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Pulse-chase analysis of the in vivo assembly of the bacteriophage T4 tail.

Abstract：

:The in vivo assembly pathway of the complex tail of bacteriophage T4 virus was determined using pulse-chase analysis as a non-invasive alternative to the in vitro experiments previously used to map assembly. Bacteriophage T4 mutants defective in head assembly were used to infect cultures of Escherichia coli in order to study tail assembly in isolation. Beginning with the onset of late protein synthesis, the cultures were labeled continuously with [(3)H]leucine to normalize against subsequent sample losses. After completed tails had begun to accumulate at a constant rate, the cultures were pulsed with [(35)S]methionine, and then chased. Completed tails were purified at one minute intervals for the next 30 minutes and their proteins separated electrophoretically and counted by liquid scintillation. Total (35)S incorporation into each protein rose and then leveled off as the chase of unlabeled methionine flushed the label through the pools of soluble proteins and assembly intermediates and into completed tails. The inflection point in the sigmoidal (35)S-incorporation curve of each protein marks the maximal uptake of (35)S within that pool just before the effect of the chase becomes apparent and the curve begins to level off. The length of the delay in the apparent chase time reflects the position of that protein in the pathway. The closer the assembly point to the end of the pathway, the sooner the chase appears, revealing the relative order of assembly. As predicted, tail completion proteins such as gp18 (tail sheath) and 19 (tail tube) show the earliest inflection, while those earlier in the pathway take longer to chase. Of the 17 tail proteins analyzed, 14 are in agreement with the established in vitro pathway. The other three, gp15, gp10 and gp53, have helped us to develop a model that offers a plausible explanation for their altered chase times.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Ferguson PL,Coombs DH

doi

10.1006/jmbi.2000.3551

keywords:

subject

Has Abstract

pub_date

2000-03-17 00:00:00

pages

99-117

issue

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(00)93551-4

journal_volume

297

pub_type

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Pulse-chase analysis of the in vivo assembly of the bacteriophage T4 tail.