280
Escherichia coli AND Salmonella typhimurium
[12]
Acknowledgments We thank Erich Six for advice on performing transduction experiments and for providing P2 sir mutants prior to publication; Dago-Arne Dimster-Denk for the construction of pDDI, pDD2, and pDD3; Giovanni Bertoni for the construction of pKGB2 and pKGB4; Alessandra Polissi for constructing P4::Tn5 AP-2; and Martin Gonzalez for cloning pMG1. Nora Linderoth and Richard Goldstein are acknowledged for careful reading of the manuscript.
[12] Special Uses o f h P h a g e for M o l e c u l a r Cloning By NOREEN E. MURRAY
Introduction In the early 1970s bacteriophage ~, was an obvious choice as a vector for cloning foreign DNA; it was, and may still be, the best understood DNA phage. Its chromosome was known to be a linear DNA molecule of approximately 50 kilobases (kb), with nearly 40% of the genome being unnecessary for the propagation of the phage. The combined use of known deletions and substitutions together with the ready selection of mutants lacking particular restriction targets led to the generation of many vectors, while the technology of packaging its DNA in vitro promoted the efficient use of h vectors for cloning the smallest DNA fragments to those in excess of 20 kb. The screening of large libraries of recombinants quickly became effective. However, despite efforts to provide essential h functions in trans, all h vectors retain at least 30 kb of the h genome in order to maintain plaque-forming ability. Inevitably, this high proportion of vector sequence complicates restriction mapping of the cloned DNA. The increased efficiencyof recovery of plasmids following electroporation of bacteria I and the arrival of vectors able to incorporate much larger fragments of DNA--45 kb for cosmids,2 100 kb for phage P13 (see also [2] in this volume), and a few hundred kilobases for yeast artificial chromosomes (YACs)4--challenge the importance of h vectors. Their current relevance probably resides in three features: (I) efficient recovery of relatively representative libraries, (2) efficient screening of plaques, and (3) ease of genetic analysis. These features suffice to maintain the usefulness of h vectors and roles in which they are used to advantage will be illustrated. 1 W. J. Dower, J. F. Miller, and C. W. Ragsdale, Nucleic Acids Res. 16, 6127 (1988). 2 j. Collins and B. Hohn, Proc. Natl. Acad. Sci. U.S.A. 75, 4242 (1978). 3 N. Sternberg, Proc. Natl. Acad. Sci. U.S.A. 87, 103 (1990). 4 D. T. Burke, G. F. Carle, and M. V. Olson, Science 236, 806 (1987).
METHODS IN ENZYMOLOGY,VOL. 204
Copyright© 1991by AcademicPress, Inc. All fightsof reproductionin any form reserved.
[12]
h PHAGE AND MOLECULARCLONING
281
Many articles review the technicalities of DNA manipulations,5-7 but the judicious choice and use of h vectors require an understanding of the phage and its interaction with Escherichia coli. I have chosen, therefore, to emphasize genetic aspects and preface this chapter with an overview of the relevant biology (see Ref. 8 for detailed reviews). Overview of Bacteriophage h The h genome is encapsidated in an icosahedral head from which projects a tail ending in a single tail fiber that adsorbs to a receptor site in the outer membrane of the bacterium. The receptor sites,which are encoded by the E. coli lamB gene, are stimulated by growth in medium containing maltose as the carbon source, and adsorption is facilitated by magnesium ions. h Phage normally remain infective for decades if kept at 4° in L (Luria) broth or phage buffer in the presence of 1-10 mM Mg2+ and the absence of detergent. A wealth of information on the sensitive handling of phage h is given in Ref. 9. On entering the cell the h genome circularizes via its complementary single-stranded 5' ends, the cohesive ends (cos), and is converted to a covalently closed circular molecule, the substrate for both replication and transcription. Being a temperate phage, h may follow either the productive (lytic) or temperate (lysogenic) pathway. In either case, transcription is initiated from two "early" promoters, PL and PR (see Fig. 1), to provide functions essential for DNA replication, genetic recombination, establishment of lysogeny, and the transcriptional activation of the late genes. In a productive (lytic) infection transition from early to late transcription is achieved, virion proteins are made, the replicated genomes are packaged, and lysis of the cell releases about 100 infective particles. In the temperate response, on the other hand, most phage functions become repressed, either directly or indirectly, by h repressor molecules bound to the operator regions associated with PL and PR. If repression occurs in time to prevent activation of the late genes, lysis is avoided and lysogeny may 5 A. M. Frischauf, N. Murray, and H. Lehrach, this series, Vol. 153, p. 103. 6 K. Kaiser and N. E. Murray, in "DNA Cloning" (D. Glover, ed.), Vol. 1, p. 1. IRL Press, Oxford and Washington, D.C., 1985. 7 j. Sambrook, E. F. Fritsch, and T. Maniatis, in "Molecular Cloning" (C. Nolan, ed.), 2nd Ed., Vol. 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. 8 R. Hendrix, J. Roberts, F. Stahl, and R. Weisberg (eds.), "Lambda II." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1983. 9 W. Arber, L. Enquist, B. Hohn, N. E. Murray, and K. Murray, in "Lambda II" (R. Hendrix, J. Roberts, F. Stahl, and R. Weisberg, eds.), p. 433. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1983.
Escherichia coli AND Salmonella typhimurium
282
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Escherichia coli AND Salmonella typhimurium
[12]
Acknowledgments We thank Erich Six for advice on performing transduction experiments and for providing P2 sir mutants prior to publication; Dago-Arne Dimster-Denk for the construction of pDDI, pDD2, and pDD3; Giovanni Bertoni for the construction of pKGB2 and pKGB4; Alessandra Polissi for constructing P4::Tn5 AP-2; and Martin Gonzalez for cloning pMG1. Nora Linderoth and Richard Goldstein are acknowledged for careful reading of the manuscript.
[12] Special Uses o f h P h a g e for M o l e c u l a r Cloning By NOREEN E. MURRAY
Introduction In the early 1970s bacteriophage ~, was an obvious choice as a vector for cloning foreign DNA; it was, and may still be, the best understood DNA phage. Its chromosome was known to be a linear DNA molecule of approximately 50 kilobases (kb), with nearly 40% of the genome being unnecessary for the propagation of the phage. The combined use of known deletions and substitutions together with the ready selection of mutants lacking particular restriction targets led to the generation of many vectors, while the technology of packaging its DNA in vitro promoted the efficient use of h vectors for cloning the smallest DNA fragments to those in excess of 20 kb. The screening of large libraries of recombinants quickly became effective. However, despite efforts to provide essential h functions in trans, all h vectors retain at least 30 kb of the h genome in order to maintain plaque-forming ability. Inevitably, this high proportion of vector sequence complicates restriction mapping of the cloned DNA. The increased efficiencyof recovery of plasmids following electroporation of bacteria I and the arrival of vectors able to incorporate much larger fragments of DNA--45 kb for cosmids,2 100 kb for phage P13 (see also [2] in this volume), and a few hundred kilobases for yeast artificial chromosomes (YACs)4--challenge the importance of h vectors. Their current relevance probably resides in three features: (I) efficient recovery of relatively representative libraries, (2) efficient screening of plaques, and (3) ease of genetic analysis. These features suffice to maintain the usefulness of h vectors and roles in which they are used to advantage will be illustrated. 1 W. J. Dower, J. F. Miller, and C. W. Ragsdale, Nucleic Acids Res. 16, 6127 (1988). 2 j. Collins and B. Hohn, Proc. Natl. Acad. Sci. U.S.A. 75, 4242 (1978). 3 N. Sternberg, Proc. Natl. Acad. Sci. U.S.A. 87, 103 (1990). 4 D. T. Burke, G. F. Carle, and M. V. Olson, Science 236, 806 (1987).
METHODS IN ENZYMOLOGY,VOL. 204
Copyright© 1991by AcademicPress, Inc. All fightsof reproductionin any form reserved.
[12]
h PHAGE AND MOLECULARCLONING
281
Many articles review the technicalities of DNA manipulations,5-7 but the judicious choice and use of h vectors require an understanding of the phage and its interaction with Escherichia coli. I have chosen, therefore, to emphasize genetic aspects and preface this chapter with an overview of the relevant biology (see Ref. 8 for detailed reviews). Overview of Bacteriophage h The h genome is encapsidated in an icosahedral head from which projects a tail ending in a single tail fiber that adsorbs to a receptor site in the outer membrane of the bacterium. The receptor sites,which are encoded by the E. coli lamB gene, are stimulated by growth in medium containing maltose as the carbon source, and adsorption is facilitated by magnesium ions. h Phage normally remain infective for decades if kept at 4° in L (Luria) broth or phage buffer in the presence of 1-10 mM Mg2+ and the absence of detergent. A wealth of information on the sensitive handling of phage h is given in Ref. 9. On entering the cell the h genome circularizes via its complementary single-stranded 5' ends, the cohesive ends (cos), and is converted to a covalently closed circular molecule, the substrate for both replication and transcription. Being a temperate phage, h may follow either the productive (lytic) or temperate (lysogenic) pathway. In either case, transcription is initiated from two "early" promoters, PL and PR (see Fig. 1), to provide functions essential for DNA replication, genetic recombination, establishment of lysogeny, and the transcriptional activation of the late genes. In a productive (lytic) infection transition from early to late transcription is achieved, virion proteins are made, the replicated genomes are packaged, and lysis of the cell releases about 100 infective particles. In the temperate response, on the other hand, most phage functions become repressed, either directly or indirectly, by h repressor molecules bound to the operator regions associated with PL and PR. If repression occurs in time to prevent activation of the late genes, lysis is avoided and lysogeny may 5 A. M. Frischauf, N. Murray, and H. Lehrach, this series, Vol. 153, p. 103. 6 K. Kaiser and N. E. Murray, in "DNA Cloning" (D. Glover, ed.), Vol. 1, p. 1. IRL Press, Oxford and Washington, D.C., 1985. 7 j. Sambrook, E. F. Fritsch, and T. Maniatis, in "Molecular Cloning" (C. Nolan, ed.), 2nd Ed., Vol. 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. 8 R. Hendrix, J. Roberts, F. Stahl, and R. Weisberg (eds.), "Lambda II." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1983. 9 W. Arber, L. Enquist, B. Hohn, N. E. Murray, and K. Murray, in "Lambda II" (R. Hendrix, J. Roberts, F. Stahl, and R. Weisberg, eds.), p. 433. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1983.
Escherichia coli AND Salmonella typhimurium
282
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