Isolation and characterization of cloned fragments of bacteriophage P1 DNA

Mural, Richard J.; Chesney, Robert H.; Vapnek, Daniel; Kropf, Martha M.; Scott, June R.

doi:10.1016/0042-6822(79)90243-5

Cited by 64 publications

(22 citation statements)

References 33 publications

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“…Gene 10 had been localized around map position 95 on the P1 chromosome (44,51,61,62,64). To study this region of the genome, the P1 EcoRI restriction fragments 6 and 20, as well as a KpnI (P1)IHindIII (A) subfragment of the XP1-76 hybrid phage (45), were cloned into pUC19 (60).…”

Section: Methodsmentioning

confidence: 99%

Bacteriophage P1 gene 10 encodes a trans-activating factor required for late gene expression

1991

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Amber mutants of bacteriophage P1 were used to identify functions involved in late regulation of the P1 lytic growth cycle. A single function has been genetically identified to be involved in activation of the phage-specific late promoter sequence P. In vivo, P1 gene 10 amber mutants fail to trans activate a lacZ operon fusion under the transcriptional control of promoter Ps. Several P1 segments, mapping around position 95 on the P1 chromosome, were cloned into multicopy plasmid vectors. Some of the cloned DNA segments had a deleterious effect on host cells unless they were propagated in a P1 lysogenic background. By deletion and sequence analysis, the harmful effect could be delimited to a 869-bp P1 fragment, containing a 453-bp open reading frame. This open reading frame was shown to be gene 10 by sequencing the amber mutation amlO.1 and by marker rescue experiments with a number of other gene 10 amber mutants. Gene 10 codes for an 18.1-kDa protein showing an unusually high density of charged amino acid residues. No significant homology to sequences present in the EMBL/GenBank data base was found, and the protein contained none of the currently known DNA-binding motifs. An in vivo trans activation assay system, consisting of gene 10 under the transcriptional control of an inducible promoter and a gene SllacZ fusion transcribed from Ps, was used to show that gene 10 is the only phage-encoded function required for late promoter activation.The temperate bacteriophage P1 propagates on several enterobacterial species, including Escherichia coli (64). The linear DNA molecule carried in a phage particle is about 100 kb in size and has a terminal redundancy of 8 to 12% (32, 65). As a prophage, P1 is a low-copy-number plasmid of about 90 kb (3, 33).The genome of P1 is organized in relatively short transcriptional units and in this respect resembles the virulent bacteriophage T4 (43) rather than the temperate lambdoid phages (57). For example, P1 genes for both immunity control and morphogenesis are widely dispersed over the genome (50,62,63). The presence of at least 14 operator sites for the P1 cI repressor protein (8,15,16,28,29,59) and the tripartite immunity system of the phage (64) indicate the degree of complexity which P1 faces in regulating and expressing its genetic information.While the lysogenic condition of P1 has been studied intensively (for a recent review, see reference 64), relatively little is known about the regulation of the lytic growth cycle. A few functions which are under transcriptional control of the P1 cI repressor have been identified (8,10). Among those is the repL function, which is essential for the lytic replication of the phage genome (9,25,52).Recently, a new class of P1 promoters, controlling the late genes encoded by the tail fiber and the dar operons, has been identified (21,22

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Section: Methodsmentioning

confidence: 99%

Bacteriophage P1 gene 10 encodes a trans-activating factor required for late gene expression

1991

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“…Purified repressor binds to this subfragment in vitro, as was revealed by a decreased mobility of the subfragment-repressor complex during gel electrophoresis. To search for other repressor binding sites, we dissected the P1 genome with EcoRI because most of the recent studies on P1 phage functions had been done with cloned EcoRI fragments (2,7,32). In preliminary experiments we tested the sensitivity of the method; whereas a decrease in the mobility of P1:3 (9.5 kb) could not be detected, the mobility of an EcoRI-Sph I subfragment of P1:3 (5.3 kb) was visibly reduced with repressor.…”

Section: Resultsmentioning

confidence: 99%

Multiple repressor binding sites in the genome of bacteriophage P1.

Velleman¹,

Dreiseikelmann²,

Schuster³

1987

Proc. Natl. Acad. Sci. U.S.A.

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After digestion of bacteriophage P1 DNA with EcoRI in the presence of P1 repressor, 6 repressor binding sites were identified in 5 of 26 EcoRI fragments. Binding sites were localized by the decreased mobility of DNA fragment-repressor complexes during electrophoresis and by DNase protection ("footprinting") analysis. The repressor binding sites, or operators, comprise a 17-base-pair-long consensus sequence lacking symmetrical elements. Three operators can be related to known genes, whereas the function of the others is still unknown. The mutant P1 bac, rendering ban expression constitutive, is identified as an operator-constitutive mutation of the ban operon.P1 is a temperate bacteriophage with a genome size of about 90 kilobases (kb). In the prophage state the proviral DNA is maintained as a plasmid, and the vegetative P1 functions are repressed. Repression is accomplished by a phage-specific repressor, the product of the cl gene, which is located at the far right side of the P1 genetic map in EcoRI restriction fragment 7 (P1:7) (1, 2). Partially purified P1 repressor binds in vitro to at least two regions near cl within BamHI fragment 9 that itself is located within P1:7 (3, 4).The binding sites close to the cl gene, however, are not the only region at which the P1 repressor acts. The latter can also repress in vivo the expression of the P1 ban gene, which is located in P1:3 (5, 6). Furthermore, P1:14 also contains a promoter repressible by the product of cl (7). Together these results reveal that the P1 cl repression system must differ from that of other temperate phages such as A, P2, and P22, in which only promoters adjacent to the repressor gene are repressed (7).During our studies on the regulation of phage P1 ban expression we have localized by indirect methods a region 5' upstream of the ban gene within P1:3 at which the P1 repressor acts (8). Highly purified P1 repressor protein binds to this region in vitro (H.S., unpublished data). Now in a systematic search for other repressor binding sites in the phage genome, EcoRI-digested P1 DNA is incubated-with repressor, and binding regions are identified by the decreased mobility of EcoRI fragment-repressor complexes during electrophoresis (9,10). Thus binding regions were detected in P1:7 and P1:14, as expected and, in addition, in P1:9 and P1:11. Moreover, the results of DNA sequence and DNase protection analyses reveal an asymmetric 17-base-pair (bp)-long consensus sequence for the P1 repressor binding site. MATERIALS AND METHODSBacteria, Phage, and Plasmids. Escherichia coli K-12 strains used included the following: C600, HB101 (recA13) (11), NY58 (dnaBI07, recA56) (12), JM101 (13), N100 (galK, recA) (14), DW101 (sup'), and DW103 (supD) (15). For the cloning of P1 DNA fragments containing the ban operon (8) the recipient bacteria C600, HB101, NY58, and JM101 carried the plasmid pKT101-P1:7 harboring the P1 repressor gene (8). Strains DW101 and DW103 were transformed by plasmid pBR325-P1:11 for marker rescue tests (2).Phage used were M13mp8/9 (16)...

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“…Work reported separately (20b) (6), PaeR7 (38), TaqI (34), and FokI (28). On (26,31). The mechanism permitting this establishment is still unknown.…”

Section: Discussionmentioning

confidence: 99%

Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium

Backer

Colson

1991

J Bacteriol

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The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res-Mod' S. typhimurium genomic library in the XEMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod' E. coli strain obtained in the first step. Attempts to transform Mod-E. coli or S. typhimurium strains with this Res' Mod' plasmid were unsuccessful, whereas transformation of Mod' strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.Restriction-modification systems (R-M systems) are biological systems that enable bacterial cells to make a distinction between their own DNA and foreign invasive DNA (phage DNA, for example). R-M systems consist of two highly specific enzymatic activities: an endonuclease and a DNA methylase which recognize the same specific sequence on DNA. After recognizing this sequence, the ei-kdonuclease cleaves both strands of the DNA within or outside the sequence. This function is called restriction. The methylase protects the resident DNA from restriction by methylating it at the specific sequence. This function is called modification. R-M systems have been classified into three groups, called types I, II, and III according to the complexity of their enzyme structures and their cofactor requirements. The three groups are also characterized by the structure of the DNA sequence recognized by the enzymes (see recent reviews in references 3, 35, 40, and 41). Mutations in the restriction-modification loci can result in two distinct phenotypes: either the loss of both restriction and modification functions (Res-Mod-phenotype) or the loss of the restriction function only (Res-Mod' phenotype).

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Isolation and characterization of cloned fragments of bacteriophage P1 DNA

Cited by 64 publications

References 33 publications

Bacteriophage P1 gene 10 encodes a trans-activating factor required for late gene expression

Bacteriophage P1 gene 10 encodes a trans-activating factor required for late gene expression

Multiple repressor binding sites in the genome of bacteriophage P1.

Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium

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