Genetic and Physiological Studies of Bacteriophage T5 I. An Expanded Genetic Map of T5

Hendrickson, Herbert E.; McCorquodale, D.J.

doi:10.1128/jvi.7.5.612-618.1971

Cited by 76 publications

(48 citation statements)

References 17 publications

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“…We think that the D7 locus is near the left end of BalI-12. T5D7 also had the highest reversion frequency of the three amber mutants shown, and those with the higher reversion (13) (Fig. 3).…”

Section: Resultsmentioning

confidence: 85%

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Physical locus of the DNA polymerase gene and genetic maps of bacteriophage T5 mutants

Fujimura

Tavtigian

Choy

et al. 1985

J Virol

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Segments of DNA that contained the DNA polymerase gene of bacteriophage T5 were isolated. The physical locus of the gene was identified by transforming Escherichia coli with purified DNA fragments generated by restriction enzyme digestions, and the transformed cells were used to rescue amber mutants of T5 with mutations in the gene for DNA polymerase. The method is applicable to any other gene that has mutations with low reversion frequencies. We studied the following mutations of the T5 DNA polymerase gene, reading from left to right by the standard convention (D. J. McCorquodale, Crit. Rev. Microbiol. 4:101-159, 1975): D7, D8, aml, ts5E-ts53, am6, and D9. These loci were found to reside within three pieces of DNA with a total length of 3,600 base pairs. Because the structural gene for T5 DNA polymerase is estimated to be 2,600 base pairs long, the whole structural gene may reside in these segments. These are located 58.3 to 61.3% of the distance from the left end of the DNA. The left-end piece of the DNA (1,100 base pairs) containing the polymerase gene has loci D7 and D8, and the right-end piece (1,600 base pairs) has locus D9, according to the results of the transformation assay. These results are consistent with the genetic map.Plaskon CTFE2300 powder and Adogen 464 were the generous gifts of the late G. D. Novelli. A column (90 by 0.6 cm) was equilibrated with 1 M NaCl in TE buffer (10 mM Tris hydrochloride [pH 7.6], 1 mM EDTA) and then with 0.5 M NaCl in TE buffer. A sample (240 ,ug) was adjusted to 0.5 M NaCl and loaded through a pump. The column was washed with 400 ml of TE buffer containing an increasing NaCl concentration (0.55 to 0.6 M) at a pressure of 300 to 400 lb/in2. The DNA was eluted with a linear gradient made with 495 on July 6, 2020 by guest

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Section: Resultsmentioning

confidence: 85%

“…MATERIALS AND METHODS Strains of bacteria and bacteriophages. The bacteriophage T5 mutants T5D7 (am40c), T5D8 (aml4b), and T5D9 (aml7c) were characterized by Hendrickson and McCorquodale (13). Bacteriophage TSts53 was described previously (9).…”

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confidence: 99%

Physical locus of the DNA polymerase gene and genetic maps of bacteriophage T5 mutants

Fujimura

Tavtigian

Choy

et al. 1985

J Virol

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“…Preparation and purification of phages T5stamN5 (T5) phages were produced on E.coli Fsuf+, a permissive host (Hendrickson and McCorquodale, 1971;Zweig and Cummings, 1973). Phage 180 was propagated in E.coli K12.…”

Section: Methodsmentioning

confidence: 99%

FhuA, a transporter of the Escherichia coli outer membrane, is converted into a channel upon binding of bacteriophage T5.

et al. 1996

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The Escherichia coli outer membrane protein FhuA catalyzes the transport of Fe3+(‐)ferrichrome and is the receptor of phage T5 and phi 80. The purified protein inserted into planar lipid bilayers showed no channel activity. Binding of phage T5 and FhuA resulted in the appearance of high conductance ion channels. The electrophysiological characteristics of the channels (conductance, kinetic behavior, substates, ion selectivity including the effect of ferrichrome) showed similarities with those of the channel formed by a FhuA derivative from which the ‘gating loop’ (delta 322–355) had been removed. binding of phage T5 to FhuA in E.coli cells conferred SDS sensitivity to the bacteria, suggesting that such channels also exist in vivo. These data suggest that binding of T5 to loop 322–355 of FhuA, which constitutes the T5 binding site, unmasks an inner channel in FhuA. Both T5 and ferrichrome bind to the closed state of the channel but only T5 can trigger its opening.

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“…Szabo et al (19) have provided some evidence supporting the proposal of Chinnadurai and McCorquodale (4) that the C2 gene product is a phage "sigma-like" factor, which, as a consequence of binding to Escherichia coli RNA polymerase, permits the host enzyme to recognize the late class of T5 DNA promoters. The gene D15 product is known to be a nuclease (6,9). Enzymatic studies of partially purified D15 nuclease have shown that the protein possesses 5'-*3' exonuclease activity on both singleand double-stranded DNA (6,14).…”

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confidence: 99%

“…Single amber mutants of T5+ in either gene C2 (T5C2amM142a) or gene D15 (T5D15amH50) were provided by D. J. Mc-Corquodale. The protein products produced by phage genes D15 and C2 are thought to be a nuclease (6,9) and a "a" factor, which interacts with E. coli RNA polymerase (4,19), respectively.…”

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Role of the T5 gene D15 nuclease in the generation of nicked bacteriophage T5 DNA

Moyer¹,

Rothe²

1977

J Virol

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The processing of newly replicated concatameric T5 DNA into both single stranded DNA chains of unit length and single-stranded fragments of sizes comparable to those found in mature T5 virion DNA occurs in the absence of late T5 protein synthesis. The formation of unit-length, single-stranded DNA chains does not require the early T5 gene D15 nuclease; however, the subsequent formation of the single-stranded fragments does require that the D15 nuclease be functional. A reexamination of the properties of the purified D15 nuclease under a variety of conditions showed that, in addition to functioning as a 5'->3' exonuclease, the enzyme can also introduce endonucleolytic scissions into mature T5 DNA in a reaction that requires duplex T5 DNA and preexisting, singlestranded interruptions.The location, origin, and function of the specific, single-stranded interruptions (nicks) contained within the genome of bacteriophage T5 DNA have attracted considerable attention for a number of years. The proposed function of the nicks currently centers around their putative role in transcription of late T5 genes. This transcription is known to require the products of both the T5 "early" genes, C2 and D15 (3, 4). Szabo et al. (19) have provided some evidence supporting the proposal of Chinnadurai and McCorquodale (4) that the C2 gene product is a phage "sigma-like" factor, which, as a consequence of binding to Escherichia coli RNA polymerase, permits the host enzyme to recognize the late class of T5 DNA promoters. The gene D15 product is known to be a nuclease (6,9). Enzymatic studies of partially purified D15 nuclease have shown that the protein possesses 5'-*3' exonuclease activity on both singleand double-stranded DNA (6,14). T5 strains containing amber mutations in gene D15 not only fail to initiate late transcription, but also fail to introduce the single-stranded interruptions into newly replicated phage DNA (7).There are at least two mechanisms by which the gene D15 product could control the introduction of nicks into replicating T5 DNA. The first, and the most direct, hypothesis would be that the nuclease encoded by gene D15 can also function as a very specific endonuclease and thereby introduce the nicks into replicating DNA. This hypothesis would also be consistent with the proposal that a nicked DNA template is required for late transcription (11). The enzyme must, however, possess very little, if any, gen-eral endonucleolytic activity because the protein is unable to cleave either doubleor singlestranded circular OX174 DNA (6). Alternatively, the introduction of nicks into T5 DNA might be under more indirect control of the D15 gene. For example, gene D15 amber mutants are unable to synthesize late T5 RNA and proteins, and nicking would then be prevented with D15 mutants if either a late protein or complex of late proteins were required for this process. Our results favor the first hypothesis, and we present evidence suggesting that the D15 nuclease plays a direct role in introducing nicks into replicating T5 DNA. Nicking ...

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Genetic and Physiological Studies of Bacteriophage T5 I. An Expanded Genetic Map of T5

Cited by 76 publications

References 17 publications

Physical locus of the DNA polymerase gene and genetic maps of bacteriophage T5 mutants

Physical locus of the DNA polymerase gene and genetic maps of bacteriophage T5 mutants

FhuA, a transporter of the Escherichia coli outer membrane, is converted into a channel upon binding of bacteriophage T5.

Role of the T5 gene D15 nuclease in the generation of nicked bacteriophage T5 DNA

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