A general method for detecting rearrangements in a bacterial genome

Proc. Natl. Acad. Sci. USA Vol. 86, pp. 5507-5511, July 1989

Genetics

A general method for detecting rearrangements in a bacterial genome (two-dimensional electrophoresis of DNA fragments/in situ DNA renaturation in gel/bacteria latent phage system)

LO-CHUN Au* AND PAUL 0. P. Ts'ot Division of Biophysics, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205

Communicated by Ronald W. Estabrook, April 24, 1989 (received for review January 3, 1989)

tion, the DNA is electrophoresed in a second dimension perpendicular to the first dimension. DNA heteroduplexes that were digested by S1 nuclease are resolved as distinct spots below a bright unresolved band of homoduplex. Here, we introduce a further method that is facile and effective in monitoring unknown genome rearrangement in a bacterial system.

An effective method was developed to moniABSTRACT tor genome rearrangement in bacteria. The whole procedure consists of five steps. (i) Genomic DNAs of reference cells and test cells are digested with the same restriction enzyme. (it) The DNA restriction fragments from the test cells are radioactively labeled. (ui) The labeled DNA fragments of test cells are mixed with unlabeled DNA fragments from reference cells that are 100- to 1000-fold in excess and the mixture is electrophoresed in an agarose gel. (iv) After electrophoresis, DNA fragments are alkali-denatured; this is followed by renaturation in situ in the gel. The labeled rearranged DNA fragments from the test cells will renature much slower, as compared with the nonrearranged fragments, since in this location of the gel these rearranged fragments do not have a counterpart in the driver DNA, which is in excess. (v) The DNA gel is electrophoresed in a second dimension perpendicular to the first dimension after renaturation. The denatured rearranged DNAs are revealed after autoradiography, since single-stranded DNA fragments have higher electrophoretic mobility than double-stranded fragments of the same sizes. This process of detection has been demonstrated in this report by using Escherichia coli HB101 as the reference strain and E. coUl HB101 carrying A phage DNA (1:1 genomic ratio) as the test strain.

MATERIALS AND METHODS Bacterial Strains. Escherichia coli HB101 (recA, hsdRB, hsdMB) is a hybrid strain of E. coli K-12-E. coli B (11). E. coli HB101 (A), a lysogenic strain of HB101, was picked up from a single cell colony that originally came from a turbid plaque caused by the infection of HB101 with A phage. Extraction of High Molecular Weight DNA. Bacteria were cultivated in 100 ml of LB broth (1.2% Bacto-tryptone/0.8% NaCl/0.6% Bacto-yeast extract, pH 7.5) at 370C with shaking to a stationary phase and harvested. Cells were washed with 50 ml of lysozyme buffer (0.05 M Tris, pH 8/1% glucose/10 mM EDTA) at 4°C and were pelleted by centrifugation. The cell pellet was frozen and thawed once. Then the cells were resuspended in 7.5 ml of lysozyme buffer containing 2 mg of lysozyme per ml. The digestion was carried out at 4°C for 35 min. After the digestion, 2.5 ml of 4x cell lysis buffer (2% SDS/40 mM EDTA/0.2 M Tris, pH 8) containing 2.5 mg of proteinase K per ml was added to the cell suspension and was mixed thoroughly. A clear lysate was obtained after 80 min of incubation at 37°C. The cell lysate was extracted three times with an equal volume of phenol and twice with chloroform with gentle shaking. The aqueous phase of the mixture containing the DNA was dialyzed in 2x SSC (lx SSC = 0.15 M NaCl/15 mM sodium citrate) overnight at 4°C. One hundred micrograms of RNase A was added to the dialyzed DNA solution and the solution was incubated at 37°C for 1.5 hr. One-third volume of 4x cell lysis buffer containing 25 ,ug of proteinase K per ml was added to the incubated DNA solution. After an incubation at 37°C for 4 hr, the DNA solution was extracted again with phenol twice and with chloroform once. Two volumes of 95% alcohol at -20°C was then overlaid on DNA solution in aqueous phase. The DNA was spooled out by a glass rod and was redissolved through dialysis against TE buffer (5 mM Tris, pH 8.0/1 mM EDTA) at 4°C overnight. Restriction Enzyme Digestion. High molecular weight bacterial DNA obtained from the above section was digested with HindIII (2.5 units/,g of DNA) in reaction buffer (50 mM Tris, pH 8/50 mM NaCl/10 mM MgCl2) at 37°C overnight. After the digestion, 1/25th volume of 0.5 M EDTA at pH 8.0 and 1/50th volume of 1 M Tris (pH 8.4) were added. The

Genome rearrangement, which includes deletion, insertion, translocation, inversion, and amplification, is commonly seen in the microbial kingdom. This process can be separated into two categories (1), unprogramed rearrangement and programed rearrangement. The random insertion of transposable elements (2-5) and spontaneous gene amplification (6) are examples of unprogramed rearrangement observed in bacteria. In contrast to unprogramed rearrangement, the nature of programed rearrangement is stereotypic. For example, phase variation in Salmonella involves a gene inversion leading to the expression of different types of flagellar protein (7). Yeast mating type interconversion is another example of the switching between two states (8). To monitor unknown genome rearrangement in prokaryotic systems is a great technical challenge. Two methods have been published previously. In the first method (9), the genomic restriction fragments were first distributed by sizes by agarose gel electrophoresis. The DNA fragments of the same size were further separated due to different partial melting characters in formamide gradient during the seconddimension polyacrylamide gel electrophoresis. A twodimensional fingerprint is thus created. In the second method (10), genomic DNAs from two variant bacterial strains are digested with four-base-recognizing restriction enzymes, mixed together, denatured, renatured, and separated on polyacrylamide gel electrophoresis. Gel strips are cut out and soaked in a buffer containing S1 nuclease. After the diges-

Abbreviation: dATP[a-35S], deoxyadenosine 5'-[a-[35S]thio]triphos-

phate. *Present address: Department of Medical Research, Veterans General Hospital, Taipei, Taiwan, Republic of China. tTo whom reprint requests should be sent.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5507

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Proc. Natl. Acad. Sci. USA 86 (1989)

Genetics: Au and Ts'o

digested DNA solution was extracted with phenol once and chloroform once. One-ninth volume of 3 M sodium acetate (pH 7.0) was added to the extracted solution. The DNA fragments in the solution were precipitated by the addition of 2.5 volumes of 95% alcohol, and the solution was kept at -200C for 3 hr. The DNA precipitate was spun down, rinsed once with -20'C 70% ethanol and once with 95% ethanol, and redissolved in TE buffer. DNA Labeling. The DNA fragments of the test cells were labeled by two methods: (i) The DNA fragments were labeled at the 5' end with ['y-32P]ATP using T4 polynucleotide kinase. The reaction mixture (10 ,ul) contained 1 ,ug of DNA, 3.2 pmol of [-32P]ATP, 50 mM Tris (pH 7.4), 5 mM MgCl2, and 15 units of polynucleotide kinase. The reaction was carried out at 37C for 1 hr. (it) By replacement synthesis, the DNA fragments were labeled with deoxyadenosine 5'-[a-[35S]thio]triphosphate (dATP[a-35S]) using T4 DNA polymerase according to Roninson (12) with some modifications: the duration for exonuclease reaction was 8 min and the duration for resynthesis reaction was 40 min. The labeled DNA fragments were purified by column chromatography using an NENsorb 20 cartridge (NEN Research Products) according to the supplier's manual. Procedure for Detecting Genome Rearrangement. The DNA fragments from the reference cells (driver) were mixed with the labeled DNA fragments from the test cells (tracer) in a ratio of 100-1000:1 (driver vs. tracer DNA) and were loaded together onto a horizontal 14 x 11 x 0.48 cm slab gel consisting of 1% ultrapure agarose (Bethesda Research Laboratories). The sample well was 0.6 cm wide and 1 mm thick. Electrophoresis was performed in TPE buffer (0.08 M Tris phosphate/ 0.008 M EDTA, pH 7.7) at 25 V for 16 hr (or 40 V for 10.5 hr). The slab gel was cut to the size of 11 cm X 11 cm and was transferred into a tray. All of the following procedures were done with constant shaking of the tray at a speed of60-80 rpm. DNA in the gel was denatured by soaking the gel in 150 ml of denaturing buffer (0.5 M NaOH/0.6 M NaCl) at room temperature. After a 30-min incubation, the buffer was removed by aspiration. The same volume of fresh denaturing buffer was added to the tray, and incubation was continued for another 30 min. This alkaline buffer was then removed by aspiration. The gel was neutralized by washing with 100 ml of renaturation buffer, containing 50% formamide and 10x SSPE (lx SSPE = 10 mM sodium phosphate, pH 7/0.18 M NaCl/1 mM EDTA), four times for 20 min each time. Following neutralization, the gel was incubated in 100 ml of the renaturation buffer with shaking at 42-45°C for 4.5 hr. The denaturing and renaturing protocols are modified from the procedure of Roninson (12). After renaturation, the gel was washed with 130 ml of TPE buffer containing 1.4 mg of ethidium bromide per liter three times, each time for 20 min. The gel slab was rotated 90°. Electrophoresis was performed in TPE buffer containing 1 mg of ethidium bromide per liter at 20 V for 15-18 hr at 4°C. After electrophoresis, the gel was dried. The autoradiography was done in a cassette containing intensifying screens with Kodak Safety ARO film. The film was exposed at -80°C for 2-5 days before development. RESULTS In the first set of experiments, we investigated the effectiveness of the in situ renaturation of denatured DNA in the gel and the subsequent separation of the renatured (double-stranded) DNA from the denatured (single-stranded) DNA in a second electrophoresis perpendicular to the direction of the first electrophoresis. In this experiment, the tracer DNA (1 ng, 5 x 103 dpm) consisted of a mixture of A phage DNA fragments obtained from HindIII restriction enzyme digestion together with a 2.9-kilobase (kb) pGEM1 plasmid linearized by HindIII

digestion. The genomic ratio of the A phage DNA and the plasmid DNA was 1:1. The reference DNA (driver, 100 ng, nonradioactive) contained only A phage DNA fragments that were obtained also from a HindIII digestion. This mixture of the tracer DNA and driver DNA was electrophoresed, denatured, renatured, and electrophoresed again in the second dimension as described in Materials and Methods. The pattern so obtained after the second electrophoresis is shown in Fig. 1. The labeled A DNA fragments renatured almost completely in a 3-hr incubation in the gel, with the nonlabeled A DNA fiagments from the driver DNA, which were 100-fold in excess. However, the pGEM1 2.9-kb fragment in the tracer DNA remained denatured in the gel since this fragment did not have a counterpart in situ. Thus, this unique fragment renatured much more slowly compared with the ADNA fragments. In the second electrophoresis, the single-stranded (denatured) DNA exhibited a higher mobility than the double-stranded DNA of the same size. These two types of DNA can be separated as shown. This difference in mobility is enhanced when ethidium bromide is present in the electrophoresis buffer, which tends to bind to the native DNA more strongly, thereby retarding the mobility. For high molecular weight DNA fragments, renaturation in the gel apparently led to multistranded aggregates that exhibited a very low mobility. The results in Fig. 1 demonstrate the effectiveness of the two adopted fundamental procedures-i.e., the renaturation of denatured DNA in the gel and the clear separation of the denatured and native form of the DNA fragments that originally were the same size. kb

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FIG. 1. In situ renaturation of denatured DNA in the gel and separation of the single-stranded (denatured) DNA from the doublestranded (renatured) DNA. Driver DNA, 0.1 gg of A phage DNA digested by HindIII; tracer DNA, 1 ng of A phage DNA + pGEM1 DNA (a 2.9-kb plasmid DNA), both digested by HindIII in the genomic ratio of 1:1. The tracer DNA was end-labeled by [y-32P]ATP to a specific radioactivity of 5 x 106 dpm/ikg. The renaturation of the alkaline denatured DNA in the gel was carried out at 45°C for 3 hr. I, First dimension; --, second dimension.

Genetics: Au and Ts'o


In the second set of experiments, the detection and separation of the A phage DNA in a mixture of E. coli DNA [genomic size, 3.5 x 106 base pair (bp)] was demonstrated. In this set of experiments, the driver DNA (5.6 ,ug) consisted of E. coli HB101 DNA fragments (nonlabeled) obtained by HindIII digestion, and the tracer DNA (56 ng, labeled) consisted of a mixture (genomic ratio, 1:1) of DNAfragments from E. coli HB101 and A phage obtained by HindIII digestion. In the control, both samples contained E. coli HB101 and A DNA fragments from HindIII digestion. As shown in Fig. 2a, all of the A DNA fragments in the test DNA appeared clearly in the single-stranded DNA region, except for the 23-kb band, which was faint. The latter could be detected only by longer exposure of the x-ray film (data not shown). Some of the DNA may be trapped with the renatured bacterial DNA or renatured in an aggregated form. The background in the single-stranded DNA region represents the labeled denatured DNA in the bacterial DNA fragments that did not renature completely in the gel with the corresponding driver DNA. As for the control (Fig. 2b), most, if not all, of the labeled A DNA fragments renatured with the nonlabeled A DNA fragments in the driver DNA sample and therefore became undetectable. Again, only a very small amount ofunrenatured or denatured DNA showed up as a background smear in the single-stranded DNA region. The results in Fig. 2 show that with the constructed mixture containing the genomic ratio of 1:1 of A DNA and E. coli DNA, the presence of A DNA can be detected without using a genomic probe for A. In the final set of experiments, the lysogenic strain of HB101 (A) containing a single copy of A DNA in its genome was used as the source of tracer DNA and the HB101 strain was used as the source of the driver DNA. As described in Materials and Methods, HB101 (A) was selected from a single cell colony originating from a turbid plaque derived from an HB101 culture infected with A phage. The strain HB101 is a

a

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hybrid of E. coli B-E. coli K-12, and E. coli K-12 is a lysogenic strain carrying a A prophage. The HB101 (A) strain is shown to be resistant to the A phage as anticipated (13). The genomic DNA of HB101 and HB101 (A) were digested by HindIII. There was no apparent difference in restriction patterns between these two strains (Fig. 3). This indicates that the inserted A phage DNA cannot be seen by its restriction pattern after first-dimension gel electrophoresis. By utilizing the A DNA as probe in a Southern blot, the following information was obtained from the results shown in Fig. 4. Lane 1 shows all of the A DNA fragments obtained from the HindIII digestion (with yeast DNA as the background). Lane 2 shows the A DNA fragments in the HB101 (A) genomic DNA. As expected from the circularization of the A DNA through ligation of the cohesive ends in the early phase of infection, 4.4-kb and 23-kb bands are missing in the digests of HB101 (A) DNA with the emergence of a new 27-kb (23 kb + 4.4 kb) fragment in comparison with the A DNA fragments in lane 1. It is known that the A DNA and the E. coli genomic DNA are recombined specifically at att sites (15), and this integration site, att, is located close to one end of the 9.4-kb fragment from the HindIII digestion. Therefore, the 9.4-kb fragment is also missing in HB101 (A) digestion (lane 2) and two new fragments were found in this digestion. One fragment, expected to be larger than 9 kb, may be represented by the band overlapping with the 27-kb fragment, as indicated by the intensity of the band in lane 2 as compared to lane 1 and lane 3. In comparing HindIII digests of HB101 DNA (lane 3) and HB101 (A) DNA (lane 2), the absence of 2.0-, 2.3-, and 6.7-kb fragments (and possibly the 4.7-kb + 23-kb fragment) in the HB101 digests is clearly evident. In addition, since HB101 carries the phenotype of recA-, this property ensures 1

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M:. FIG. 2. Detection of A phage DNA fragments in the DNA mixture containing genomic DNA fragments of HB101. (a) Driver DNA, 5.6 of HB101 DNA digested by HindIII; tracer DNA, 56 ng of E. coli j.g HB101 DNA + A phage DNA, both digested by HindIll, in the genomic ratio of 1:1. (b) Driver DNA (5.6 ,ug) and tracer DNA (56 ng) were identical-i.e., E. coli HB101 DNA + A phage DNA, both digested by HindIll, in the genomic ratio of 1:1. The tracer DNA was labeled as described in the legend to Fig. 1. Renaturation of the alkaline denatured DNA was carried out at 42°C for 4.5 hr in the gel.

FIG. 3. Pattern of genomic DNA digested with HindIl. Lane 1, DNA size marker; lanes 2 and 3, 5.6 ,Ug of HindIlI-digested genomic DNA of HB101 (lane 2) and HB101 (A) (lane 3) were loaded onto a 1% agarose gel.

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Proc. Natl. Acad. Sci. USA 86 (1989) 3

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FIG. 4. Detection of A phage DNA in the Southern blot. Lane 1, 4.7 ng of A phage DNA digested by HindI11 mixed with 7 ,g of yeast DNA digested by HindIII; lane 2, 7 ,tg of HB101 (A) DNA digested by HindI1; lane 3, 7 ,ug of HB101 DNA digested by HindIll. The Southern blot was done according to the procedure given in ref. 14. The probe was A DNA digested by HindI11 and was labeled with dATP[a-35S] by T4 DNA polymerase to the specific radioactivity of 1 x 107 dpm/,g. One microgram of probe has been used in the hybridization.

that the prophage would not be spontaneously induced in HB101 (A) (16), and genomic ratio of HB101 andA is 1:1 in the DNA sample. Now we do have the proper genomic constructs of the two bacterial strains, HB101 and HB101 (A). The difference has been shown by the use of the A probe. The question now is whether this difference in the genomic DNA of a single copy between these two strains can be demonstrated without the use of the A probe. In this set of experiments, DNA from HB101 (A) was used as tracer (labeled) DNA, and DNA from HB101 was used as the driver DNA. The ratio of test DNA to driver DNA is 1:1000. As the control, DNA from HB101 (A) was used as both tracer and driver DNA. Fig. Sa illustrates that there are three bands with the sizes of 2.0-, 2.3-, and 6.7-kb fragments standing out over the smear background in the singlestranded DNA region. There is a suggestion of a faint band of 27 kb. These bands are clearly absent from the control experiments shown in Figure Sb. The results from Fig. 5 agree completely with those from Fig. 4, except that the data for Fig. 4 were obtained from the use of A DNA as the specific probe, whereas the results in Fig. 5 were obtained without the use of any probe.

DISCUSSION The effectiveness of two fundamental procedures in the general method for detecting rearrangement in genomic DNA

(A) in comparison with the strain HB101 as reference cells (a) Driver DNA, 5.6 gg of HB101 DNA digested by Hind~ll; tracer DNA, 5.6 ng of HB101 (A) DNA digested by Hind~lil (b) Driver DNA, 5.6 k~g of HB101 (A) DNA digested by HindlIIl tracer DNA, 5.6 ng of HB101 (A) DNA digested by Hind~ll. The tracer DNA was labeled with dATP[a_35S] by replacement synthesis using T4 DNA polymerase. The specific radioactivity was about 2 x 107 dpm/,tg. The renaturation of the alkaline denatured DNA was carried out at 450C for 4.5 hr in the gel. of bacteria has been clearly demonstrated in Results. The first procedure is the in situ DNA denaturation and renaturation in the gel after the first electrophoresis. Here we utilize the difference in DNA concentration as the crucial ratedetermining factor of the complementary DNA renaturation process (i.e., the factor of Cot). It is logical to assume that after genome rearrangement (e.g., insertion, deletion, translocation, inversion, or amplification), the DNA restriction fragments obtained from the rearranged regions by an appropriate restriction enzyme would have different sizes as compared to those from the unaltered genome. Thus, the altered DNA restriction fragments (previously unknown or nonexistent) from the rearranged genome of the test cells will most likely have a different electrophoretic mobility as compared to the fragments of the same sequence in the reference cells. If this situation is not observed by the use of one restriction enzyme, another restriction enzyme can be used to generate another set of restriction fragments to show the difference in electrophoretic mobility. Finally, an appropriate restriction enzyme can be found that would generate two different sets of restriction fragments from the test cells and from the reference cells. The second procedure is to separate this denatured (single-stranded) DNA from the renatured (double-stranded) DNA of the same fragment by electrophoresis, particularly in the presence of ethidium bromide. Compared to the two previous methods (9, 10) mentioned in the Introduction, our method shows the following advantages: (i) the result is clear and the detection is easier without doing two-dimensional mapping and searching; (ii) low copies of repetitive or homologous sequences will not interfere with the detection; and (iii) the detectable length of DNA is in the suitable range (2-20 kb). Although the broad application of this general method for monitoring unknown genomic changes is self-evident, a dis-

Genetics: Au and Ts'o cussion of the limitation of this approach is in order. In general, there are two main factors of limitation. The first limiting factor is the size and the complexity of the genome. Since the loading of the DNA in the gel is nearly saturated in the current experiment, the larger the genomic size, the less the copy number of genomic fragments that can be loaded. Moreover, higher complexity of the genome implies a greater variety of restriction fragments. In those cases, the rate of renaturation will decrease and the background in the singlestranded region will increase proportionally. To circumvent the problem, we need (i) to prolong the renaturation time and (ii) to use a higher ratio of tracer/driver DNA (e.g., 1:1000); thus, a genome size under 1 x 107 bp would not be perceived as problematic. For the bacterial genome, a ratio of tracer/ driver DNA of 1:100 (Figs. 1 and 2) and a ratio of tracer/ driver DNA of 1:1000 (Fig. 5) were used, and both were found to be applicable. The second limiting factor is the presence of repetitive or highly repetitive sequence of the same family in situ. Part of this problem can be reduced by adopting a more stringent condition for hybridization, but this more stringent procedure will increase the time needed for renaturation. The above two factors of limitation eliminate the application of this general procedure to monitor DNA rearrangement in the mammalian genome. A further procedure has been developed in our laboratory for this purpose. This current


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method, however, may also be applicable to the study of DNA rearrangement in yeast. 1. Simon, M. & Herskowitz, I. (1985) Genome Rearrangement (Liss, New York). 2. Berg, D. E. & Berg, C. M. (1983) Biotechnology 1, 417-435. 3. Morisato, D. & Kleckner, N. (1984) Cell 39, 181-190. 4. Harshey, R. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 273-278. 5. Roeder, G. S. & Fink, G. R. (1982) Proc. Natl. Acad. Sci. USA 79, 5621-5625. 6. Anderson, R. P. & Roth, J. R. (1977) Annu. Rev. Microbiol. 31, 473-504. 7. Silverman, M. & Simon, M. (1980) Cell 19, 845-854. 8. Kushner, P. J., Blair, L. C. & Herskowitz, I. (1979) Proc. Natl. Acad. Sci. USA 76, 5264-5268. 9. Fisher, S. G. & Lerman, L. S. (1979) Cell 16, 191-200. 10. Yee, T. & Inouye, M. (1984) Proc. Natl. Acad. Sci. USA 81, 2723-2727. 11. Boyer, H. W. & Roulland-Dussoix, D. (1969) J. Mol. Biol. 41, 459-472. 12. Roninson, I. B. (1983) Nucleic Acids Res. 11, 5413-5431. 13. Au, L.-C. (1988) Thesis (Johns Hopkins University, Baltimore, MD). 14. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold

Spring Harbor, NY). 15. Landy, A. & Ross, W. (1977) Science 197, 1147-1160. 16. Roberts, J. W., Roberts, C. W. & Craig, N. (1978) Proc. Natl. Acad. Sci. USA 75, 4714-4718.