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

Abstract

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.

Maturation of the head of bacteriophage T4. V. A possible DNA packaging mechanism: In vitro cleavage of the head proteins and the structure of the core of the polyhead

“…Other data suggest that gene 2 1 codes for a protein which may be the proteolytic enzyme which cleaves protein P22 (19,23) (see below: In vitro cleavage of the head proteins of partially purified polyheads). and also P22, IPIII, IPII, IPI, and P20 (24,38,40). The particles isolated from 24 defective cells have a similar composition (Laemmli, unpublished).…”

“…[Similar experiments with 2 1 infected cells were negative (24,38,40)1. An important control established that the 7-particles remain intact and do not disperse and reassemble following activation of gene 24 (43).…”

“…The 7-particles isolated from 2 1 defective cells have a sedimentation coefficient of about 420 S and contain mostly the uncleaved protein P23, and the minor components P22, IPIII, and P20 (24,38,40). They appear t o be bound to the inner membrane ( l ) , and a detergent (Nonidet P40) is required t o release these particles (21,38).…”

Isolation and characterization of bacteriophage T4 mutant preheads

“…Mutations in two T4 head genes 21 and 24 are known to accumulate normal-appearing prehead-like particles (4, 8) which contain no DNA (13,16). Unfortunately, these particles are membrane bound and fragile, and until now it has not been possible to isolate them in sufficient quantity and in a sufficiently good state of preservation to determine accurately their protein composition and detailed structure.…”

Conditional-lethal mutations that suppress genetic defects in morphogenesis by altering structural proteins.

“…The conditional-lethal phenotypes of the new mutations, in turn, allowed us to show by standard genetic methods that in many instances the new mutation is in a gene other than the one in which the original mutation lies. Having confined our attention to conditional-lethal mutations, we are in a position to take advantage of their additional virtues: they can be crossed into diverse genetic backgrounds at will, they can be used in temperature-shift experiments in vivo and in vitro (15), and they can-be used in schemes of enzyme or protein-complex purification (16,17).…”