DOCSLIB.ORG

Analysis of Bacteriophage P1 Immunity by Using X-P1 Recombinants

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Analysis of Bacteriophage P1 Immunity by Using X-P1 Recombinants Proc. Natl. Acad. Sci. USA Vol. 75, No. 11, pp. 5594-5598, November 1978 Genetics Analysis of bacteriophage P1 immunity by using X-P1 recombinants constructed in vitro (P1 repressor genes/PI antirepressor gene/PI dnaB analog gene/DNA cloning) NAT STERNBERG, STUART AUSTIN, DANIEL HAMILTON, AND MICHAEL YARMOLINSKY Cancer Biology Program, National Cancer Institute, Frederick Cancer Research Center, P.O. Box B, Frederick, Maryland 21701 Communicated by Gary Felsenfeld, August 10, 1978 ABSTRACT We describe the dissection and reconstruction MATERIALS AND METHODS of a complex control circuit, the P1 immunity system, by a method that involves inserting EcoRI-generated fragments of Bacterial and Phage Strains. The Escherichia coli strains P1 DNA into X vectors that can then be sequentially inserted used were NS985 (HfrH, sup+), RW842 [HfrH XA(int-FII)gajT] into a bacterial cell. Using these techniques we have isolated (14), YMC (supF) (15), K175 (supD, Xr) (10), Q1508 X-P1 hybrid phages that express the products of P1 genes cl, c4, [dnaB70(ts), thyAl (12), NS91 (C600 groPA15) (16), and N3072 ant, and ban and, in appropriately constructed lysogens, con- [HfrH, sup+, A (pro-lac) Xlii] (6). The X vector X firmed the roles played by the first three of these products in Dam15b538sr1X3c1857(ts)nin5 contains a single EcoRI site for phage immunity. In addition we have'localized to particular inserting EcoRI fragments of P1 DNA (11).' Hybrid phages P1 fragments the sites requisite for expression and repression obtained by inserting P1 fragments (11) are designated X-Pl:c4, of these gene products. The analysis leads to the conclusion that :cl gpant acts in trans to antagonize repression mediated by gpcl, X-Pl:c1, etc.; :c4 or refers to pertinent genes located on the in support of' one of two proposed models for gpant action. inserted P1 fragment. Other phages used were PlCm, Moreover, two features of the,immunity system are revealed: PlCmcl.100(ts), PlCmcl.55(am), PlCmc4.32, PlCmelrs, and (f) a hitherto unknown component that effects gpcl repression; Plvdrsban-l. Unless otherwise indicated, all of the P1 phages and (if) an unexpected ability of gpc4 to channel a superin- carried the Tn9 transposon (Cm). PlCmvirsant-10 and fecting cl'+ phage into the lysogenic state, which suggests that PlCmc4.32ant-17 were isolated from P1 lysogens made by gpc4 activity regulates the establishment phase of lysogeny. infecting strain NS985 with either PlCmvdrs or PlCmc4.32. The presence of the ant- mutation in these phages was con- Analysis of complex genetic'controls requires methods that firmed by their inability to make plaques on strain.K175(Plcry) permit parts of the control circuits to be isolated, characterized, (5). Mutational defects are indicated here by a minus sign su- and variously reassembled. We show here how the insertion of perscript unless an allele numbei is specified. defined DNA segments into X vectors that can be successively Media and Growth Conditions. Media and commonly used integrated into the chromosome of a suitable bacterium permits microbiological procedures for P1 and N were as described (13, the analysis of several interacting genes by reconstruction as 17). Because the attX-int region of the X vector has been re- well as dissection experiments. Because of the phenomenon of moved' by the b538 deletion (18), homologous recombination incompatibility, this type of analysis would not be possible if with the cryptic X prophage of 'strain RW842 was used to ly- the genes to be studied were located in a plasmid. sogenize with primary X-P1 hybrids. The subject of our analysis is the immunity to superinfection The experiments described in this report were carried out conferred by the plasmid prophage of coliphage Pl. This im- at an EKi-Pi containment level in accordance with the'stipu- munity is thought to' require the presence of two repressor lations specified in FCRC MUA# 1. proteins, the products of genes ci (1) and c4 (2) (gpcl and gpc4) Construction of Xatt+imm21-P1 and Xatt+imm434-P1 that are located in the separate immC and immI regions of the Hybrid Phages. The Xb538cI857(ts)nin5-P1 hybrids (X-P1 phage genetic map (Fig. 1). Gpcl represses the viral lytic hybrids) -were restructured first by recombination with functions. Gpc4 is thought to repress a second immI' gene, ant Xfmm21nin5 or Ximm434nin5 to replace the immX' region (or reb). The product of the ant gene has been postulated to act with'imm2l or imm434 and then by recombination with either in trans to antagonize gpcl-mediated repression (9) or Xb515b519Nam7cI857(ts) to replace the b538 deletion with in cis to replicate DNA of the superinfecting phage even in the the two smaller deletions, b515 and b519, which leave attX and presence of gpcl (i.e., it acts as a repressor bypass) (10). Phage the X int gene intact (18). The restructured hybrids (designated P7, which has a ci gene functionally identical to the ci gene Ximm2l-Pl or Ximm434-Pl) can lysogenize standard strains of P1 but differs from P1 in the specificity of its c4 repressor at 32 or 42°C. (9), is able to grow in a P1 lysogen because gpant of the in- Isolation of a X-P1:ban Hybrid. We took advantage of the fecting P7 phage is not repressed by gpc4 of the prophage. ability'of the P1 ban gene product to substitute for the dnaB and inter- protein of the host (12) in order to isolate a X-Pl:ban hybrid. We describe here the isolation, characterization, Strain Q1508(X) was infected with a pool of \-P1 hybrids (de- actions of individual X-P1 hybrids bearing EcoRI-generated rived from 1000 plaques). The infected cells were spread on fragments of P1 (11) that express each of the known components thymine-supplemented'Ll plates and incubated for 36 hr at of the P1 immunity system. An additional hybrid that'expresses 42°C. The frequency of infected cells that survived to form the P1 ban gene (12), known to be directly under cl control (13) colonies (3 X 10-6) was about 30-fold greater than the (Fig. 1), provides'an indicator of cl repression. dnaB70(ts) reversion frequency. Phage that formed turbid plaques at 320C and clear plaques at 42°C [phage with the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: Cm, chloramphenicol resistance transposon Tn9; am, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate amber, ts, temperature sensitive; gpc4, gpcl, etc., gene product of gene this fact. c4, cl, etc. 5594 Downloaded by guest on September 27, 2021 Genetics: Sternberg et al. Proc. Natl. Acad. Sci. USA 75 (1978) 5595 2 1122 7 expresses should a riE n gpc4 lysogenization-deficient Plc4- I EoRI fragments r-,n i m permit phage to lysogenize. Of 50 independent RW842 (X-P1) lysogens Cm imml immC v virs screened, 2 showed a 100- to 1000-fold increase in the capacity l /~~~~~~~/\,'fl for being lysogenized by Plc4- (PlCmc4.32). The X-P1 phages gpf2 gpc4 gpant-.- gpban gpc1 (X-Pl:c4a and X-Pl:c4b) obtained from these lysogens were made att + int + and then integrated into another host (NS985). In NS985, as in the original host, they conferred proficiency to be lysogenized by Plc4.32 at high efficiency. Because 90-95% O1la ~ of the P1 phage recovered from the NS985 (X-Pl:c4, FIG. 1. P1 genome with the positions ofthe two immunity regions PlCmc4.32) lysogens still carried the c4.32 allele, lysogenization and the Cm (Tn9) transposon (3,4) above the bar and pertinent genes, by Plc4.32 was not due to recombination between the c4+ gene whose products are designated in the figure, below the bar. The extent of the X-P1 prophage and the c4- gene of the P1. (The re- of the deletions in the Plcry (5), Pldpro8a (6), and Pldprolla (6) maining 5-10% of the recovered P1 phage were Plc4+, by prophages are indicated by the black areas. The location of EcoRI- rescue of c4.32+ from the X-Pl:c4 prophages). Thus, the generated P1 fragments is based in part on the map of Bachi and Arber (7) and on our own marker rescue experiments using am X-Pl:c4 phages express gpc4 constitutively in the prophage state mutations (8). The virs mutation, located in the immI region (2, 9), and this product can act in trans to permit Plc4- to form stable permits the phage to express gpant constitutively even in the presence lysogens. of gpc4. Thus, when a P1 lysogen is infected with Plvirs, gpant is X-Pl:c4 Lysogens Are Immune to P1. Although expressed, repression of lytic functions is lifted, and the infecting Plcl.55(am) formed plaques efficiently and yielded a signif- phage grows. For the same reason, P7, whose ant gene expression is icant number of phage after infection of lysogens of either insensitive to repression by gpc4 of P1, will grow in a P1 lysogen. A-Pl:c4a or X-Pl:c4b, P1 wild type did not (Table 1, line 2). This immunity is probably a consequence of gpc4-mediated c1857(ts) allele] were isolated from these lysogens. Infection repression of the ant gene of the superinfecting phage, because with these phages (X-Pl:ban) increased by about 300-fold the PlIrs and P7, whose ant gene is not sensitive to repression by survival of Q1508(X) at 42°C. A Xatt +imm21-Pl:ban phage the gpc4 of P1, grew well in the X-Pl:c4 lysogens was constructed, and Q1508 lysogens containing this prophage (Table 1, line survived with unit efficiency at 42°C.
Load more

Recommended publications
  • JOURNAL of VIROLOGY VOLUME 54 * JUNE 1985 * NUMBER 3 Arnold J
    JOURNAL OF VIROLOGY VOLUME 54 * JUNE 1985 * NUMBER 3 Arnold J. Levine, Editor in Chief Michael B. A. Oldstone, Editor (1988) (1989) Scripps Clinic & Research Foundation Princeton University La Jolla, Calif. Princeton, N.J. Thomas E. Shenk, Editor (1989) David T. Denhardt, Editor (1987) Princeton University University of Western Ontario Princeton, N.J. London, Ontario, Canada Anna Marie Skalka, Editor (1989) Bernard N. Fields, Editor (1988) Hoffmann-La Roche Inc. Harvard Medical School Nutley, N.J. Boston, Mass. Robert A. Weisberg, Editor (1988) Robert M. Krug, Editor (1987) National Institute of Child Health Sloan-Kettering Institute and Human Development New York, N.Y. Bethesda, Md. EDITORIAL BOARD David Baltimore (1987) Hidesaburo Hanafusa (1986) Lois K. Miller (1985) Priscilla A. Schaffer (1987) Amiya K. Banerjee (1985) William S. Hayward (1987) Peter Model (1986) Sondra Schlesinger (1986) Andrew Becker (1985) Roger Hendrix (1987) Bernard Moss (1986) June R. Scott (1986) Tamar Ben-Porat (1987) Martin Hirsch (1986) Fred Murphy (1986) Bart Sefton (1985) Kenneth I. Berns (1985) John J. Holland (1987) Nancy G. Nossal (1987) Phillip A. Sharp (1985) Michael Botchan (1986) Ian H. Holmes (1986) Abner Notkins (1986) Charles J. Sherr (1987) David Botstein (1985) Robert W. Honess (1986) J. Thomas Parsons (1986) Saul J. Silverstein (1985) Barrie J. Carter (1987) Nancy Hopkins (1986) Ulf G. Pettersson (1986) Purnell Choppin (1986) Peter M. Howley (1987) Lennart Philipson (1987) Patricia G. Spear (1987) John M. Coffin (1986) Alice S. Huang (1987) Lewis I. Pizer (1987) Nat Sternberg (1986) Geoffrey M. Cooper (1987) Tony Hunter (1986) Craig R. Pringle (1986) Mark F. Stinski (1986) Donald Court (1987) Masayori Inouye (1985) Carol Prives (1986) James Strauss (1987) Richard Courtney (1986) Robert Kamen (1985) Robert Purcell (1986) F.
    [Show full text]
  • Interactions in Escherichia Coli NAT STERNBERG Central Research and Development Department Experimental Station, E
    JOURNAL OF BACTERIOLOGY, OCt. 1985, p. 490(493 Vol. 164, No. 1 0021-9193/85/100490-04$02.00/0 Copyright © 1985, American Society for Microbiology Evidence that Adenine Methylation Influences DNA-Protein Interactions in Escherichia coli NAT STERNBERG Central Research and Development Department Experimental Station, E. l. Du Pont de Nemours and Co., Inc., Wilmington, Delaware 19898 In this review, most of the information presented will be functional recA gene. K. R. Peterson, K. F. Wertman, derived from studies with Escherichia coli K-12 (E. coli) and D. W. Mount, and M. G. Marinus have recently found that its related bacteriophages, simply because more is known the genes of the SOS regulon could be induced (1.7- to about methylation in these organisms than in any other. The 6-fold) by introducing a dam mutation into E. coli. The two methylated bases that have been detected in E. coli are affected genes include recA, lexA, uvrA, uvrB, sulA, dinD, 6-methyladenine (6-meAde) and 5-methylcytosine (7, 18). and dinF. The only SOS gene not effected is uvrD. Except Since little is known about the biological function of 6- for lexA and uvrB, none of the genes are fully induced. In methylcytosine, I will deal exclusively here with the studies essence, the cell adjusts the basal level of the SOS response on 6-meAde. to that needed without fully inducing the regulon. In this 6-meAde is the product of a reaction catalyzed by a case, the control of gene expression is probably an indirect methylase coded for by the bacterial dam gene (21).
    [Show full text]
  • Everything You Always Wanted to Know About Rabies Virus ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ (But Were Afraid to Ask) Benjamin M
    ANNUAL REVIEWS Further Click here to view this article's online features: t%PXOMPBEmHVSFTBT115TMJEFT t/BWJHBUFMJOLFESFGFSFODFT t%PXOMPBEDJUBUJPOT Everything You Always Wanted t&YQMPSFSFMBUFEBSUJDMFT t4FBSDILFZXPSET to Know About Rabies Virus (But Were Afraid to Ask) Benjamin M. Davis,1 Glenn F. Rall,2 and Matthias J. Schnell1,2,3 1Department of Microbiology and Immunology and 3Jefferson Vaccine Center, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, 19107; email: [email protected] 2Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 Annu. Rev. Virol. 2015. 2:451–71 Keywords First published online as a Review in Advance on rabies virus, lyssaviruses, neurotropic virus, neuroinvasive virus, viral June 24, 2015 transport The Annual Review of Virology is online at virology.annualreviews.org Abstract This article’s doi: The cultural impact of rabies, the fatal neurological disease caused by in- 10.1146/annurev-virology-100114-055157 fection with rabies virus, registers throughout recorded history. Although Copyright c 2015 by Annual Reviews. ⃝ rabies has been the subject of large-scale public health interventions, chiefly All rights reserved through vaccination efforts, the disease continues to take the lives of about 40,000–70,000 people per year, roughly 40% of whom are children. Most of Access provided by Thomas Jefferson University on 11/13/15. For personal use only. Annual Review of Virology 2015.2:451-471. Downloaded from www.annualreviews.org these deaths occur in resource-poor countries, where lack of infrastructure prevents timely reporting and postexposure prophylaxis and the ubiquity of domestic and wild animal hosts makes eradication unlikely. Moreover, al- though the disease is rarer than other human infections such as influenza, the prognosis following a bite from a rabid animal is poor: There is cur- rently no effective treatment that will save the life of a symptomatic rabies patient.
    [Show full text]
  • 1987 June, American Daffodil Society Journal
    The Daffodil Journal VOLUME 23 NUMBER 4 JUNE 1987 AMERICAN DAFFODIL SOCIETY, INC. The Daffodil Journal ISSN 0011-5290 Quarterly Publication of the American Daffodil Society, Inc. Vol. 23 JUNE 1987 Number 4 OFFICERS OF THE SOCIETY DR. THEODORE SNAZELLE, President 418 McDonald Dr., Clinton, MS 39056 MRS. MARVIN V. ANDERSEN, First Vice President 7 Perth Drive, Wilmington, DE 19803 J. S. ROMINE, Second Vice President 2065 Walnut Blvd., Walnut Creek, CA 94596 MS. MARILYNN HOWE, Secretary 11831 Juniette, Culver City, CA 90230 MRS. P. R. MOORE, JR., Treasurer 16 Maple Ave., Newport News, VA 23607 Executive Director — MISS LESLIE E. ANDERSON Rt. 3, 2302 Byhalia Rd., Hernando, MS 38632 (Tel. 601-368-6337) All correspondence regarding memberships, change of address, receipt of publications, supplies, ADS records, and other business matters should be addressed to the Executive Director. THE DAFFODIL JOURNAL is published quarterly (March, June, September, and December) by the American Daffodil Society, Inc., Hernando, MS 38632. Second class postage paid at Hernando, MS, and additional mailing office. Subscription price (including membership) is $10.00 per year, $27.50 for three years. Single copies of current or back numbers are $2.00 © 1987 American Daffodil Society, Inc. Chairman of Publications Editor, Daffodil Journal Mr. David Karnstedt Mrs. Richard Frank, Jr. 1790 Richard Circle 1018 Stonewall Dr. West St. Paul, MN 55118 Nashville, TN 37220 (Tel. 612-455-6177) (Tel. 615-383-7058) Articles and photographs (glossy finish for black and white, transparency for color) on daffodil culture and related subjects are invited from members of the Society. Manuscripts should be typewritten double-spaced, and all material should be addressed to the Editor.
    [Show full text]
  • P1 Site-Specific Recombination: Nucleotide Sequence of the Recombining Sites (Dyad Symmetry/Lox Site/BAL-31 Deletion/Plasmid Recombination) RONALD H
    Proc. NatL Acad. Sci. USA Vol. 79, pp. 3398-3402, June 1982 Biochemistry P1 site-specific recombination: Nucleotide sequence of the recombining sites (dyad symmetry/lox site/BAL-31 deletion/plasmid recombination) RONALD H. HOESS, MARCIA ZIESE, AND NAT STERNBERG Cancer Biology Program, NCI-Frederick Cancer Research Facility, Frederick, Maryland 21701 Communicated by Philip Leder, February 8, 1982 ABSTRACT Site-specific recombination between molecules ofbacteriophage P1 DNA occurs at sites called loxP and requires '--BamHI-9-- the action of a protein that is the product of the P1 cre gene. Al- A cre loxP EcoPJ BamHI BamHI EcoRI though recombination between two loxP sites is very efficient, a _ 0 l recombination between loxP and a unique site in the bacterial -2600 -800 -0 +700 + 3950 chromosome (loxB) is inefficient and generates two hybrid lox sites called loxR and lozL. We present here the nucleotide sequences of all four lox sites. Analysis of these sequences indicates that (i) -100 0 +100 a region of extensive homology is not present at the loxP x loxB crossover point, in contrast to the 15-base pair common-core se- Sau3a PvuH HindI - quence in the bacteriophage A aft sites, and (ii) the sites contain a region ofdyad symmetry with 8- to 13-base pair inverted repeats B X loB EcoRI BamHI separated by an 8- to 9-base pair sequence. The loxP cross- MMM over point falls in the sequence that separates the inverted re- -1950 /° \ 700 peats, and deletions that remove either the left or the right in- verted repeat of loxP inactivate the site.
    [Show full text]
  • Site-Specific DNA Recombinationin Mammalian Cells by the Cre
    Proc. Nail. Acad. Sci. USA Vol. 85, pp. 5166-5170, July 1988 Genetics Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1 (genome rearrangement/herpesviruses/Cre-lox/metalothionein) BRIAN SAUER AND NANCY HENDERSON E. I. du Pont de Nemours and Company, Inc., Central Research and Development Department, Experimental Station, Wilmington, DE 19898 Communicated by Robert A. Weinberg, March 28, 1988 ABSTRACT The Cre protein encoded by the coliphage P1 give inversion of the intervening DNA segment. Both intra- is a 38-kDa protein that efficiently promotes both intra- and and intermolecular recombination is catalyzed by Cre with intermolecular synapsis and recombination of DNA both in either supercoiled or linear DNA. Recently we have shown Escherichia coli and in vitro. Recombination occurs at a specific that the Cre-lox system can function efficiently in yeast to site, called lox, and does not require any other protein factors. cause recombination on chromosomes present in the nucleus The Cre protein is shown here also to be able to cause synapsis (5). We now demonstrate that this site-specific recombination of DNA and site-specific recombination in a mammalian cell system also functions in a mammalian cell. line. A stable mouse cell line was established that expresses the Cre protein under the control of the Cd2+-inducible metallo- MATERIALS AND METHODS thionein I gene promoter. DNA recombination was monitored with DNA substrates containing two directly repeated lox sites. Plasmids. The Cre expression plasmid pBS31 and its One such substrate is a circular plasmid with two directly parent, pBMTx (obtained from G.
    [Show full text]
  • The Genesis of Cre-Lox Technology
    VI02CH02-Yarmolinsky ARI 1 October 2015 7:19 ANNUAL REVIEWS Further Click here to view this article's online features: • Download figures as PPT slides • Navigate linked references • Download citations The Legacy of Nat Sternberg: • Explore related articles • Search keywords The Genesis of Cre-lox Technology Michael Yarmolinsky1 and Ronald Hoess2 1Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; email: [email protected] 2Chadds Ford, Pennsylvania 19317; email: [email protected] Annu. Rev. Virol. 2015. 2:25–40 Keywords The Annual Review of Virology is online at bacteriophage P1, plasmid, site-specific recombination, chromosome virology.annualreviews.org segregation, genetic engineering This article’s doi: 10.1146/annurev-virology-100114-054930 Abstract c Annu. Rev. Virol. 2015.2:25-40. Downloaded from www.annualreviews.org Copyright 2015 by Annual Reviews. Cre-lox of bacteriophage P1 has become one of the most widely used tools All rights reserved Access provided by Johns Hopkins University on 12/12/15. For personal use only. for genetic engineering in eukaryotes. The origins of this tool date to more than 30 years ago when Nat L. Sternberg discovered the recombinase, Cre, and its specific locus of crossover, lox, while studying the maintenance of bac- teriophage P1 as a stable plasmid. Recombinations mediated by Cre assist in cyclization of the DNA of infecting phage and in resolution of prophage multimers created by generalized recombination. Early in vitro work demon- strated that, although it shares similarities with the well-characterized bac- teriophage λ integration, Cre-lox is in many ways far simpler in its require- ments for carrying out recombination.
    [Show full text]
  • A Positive Selection Vector for Cloning High Molecular Weight DNA
    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2056-2060, March 1992 Biochemistry A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: Improved cloning efficacy (high molecular weight genomic DNA/bacteriophage P1 cloning/sacB gene/positive selection) JAMES C. PIERCE, BRIAN SAUER, AND NAT STERNBERG The Du Pont Merck Pharmaceutical Company, Wilmington, DE 19880-0328 Communicated by James D. Watson, November 13, 1991 ABSTRACT The bacteriophage P1 cloning system can Analysis of the P1 human library revealed that a small package and propagate DNA inserts that are up to 95 kilobases. number of the initial clones (10-20%6) contained P1 vector Clones are maintained in Escherichia coil by a low-copy DNA without inserts and that these clones grew significantly replicon in the P1 cloning vector and can be amplified by faster than those with inserts. Consequently, when the var- inducing a second replicon in the vector with isopropyl (3-D- ious pools were amplified to prepare DNA for subsequent thiogalactopyranoside. To overcome the necessity of screening analysis, the majority of the vector molecules (as much as clones for DNA inserts, we have developed a P1 vector with a 80%) had no inserts. This made it more difficult than expected positive selection system that is based on the properties of the to isolate unique copy sequences from the library and to use sacB gene from Bacilus amyloliquefaciens. Expression of that those sequences in genome mapping strategies. Moreover, gene kills E. coli cells that are grown in the presence ofsucrose. the problem might be more pronounced if the ligation reac- In the new P1 vector (pAdlOsacBII) sacB expression is regu- tion used to generate a library of cloned inserts was less than lated by a synthetic E.
    [Show full text]
  • Bacteriophage a (Phage Display/Biopanning/D Protein/Site-Specific Recombination/Libraries) NAT STERNBERG and RONALD H
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1609-1613, February 1995 Genetics Display of peptides and proteins on the surface of bacteriophage A (phage display/biopanning/D protein/site-specific recombination/libraries) NAT STERNBERG AND RONALD H. HoEss Dupont-Merck Pharmaceutical Company, Experimental Station E328/B33, Wilmington, DE 19880-0328 Communicated by Sankar Adhya, National Cancer Institute, Bethesda, MD, October 14, 1994 (received for review August 12, 1994) ABSTRACT The display of peptides or proteins on the age processing has also shown that D protein is assembled as surface of viruses is an important technology for studying trimers, which appear as prominent protrusions on the capsid peptides or proteins and their interaction with other mole- surface (8). This feature suggests that peptides or proteins cules. Here we describe a display vehicle based on bacterio- fused to D protein are likely to be accessible for binding to phage A that incorporates a number of features distinct from target molecules. Recently, a display system using the V tail other currently used display systems. Fusions of peptides or protein of bacteriophage A has been reported (9). protein domains have been made to the amino terminus ofthe 11-kDa D protein ofthe A capsid. These fusions assemble onto the viral capsid and appear to be accessible to ligand inter- MATERIALS AND METHODS actions, based on the ability of a monoclonal antibody to Construction and Use of Bacterial Strains and Phage*. recognize an epitope fused to the D protein on phage heads. Escherichia coli strain YMC supF was used for titering plaque- To produce large D fusion display libraries and yet avoid the forming units (pfu).
    [Show full text]
  • Participation of the Lytic Replicon in Bacteriophage P1 Plasmid Maintenance MICHAEL B
    JOURNAL OF BACTERIOLOGY, Sept. 1989, p. 4785-4791 Vol. 171, No. 9 0021-9193/89/094785-07$02.00/0 Copyright X3 1989, American Society for Microbiology Participation of the Lytic Replicon in Bacteriophage P1 Plasmid Maintenance MICHAEL B. YARMOLINSKY,1* EGON B. HANSEN,'t SAMINA JAFRI,'t AND DHRUBA K. CHATTORAJ2§ Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892' and Laboratory of Molecullar Biology, LBI Basic Research Program, NCI-Frederick Cancer Research Facility, Frederick, Maryland 217012 Received 7 February 1989/Accepted 15 June 1989 P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomplete repression of the lytic replicon by the ci immunity repressor. Incomplete repression was particularly evident in lysogens of the thermoinducible P1 cl.100 prophage, whose replication at 32°C remained almost unaffected when use of the plasmid replicon was prevented. Moreover, the average plasmid copy number of P1 in a P1 cl.100 lysogen was elevated with respect to the copy number of P1 c1+. The capacity of the lytic replicon to act as an auxiliary in plasmid maintenance may contribute to the extraordinary stability of P1 plasmid prophage. P1 prophage replicates in Escherichia coli as a stable, the bacterial dnaA function in a host whose own dnaA low-copy-number, 90-kilobase (kb) plasmid.
    [Show full text]
  • Site-Specific Insertion of DNA Into a Pseudorabies Virus Vector
    Proc. Nad. Acad. Sci. USA Vol. 84, pp. 9108-9112, December 1987 Genetics Site-specific insertion of DNA into a pseudorabies virus vector (DNA recombination/herpesviruses/glycoprotein/phage Pl/Cre-lox) BRIAN SAUER, MARY WHEALY, ALAN ROBBINS, AND LYNN ENQUIST Central Research and Development Department, Experimental Station, E. I. du Pont de Nemours and Company, Wilmington, DE 19898 Communicated by I. Robert Lehman, August 24, 1987 ABSTRACT A simple, efficient method for introducing 34-base pair (bp) sequence composed of two 13-bp inverted recombinant DNA into a herpesvirus vector and retrieving it at repeats separated by an asymmetric 8-bp core sequence. a later time has been developed. By using the Cre-lox site- Recombination between loxP sites (i) can occur either specific recombination system of coliphage P1, DNA can be intermolecularly or intramolecularly, (ii) can occur when the readily inserted in vitro into a pseudorabies virus (PRV) vector sites are present on either supercoiled or linear DNA, and (iii) containing the lox recombination site. The vector PRV42 is independent of the relative orientation of the loxP sites on contains a lox site within the nonessential gIII gene, which the DNA molecule. Recombination between two directly encodes a virion envelope glycoprotein. Incubation in vitro of repeated sites on the same DNA molecule excises the DNA PRV42 DNA with Cre protein and a circular plasmid contain- segment lying between the sites. Conversely, Cre can inte- ing a lox site generates approximately 5% recombinant mole- grate a circular DNA molecule into a second DNA molecule cules having the plasmid integrated into the PRV genome at the if each contains a lox site.
    [Show full text]
  • "Small Genomes: New Initiatives in Mapping and Sequencing"
    Workshop Summary Report "Small Genomes: New Initiatives in Mapping and Sequencing" Sponsored by: The Office of Health and Environmental Research of the United States Department of Energy Workshop Report WORKSHOP TITLE "Small Genomes: New Initiatives in Mapping and Sequencing" Sponsored by the Ofice of Health and Environmental Research of the United States Department of Energy Hosted by the University of Maryland Biotechnology Institute (UMBI) and the National Institute of Standards and Technology (NIST). Held at the Center for Advanced Research in Biotechnology 9600 Gudelsky Drive Rockville, Maryland 20850 July 57,1993 Report prepared by Keith McKenney Biotechnology Division National Institute of Standards and Technology Gaithersburg, MD. 20899-0001 301-975-4872 Frank Robb Center of Marine Biotechnology University of Maryland Biotechnology Institute Baltimore, MD. 410-783-4821 1 DISCLAIMER Portions of this document may be illegible in electronic image products. Images are produced from the best available original document. DISCLAIMER This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employes, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or use- fulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any spe- cific commercial product, process, or service by trade name, trademark, manufac- turer, or otherwise does not necessarily constitute or imply its endorsement, recom- mendation, or favoring by the United States Government or any agency thereof.
    [Show full text]