ABCA B CAB. D ft a ..o a. FIG. 2. FIGEofSmaI (part 1), BamHI (part 2), and BglII (part 3) restriction enzyme digests of strain A13 DNA. Lane A shows an ethidium ...
JOURNAL
OF BACTERIOLOGY, Apr. 1989, p. 1898-1903 0021-9193/89/041898-06$02.00/0 Copyright © 1989, American Society for Microbiology
Vol. 171, No. 4
Amplification of DNA at a Prophage Attachment Site in Haemophilus influenzae LESZEK KAUCt AND SOL H. GOODGAL*
Department
of Microbiology,
University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076 Received 26 July 1988/Accepted 29 December 1988
The Escherichia coli plasmids pBR322 and pBR327 can be taken up by Haemophilus influenzae but do not replicate in this organism; however, integration of pBR into the H. influenzae chromosome was achieved by ligation to a fragment of the Haemophilus phage S2 that carried a phage attachment site (attP). Once these sequences were integrated, they could serve as sites of recombination and amplification for homologous (pBR or phage) DNA. Amplification appeared to occur in one of two prophage sites (attB) present in the H. influenzae chromosome. The extent of amplification was different in different cells and reflected the ability of these sequences to undergo rearrangement leading to the formation of a DNA ladder. The ladder was obtained by treatment of DNA with restriction enzymes that cut outside of the inserted DNA, i.e., did not cut in the repeat sequence, and represented different numbers of repeat elements. Reversed-field gel electrophoresis was instrumental in resolving amplified structures. Inasmuch as single-cell isolates gave rise to the same ladder structure, it was assumed that amplification was under regulatory control and that it reproduced the same equilibrium of repeat structures. Transformation of E. coli with the amplified H. influenzae DNA resulted in precise excision and replication of the original monomeric plasmids. This excision was independent of the recA and recBC genes. Gene amplification has been reported for many procaryotic systems (1, 14, 18, 25, 32-34, 38) and is well recognized for both chromosomal and extrachromosomal elements. Amplification in all systems studied involves DNA sequences flanked by repeat elements. In the procaryotic systems reported, the repeat elements are direct repeats (14, 17, 25, 37, 38); however, inverted repeats have been shown to play a role in amplification in a eucaryotic system (24). Several models for the mechanism of amplification have been proposed. The most prominent of these involve unequal recombination between homologous elements (2), excision and reintegration of DNA (27, 39), or replication of DNA to yield a multicopy repeat structure followed by recombination (40). Plasmids containing a selectable marker can integrate into the host chromosome even though the plasmid is unable to replicate in the recipient (10, 13, 15, 23, 36). The presence of sequences on the plasmid homologous to recipient DNA is necessary and responsible for integration and can lead to gene amplification in the chromosome when the inserted sequence is flanked by direct repeats (14, 17, 25, 38). In most cases of gene amplification in procaryotes, selection pressure is required to maintain the amplified structure (17, 28, 29, 38); however, stable amplification has been observed in the absence of selection (18). Standard gel electrophoresis is usually insufficient to resolve amplified DNA sequences larger than 30 kilobase pairs (kbp). Pulsed gel electrophoresis, on the other hand, is a powerful tool to separate amplified DNA sequences (5), and it has been possible to utilize field inversion gel electrophoresis (FIGE) (7) to study the structure and properties of amplified DNA. In this report we describe the integration and stable amplification of a hybrid pBR327 plasmid DNA that contains a cloned fragment of Haemophilus influenzae phage S2 DNA. A surprising feature of this amplified inte-
grate is the observation that excised amplified sequences consisted of a family of direct repeats that, upon FIGE,
formed the rungs of a DNA ladder. (A preliminary report of this work was presented at the 30th Annual Wind River Conference on Genetic Exchange, Estes Park, Colo., 8 through 13 June, 1986.) MATERIALS AND METHODS Bacterial strains. Escherichia coli HB101 (4) and C600 (3) have been described previously. H. influenzae Rd3O has also been described (11). H. influenzae A13 and A13N are
described in this paper. Plasmids. The cloning of the BglII fragments of phage S2 into pBR327 has been described previously (31). We used plasmid pKS6, which carries an attP site in the phage portion of its DNA (10-kbp BglII fragment A, Fig. 1A). Plasmid PiBR was constructed by cloning the small (885-bp) plasmid PiAN7 (30) into the PstI site of pBR327 (26). The PiBR vector contains a unique BglII site and has a selectable marker for tetracycline resistance. Plasmid pNBR was constructed by cloning a 10-bp NotI polylinker (New England BioLabs) into the PstI site of pBR327. This plasmid carries a tetracycline resistance marker. Transformation. H. influenzae cells were made competent by either the M IV procedure (16) or the aerobic-anaerobic method (12). Competent cultures were transformed by a standard assay procedure (22). E. coli transformations were performed by the Mandel and Higa procedure (20). Unless otherwise noted, selection for ampicillin resistance was at 50 ,g/ml and selection for tetracycline was at 10 ,ug/ml. DNA isolation and digestion with restriction enzymes. The preparation of phage DNAs and H. influenzae chromosomal DNA embedded in agarose beads has been described elsewhere (L. Kauc, M. Mitchell, and S. H. Goodgal, J. Bacteriol., in press). Plasmid DNA was isolated by the method of Clewell (9). Restriction enzymes were obtained from New England BioLabs, Pharmacia, Bethesda Research Laboratories, Inc., and the Institute of Microbiology of the University
* Corresponding author. t Permanent address: Institute of Microbiology, University of
Warsaw, Warsaw, Poland. 1898
AMPLIFICATION OF DNA IN H. INFLUENZAE
VOL. 171, 1989
A EBg 0
X 2
Bg
X H B 4
6
8
1
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3
2
ABCA B CAB
D
10
B ft a
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I
FIG. 2. FIGE of SmaI (part 1), BamHI (part 2), and BglII (part 3) restriction enzyme digests of strain A13 DNA. Lane A shows an ethidium bromide-stained gel. For lanes B, C, and D, the gel of lane A was transferred to nylon membranes and hybridized with the following 32P-labeled probes: B, phage fragment A; C, pBR327; D, the 1.5-kbp fragment of pYS between the HindIII and BamHI sites at 4 kbp (Fig. 1).
^X FIG. 1. Restriction map of (A) H. influenzae phage S2 BgllI fragment A and (B) plasmid pYS DNAs. Symbols: (I i) pBR327 and/or PiBR DNA, ( ) S2 phage DNA, (_) H. influenzae. The open bar (Ei ) in panel A indicates the position of phage att site according to reference 35. B, BamHI; Bg, BglII; E, EcoRI; H, HindIll; X, XbaI. The sizes of fragment A and plasmid pYS are given in kilobase pairs.
of Warsaw; the reaction conditions were those recommended by Bethesda Research Laboratories. The digestion of chromosomal DNA in agarose beads employed concentrations of restriction enzymes equivalent to those used for DNA in buffer solutions. Electrophoresis and hybridization. Normal gel electrophoresis and Southern blot hybridization were performed essentially as described by Maniatis et al. (21). FIGE was a modification of the technique of Carle et al. (7) and has been described previously in detail (Kauc et al., in press). Orthogonal field alternating gel electrophoresis (6) was performed with 1% agarose gel and fields of 10 V/cm with an 80-s pulse time at 8°C for 20 h.
RESULTS Construction and analysis of H. influenzae DNA containing pBlI and phage sequences. The BglII-digested phage S2 DNA fragments were cloned into the BamHI site of pBR327 (31). The resulting plasmid DNAs were used to transform competeht H. influenzae Rd30 to ampicillin resistance with selection at 5 jig of ampicillin per ml. Transformants (101/,xg of DNA) were obtained after transformation with pKS6 DNA, the plasmid that contains phage S2 BglII fragment A (Fig. 1A). Transformants were checked for the presence of extrachromosomal DNA; however, no free plasmid DNA was detected. One of the ampicillin-resistant transformants of H. iAfluenzae, designated A13, was used for further study. Although no free plasmid DNA was found in A13, plasmid
sequences were readily observed after restriction enzyme digestion of A13 followed by electrophoresis, Southern blotting, and hybridization to specific probes: pBR327 and the A fragment of phage S2 DNA (Fig. 2). Structure of amplified DNA in H. influenzae. Recent advances in the resolution of high-molecular-weight DNA (6-8) have made it possible to analyze the size and composition of the large BglII fragments of H. influenzae DNA. Since there were no BglII sites in the pKS6 plasmid used to transform H. influenzae, we expected that all DNA fragments appearing in the gel were a result of BglII recognition sites in the H. influenzae chromosome. The results of one such experiment are presented in Fig. 3 (lanes 5 and 5'). These results led to the observation that upon hybridization with the pBR or appropriate phage probe there was a graded series of DNA fragments forming an amplification ladder. Figure 4 presents a comparison of the BglII digestions of DNA isolated by the normal procedure for purifying DNA from solution or DNA from cells embedded in agarose beads. The largest size of the DNA isolated from solution was on the order of 50 kbp, whereas the DNA obtained from agarose beads and separated by FIGE yielded fragments that extended to hundreds of kilobases. Three different conditions used for running FIGE demonstrated that FIGE employing a time gradient was the best method for separating large fragments of H. influenzae DNA. In normal agarose gel electrophoresis the ladderlike structure could not be detected. To elucidate the nature of the ladder, a series of experiments was performed, and the results are summarized as follows. (i) Several strains of H. influenzae were compared, and only those containing pBR-S2 phage hybrids integrated into the chromosome produced a ladder. The hybridization data presented in Fig. 4 demonstrate that changing the FIGE programs resulted in an increase in the resolution of bands detected with appropriate probes. The BglII fragments of A13 DNA began at about 50 kbp and extended in 13-kbp intervals up to approximately 170 kbp (Fig. 2, part 2; Fig. 3). (ii) Treatment of A13 DNA with an enzyme that cut into the
KAUC AND GOODGAL
1900
1
2
3
4
J. BACTERIOL.
5 6
3 41 51 6
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40
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FIG. 3. FIGE of Rd (lanes 1 through 3) and A13 (lanes 4 through 6) DNA digested with ApaI (lanes 1 and 4), BgII (lanes 2 and 5), and SmaI (lanes 3 and 6) restriction enzymes. The gel was transferred to nylon membranes and hybridized with 32P-labeled pBR327 DNA probe (lanes 3' through 6'). The size of Apal fragments 5 through 14 are given in kilobase pairs. The arrows indicate SmaI fragments 7, 8, and 9.
plasmid or phage fragment A once gave rise to a hybridization band that corresponded to unit length of the plasmid. For example, BamHI-treated A13 DNA did not produce a ladder; instead, the treatment gave rise to a heavy band at about 14 kbp (10.2 kbp of A fragment plus 3.3 kbp of pBR) (Fig. 2, part 2). (iii) For eight isolated subclones tested, the DNAs digested with BglII gave the same banding patterns. Considering the number of bands in the ladder, it appeared that each cell had the potential to form a ladder (Fig. 5). (iv) Incubation of strain A13 in the absence or presence of ampicillin in different concentrations, up to 500 ,ug/ml, had no effect on the banding pattern (Fig. 5). Evidence that the amplified segment is located in a phage attachment site. BglII digestion of the H. influenzae chromo-
SS
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2
3
4
5
1'
2'
3'
4'
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FIG. 4. Comparison of different electrophoresis procedures used for the improved resolution of the DNA amplification ladder. Two different strains containing amplified DNA were digested with BgIII and run under the conditions noted below. Lanes: 1 through 5, ethidium bromide-stained gels; 1' through 5', gels 1 through 5 transferred to nylon membranes and hybridized with 32P-labeled pBR327 DNA; 1 and 1', regular forward electrophoresis; 2 and 2', pulse-gel electrophoresis for 3 s forward, 1 s in reverse at 7 V/cm overnight; 3 and 3', FIGE for 3 to 20 s forward, 1 to 6 s in reverse, 300 cycles at 7 V/cm overnight; 4 and 4', FIGE for 9 to 60 s forward, 3 to 20 s in reverse, 851 cycles at 7 V/cm overnight; 5 and 5', FIGE for 9 to 60 s forward, 0.01 to 3 s in reverse, 300 cycles at 10 V/cm overnight.
FIG. 5. Comparison of the amplification ladders from strain A13 DNAs isolated from a single colony, cultured in different concentrations of ampicillin. The DNAs were digested with ApaI and hybridized with 32P-labeled pBR327 DNA. Lanes: A, without ampicillin; B, 50 jig/ml; C, 200 jig/ml; D, 500 jig/ml.
some produced a large number of fragments that were difficult to resolve by regular agarose gel electrophoresis. However, digesting H. influenzae DNA with restriction enzymes SmaI and ApaI gave rise to a manageable number of fragments that could be resolved by FIGE (Fig. 2 through 4). Two of these fragments, Smal fragment 7 (ApaI fragment 11) and SmaI fragment 9 (ApaI fragment 12), contained S2 phage attachment sites (L. Kauc and S. H. Goodgal, submitted for publication). These attB sites contained regions of homology with the phage attP site. Therefore, a SmaI digest of integrated DNA is predicted to affect SmaI fragment 7 or 9 or both, depending upon the site of integration. The presence of the pKS6 plasmid DNA in the chromosome should increase the size of the DNA fragment by at least 13 kbp. SmaI and ApaI restriction enzyme digestions of DNA of H. influenzae Rd and A13 are shown in Fig. 2 and 3. Strain A13 lost SmaI fragments 7 and 9 (ApaI fragments 11 and 12, respectively). In addition, the A13 SmaI fragment 8 had an increased intensity, suggesting that the 90-kbp fragment 9 incorporated one copy of the plasmid to yield a new 103-kbp fragment (fragment 8 is 105 kbp). Similarly, A13 ApaI fragment 12 (51 kbp) gave rise to a new fragment (64 kbp), whereas ApaI fragment 11 was missing in the gel. The disappearance of SmaI fragment 7 (and ApaI fragment 11) suggested that the pKS6 plasmid DNA integrated into the bacterial chromosome. These observations were confirmed by hybridization to pBR (Fig. 2, lanes C; Fig. 3), phage S2 BglII fragment A (Fig. 2, lanes B), and chromosomal fragments with a bacterial attachment site (SmaI fragment 7, ApaI fragment 11) (Fig. 2, lane D). The hybridization revealed the presence of a ladder of DNA fragments. No other SmaI or ApaI fragments of the H. influenzae chromosome hybridized to the ladder fragments (data not presented), i.e., the ladder was a reflection-of amplified sequences. The striking feature of the ladder observed after hybridization of A13 was its regularity. The increase in apparent molecular weight of the fragments appeared to be on the order of 13
VOL. 171, 1989
kbp for most of the ladders, which was close to the sum of the pBR327 and phage S2 fragment A. Cloning of an amplified structure. One way to analyze the amplified DNA of strain A13 was to clone this material into an E. coli vector and determine its organization. Since there were no remaining BglII sites in the plasmids that were used to produce A13, it was possible to excise amplified DNA units with the BglII restriction enzyme. One of these structures was isolated from strain A13 DNA and cloned into the unique BglII site of the vector PiBR. A recombinant plasmid, pYS, was isolated, and a restriction site analysis was performed. The restriction map (Fig. 1B) was about 48 kbp and contained two tandem copies of pKS6 linked to about 12 kbp of the H. influenzae chromosomal DNA. A fragment of 1.5 kbp between a HindIII site and a BamHI site of the H. influenzae DNA was removed and used to prepare a 32p_ labeled probe. This probe was hybridized to SmaI DNA digests of wild-type strain Rd and strain A13. The results (Fig. 2, lane D) revealed that the 1.5-kbp fragment was homologous to the strain Rd SmaI fragment 7 and produced a ladder with digested A13 DNA. Introduction of a NotI site into the bacterial chromosome. Heterologous pBR sequences present in the H. influenzae A13 chromosome provide a region of homology for the integration of pBR-derivative plasmids. An example of integration and amplification of one such plasmid is presented in Fig. 6. Since the H. influenzae chromosome does not contain a NotI site (19; Kauc et al., in press), we introduced this restriction site into the bacterial chromosome of strain A13. Plasmid pNBR DNA (a 10-bp NotI polylinker cloned into the Pstl site of pBR327 DNA) was used to transform H. influenzae A13, and Ampr Tetr transformants were selected (0.5 x 104 to 1 x 104I/,g of DNA). One of the transformants was designated A13N. Digestion of its DNA with the Notl restriction enzyme followed by FIGE or orthogonal field alternating gel electrophoresis and hybridization with specific probes revealed four bands (Fig. 6). The two larger ones contained H. influenzae DNA, and the other two contained phage fragment A and pBR DNA. Undigested H. influenzae chromosomal DNA did not migrate and remained in the agarose beads (Fig. 6A, lanes 4 and 5). Excision of the integrated sequences in E. coli. Transformation of chromosomal A13 DNA into E. coli gave rise to ampicillin-resistant transformants (104I/,g of DNA). Plasmids extracted from these transformants corresponded to the original pKS6 plasmid used to transform H. influenzae Rd3O. Excision did not depend upon the E. coli recA or recBC gene, since recipients deficient in these functions also exhibited precise excision of the pKS6 plasmid from the amplified structure (data not presented). DISCUSSION The ColEl plasmids, such as pBR322 or pBR327, can be
taken up by competent cells of H. influenzae but do not replicate autonomously in this organism. Nevertheless, these plasmids can be ligated to fragments of the H. influenzae chromosome and subsequently bind to competent cells and integrate into the chromosome (13). We found that a similar phenomenon occurred after ligation of the plasmid pBR327 to a fragment of Haemophilus phage S2 (HPlcl) DNA that carries the phage attachment site (attP). Recombination presumably proceeds via the same Campbell-like mechanism as whole-phage integration, i.e., the integration of plasmid pKS6 in strain A13 occurs in both SmaI fragments that carry bacterial attB sites (SmaI fragments 7 and
1901
AMPLIFICATION OF DNA IN H. INFLUENZAE
A
B
1 2 3 4 5 1 2 3 4 5 6 W r -w -X WI
Is
a--
c
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-d ,3.1 FIG. 6. Orthogonal field alternating gel electrophoresis of H. influenzae A13N DNA (A) and FIGE (B) of H. influenzae Rd and A13N DNAs. (A) Lanes: 1, A13N DNA digested with NotI (the smallest fragment has run off the gel); 2, chromosomes of Saccharomyces cerevisiae AB1380; 3, lambda ladder; 4, undigested A13N DNA; 5, undigested A13 DNA. (B) A13N (lanes 1, 3, and 5) and Rd (lanes 2, 4, and 6) DNAs were digested with Notl (lane 5) and EagI (lane 6) restriction enzymes, respectively, transferred to nylon membranes, and hybridized with pBR327 (lanes 1 and 2) and phage S2 BgII fragment A (lanes 3 and 4) probes. The sizes of the EagI fragments are given in kilobase pairs. The four fragments of Notldigested A13N DNA are labeled a, b, c, and d. 9). These same sites are used by the whole-phage DNA (Kauc and Goodgal, submitted). Integration of the pKS6 plasmid into the bacterial chromosome in attB sites is followed by recombinational integration of additional pKS6 plasmids into the phage or pBR regions of homology, leading to amplification of the integrated structure. This hypothesis is supported by the analysis of plasmid pYS, which contains a cloned dimer of pSK6 DNA derived from an amplified DNA structure. Integration of a plasmid that contains a phage att site (pKS6) occurred readily. Although the efficiency of transformation was initially low, it could be significantly improved by electroporation with an increase in the efficiency of transformation of 2 to 3 orders of magnitude (K. Skowronek, L. Kauc, and S. Goodgal, submitted for publication). On the other hand, pBR327 plasmids with inserts of phage fragments C, D, B, and E that do not contain attP sites have not been found to integrate independently. Once pBR327 has integrated into the chromosome, its sequences can serve as a source of homology for the integration of other DNA into the bacterial chromosome. Strain A13N was derived by transformation of strain A13 with pNBR plasmid DNA (pBR327 containing a NotI site). A NotI restriction enzyme digest of its DNA yields two structures consisting of pBR327 and phage fragment A and
1902
KAUC AND GOODGAL
divides the rest of the chromosome into two uneven fragments. The smaller one (105 kbp) corresponds to the distance between SmaI fragments 7 and 9 (ApaI fragments 11 and 12, respectively), i.e., the distance between the two bacterial att sites into which the plasmid intesrated (Kauc and Goodgal, submitted). The top band of lane' 1 iq Fig. 6A represents the rest of the bacterial chron,psome and has a molecular size greater than 1,600 kbp (the size of the largest S. cerevisiae chromosome). These data are in agreement with previous observations that suggest a size of 1,900 to 2,000 kbp for the H. influenzae chromosome (19; Kauc et al., in press). The introduction of pB1k327 with a Notl linker appears to replace repeat units containing phage fragment A and also leads to a stable amplified structure. There is no evidence qf a ladder in strain A13N, suggesting that the phage fragment may contribute a sequence or function necessary for amplification.
The appearance of a series of amplified structures, ladder formatiop, was an interesting feature of strains containing amplified DNA, and FIGE was instrumental in resolving these structures. The amplified DNA was quite stable in cell populations in the presence or absence of ampicillin selection. The simplest interpretation of the ladders is that the population contains cells with different numbers of repeats that are produced by insertion or deletion of plasmid repeat units. An additional assumption is required to explain the same distribution of ladders from eight different single colony isolates: the ladder must be in a state of flux leading to equilibrium. In ethidium bromide-stained gels, after FIGE, the ladder was not apparent because only a small fraction of the amplified DNA was in each band; nonetheless, they were readily discerned by hybridization. Ladder rungs represent linear structures excised from the H. influenzae chromosomes by restriction enzymes that did not recognize sites within the amplified' DNA but only in the adjacent chromosomal DNA. The 13-kbp periodicity indicates that the pKS6 plasmid serves as the only repeat unit. The size of the fragment containing the phage attachment site affected the distribution of rung sizes in the ladder. The SmaI fragment 7 was 140 kbp, the ApaI fragment 11 was 57 kbp, and the BglII fragment was approximately 12 kbp (the latter was determined from the insertion of this fragment into the plasmid pYS; see above). The visible hybridization rungs extended from about 50 to 180 kbp for the BglII digests, from 80 to 170 kbp for the ApaI digests, and from 170 to 250 kbp for the SmaI digests. Although unlikely, the possibility has not been rigorously excluded that the ladder may be the consequence of a single amplified sequence that assumed different structural configurations that can be differentiated by FIGE. It is important to emphasize that DNAs from A13-as well as the other H. influenzae strains with amplified structures transformed into E. coli-precisely excised the plasmids originally used for amplification. Only monomeric plasmids were isolated. We assume that the regeneration of the original plasmid represents recombinational events in which an unknown number of repeats are deleted. ACKNOWLEDGMENTS We thank Thomas G. Larson for reading the manuscript and Marilyn Mitchell for technical assistance during this study. This work was supported by the Polish Academy of Sciences research project 09.7.2 and by Public Health Service grant GM24915 from the National Institutes of Health. LITERATURE CITED 1. Albertini, A. M., and A. Galizzi. 1985. Amplification of a chromosomal region in Bacillus subtilis. J. Bacteriol. 162:
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1203-1211. 2. Anderson, R. P., and J. R. Roth. 1977. Tandem genetic duplications in phage and bacteria. Annu. Rev. Microbiol. 31: 473-505. 3. Appleyard, R. K. 1954. Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12. Genetics 39:440 446. 4. Bolivar, F., and K. Backman. 1979. Plasmids of E. coli as cloning vectors. Methods Enzymol. 68:245-280. 5. Borst, P., A. M. Van Der Bliek, T. Van Der Yelde-Koerts, and E. Hes. 1987. Structure of amplified DNA, analyzed by pulsed field gel electrophoresis. J. Cell. Biochem. 34;247-258. 6. Cantor, C. R., C. L. Smith, and M. K. Mathew. 1988. Pulsedfield gel electrophoresis of very large DNA molecules. Annu. Rev. Biophys. Bjophys. Chem. 17:287-304. 7. Carle, G. F., M. Frank, and M. V. Olson. 1986. Electrophoretic separation of large DNA molecules by periodic inversion of the electric field. Science 232:65-68. 8. Carle, G. F., and M. V. Olson. 1904. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alteration gel electrophoresis. Nucleic Acids Res. 12:5647-5664. 9. Clewell, D. B. 1972. Nature of Col E1 plasmid replication in Eschericbia coli in the presence of chloramphenicol. J. Bacteriol. 110:667-676. 10. Ferrari, F. A., A. Nguyen, D. Lang, and J. A. Hoch. 1983. Construction and properties of an integrable plasmid for Bacillus subtilis. J. Bacteriol. 154:1513-1515. 11. Glover, S. W., and A. Piekarowicz. 1972. Host specificity of DNA in Haemophilus influenzae: restriction and modification in strain Rd. Biochem. Biophys. Res. Commun. 46:1610-1618. 12. Goodgal, S. H., and M. Herriott. 1961. Studies on transformations of Haemophilus influenzae. Competence. J. Gen. Physiol. 44:1201-1227. 13. Grundy, F. J., A. Plaut, and A. Wright. 1987. Haemophilus influenzae immunoglobulin Al protease genes: cloning by plasmid integration-excision, comparative analyses, and localization of secretion determinants. J. Bacteriol. 169:4442-4450. 14. Gutterson, N. I., and D. E. Koshland. 1983. Replacement and amplification of bacterial genes with sequences altered in vitro. Proc. 'atl. Acad. Sci. USA 80:4894-4898. 15. Haldenwang, W. G., C. D. Banner, J. F. Olington, R. Losick, J. A. Hoch, M. B. O'Connor, and A. L. Sonenshein. 1980. Mapping a cloned gene under sporulation control by insertio'n of a drug resistance marker into the Bacillus subtilis chromosome. J. Bacteriol. 142:90-98. 16. Hgrriott, R. M., E. M. Meyer, and M. Vogt. 1970. Defined non-growth media for stage II development of competence in Haemophilus influenzae. J. Bacteriol. 101:517-524. 17. Janniere, L., B. Niaudet, and S. D. Erlich. 1985. Repeated DNA sequences recombine 1,000 times more frequently in a plasmid than in the chromosome of Bacillus subtilis. Basic Life Sci. 30:93-103. 18. Janniere, L., B. Niaudet, E. Pierre, and S. D. Erlich. 1985. Stable gene amplification in the chromosome of Bacillus subtilis. Gene 40:47-55. 19. Lee, J. J., and H. 0. Smith. 1988. Sizing of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis. J. Bacteriol. 170:4402-4405. 20. Mandel, M., and A. Higa. 1970. Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53:159-162. 21. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 22. Michalka, J., and S. H. Goodgal. 1969. Genetic and physical map of the chromosome of Haemophilus inflaenzae. 45:407421. 23. Niaudet, B., and S. D. Erlich. 1979. In vitro genetic labeling of Bacillus subtilis cryptic plasmid pHV400. Plasmid 2:48-58. 24. Passananti, C., B. Davies, M. Ford, and M. Fried. 1987. Structure of an inverted duplication formed as a first step in a gene amplification event: implications for a model of gene amplification. EMBO J. 6:1697-1703. 25. Peterson, B. C., and R. H. Rownd. 1983. Homologous sequences
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33. Spies, T., R. Laufs, and F.-C. Riess. 1983. Amplification of resistance genes in Haemophilus influenzae plasmids. J. Bacteriol. 155:839-846. 34. Tlsty, T. D., A. M. Albertini, and J. H. Miller. 1984. Gene amplification in the lac region of E. coli. Cell 37:217-224. 35. Waldman, A. S., W. P. Fitzmaurice, and J. J. Scocca. 1986. Integration of the bacteriophage HP1c1 genome into the Haemophilus influenzae Rd chromosome in the lysogenic state. J. Bacteriol. 165:297-300. 36. Walter, R. B., and J. H. Stuy. 1982. Effect of linked-point mutations on additive transformation in Haemophilus influenzae, p. 179-185. In U. N. Streips et al. (ed.), Genetic exchange. Marcel Dekker, Inc., New York. 37. Young, M. 1983. The mechanism of insertion of a segment of heterologous DNA into the chromosome of Bacillus subtilis. J. Gen. Microbiol. 129:1497-1512. 38. Young, M. 1984. Gene amplification in Bacillus subtilis. J. Gen. Microbiol. 130:1613-1621. 39. Young, M. 1984. Gene amplification in Bacillus subtilis: the establishment of multiple tandemly-repeated copies of a heterologous DNA segment in the bacterial chromosome, p. 89-102. In A. T. Ganesan and J. A. Hoch (ed.), Genetics and biotechnology of bacilli. Academic Press, Inc., New York. 40. Young, M., and J. Cullum. 1987. A plausible mechanism for large-scale chromosomal DNA amplification in streptomycetes. FEBS Lett. 212:10-14.
