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Part 4

Phage T4 was the first organism for which all the essential genes have been described. This was possible through the use of two kind of conditional lethal mutations that can occur in practically any gene. Amber mutations introduce the UAG stop codon. Two factors contribute to the general use of amber mutations. Many strains derived from the original K-12 strain carry amber suppressors and their efficiency is very high. Temperature-sensitive mutations only allow growth at the permissive temperature. They occur in most proteins that unfold at the restrictive temperature and are often degraded; they also occur in tRNA genes, where they destabilize the structure by preventing base pairing. Cells infected under non permissive conditions were analyzed biochemically and by electron microscopy. Mutations that prevent replication of the viral DNA all prevented the synthesis of viral components; they define the T4 early genes. Mutations in structural genes allow DNA replication but prevent the appearance of one or more phage components. However, mutations in head genes allow formation of tails and fibers and reciprocally, tail mutants accumulate heads and fibers. With fibers mutants, head and tail assemble but are not infectious.When - particles are incubated with an extract containing fibers but no heads (or no tails), the components rapidly, spontaneously and efficiently reassemble to yield infectious viral particles. Extracts form mutant infections can be classified as head-donor or tail-donors. In all cases, the genotype of the active virus is determined by the heads, as expected. These experiments were instrumental in defining the assembly process of phage T4: three independent assembly lines (head, tail and fiber) converge to form an infectious virus. They also provided a functional assay for the purification of structural components of the phage.

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