Proc. Nat!. Acad. Sci. USA

Vol. 76, No. 5, pp. 2195-2198, May 1979 Biochemistry

Organization of a hybrid between phage fl and plasmid pSC 101 (DNA sequencing/insertion sequences/in vivo recombinant/in vitro protein synthesis)

JEFFREY V. RAVETCH, MARIKO OHSUMI, PETER MODEL, GERALD F. Vovis, DAVID FISCHHOFF, AND NORTON D. ZINDER The Rockefeller University, New York, New York 10021

Contributed by Norton D. Zinder, February 16, 1979

ABSTRACT We have characterized the 200-nucleotide-long insertion found in fl after segregation of a chimeric phage containing the genomes of fI and pSC1O1 [Ohsumi, M., Vovis, G. F. & Zinder, N. D. (1978) Virology 89,438-449]. The insertion in this novel fI species, called f 1', is derived from pSC1O1 and has the potential to form an extended base-paired secondary structure, as determined by nucleotide sequence analysis. A five-nucleotide direct repeat, derived from fI sequences, is present in f 1'. The 200 additional nucleotides that are inserted into the DNA sequence coding for the carboxy terminus of fI gene IV protein have generated a novel carboxy terminus for the f 1' gene IV protein. In vitro transcription-translation studies demonstrate that a read-through protein can be expressed, as predicted from the f ' nucleotide sequence results. This 200nucleotide-long sequence appears to be a transposable element found within pSCO1 and is similar in sequence to the inverted repeat found in Tn3. Restriction enzyme analysis of the chimeric phage DNA, coupled with the nucleotide sequencing results, allows us to predict a structure for the genomic organization of this chimera.

The unique properties of the filamentous, single-stranded DNA bacteriophage fl have recently been exploited to utilize it as a molecular cloning vector (1-5). Recently, we described (6) the isolation and characterization of an in vivo recombinant between bacteriophage fi and the tetracycline-resistanceconferring plasmid pSC101. The resulting chimeric phage, called VO-1, contains the genomes of fi and pSC101 in their entirety and is capable of transducing Escherichia coli to tetracycline resistance. However, the VO-1 molecule segregates 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.

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MATERIALS AND METHODS The bacterial strains have been described (6). VO-1 and f 1' replicative form I DNA were isolated and purified as described (6). Restriction endonucleases Hae III and Hinfl were prepared as described (9, 10). Taq I was purchased from Bethesda Research Laboratories (Rockville, MD). Preparation of restriction fragments for DNA sequence analysis has been described (10). DNA sequence analysis was performed by the base-specific modification method of Maxam and Gilbert (11). In vitro transcription and translation of replicative form I DNA was performed as described (12). Spreading for electron microscopy was performed by the method of Davis et al. (13).

X/G

Y/Z Z/X Taq Taq I

within E. coli to yield a molecule identical to pSC101 and a molecule slightly larger than fI called f 1'. f 1' contains 200 extra nucleotides located at the site where pSC101 was inserted into fi to form VO-1. In the present study we have investigated this novel fl species further by DNA sequence analysis and in vitro transcription-translation. The additional nucleotides in f ' are inserted into the DNA sequence coding for the carboxy terminus of gene IV protein. As a result, f 1' has a novel gene IV protein carboxy terminus. These extra nucleotides found in f ' have the potential to form an extended base-paired structure. The sequencing studies of f ' coupled with restriction enzyme analysis of VO-1 replicative form I suggest a structure for these chimeric molecules. Our results indicate that there is a transposable element in pSC101 whose terminal sequences are similar to the inverted repeat flanking the ampicillin-resistance-conferring gene of the Tn3 transposon (7, 8) and further define the requirements for a transposable element of DNA.

Intergenic space

pSC101

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73

FIG. 1. (Upper) Detailed physical map of the region of fi' containing the 200-nucleotide insertion. The sequencing experiments are shown by arrows, with * indicating the 5'-terminal label. The length of the arrow indicates that portion of the restriction fragment whose sequence was determined in each experiment. Arrows above the line denote sequences of the complementary strand (-); arrows below the line denote sequences of the viral strand (+). Repeated sequencing experiments of the same restriction fragment are not shown. (Lower) Corresponding genetic map of this region. 2195

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RESULTS Sequence of Insertion in f 1'. The experiments performed to determine the sequence of the insertion in f ' are diagrammed in Fig. 1. Hae III digestion of f 1' yielded two restriction fragments, called X and F', not found in fI digests; Taq digestion yielded four fragments (A', X, Y, and Z) not found in digests of fi (14). We determined the sequences of these novel restriction fragments. Comparison of these sequences to the known sequence of fi in this region (14) demonstrated that the inserted DNA in f 1' is not derived from fi sequences. Recent results (unpublished) indicate that this inserted sequence is found in pSC101. Fig. 2 presents the nucleotide sequence for the insertion in f 1' drawn into a potential secondary structure. Immediately preceding the transition from fi to pSC101 in f ' is the five-base nucleotide sequence T-A-G-T-A which is repeated at the point of transition from the pSC101-like sequences back to the fl-like sequences. Transposable elements induce a direct repeat in the target DNA. Tn3 induces a fivebase direct repeat (7), whereas insertion of IS1 into a host DNA induces a nine-base direct repeat (15, 16). The presence of this five-base repeat in f ' suggests that the extra DNA by which f 1' differs from fi may be a transposable element similar to Tn3. The transition from fI to pSC101 occurs in the region of the genome that codes for the carboxy terminus of gene IV protein (14). The predicted carboxy-terminal amino acids Arg-Ala-Leu in wild-type fI would be absent in f1' and would be replaced by Gly-Val. Thus, f ' would contain a novel gene IV protein one amino acid shorter than that of wild-type f 1. f 1', as well as VO-1, is viable without a helper virus but forms minute plaques when compared to wild-type fI plaques (6). Single-step growth curves of fi as compared to f 1' (unpublished data) demonstrated diminished growth properties for f1' phage. The molecular basis for these differences may be due to the novel gene IV protein predicted from the DNA sequence of f 1'. Based on the DNA sequence (see Fig. 2), the mRNA of f 1' is predicted to contain UGA as the chain-terminating codon for its gene IV; the wild-type fi uses UAG to terminate gene IV. The UGA codon is leaky in vitro (17) and thus could result in a read-through protein for gene IV in f 1', 11 amino acids longer and terminating at a UAA. Comparison of in vitro transcription-translation of replicative form I of f 1' to that of f 1, shown in Fig. 3, demonstrated that, along with the expected gene IV protein, a slightly larger species was present in f ' (lane c). Addition of tryptophan tRNA, which suppresses UGA termination (ref. 18; J. Atkins and R. Gesteland, personal communication), shifted the f ' gene IV product to the larger species (lane d). Similarly, when the chimeric VO-l DNA was used to direct the in vitro system, the same pattern of gene IV products was obtained (lanes e and f). We conclude that the f1' gene IV protein is terminated by a UGA codon rather than by a UAG codon, and a read-through protein can be translated from f 1' mRNA. f 1' Dimers. When f 1' is used to infect E. coli, circular single-stranded dimeric molecules twice the length of f 1' DNA are included in the progeny yield. Similar dimers can be demonstrated for fl-infected cells as well. Fig. 4 shows one such single-stranded f 1' dimer. The structure suggests that the dimer consists of two f ' molecules and that intramolecular basepairing is formed through the pSC101 sequence, which is highly self-complementary as shown in Fig. 2. Restriction enzyme analysis of the dimer replicative form molecules yielded only f ' fragments (data not shown). These results imply that the dimer is a tandem repeat of the f 1' monomer, as opposed to an inverted repeat.

Proc. Natl. Acad. Sci. USA 76 (1979)

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Proc. Natl. Acad. Sci. USA 76 (1979)

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Fic. 3. of the products of in vitro synthesis of protein directed by fi DNA (replicative form I) (lane a), fi DNA + tryptophan tRNA (b), fi' DNA (replicative form I) (c), fi' DNA + tryptophan tRNA (d), VO-1 DNA (replicative form I) (e), and VO-1 DNA + tryptophan tRNA (f). The protein products were fractionated on a sodium dodecyl sulfate/polyacrylamide exponential gradient gel (10-18%/ polyacrylamide). IV, Gene IV protein of fi; IV' read-through gene IV protein of fi' (see Fig. 2 and text).

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FIG. 5. Schematic representation of the genomic structures of the chimeric phage VO-1. The genome of fl is indicated at the bottom. The Roman numerals show the eight fl genes. pSC101 is indicated at the top of the figure. The 200-nucleotide imperfect inverted repeat, shown in Fig. 2, is represented by A-B, with the complementary sequences indicated by an open box (B) and a shaded box (A) connected by a region without complementarity. Z refers to the five-nucleotide direct repeat derived from fl sequences (T-A-G-T-A). The orientation of each inverted repeat is in a direct repeat configuration as indicated by the long arrow on either side of the diagram. The dashed line indicates the structure of fl', yielding a molecule with a 200-nucleotide insertion of pSC101 into fl and a 5-base direct repeat derived from fl (Z). The numbers in parentheses refer to the number of nucleotides for each region.

fragment present in the pSClOI digest but it contained two novel fragments (f has a single HinclI site). If VO-1 were formed by the insertion of the entire pSC1O1 genome directly into the fI genome, these two novel VO-1 HincII fragments would contain the missing pSC1O1 HincII fragment and the entire fI genome. Each of the fragments would consist of some part of the missing pSC101 HinclI fragment and some portion of the fit genome. The transition point between pSC1O1 -and fi in each of these two fragments would consist of a novel sequence not present in pSC101 or f 1. Consequently, restriction enzyme digestion of each of these VO-1 HinclI fragments should yield a novel restriction fragment not found in the relevant pSC1O1 HincIl fragment nor in the fI genome. Hae III digestion of the two fragments yielded all three Hae III fragments contained within the missing pSC101 HinclI fragment and all of the fragments obtained from a combined HincII/Hae III digestion of f 1'. Thus, the chimeric molecule VO-1 contains the pSC101 and f 1' genomes rather than the pSC101 and fi genomes. These results are compatible with the structure of VO-1 shown in Fig. 5. This model proposes that the inverted repeat present in pSC1O1 is duplicated in VO-1 and that each inverted repeat constitutes a region of transition between the two genomes contained within VO-1. one

DISCUSSION The in vivo recombinant formed between fI and pSC1O1 is believed to be the result of the insertion of the pSC1O1 genome into the fI genome. Segregation of this species yields a novel form of f 1, called f 1', which has retained a portion of pSC101. The DNA sequencing studies of f 1' have demonstrated that the terminal portions of the pSClO1 DNA inserted into fi are in the form of an imperfect inverted repeat. Inverted repeats are apparently found at the termini of transposable elements (19). The sequence of the inverted repeat flanking the gene con-

2198

Biochemistry: Ravetch et al.

5' GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG 3 f l' + 5' GGGGTCTGAGGGCCAATGGAACGAAAACGTACGTTAGT 3 Fi(.. 6. Comparison of a portion of the inserted sequence found in fi' (fl'+) with the inverted repeat found in Tn3 (IR-L) as determined by Ohtsubo et al. (7). The underlined nucleotides indicate sequence homology between the fl' sequences and the Tn3 sequence. IR-L

ferring ampicillin resistance in Tn3 has recently been reported (7, 8). Fig. 6 compares the inverted repeat sequence found in f 1' with that of Tn3. The similarity in sequence as well as the fact that a 5-base repeat flanks this inverted repeat in fI suggest that the approximately 200 extra nucleotides by which f 1' differs from fI constitute an insertion sequence. When grown in E. coli, f 1' appears to be a stable species. Reversion to f I has not been seen even though the emergence of a wild-type fI would rapidly overtake any f 1' species. Growth of f 1' in E. coli containing pSC101 does not appear to lead to enhanced production of a chimeric molecule or to regeneration of wild type fI (unpublished data). Presumably, the transposable element from pSC101 is no longer mobilizable even though pSC101 is provided in trans. This suggests that the requirements for a transposable element extend beyond the inverted repeat region-i.e., a transposable element requires both cis- and trans-acting functions as has been described for TnA (19, 20). Restriction enzyme analysis of VO-1, coupled with the sequence data for f 1', has yielded a model for the organization of VO-1. pSC101 appears to have a single transposable element identical to that found in f 1' (unpublished data). Although each contains an inverted repeat, these two insertion sequence-like pieces are in an overall direct repeat order. Presumably, this fact accounts for the instability of VO-1 because reciprocal recombination between the two direct repeat sequences would yield f 1' and pSC101. We do not know whether the insertion sequence-like piece is duplicated prior to or during the original insertion process. At the moment all we can say is that, after the insertion, 200 bases of pSC 101 and 5 bases of fI were found to be duplicated. Analysis of similar chimeras and their properties may shed some light on the duplication and migration of DNA sequences.

Proc. Natl. Acad. Sci. USA 76 (1979) We thank C. Yehle for his generous gifts of T4 kinase, Ming-Ta Hsu for his assistance and expertise in the electron microscope studies, and Kensuke Horiuchi for his advice and critical reading of this manuscript. This work was supported in part by grants from the National Science Foundation and the National Institutes of Health.

1. Vovis, G. F., Ohsumi, M. & Zinder, N. D. (1977) in Molecular Approaches to Eukaryotic Genetic Systems, eds. Wilcox, G., Abelson, J. & Fox, C. F. (Academic, New York), pp. 55-61. 2. Messing, J., Gronenborn, B., Muller-Hill, B. & Hofschneider, P. H. (1977) Proc. Natl. Acad. Sci. USA 74,3642-3646. 3. Nomura, N., Yamagishi, H. & Oka, A. (1978) Gene 3,39-51. 4. Hermann, R., Neugebauer, K., Schaller, H. & Zentgraf, H. (1978) in The Single-Stranded DNA Phages, eds. Denhardt, D., Dressler, D. & Ray, D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 473-476. 5. Ray, D. S. & Kook, K. (1978) Gene 4, 109-119. 6. Ohsumi, M., Vovis, G. F. & Zinder, N. D. (1978) Virology 89, 438-449. 7. Ohtsubo, H., Ohmori, H. & Ohtsubo, E. (1978) Cold Spring Harbor Symp. Quant. Biol., in press. 8. Cohen, S. N., Casadaban, M. J., Chou, J. & Tu, C.-P. D. (1978) Cold Spring Harbor Symp. Quant. Biol. 43, in press. 9. Horiuchi, K., Vovis, G. F., Enea, V. & Zinder, N. D. (1975) J. Mol. Biol. 95, 147-165. 10. Ravetch, J. V., Horiuchi, K. & Zinder, N. D. (1977) Proc. Natl. Acad. Sci. USA 74, 4219-4222. 11. Maxam, A. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74, 560-564. 12. Model, P. & Zinder, N. D. (1974) J. Mol. Biol. 83, 231-251. 13. Davis, R. W., Simon, M. & Davidson, N. (1971) Methods Enzytool. 21, 413-428. 14. Ravetch, J. V., Horiuchi, K. & Zinder, N. D. (1979) J. Mol. Biol. 128, 305-318. 15. Grindley, N. D. F. (1978) Cell 13, 419-426. 16. Calos, M. P., Johnsrud, L. & Miller, J. H. (1978) Cell 13, 411418. 17. Model, P., Webster, R. E. & Zinder, N. D. (1969) J. Mol. Biol. 43, 177-190. 18. Hirsch, D. & Gold, L. (1971) J. Mol. Biol. 55, 459-468. 19. Kleckner, N. (1977) Cell 11, 11-23. 20. Heffron, F., Bedinger, P., Champoux, J. J. & Falkow, S. (1977)

Proc. Natl. Acad. Sci. USA 74, 702-709.

Organization of a hybrid between phage f1 and plasmid pSC101.

Proc. Nat!. Acad. Sci. USA Vol. 76, No. 5, pp. 2195-2198, May 1979 Biochemistry Organization of a hybrid between phage fl and plasmid pSC 101 (DNA s...
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