John W. Drake

Properties of ultraviolet-induced rII mutants of bacteriophage T4

The induction of r II mutants by u.v. irradiation of intracellular bacteriophage T4 and the u.v.-induced reversion of r II mutants are described. A collection of induced mutants has been mapped, revealing a characteristic distribution which is related to other properties of the mutants. The mutants were classified according to their susceptibilities to induced reversion by 2-aminopurine, 5-bromouracil, hydroxylamine, profiavin and u.v. irradiation. Approximately half of the mutants were induced to revert by the base analogues (transition mutants) and nearly all of these behaved as if they contained an adenme–thymine base pair at the mutant site. The guanine– hydroxymethylcytosine base pair is therefore the major mutagenic target in the induction of transition mutations by u.v. irradiation. Most of the remainder (non-transition mutants) were induced to revert by proflavin, but did not respond to base analogues. They resemble the acridine-type mutants of Crick, Barnett, Brenner & Watts-Tobin (1961) ; they behaved as if they contained very small additions or deletions at the mutant sites. Revertants induced by the test mutagens were screened for suppressor mutations by examination of phenotypes and by backcrosses. False revertants occurred in many cases. The false revertants arising among transition mutants were of a different type from those arising among non-transition mutants.

MUTAGENIC EFFECTS OF THYMINE DIMERS IN BACTERIOPHAGE T4.

Sensitized irradiation of bacteriophage T4 v , which forms, almost exclusively, thymine dimers in the phage DNA, produces 2.4 × 10 −4 r mutants per lethal hit. Nearly all of the mutations can be reversed by photoreactivation. The rII mutants produced by sensitized irradiation are primarily frameshift mutants and G·C → A·T transitions. Thymine dimers must be premutational lesions for both types of mutations. These results are inconsistent with models for ultraviolet mutagenesis involving base changes opposite the photochemical lesions but do support more indirect mechanisms.

A genetic assay for transversion mutations in bacteriophage T4

SummaryA method is described for recognizing many transversion mutations arising within an ochre (UAA) codon in therII gene of bacteriophage T4. Most of therII ochre mutants used revert only by base pair substitution mutations in their DNA. Transition mutations within the UAA codon produce either an amber (UAG) mutant, an opal (UGA) mutant, or a glutamine (CAA) revertant. All three can be isolated and identified when the ochre mutant is treated with a mutagen which produces transitions. On the other hand, many nonsuppressed revertants with phenotypes different from the CAA revertant arose as transversions. The method has been used to score the spontaneous production of transversions at A:T base pair sites.

Host-controlled variation in NDV.

Abstract Newcastle disease virus grown in embryonic chicken fibroblasts (Fb-V) differed significantly from chorioallantoic membrane-grown virus (CAM-V) in its sensitivity to inactivation by heat, acid, and ultraviolet irradiation. Alteration of CAM-V to Fb-V, and reversion of Fb-V to CAM-V, occurred under conditions of normal yield in a single growth cycle. The selection of pre-existing mutants in the two populations was excluded. Medium or lysate components, as well as the mode of tissue culture growth, did not contribute to the modification. It is suggested that the host-induced characteristics of the virus may be due to inclusion of materials in the virus particle which are host specific.

Cultivation of duck hepatitis virus in tissue culture.

Summary Duck hepatitis virus was passed serially 7 times through monolayer chicken embryo liver cell cultures. Multiplication of virus was demonstrated by assay in embryonated chicken eggs and by gel diffusion precipitin test. Microscopic examination of stained tissue culture cells failed to reveal CPE.

Bacteriophage T4 transformation: An assay for mutations induced in vitro☆

Abstract A substantially improved method has been developed to assay bacteriophage T4 transformation with native DNA. The most important change was the substitution of 0.02 M Tris for the phosphate buffer. The efficiency of joint transformation (transformants/DNA molecule) of amH17amN116 , which spans about 7% of the T4 genome and includes both r II genes, is about 10 −3 . The assay was designed to detect in vitro mutagenesis in the r II genes of T4. To demonstrate this aspect of the assay, wild-type T4 DNA was mutagenized with hydroxylamine, then assayed for transformation of amH17amN116 . The frequency of induced r mutants among the am + transformants increased linearly with increasing hydroxylamine dose, while the transforming DNA was inactivated at a progressively decreasing rate. Under conditions that resulted in 3.5% survival, 20% of the am + transformants were r mutants, a 200-fold increase in r frequency over background. Some of the r mutants induced by hydroxylamine were actually r I, lying well outside of the selected region, but the fraction of r I mutants ( r I total r ) decreased markedly with increasing lethal hits. This phenomenon, together with the inactivation kinetics, is explained in terms of a diminishing target size.

Mutagen Screening with Virulent Bacteriophages

Virus systems, and particularly those employing the T-even bacteriophages, the single-stranded DNA bacteriophages, and the temperate bacteriophage λ, have occupied the center of the molecular biologist’s arena for up to three decades. The reasons for the popularity of virus systems are as relevant now as they were in the 1930s. Viruses often excel in those properties which make microorganisms in general highly useful for genetic studies. They are easy to grow. They exhibit extremely short generation times. (The bacteriophage T4 growth cycle, encompassing about 30 min at 37°C and resulting in the release of several hundred progeny particles, corresponds to an average doubling time of about 3 to 4 min.) Genome storage is extremely simple. Virus stocks are stable for many years in the refrigerator and do not accumulate many spontaneous mutations (Drake, 1966). Extremely large populations (for instance, 1011 to 1014 particles) are easily obtained, and a variety of techniques have been developed for selecting rare particles representative of events such as mutation. The relative chemical simplicity of bacteriophages, together with certain chemically unique aspects of their reproduction, has made possible a more detailed dissection of their molecular genetics than has been achieved even in the equally well-studied bacterium Escherichia coli. The formal genetics of several virus systems has been developed to a highly detailed and sophisticated level, making possible numerous genetic manipulations which render the analysis of processes such as mutation highly profitable. Finally, the pursuit of bacteriophage genetics, including even many aspects of their molecular genetics, is relatively inexpensive, particularly in comparison with metazoan systems.

Is bacteriophage T4 DNA polymerase involved in the repair of ultraviolet damage

Abstract Defects in bacteriophage T4 gene 43 (DNA polymerase) do not enhance the ultraviolet sensitivity of the virus, even when the host DNA polymerase I is also defective. Methods are described for markedly enlarging T4 plaque sizes.