is directly adjacent to the new DNA sequences (or novel joint) in hdv214, in agreement with the three rules given to explain the genetic results in Table 4. The measurements listed in Table 6 can be displayed as a physical map of /\dv214, which is shown in Figure 3. One assumption that is made in constructing this map is that only two genetic rearrangements occurred when hdv214 was generated from hpk3 red-. These are the novel joint formed through Tn903, and the novel joint Tn 903 f

\ e

e

FIG. 3. The physical map of Adv214. The co-ordinates on this map were determined by the measurements given in Table 6. In addition, the size of each of the EcoRI fragments was measured directly from electron micrographs of hdv214 DNA digested with EcoRI. Two sizes, one of 0.0995 h length and the other 0.322 h based on an external size standard lengths were seen. If the greater length is actually 0.306 units, then the shorter is 0.0946 units.

12

M.

SYVSNEN

formed at the 0.78/0.74 junction, shown in Figure 3. A clarifieat,ion of the term novel joint is in order. Normally, novel joint refers to a nucleotide sequence that is produced by an illegitimate recombination event where a new sequence of nucleotides is created that does not occur in the ancestral molecule. The junction at @78/0.74 defines such a novel joint. However, if one considers the nucleotide sequences in the region of Tn903, there is no new point that did not necessarily exist in the ancestral molecule. One must examine a region of great,er than 1130 base-pairs (the size of 70) to see new nucleotide arrangements. Hence. in this case. the novel joint refers to a rather extensive region. (d) hdvl50,

Adwl51 and Xdv153 have inverted duplications about Tn.903

of h sequences

I was able to show that Xdvl50, Xdv151 and hdv153 also carry inverted duplications of lambda sequences about the Tn903 insertion, as does Xdv214. The simplest test to show this is to take purified plasmid DNA, digest with EcoRI endonuclease, melt and reanneal, and then examine in the electron microscope. In each case, a stem and loop structure could be found with a loop of the size expected for the Tn903 unique region. Furthermore, the size of the stem is what is expected for an inverted duplication which originates at the stem of Tn903, in the ancestral XTn903, and extends toward genes 0 and P to the first EcoRI cutting site. The data are given in Table 7. The choice of the EcoRI site in calculating the expected distances is clear if one considers that hdvl50, hdv151 and Adv214 each derive from Xpk3, which has the Tn903 insertion at O-93 on lambda, and that Xdv214 derives from /\pk21, where the insertion is at 0.993 (see Fig. 1). A second line of evidence that is consistent with extensive inverted duplications in these plasmids is the pattern of DNA fragments produced when the plasmids are

TABLE

Xize of the inverted duplication Stem Observed

size Expected

7

in four TnY03Xdv plusmids Loop Observed

size Expected

hdv214

o-149

0.143

0~018

0.019

hdvl50

0.143

0.143

0.019

0,019

hdvl51

0.130

0.143

0.018

0,019

Xdv153

0.085

0.084

0.016

0,019

Each of the 4 purified plasmids was digested with ICcoRI, denatured, renatured and electron micrographs prepared of the stem and loop structures. The fraction that contained a loop closest in circumference to the Tn903 unique region is given in the Table. The size of the Tn903 loop region is taken as 900 base-pairs or 0.019 h units. The length of each stem represents an average of at least 15 measurements, whose standard deviation in 5%. The measurements are normalized to PM-2 DNA taken as 0.213 X units. The expected value for the stem size is the distance from an EcoRI site on the ancestral hTn903 molecule and the site of the Tn903 insertion plus the distance of 770 taken as 1130 base-pairs or 0.025 h units. The EcoRI site at 0.806 was chosen for hdv214, hdvl50 and hdvl51 and that at 0.931 for Xdvl.53.

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digested with EcoRI and separated by agarose gel electrophoresis. Table 8 gives the sizes of the fragments produced for each of the four plasmids. For Xdvl50, Xdvl51 and Xdv214, the marker rescue tests indicate that only one EcoRI site at 0.806 is present (see Table 4, compare with Fig. 1). However, based on the appearance of two fragments, each of these three plasmids must have two sites. Thus, the EcoRI site is duplicated. For hdv153, three fragments are found. But based on the marker rescue tests, there are only two EcoRI sites and only two fragments expected. Hence, at, least, one of the EcoRI sites is duplicated. Based on the intensity of the bands in the agarose gel, it appears that two EcoRl sites in Xdv153 are duplicated, since the 0.062 band (see Table 8) appeared in the gel with greater intensity than did the 0.096 band. We infer that the entire 0.062 fragment is duplicated in hdv153. The 0.062 fragment represents the region of DNA contained between the EcoRI sites at O-806 and 0.931 on the lambda map. (Note that Xpk21, the ancestral phage, contains the nin5 deletion, which removes a sequence equal to O-058 of lambda.) Each of the four Tn903 Xdv plasmids examined has an inverted duplication of lambda sequences that extends from the Tn903 insertion through genes 0 and P. 1 next asked whether or not the second novel joint that is found in the Tn903hdv plasmids is at a specific site. The data in Table 8 suggest that it is not. It can be seen that the EcoRI fragment that contains the second novel joint in /\dv214, Xdvl50 and hdvl51 are each of different molecular weight. If there is only a single novel joint in these EcoRI fragments, this difference in molecular weight necessarily means that each novel joint is at a different site. I also determined the position of the joint in hdvl50 using the same procedure as was used to characterize hdv214 (data not shown). It was found that the novel joint in hdvl50 is at 0.77/0*73 of the lambda co-ordinates,

TABLE 8 Molecular

size of EcoRI endonuclease digestion fragments four Tn903hdv plasmids Plasmids

Size of EcoRI digestion fragments (fraction of h DNA)

hdv214

0.37t 0,098

hdvl50

0.32 0.103t

hdvl51

0.37 0.05t

Xdv1.53

0.21 0.096t 0.062

Molecular sizes were determined and Methods). The larger fragments are given to indicate size class. i Contains the non-specific novel

of

by the agarose gel electrophoresis technique cannot br measured accurately by this method, joint.

(see Materials their weights

14

M.

SYVANEN

compared to the O-78/0*74 location for the second novel joint in hdv214. I cannot exclude the possibility that Xdv153 and Xdv214 have identical novel joints, given that the EcoRI fragments containing the novel joint are nearly the same size (see Table 8).

4. Discussion The major point of this report is that the transposable element, Tn903, when carried in the bacteriophage lambda chromosome causes a rather profound genetic rearrangement of the lambda chromosome. These rearranged chromosomes are detected as defective lambda plasmids. Defective lambda plasmids, as such, have been described and are called Xdv (Matsubara, 1975 ; Berg, 1974). The ATn903-induced hdv plasmids are distinguished from those described earIier in a number of ways. One of the more striking differences is the frequency with which ATn903 gives rise to hdv carriers, as compared to lambda itself. For example, Matsubara (1975) reports that hcI- will produce one Adv-carrying strain for every lo6 infected bacteria ; whereas hTn903 can give one to three hdv-Tn903-carrying strains for every lo4 infected bacteria (see Table 2). A second important difference is that the site of Tn903 insertion in the ancestral /\Tn903 determines a novel joint in the nucleotide rearrangement which gives rise to Tn903hdv. This is apparent from the genetic analysis, shown in Table 4, where in 65 independently isolated Tn903Xdv plasmids all have a novel joint adjacent to Tn903 in the respective ancestral Tn903. In addition, the physical structures of four different Tn903Xdv plasmids were determined and it was shown directly that, the Tn903 sequences in each of these four plasmids defined the novel joint. In contrast, Chow et al. (1974) have shown quite clearly from t,he comparison of the physical structures of over ten Xdv plasmids that none shared the same novel joint’. Of the four Tn903Xdv plasmids examined, all contained inverted duplications of lambda sequences of the form - -0 P - - Tn903 -- P 0 - -. The Tn903 sequences are not duplicated and the position of Tn903 with respect to genes 0 and P is the same in each of the four Tn903/\dv plasmids as they are in the ancestral lambda Tn903 (Table 7). Chow et al. (1974) have reported that hdv plasmids can have inverted duplications of lambda sequences. The appearance of inverted duplications of DNA in the Tn903hdv plasmids almost certainly arises by a different mechanism. This conclusion is based on two arguments. First, the Tn903Xdv plasmids contain a novel joint at a, specific sequence, namely Tn903. Second, the inverted duplications of lambda, sequences were seen in all four tested. On the other hand, the Xdv plasmids examined by Chow rt al. (1974) contained inverted repeat,s only rarely when the ancestral phagc was hnin’ ; more often when Xnin5 was used. hpk3 is nin+ and the Tn903hdv plasmids 150, 151 and 214 were derived from t,his phage. There is one important similarity between the inverted duplications in the Tn903Xdv plasmids and the earlier Xdv plasmids with inverted duplications. The duplication always extends through the origin of replication. Thus there is probably some feature in common in the formation of these two classes of hdv plasmids. Granted then that Tn903 induces the formation of these plasmids, we may next consider by what type of mechanism. I wish to consider two models; one involves a genetic recombination or a crossing-over event. and the second involves abnormal replication. I will discuss these models within the framework Chow et al. (1974) used

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15

to explain how the inverted duplications within the Xdv plasrnids they observed may have arisen. To summarize their model: early in infection, the circular bacteriophage lambda chromosome replicates in a bidirectional manner from the origin to give the theta intermediate (see top structure in Fig. 4). If an illegitimate recombination event occurs between the parental strands in front of the replication fork, this will cause the polymerase to change direction at this point and new synthesis will be made on the opposit*e strand. If this happens at each replication fork, the two progeny duplexes within the theta structure will be covalently linked into an inverted duplicated

-non

Formation of -spec,r,c ~---‘*‘- el joint

FIG. 4. Schemes by which Tn9OS converts X to Xdv plasmid. After infoct,ion, the circular lambda molecule replicates bidirectionally by theta form replication. The left-hand pathway indicates a crossing-over mechanism for the generation of both novel joints. The non-specific novel joint,, shown here to bc on opposite sides of the c1 gene, is made by the breakage of both the + strand and ~ strand in front of the replicating fork (indicated by the double-headed arrow) and the sealing together of the opposite strands. Continued replication past this point will complete the novel joint. The Tn903 (shown as +0~) sequences have been replicated. The T# sequence in one daughter strand proximal to ori recombines with a second 70 sequence on the other daughter strand distal to ori, by virtue of sequence homology. Doublestranded crossing-over is shown by the double-headed arrow. The non-specific novel joint in thP abnormal replication scheme (the right-hand pathway) simply has the polymerase losing ant parental strand and gaining the second. The daughter strand that results from this error will necessarily be contained in an inverted duplication, because polvmerases replicatr from 5’ to 3’. Det,ails of this model for abnormal replication at. Tn903 at-e givenln Fig. 5.

16

hf.

SYVANEN

structure and will be deleted from the remainder of the parental molecule. Alternatively, the polymerase may simply err and copy the opposite strand to give the same result. The unique sequences at the t.erminus of the duplication are produced because the illegimate recombination event or aberrant replication does not necessarily occur across homologous sequences of the parental strands. Either of these events occurs randomly near the replication forks. This is basically the model proposed by Chow et al. (1974) and I will use it to explain how the non-specific novel joint in the Tn903hdv plasmids are generated. These are shown in Figure 4. Now I would like to consider how Tn903 specifically causes its rearrangement. The simplest explanation is that homologous recombination occurs between the two 70 as shown in sequences on the progeny duplexes, subsequent to their replication, Figure 4. The 70 sequences proximal to ori on one progeny recombine with the 70 sequence distal to ori on the second progeny. This will give an inverted duplication of the lambda sequences about Tn903. At present, there is no evidence in support of such a crossing-over hypothesis. For example, if homologous recombinat’ion at the 76 sequences was contributing t’o Tn903/\dv formation, then elimination of the lambda and the host general recombination systems should cause a substantial decrease in the frequency of their appearance. However, the data in Table 3 show that’ this is not so. In addition, hdv214 was formed under the recombinat,ion-deficient conditions: and its structure is basically the same as the others. The possibility remains that the special features of Tn903 that allow transposition under recombination-deficient conditions can lead to recombination through the 70 sequences. Now let us consider abnormal replication as the mechanism by which the inverted duplications about Tn903 may arise. Figure 5 outlines the scheme. Normally, when the replication fork reaches the edge of Tn903, it will proceed through the region and give a faithful replica of the parental molecule. Occasionally replication stops. We can attribute this to a specific sequence at 77with, perhaps, associated proteins. At this point, the replication complex recognizes 7 at the opposite side of Tn903, is physically

(a)

(b)

(cl

FIG. 5. The model for how abnormal replication causes the inverted duplication of lambda sequences about Tn903. This is an enlargement of the replicating fork shown in Fig. 4. The migrating fork pauses at the 70 sequences in the Tn903 stem, proximal to ori (a). The replication complex then loses this site and invades the duplex at the other 7 sequence distal to ori (b). If replication continues in the direction from 7 to 8, closure of the two daughter strands will be complete (c). This model requires breaking each parental strand. Possible cutting sites are indicated 0. Repair of the in (b) as -O--and the free ends produced are shown in (c) as ____ structure in (c) can be achieved with polymernsc and ligase.

Tit903

INDUCES

INVERTED

DUPLICATIONS

IN

h

17

transferred to that site, and replicates from 7 to B with the same polarity it had before leaving the original 7 site. After replication of the entire Tn903 region, but in the opposite direction from the original fork movement, covalent closure of the two progeny is complete. The two progeny would necessarily be covalently attached but in an inverted orientation. The second non-specific novel joint is formed by an unrelated mechanism as outlined above. There is no known enzymology of the DKA polymerases and replication complexes to support such a replication fork transfer mechanism. However, it is the simplest mechanism involving abnormal replication. Therefore, it is a possibility that should be considered in the absence of clear evidence in support of genetic crossing-over through the 70 sequences. I would like to point out that if the 70 sequences can cause replication fork transfer, it would be easy to use this property to develop a model to expIain how the transposition process itself mav occur. I thank J. Geisselsoder, J. Hopkins and T. Silhavy for careful reading of early drafts of tliis manuscript. This work was supported by a grant from the National Institutes of Health (no. 5ROlCA18217). REFERENCES Armstrong, K., Hershfield, D. & Helinski, D. (1977). Science, 196, 172-174. Berg, D. (1974). Virology, 62, 224-333. Berg, D. (1977). DXA Insertion Elements, Plasmids and Episomes (Bukhari, Shapiro & Adhya, eds), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Berg, D., Davies, J., Allet, B. & Rochaix, J. D. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 3628-3633. Berg, D. E., Jorgensen, R. & Davies, J. (1978). Microbiology-1978, pp. 13-15, American Societ,y for Microbiology, Washingt,on, D.C. Rotstein, D. & Kleckner, N. (1977). DNA Insertion Elements, Plasmids and Episomes (Bukhari, Shapiro & Adhya, eds), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Chow, L., Davidson, N. & Berg, D. (1974). J. Mol. Biol. 86, 69-89. Cohen, S. N. (1977). DNA Insertion Elements, Plasmids and Episomes (Bukhari, Shapiro & Adhya, eds), p. 672, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Davis, R., Simon, M. & Davidson, N. (1971). Methods Enzymol. 21, 413-418. Feunt’cun, J., Sompayrac, L., Fluck, M. & Benjamin, T. (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 4169-4173. Gott,esman, M. & Rosner, .J. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 5041-5045. Greer, H. (1975). Ph.D. thesis, Massachusetts Inst.itute of Technology, Cambridge, Mass. Kleckner, N., Chan, R. K., Tye, B. K. & Botstein, D. (1975). J. Mol. Biol. 97, 561. Kleckner, N., Reichardt, K. & Botstein, D. (1979). J. Mol. Biol. 127, 89-115. Matsubara, K. (1975). J. Mol. Biol. 102, 427-439. Matsubara, K. & Kaiser, A. (1968). Cold Spring Harbor Symp. f&ant. Biol. 33, 269-775. Nomura, N., Yomagish, H. & Okn, -4. (1978). Gene, 3, 39-51. Ohtsubo, H. & Ohtsubo, E. (1977) DNA Insertion Elements, Plasmids and Episomes (Bukhari, Shapiro & Adhya, eds), Cold Spring Harbor, New York. Sharp, P., Hsu, M. T., Ohtsubo, E. & Davidson, N. (1972). J. Mol. Biol. 71, 471-497. Sharp, P., Sugden, B. & Sambrook, S. (1973). Biochemistry, 12, 3055-3063. Young, R., Way, J., Yin, J., Way, S. & Syvanen, M. (1979). J. Mol. Biol. 132, 307-322.