Physical mapping of BglII, BamHI, EcoRI, HindIII and PstI Restriction fragments of bacteriophage P1 DNA

Bächi, Brigitte; Arber, Werner

doi:10.1007/bf00431596

Cited by 162 publications

(48 citation statements)

References 26 publications

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“…The processive headful packaging from a specific cut in a concatemer that produces cyclically permuted, terminally redundant DNA in T4 phage populations was originally proposed to start from a specific cut in a concatemeric molecule. T4 was later shown not to be specifically cut for packaging, but certain other phages were, including P1 (22).…”

Section: Vol 186 2004 Bacteriophage P1 7053mentioning

confidence: 99%

Genome of Bacteriophage P1

et al. 2004

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P1 is a bacteriophage of Escherichia coli and other enteric bacteria. It lysogenizes its hosts as a circular, low-copy-number plasmid. We have determined the complete nucleotide sequences of two strains of a P1 thermoinducible mutant, P1 c1-100. The P1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. Protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four are decisive for the choice between lysis and lysogeny. Four others ensure plasmid maintenance. The majority of the remaining 37 operons are involved in lytic development. Seventeen operons are transcribed from 70 promoters directly controlled by the master phage repressor C1. Late operons are transcribed from promoters recognized by the E. coli RNA polymerase holoenzyme in the presence of the Lpa protein, the product of a C1-controlled P1 gene. Three species of P1-encoded tRNAs provide differential controls of translation, and a P1-encoded DNA methyltransferase with putative bifunctionality influences transcription, replication, and DNA packaging. The genome is particularly rich in Chi recombinogenic sites. The base content and distribution in P1 DNA indicate that replication of P1 from its plasmid origin had more impact on the base compositional asymmetries of the P1 genome than replication from the lytic origin of replication.

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Section: Vol 186 2004 Bacteriophage P1 7053mentioning

confidence: 99%

Genome of Bacteriophage P1

et al. 2004

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“…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).…”

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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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“…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. The EcoRI digest of P1 DNA (='90 kb) yields 26 bands from 15 to 0.05 kb in size (33). When P1 DNA was digested with EcoRI in the presence of repressor, complex formation with P1:7 (6.0 kb), P1:9 (3.7 kb), P1:11 (3.1 kb), and P1:14 (1.8 kb) was revealed by the slower movement of only these fragments during electrophoresis.…”

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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Physical mapping of BglII, BamHI, EcoRI, HindIII and PstI Restriction fragments of bacteriophage P1 DNA

Cited by 162 publications

References 26 publications

Genome of Bacteriophage P1

Genome of Bacteriophage P1

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

Multiple repressor binding sites in the genome of bacteriophage P1.

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