Some steps in the assembly of bacteriophage T4

Edgar, R. S.; Lielausis, I.

doi:10.1016/0022-2836(68)90008-9

Cited by 139 publications

(55 citation statements)

References 7 publications

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Section: Introductionmentioning

confidence: 99%

“…Thus, for bacteriophage T4, we know a great deal about the pathways of assembly of tail fibers (1-4), baseplates, sheaths, and cores of the tail (5-9) and assembly of the head (10). These advances have been aided greatly by the development by Edgar and Wood of methods for performing these reactions in cell-free extracts (11,12).The DNA of several bacteriophages (T4, T7, X, and P22) has been shown to replicate as molecules that are several times longer than the DNA found inside the head of the mature virus (13)(14)(15)(16)(17). It is also known that maturation of these long chains of DNA to monomeric units is closely coupled to the formation of the phage head, since cells infected with mutants that are defective in head formation accumulate these long molecules of immature DNA (18-21).…”

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confidence: 99%

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Packaging and Maturation of DNA of Bacteriophage T7 In Vitro

Kerr¹,

Sadowski²

1974

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

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We have developed an in vitro complementation assay to demonstrate packaging and maturation of DNA of phageT7. Cells of Escherichia coli B infected with an appropriate T7 amber mutant are concentrated 200-fold and iysed by freezing and thawing. Two extracts from cells infected with different amber mutants are mixed and incubated at 300. Positive complementation results in a 100-fold increase in phage titer.Using this assay we have demonstrated the packaging of phage DNA from an extract that contains no phage heads (gene 9-, 10-), within head structures present in an extract that contains no phage DNA (gene 5-). We have also demonstrated an activity in extracts that contain no phage DNA or heads (gene 5-,9, 10-), which complements gene 19-infected cells. We have proven that this activity is due to the gene-19 product by showing that the activity is temperature-sensitive if the extract is made from cells infected with a mutant having a temperature-sensitive mutation in gene 19. This assay should be useful in elucidating the mechanism of packaging and maturation of DNA of phage T7.In recent years there has been rapid progress in our understanding of the mechanisms by which complex bacteriophages are assembled. Thus, for bacteriophage T4, we know a great deal about the pathways of assembly of tail fibers (1-4), baseplates, sheaths, and cores of the tail (5-9) and assembly of the head (10). These advances have been aided greatly by the development by Edgar and Wood of methods for performing these reactions in cell-free extracts (11,12).The DNA of several bacteriophages (T4, T7, X, and P22) has been shown to replicate as molecules that are several times longer than the DNA found inside the head of the mature virus (13)(14)(15)(16)(17). It is also known that maturation of these long chains of DNA to monomeric units is closely coupled to the formation of the phage head, since cells infected with mutants that are defective in head formation accumulate these long molecules of immature DNA (18-21).However, our knowledge of the molecular mechanisms whereby the immature phage DNA enters the head ("DNA packaging") and is cleaved to monomeric length ("DNA maturation") has been limited, in part, by the inability to perform these reactions in vitro. Recently, however, assays have been developed to demonstrate packaging of phage DNA in vitro. Kaiser and Masuda (22) showed that exogenous lambda DNA could be packaged by extracts from induced lysogens and Hohn and Hohn (23) have demonstrated that head-related particles containing no DNA ("petit lambda") can be filled with DNA in vitro. Finally, the experiments of Pruss, Goldstein, and Calendar (24) showed that the size of the head of phage P2 is determined solely by the proteins involved and not by the phage DNA.Bacteriophage T7 may be a convenient system in which to study DNA packaging and maturation because it is a 3545 relatively simple virus for which there is a great deal of information about its genetics and physiology (25). The DNA of phage T7 is a unique, terminall...

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Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

Packaging and Maturation of DNA of Bacteriophage T7 In Vitro

Kerr¹,

Sadowski²

1974

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

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“…In bacteriophage T4D+, these mutants have allowed the role of specific structures in the assembly pathway to be determined (4,5). Several potential intermediates in head morphogenesis (6)(7)(8)(9) that are found at the terminal steps of head maturation have been isolated. These intermediates are the gene 49-defective unfilled head particle, blocked in DNA packaging (8,9) and the gene 13-defective filled head particle, blocked in tail attachment (6,7).…”

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confidence: 99%

Bacteriophage T4 Head Morphogenesis. Isolation, Partial Characterization, and Fate of Gene 21-Defective Tau-Particles

Luftig

Lundh

1973

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

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A lysozyme-detergent procedure was developed for isolation of tau-particles from cells infected by gene-21 mutants of T4 bacteriophage. These particles have a sedimentation coefficient of 440 h 10 S. They contain less than 1% detectable nuclease-resistant DNA, are smaller (650 X 850 A) than normal bacteriophage heads (800 X 1100 A), and exhibit two major bands on 7.5% Na dodecyl sulfate-acrylamide gels. The more prominent band (55,000 daltons) corresponds to the uncleaved, major capsid polypeptide (P23); the other band (32,000 daltons) corresponds to the gene-22 product (P22). Temperatureshift experiments with cells infected with tsN8 (gene 21) mutants were used to study the fate of tau-particles accumulated under nonpermissive conditions. 50 Min after ts N8-infected cells were shifted from the nonpermissive (41.50) to the permissive (250) temperature, a phage burst occurred that was 75% of that observed with wild-type phage. However, in "pulse-chase" temperature-shift experiments, the radioactive tau-particle peak only slightly decreased (by 10-14%) by 50 min after the shift, whereas an increased amount of radioactivity (about four times as much as the tau-particle decrease) appeared in phage particles. The results suggest that at least two pools of head polypeptides coexist in cells infected with gene-21 mutants. One pool is composed of head subunits assembled into tau-particles, which are mostly aberrant structures; the second pool is composed of head subunits that are incorporated into mature phage when the gene-21 product becomes functional.

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“…The tail fails to join the head in the absence of gp13 and gp14 as well as gp3 and gp15 (7,10). Coombs and Eiserling have observed that some knob-like structure, which contains gp13 and gp14, is formed on top of the tail during the prolonged cultivation of the cells infected by head-deficient mutants (6).…”

Section: Downloaded Frommentioning

confidence: 99%

“…Completion of the assembly of the tail of bacteriophage T4 requires two gene products, gp15 and gp3 (7,10). Both 15-lacking and 3-lacking tails have an unstable tail sheath, lack the connector, and are consequently incompetent to join the head (10,11).…”

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confidence: 99%

P15 and P3, the Tail Completion Proteins of Bacteriophage T4, Both Form Hexameric Rings

Kanamaru

Chaidirek

et al. 2003

J Bacteriol

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Two proteins, gp15 and gp3 (gp for gene product), are required to complete the assembly of the T4 tail. gp15 forms the connector which enables the tail to bind to the head, whereas gp3 is involved in terminating the elongation of the tail tube. In this work, genes 15 and 3 were cloned and overexpressed, and the purified gene products were studied by analytical ultracentrifugation, electron microscopy, and circular dichroism. Determination of oligomerization state by sedimentation equilibrium revealed that both gp15 and gp3 are hexamers of the respective polypeptide chains. Electron microscopy of the negatively stained P15 and P3 (P denotes the oligomeric state of the gene product) revealed that both proteins form hexameric rings, the diameter of which is close to that of the tail tube. The differential roles between gp15 and gp3 upon completion of the tail are discussed.

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Some steps in the assembly of bacteriophage T4

Cited by 139 publications

References 7 publications

Packaging and Maturation of DNA of Bacteriophage T7 In Vitro

Packaging and Maturation of DNA of Bacteriophage T7 In Vitro

Bacteriophage T4 Head Morphogenesis. Isolation, Partial Characterization, and Fate of Gene 21-Defective Tau-Particles

P15 and P3, the Tail Completion Proteins of Bacteriophage T4, Both Form Hexameric Rings

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