Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6240-6244, December 1979
Biochemistry
Cell-cycle-associated rearrangement of inverted repeat DNA sequences (Caulobacter crescentus/chromosomal rearrangement/translocation/differentiation)
PERRY NISEN, RUSSELL MEDFORD, JAMES MANSOUR, MARY PURUCKER, ANN SKALKA*, AND LUCILLE SHAPIROt Departments of Molecular Biology and Cell Biology, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461; and the *Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110
Communicated by Harry Eagle, September 17, 1979
ABSTRACT Inverted repeat DNA sequences of Caulobacter crescentus have been isolated, characterized, and cloned in a bacteriophage X vector. Both whole populations and individual clones of these sequences were hybridized to restriction endonuclease-generated fragments of chromosomal DNA isolated from cells that were in different stages of the cell cycle. Some inverted repeat DNA sequences were observed to hybridize to different regions of the chromosomal DNA isolated from the morphologically and biochemically distinct swarmer cell and stalked cell populations. These results suggest that the inverted repeat sequences have the capacity to rearrange and thus be located at different sites on thie genomes of the different cell types.
that IR DNA sequences would be a logical probe to use in the analysis of nucleotide sequence continuity during a prokaryotic cell cycle. Caulobacter crescentus is a logical test organism because the cell cycle includes two biochemically and structurally distinct cell types that are easily separable: stalked cells and swarmer cells (15, 16). In addition, at least 3% of the C. crescentus genome is composed of IR DNA sequences (17), which are of two heterogeneous size classes, 100-600 base pairs and 1500-3000 base pairs. Sequence homology between insertion sequences of Escherichia coli (IS1, IS2, and IS5) and the IR sequences from C. crescentus could not be detected although regions of homology were observed between the IS elements and several of the 19 genomes tested (18). Homologies between IR DNA sequences from C. crescentus and 11 bacterial chromosomes were not detected. However, IR DNA sequences from two different strains of C. crescentus were homologous. We show here that an individual clone of a C. crescentus IR DNA sequence hybridized to different regions of the stalked and swarmer cell genome.
Chromosomal rearrangement as a means for the regulation of gene expression was proposed almost 30 years ago when McClintock (1, 2) described controlling elements in maize. More recently it has been proposed that rearrangement of DNA sequences coding for immunoglobulins plays a major role in antibody diversity and expression (3). In prokaryotes phase variation of flagellin antigens in Salmonella typhimurium is controlled by the inversion of a regulatory nucleotide sequence adjacent to a gene that codes for one of those flagellin antigens (4, 5). Similarly, inversion of the G-loop DNA sequence in bacteriophage Mu controls phage infectivity (6). Additional translocatable DNA elements have also been described, although no function has been assigned to these sequences. These include classes of nontandem repeated DNA sequences in yeast (7) and Drosophila (8, 9). The inverted repeat (IR) DNA of Xenopus laets may be translocatable as well. This conclusion was based on the observation that foldback DNA (IR DNA sequences with adjacent nonself-complementary tails) reassociated with all of the single-copy complement of the entire genome (10). Prokaryotic insertion sequences (IS) and antibiotic resistance (Tn) elements have been shown to translocate from one genome to another. The IS elements can carry signals from the initiation or termination of transcription, but are otherwise phenotypically cryptic (for review, see ref. 11), whereas the Tn elements carry genes for resistance to one or more antibiotics (for review, see refs. 12 and 13). A property common to translocatable elements are repeated nucleotide sequences at the termini. The necessity of these sequences for translocation was demonstrated by deleting one of the repeats in the antibiotic resistance element Tn3, which precluded further translocation (14). The known translocatability of IR-terminated IS and Tn elements in prokaryotes (11, 12), combined with the possible translation of large IR sequences in Xenopus (10), suggested
MATERIALS AND METHODS Bacterial Strains and Growth Conditions. Cultures of C. crescentus CB13 were grown in minimal M2 glucose broth (19) at 30'C to late logarithmic phase. Stalked and swarmer cells were separated by Ludox density gradient centrifugation, as described by Evinger and Agabian (20). The separated swarmer cells were greater than 90% pure in all cases. The cell population we refer to here as stalked cells, obtained by the Ludox procedure, was a mixture of stalked predivisional cells and young stalked cells and contained less than 3% swarmer cells. Preparation and Labeling of DNA. Separated populations of stalked cells and swarmer cells were washed five times in 0.1 M Tris-HCl, pH 7.5/10 mM EDTA. The final cell pellets were then resuspended in 10 ml of the wash buffer. Lysozyme (10 mg/ml) was then added and the mixture was incubated at 0°C for 5 min. Sarkosyl was added to a final concentration of 0.2%, followed by 1 mg of autodigested Pronase per ml. The mixture was incubated at 37°C for 2 hr, then extracted with a mixture of redistilled phenol/chloroform, 1:1 (vol/vol), that had been saturated with 0.30 M NaCl/0.030 M sodium citrate. The aqueous phase was then dialyzed overnight against 40 mM NaCl/20 mM Na2EDTA/0.2% ethanol/10 mM Tris buffer, pH 7.5 (buffer A). Pancreatic RNase (50 ,g/ml, heated at 650C for 2 min) was added and the mixture was incubated for 30 min at 37°C. Pronase (100 ,ug/ml) was then added and incubation was continued for 1.5 hr at 370C. The DNA was extracted with phenol and dialyzed extensively against buffer A. The DNA
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.
Abbreviations: IR, inverted repeat; IS, insertion sequence. t To whom all correspondence should be addressed.
6240
Biochemistry:
Nisen et al.
preparations were then purified by density banding in CsCl gradients (21). 32P-Labeled DNA was prepared by the nielk translation procedure of Rigby et al. (22), with [32P]dCTP. 3H-Labeled DNA was prepared as described (23); cells were grown in the presence of [3H]deoxyadenosine. Isolation of IR DNA Sequences. 32P-Labeled stalked or swarmer cell DNA was extracted first with phenol and then with ether. The DNA was then precipitated with ethanol. The precipitated DNA was resuspended in 10 mM Tris, pH 8.0/1 mM Na2EDTA and purified by Bio-Gel P-60 chromatography. The purified DNA was denatured by boiling for 10 min and then quick-cooled in an ice bath. The denatured DNA, which still retained the double-stranded IR sequences, was then digested with S1 nuclease (125 units/ml) for 45 min at 450C in a buffer that contained 0.1 M sodium acetate (pH 4.5), 1 mM ZnSO4, and 10 ,g of denatured calf thymus DNA per ml (for 32P-labeled DNA only). For large-scale DNA preparations, 0.2 M NaCl and 2 mM ZnSO4 were added to the buffer to reduce single-strand nicking of double-stranded DNA. The rate and yield of SI nuclease digestion of denatured DNA, as well as a native DNA control, was determined by measuring trichloroacetic acid-insoluble radioactivity during the reaction. Exo VII digestions were as described (24). 32P-End-labeled IR DNA was prepared by the method of Van de Sande et al. (25). Cloning of C. crescentus Inverted Repeat DNA in a Bacteriophage XWES Vector. Following the procedures described by Maniatis et al. (26), IR DNA sequences were blunt-endligated to EcoRI linkers at 150C for 16 hr and then on ice for 2 days. After digestion with the restriction endonuclease EcoRI, the IR DNA carrying EcoRI linkers was ligated to the purified arms of XWES-B that had been generated by EcoRI cleavage. The ligated recombinant DNA was then packaged in vitro by the procedures described by Blattner et al. (27). The host used to propagate the recombinant XWES molecules was a recAstrain of E. coli ED8767 (28). In situ plaque hybridization with a labeled IR DNA probe (29) was used to detect clones containing IR DNA sequences. Phage and DNA of XWES-IR-1 and XWES.IR-2 were prepared as described (18). These experiments were performed according to the National Institutes of Health guidelines with an EK-2 vector under P-2 containment facilities. Gel Electrophoresis of Restriction Endonuclease-Generated DNA Fragments. Conditions for the use of various restriction endonucleases were as described in the following references: EcoRI (30), BamHI (31), HindIl (32), Msp 1 (33), Sau IIla (34), and Hpa II (35). To ensure that complete restriction endonuclease digestion had occurred, X DNA was always included with Caulobacter DNA in parallel experiments. Agarose gel electrophoresis (36) of the restriction digests was followed by Southern blotting (37) and hybridization to a 32P-labeled X DNA probe. 32P-End-labeled IR DNA was subjected to electrophoresis on 8% acrylamide (38) or 2% agarose gels. Materials. BamHI and EcoRI restriction endonucleases and Aspergillus orzae nuclease S1 were obtained from Miles Laboratories. Msp I and Sau IIIa were obtained from New England Biolabs. DNA polymerase I was obtained from Boehringer Mannheim. Pancreatic RNase A was purchased from Worthington, and Pronase grade A from Calbiochem. Exo VII was the kind gift of J. Chase. [32P]dCTP (>300 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) was obtained from New England Nuclear. Ludox LSU was obtained from DuPont Nemours. BA85 nitrocellulose filter paper sheets were from Schleicher and Schuell. Nitrocellulose filter circles were from Enzo.
Proc. Natl. Acad. Sci. USA 76 (1979)
6241
RESULTS Characterization and Cloning of IR DNA of C. crescentus. At least 3% of the denatured and quick-cooled C. crescentus chromosome was resistant to digestion by endonuclease Si under conditions where single-stranded DNA was digested to completion. This percentage of fast-reassociating DNA is consistent with the IR DNA population obtained by Wood et al. (17), who used hydroxylapatite chromatography and SI digestion of denatured and randomly sheared DNA. Upon denaturation and redigestion of the IR DNA thus obtained, less than 1% remained acid insoluble. This implies that 99% of the IR DNA structures contained Sl-sensitive, single-stranded hairpin structures and that the original Sl-resistant fraction was due to unimolecular reassociation. Digestion of denatured and quick-cooled C. crescentus chromosomal DNA with E. coli exonuclease VII, which specifically digests single-stranded DNA in both the 5' - 3' and 3' 5' directions, leaves 7.4% of the chromosome acid insoluble. This represents both the doublestrained IR DNA stem and the intervening single-stranded loops. Thus, at least 4.5% of the C. crescentus chromosome is contained between IR DNA sequences. IR DNA sequences end-labeled with 32p separated into two size classes on either an 8% acrylamide DNA sequencing gel (38) or a 2% agarose gel, one
Biochemistry
Cell-cycle-associated rearrangement of inverted repeat DNA sequences (Caulobacter crescentus/chromosomal rearrangement/translocation/differentiation)
PERRY NISEN, RUSSELL MEDFORD, JAMES MANSOUR, MARY PURUCKER, ANN SKALKA*, AND LUCILLE SHAPIROt Departments of Molecular Biology and Cell Biology, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461; and the *Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110
Communicated by Harry Eagle, September 17, 1979
ABSTRACT Inverted repeat DNA sequences of Caulobacter crescentus have been isolated, characterized, and cloned in a bacteriophage X vector. Both whole populations and individual clones of these sequences were hybridized to restriction endonuclease-generated fragments of chromosomal DNA isolated from cells that were in different stages of the cell cycle. Some inverted repeat DNA sequences were observed to hybridize to different regions of the chromosomal DNA isolated from the morphologically and biochemically distinct swarmer cell and stalked cell populations. These results suggest that the inverted repeat sequences have the capacity to rearrange and thus be located at different sites on thie genomes of the different cell types.
that IR DNA sequences would be a logical probe to use in the analysis of nucleotide sequence continuity during a prokaryotic cell cycle. Caulobacter crescentus is a logical test organism because the cell cycle includes two biochemically and structurally distinct cell types that are easily separable: stalked cells and swarmer cells (15, 16). In addition, at least 3% of the C. crescentus genome is composed of IR DNA sequences (17), which are of two heterogeneous size classes, 100-600 base pairs and 1500-3000 base pairs. Sequence homology between insertion sequences of Escherichia coli (IS1, IS2, and IS5) and the IR sequences from C. crescentus could not be detected although regions of homology were observed between the IS elements and several of the 19 genomes tested (18). Homologies between IR DNA sequences from C. crescentus and 11 bacterial chromosomes were not detected. However, IR DNA sequences from two different strains of C. crescentus were homologous. We show here that an individual clone of a C. crescentus IR DNA sequence hybridized to different regions of the stalked and swarmer cell genome.
Chromosomal rearrangement as a means for the regulation of gene expression was proposed almost 30 years ago when McClintock (1, 2) described controlling elements in maize. More recently it has been proposed that rearrangement of DNA sequences coding for immunoglobulins plays a major role in antibody diversity and expression (3). In prokaryotes phase variation of flagellin antigens in Salmonella typhimurium is controlled by the inversion of a regulatory nucleotide sequence adjacent to a gene that codes for one of those flagellin antigens (4, 5). Similarly, inversion of the G-loop DNA sequence in bacteriophage Mu controls phage infectivity (6). Additional translocatable DNA elements have also been described, although no function has been assigned to these sequences. These include classes of nontandem repeated DNA sequences in yeast (7) and Drosophila (8, 9). The inverted repeat (IR) DNA of Xenopus laets may be translocatable as well. This conclusion was based on the observation that foldback DNA (IR DNA sequences with adjacent nonself-complementary tails) reassociated with all of the single-copy complement of the entire genome (10). Prokaryotic insertion sequences (IS) and antibiotic resistance (Tn) elements have been shown to translocate from one genome to another. The IS elements can carry signals from the initiation or termination of transcription, but are otherwise phenotypically cryptic (for review, see ref. 11), whereas the Tn elements carry genes for resistance to one or more antibiotics (for review, see refs. 12 and 13). A property common to translocatable elements are repeated nucleotide sequences at the termini. The necessity of these sequences for translocation was demonstrated by deleting one of the repeats in the antibiotic resistance element Tn3, which precluded further translocation (14). The known translocatability of IR-terminated IS and Tn elements in prokaryotes (11, 12), combined with the possible translation of large IR sequences in Xenopus (10), suggested
MATERIALS AND METHODS Bacterial Strains and Growth Conditions. Cultures of C. crescentus CB13 were grown in minimal M2 glucose broth (19) at 30'C to late logarithmic phase. Stalked and swarmer cells were separated by Ludox density gradient centrifugation, as described by Evinger and Agabian (20). The separated swarmer cells were greater than 90% pure in all cases. The cell population we refer to here as stalked cells, obtained by the Ludox procedure, was a mixture of stalked predivisional cells and young stalked cells and contained less than 3% swarmer cells. Preparation and Labeling of DNA. Separated populations of stalked cells and swarmer cells were washed five times in 0.1 M Tris-HCl, pH 7.5/10 mM EDTA. The final cell pellets were then resuspended in 10 ml of the wash buffer. Lysozyme (10 mg/ml) was then added and the mixture was incubated at 0°C for 5 min. Sarkosyl was added to a final concentration of 0.2%, followed by 1 mg of autodigested Pronase per ml. The mixture was incubated at 37°C for 2 hr, then extracted with a mixture of redistilled phenol/chloroform, 1:1 (vol/vol), that had been saturated with 0.30 M NaCl/0.030 M sodium citrate. The aqueous phase was then dialyzed overnight against 40 mM NaCl/20 mM Na2EDTA/0.2% ethanol/10 mM Tris buffer, pH 7.5 (buffer A). Pancreatic RNase (50 ,g/ml, heated at 650C for 2 min) was added and the mixture was incubated for 30 min at 37°C. Pronase (100 ,ug/ml) was then added and incubation was continued for 1.5 hr at 370C. The DNA was extracted with phenol and dialyzed extensively against buffer A. The DNA
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.
Abbreviations: IR, inverted repeat; IS, insertion sequence. t To whom all correspondence should be addressed.
6240
Biochemistry:
Nisen et al.
preparations were then purified by density banding in CsCl gradients (21). 32P-Labeled DNA was prepared by the nielk translation procedure of Rigby et al. (22), with [32P]dCTP. 3H-Labeled DNA was prepared as described (23); cells were grown in the presence of [3H]deoxyadenosine. Isolation of IR DNA Sequences. 32P-Labeled stalked or swarmer cell DNA was extracted first with phenol and then with ether. The DNA was then precipitated with ethanol. The precipitated DNA was resuspended in 10 mM Tris, pH 8.0/1 mM Na2EDTA and purified by Bio-Gel P-60 chromatography. The purified DNA was denatured by boiling for 10 min and then quick-cooled in an ice bath. The denatured DNA, which still retained the double-stranded IR sequences, was then digested with S1 nuclease (125 units/ml) for 45 min at 450C in a buffer that contained 0.1 M sodium acetate (pH 4.5), 1 mM ZnSO4, and 10 ,g of denatured calf thymus DNA per ml (for 32P-labeled DNA only). For large-scale DNA preparations, 0.2 M NaCl and 2 mM ZnSO4 were added to the buffer to reduce single-strand nicking of double-stranded DNA. The rate and yield of SI nuclease digestion of denatured DNA, as well as a native DNA control, was determined by measuring trichloroacetic acid-insoluble radioactivity during the reaction. Exo VII digestions were as described (24). 32P-End-labeled IR DNA was prepared by the method of Van de Sande et al. (25). Cloning of C. crescentus Inverted Repeat DNA in a Bacteriophage XWES Vector. Following the procedures described by Maniatis et al. (26), IR DNA sequences were blunt-endligated to EcoRI linkers at 150C for 16 hr and then on ice for 2 days. After digestion with the restriction endonuclease EcoRI, the IR DNA carrying EcoRI linkers was ligated to the purified arms of XWES-B that had been generated by EcoRI cleavage. The ligated recombinant DNA was then packaged in vitro by the procedures described by Blattner et al. (27). The host used to propagate the recombinant XWES molecules was a recAstrain of E. coli ED8767 (28). In situ plaque hybridization with a labeled IR DNA probe (29) was used to detect clones containing IR DNA sequences. Phage and DNA of XWES-IR-1 and XWES.IR-2 were prepared as described (18). These experiments were performed according to the National Institutes of Health guidelines with an EK-2 vector under P-2 containment facilities. Gel Electrophoresis of Restriction Endonuclease-Generated DNA Fragments. Conditions for the use of various restriction endonucleases were as described in the following references: EcoRI (30), BamHI (31), HindIl (32), Msp 1 (33), Sau IIla (34), and Hpa II (35). To ensure that complete restriction endonuclease digestion had occurred, X DNA was always included with Caulobacter DNA in parallel experiments. Agarose gel electrophoresis (36) of the restriction digests was followed by Southern blotting (37) and hybridization to a 32P-labeled X DNA probe. 32P-End-labeled IR DNA was subjected to electrophoresis on 8% acrylamide (38) or 2% agarose gels. Materials. BamHI and EcoRI restriction endonucleases and Aspergillus orzae nuclease S1 were obtained from Miles Laboratories. Msp I and Sau IIIa were obtained from New England Biolabs. DNA polymerase I was obtained from Boehringer Mannheim. Pancreatic RNase A was purchased from Worthington, and Pronase grade A from Calbiochem. Exo VII was the kind gift of J. Chase. [32P]dCTP (>300 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) was obtained from New England Nuclear. Ludox LSU was obtained from DuPont Nemours. BA85 nitrocellulose filter paper sheets were from Schleicher and Schuell. Nitrocellulose filter circles were from Enzo.
Proc. Natl. Acad. Sci. USA 76 (1979)
6241
RESULTS Characterization and Cloning of IR DNA of C. crescentus. At least 3% of the denatured and quick-cooled C. crescentus chromosome was resistant to digestion by endonuclease Si under conditions where single-stranded DNA was digested to completion. This percentage of fast-reassociating DNA is consistent with the IR DNA population obtained by Wood et al. (17), who used hydroxylapatite chromatography and SI digestion of denatured and randomly sheared DNA. Upon denaturation and redigestion of the IR DNA thus obtained, less than 1% remained acid insoluble. This implies that 99% of the IR DNA structures contained Sl-sensitive, single-stranded hairpin structures and that the original Sl-resistant fraction was due to unimolecular reassociation. Digestion of denatured and quick-cooled C. crescentus chromosomal DNA with E. coli exonuclease VII, which specifically digests single-stranded DNA in both the 5' - 3' and 3' 5' directions, leaves 7.4% of the chromosome acid insoluble. This represents both the doublestrained IR DNA stem and the intervening single-stranded loops. Thus, at least 4.5% of the C. crescentus chromosome is contained between IR DNA sequences. IR DNA sequences end-labeled with 32p separated into two size classes on either an 8% acrylamide DNA sequencing gel (38) or a 2% agarose gel, one
