rII Mutants Research Articles

X-ray-induced Mutations in Extracellular Phage

IONIZING radiation produces mutations in a wide variety of living organisms, but its effectiveness as a mutagenic agent for extracellular virus particles is uncertain1–4. We investigated the mutagenic effects of ionizing radiation on free virus particles using the sensitive screening system provided by rII mutants of bacteriophage T4 (ref. 5). In this report, results will be described which show that r+II reverse mutations in T4B rII phage can be induced by X-irradiation of aqueous suspensions of the phage.

Suppression of amber and ochre rII mutants of bacteriophage T4 by streptomycin.

Orias, E. (University of California, Santa Barbara), and T. K. Gartner. Suppression of amber and ochre rII mutants of bacteriophage T4 by streptomycin. J. Bacteriol. 91:2210-2215. 1966.-Streptomycin-induced suppression of amber and ochre rII mutants of phage T4 was studied in a streptomycin-sensitive strain of Escherichia coli and four nearly isogenic streptomycin-resistant derivatives of this strain, in the presence and in the absence of an ochre suppressor. Most of the 12 rII mutants tested were suppressed by streptomycin in the streptomycin-sensitive su(-) strain. This streptomycin-induced suppression in the su(-) strain was eliminated by the independent action of at least two of the four nonidentical mutations to streptomycin resistance. In two of the su(+)str-r strains, streptomycin markedly augmented the suppression caused by the ochre suppressor. In those su(-)str-r hosts in which significant streptomycin-induced suppression could be measured, the amber mutants were more suppressible than the ochre mutants.

Studies on the physiological defect in rII mutants of bacteriophage T4

The inability of r II mutants of bacteriophage T4 to multiply in strains of Escherichia coli lysogenio for λ is shown to be due to an inhibition by monovalent cations usually present in culture media. If monovalent cations are omitted from the medium, r II mutants and the wild-type phage multiply normally, provided sufficient osmotic pressure is maintained by, for example, salts of divalent cations or sucrose. One effect of inhibitory concentration of monovalent cations is to avert the uptake of magnesium ions by r II-infected cells early in the latent period. Evidence is presented that a deficit in phosphorylation might be the primary cause of cessation of phage development in r II-infected E. coli KB: (1) Incorporation of 32 P into ATP (as well as into other phosphate-containing compounds) drops after r II infection, while the uptake of inorganic phosphate by cells continues normally. (2) Simultaneous labeling by 32 P and [ 14 C]uridine showed that the failure in phosphorylation of nucleotides precedes the failure in the synthesis of nucleid acid. (3) The ATP level of bacteria drops after infection with r II. (4) The drop in phosphorylation activity in r II-infected cells appears immediately after infection, whereas other metabolic activities continue normally for about 10 minutes. Various enzymes concerned in ATP metabolism were examined, but all showed about the same activity levels in r + -and r II-infected cells. An extensive leakage of NAD from r II-infected cells was observed, but the relation between loss of NAD and the failure in phosphorylation is not clear.

Absence of mutagenic effects of 3- and 6-alkyl-5-bromouracil herbicides on a bacteriophage.

SINCE the 1962 report by Bucha et al.1, who described the herbicidal activity of certain uracils substituted in the 3 and 6 positions, one of this class of chemicals, 5-bromo-3-sec-butyl-6-methyluracil (bromacil), has gained wide acceptance as an industrial herbicide. Investigations of the mode of action of this herbicide2,3 have shown that it is a potent direct inhibitor of photosynthesis acting at the chloroplast level. The chemical analogy between 5-bromouracil and the foregoing 3,6-substituted 5-bromouracil suggested the possibility that it might be incorporated into DNA and thereby act as a mutagen. McGahen and Hoffmann4,5 were unable to demonstrate incorporation of 5-bromo-3-sec-butyl-6-methyluracil (IV) either into the DNA of a thymineless Escherichia coli (15T−) or into mouse liver DNA under conditions in which 5-bromouracil (5-BU) was incorporated. Their investigations, however, did not rule out the possibility of indirect mutagenic activity. The subject of this report is a comparison of the effects of 5-BU and 5-bromouracils with several different alkyl substituents at the 3 and/or 6 positions on the back-mutation of an aminopurine-induced rII mutant of coliphage T4.

Mutagenesis of super-suppressors in yeast.

A new class of mutations, referred to as “nonsense” mutations, has been described in phages and bacteria in recent years (Benzer and Champe, 1962; Garen and Siddiqi, 1962). It is characterized by two major properties: a drastic effect on protein synthesis (A cistron of the rII region in phage T4, Benzer and Champe, 1962; alkaline phosphatase in E. coli, Garen and Siddiqi, 1962) produced by a polypeptide chain termination at the site of the mutational lesion (Sarabhai et al., 1964), and by responsiveness to external suppressors which can give rise to a wild or pseudo-wild phenotype (ambivalent rII mutants, Benzer and Champe, 1961; host-dependent defective mutations in phage λ, Campbell, 1961; alkaline phosphatase in E. coli, Garen and Siddiqi, 1962; amber mutations in phage T4, Epstein et al., 1963).

Bacteriophage-induced functions in Escherichia coli K (lambda) infected with rII mutants of bacteriophage T4.

Rutberg, Blanka (Karolinska Institutet, Stockholm, Sweden), and Lars Rutberg. Bacteriophage-induced functions in Escherichia coli K(lambda) infected with rII mutants of bacteriophage T4. J. Bacteriol. 91:76-80. 1966.-When Escherichia coli K(lambda) was infected with rII mutants of phage T4, deoxycytidine triphosphatase, one of the phage-induced early enzymes, was produced at initially the same rate as in r(+)-infected cells. Deoxyribonuclease activity was one-third to one-half of that of r(+)-infected cells. This lower deoxyribonuclease activity was observed also in other hosts or when infection was made with rI or rIII mutants. Presence of chloramphenicol did not allow a continued synthesis of phage deoxyribonucleic acid in rII-infected K(lambda). No phage lysozyme was detected nor was any antiphage serum-blocking antigen found in rII-infected K(lambda). It is suggested that the rII gene is of significance for the expression of phage-induced late functions in the host K(lambda).

The effects of polyamines on the replication of T4 rII mutants in Escherichia coli K12 (λ)

While rII mutants of bacteriophage T4 can replicate in Escherichia coli K12S, replication is blocked when this host is lysogenized by λ phage. Polyamines, Mg ++, and streptomycin increase the yield of rII deletion mutants in K12(λ) to varying degrees below the normal yield obtained with wild-type phage. Of a homologous series of diamines, 1,7-diaminoheptane is about 7 times more effective than 1,4-dia-aminobutane (putrescine) or Mg ++. The critical portion of the latent period for diamine stimulation is between 10 and 20 minutes after infection. In the absence of diamine, UV induction of λ replication permits subsequent rII replication; further stimulation by 1,7-diaminoheptane still occurs under these conditions. Diamines and Mg ++, unlike UV irradiation, do not bring about induction of λ phage. Uninfected cells of K12(λ) and K12S were found to contain nearly identical amounts of putrescine. Several independent methods were used to examine permeability differences between the two types of infected cells; in all cases, no significant differences in permeability to large or small molecules were found. Therefore, the inability of rII mutants to replicate in K12(λ) cannot be attributed to generalized leakage resulting from phage infection. Several alternatives are suggested for the mode of action of these substances.

MARKER STIMULATION OF CROSSING OVER: A TEST OF THE THEORY YIELDING NEGATIVE RESULTS AND USING rII MUTANTS OF PHAGE T4

MARKER STIMULATION OF CROSSING OVER: A TEST OF THE THEORY YIELDING NEGATIVE RESULTS AND USING <i>rII</i> MUTANTS OF PHAGE T4

A type of λ prophage unable to confer immunity

A “cryptic” λ prophage has been detected in an apparently cured strain of K12 (λ). This strain, called U173, is normally sensitive to λ mutants, to other phages related to λ and to rII mutants of T4. Induction with ultraviolet light has no effect either in producing active phages, or in promoting lysis of the culture. Rescue of λ wild-type alleles has been possible upon infection of this strain with certain λ mutants. The extension of the “cryptic” fragment covers about one-half or more of the map of λ.

Molecular mechanisms of genetic recombination in bacteriophage: I. Effect of KCN on genetic recombination of phage T4

The effects of various metabolic inhibitors on genetic recombination of bacteriophage T4 were studied. Treatment of mixedly-infected cells with 8 × 10−3 M -KCN increased the frequency of recombination between two rII mutants in progeny formed after release from inhibition. During a prolonged treatment with these inhibitors the infected cells produced progeny particles which included recombinants in high proportions. A similar effect was observed by treatment with 8 × 10−3 M -As205, 2 × 10−3 M -NaN3 or 10−2 M -2,4-dinitrophenol. Keeping the infected cells in buffer also increased the frequency of recombinants in progeny subsequently formed after addition of nutrients. During treatment of infected cells with KCN very little DNA synthesis took place. After treatment of cells infected with 32P-labelled phage particles with KCN, infective centres did not change in sensitivity to decay of incorporated 32P. It is therefore concluded that phage chromosomes tend to recombine rather than to grow during the treatment. These inhibitors could be used to separate recombination from replication of phage chromosomes

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.

A change from nonsense to sense in the genetic code.

For the genetic information in a cistron to be translated into a polypeptide chain each coding unit in the nucleotide sequence must correspond to one of the twenty or so amino acids. If not every possible sequence corresponds to an amino acid, mutations that substitute one base for another could, in certain cases, cause the continuity of the information to be interrupted, and such mutations would block completion of the polypeptide chain. By virtue of a mutant with special properties, it is possible to identify nonsense mutations within the A cistron of the rII region of phage T4. In this paper the criterion for nonsense is applied to certain rII mutants [1], i.e., ones whose phenotypes can be reversed by suppressor mutations in the bacterial host, Escherichia coli. The results show that an ambivalent mutation that behaves like nonsense in one bacterial host may nevertheless make sense in a second (suppressor containing) host. This suggests that a suppressor mutation in the bacterium can result in addition to the cell's dictionary of a new sensible coding unit, constituting a change in the genetic code of the bacterial cell.

Reversal of mutant phenotypes by 5-fluorouracil: an approach to nucleotide sequences in messenger-RNA.

Genetic information in DNA is apparently expressed via transcription into RNA messengers [1-5,10] which in turn act as the templates for protein synthesis. Thus, incorporation of base analogues into messenger-RNA could lead to errors in the reading of the message into an amino acid sequence. The effect would be an alteration of phenotype without a permanent change in the DNA genotype. A promising analogue for this purpose is 5-fluorouracil (5FU), which is readily incorporated into RNA, mostly in place of uracil [6]. Modification of proteins by 5FU has been reported by Naono and Gros [7] and by Bussard et al. [8] who found that the enzymes alkaline phosphatase and β-galactosidase synthesized in the presence of the analogue are abnormal. A very sensitive method for the detection of induced errors in the translation of genetic information is to begin with a mutant that is defective in some function so that errors causing the appearance of a small amount of activity can be easily observed. This approach has been successful [9] using rII mutants of phage T4, which are ordinarily unable to grow on strain K of E. coli. Addition of 5FU after infection of the cell can partially reverse the phage mutant phenotype, leading to active development of the phage. The response is highly specific, occurring only with certain rnI mutants and not others within the same cistron. Since, after phage infection, synthesis of ribosomal RNA and S-RNA is almost totally arrested [10], it is plausible that the effect of 5FU on phage mutants is due to its incorporation into messenger-RNA.

An active cistron fragment

An unusual rII mutant of phage T4 is described, in which a terminal portion of a cistron has been deleted without impairing the function of the remaining fragment. This suggests that the deleted section is non-essential. Nevertheless, mutations within this segment which do impair the function can be observed.

Further studies on induced reverse mutations of phage T4 rII mutants

Further studies on induced reverse mutations of phage T4 rII mutants

Detecting reversion in T4rII bacteriophage to r plus induced by ultra-violet irradiation.

Details of a test developed for estimating the extent of reversion of bacteriophage T4B from rII to r+ induced by ultravolet irradiation are given. T4rII mutants were obtained by ultraviolet irradiation of T4Br phage, and 12 hr cultures of E. coli and K-12 ( lambda ) were used in assays of rII and r+ phage, respectively. The phage were diluted and irradiated in media which contained, per liter ion-free water, 10 g tryptone, 8 g sodium chloride, and 1 g glucose. Each of 12 rII mutant stocks was assayed on B and on K-12 ( lambda ) to determine accurately the titer of total and of r+ phage. It was found that 5 of the 12 mutants revert to r+ when irradiated with ultravolet. The extent of reversion ranges from 1.81 to 4.2 x 105 r+ per 106 survivors.

AMBIVALENT rII MUTANTS OF PHAGE T4

The rII mutants of phage T4 differ from the standard type in being inactive on strains of Escherichia coli lysogenic for phage X. While the mutants attach to the cells and inject their DNA, progeny fail to appear. In this paper it is shown that one or another subset of the rII mutants can become active as a result of modification of the host, either by mutation or by the action of 5-fluorouracil. The effects are characterized by extreme specificity, applying only to certain rII mutations at certain points within the two rII cistrons. The phenomenon resembles what has been called, in many other systems, allele-specific suppression [1], in which a mutation outside a genetic region controlling a particular function suppresses the effects of some of the mutations within the region. An advantage of the phage system is that a given genetic message, in the form of a phage mutant, can be readily inserted into different hosts. The stimulus for the present investigation derives from the possibility that modifications of the host affecting the expression of specific phage mutations might be a key to the detection of alterations in the components of the cellular system for translating genetic information.

DNA synthesized in Escherichia coli K12 (lambda) after infection with an rII mutant of bacteriophage T4.

Abstract Deoxyribonucleic acid (DNA) synthesized in Escherichia coli K12 (λ) after abortive infection with rII mutants of phage T 4 was found to contain 5-hydroxymethylcytosine without any cytosine, as characteristic of phage DNA. It appears that physiological defect associated with the rII mutation is not a failure to form deoxycytidylate hydroxymethylase.

Physiological effects of rII mutations in bacteriophage T4.

Abstract The rII mutants of coliphage T4 are unable to produce progeny in host cells lysogenic for phage λ, if the infected cells are incubated in a medium containing a low concentration of Mg ++ ions. If a high Mg ++ concentration (0.05 M ) is provided, a normal yield of phage is obtained. In the low Mg ++ medium, phage development proceeds normally during the first 10 minutes after infection, as evidenced by the appearance of various phage-specific activities; also, deoxyribonucleic acid (DNA) and protein are synthesized at the normal rate during this period. Phage development, including DNA and protein syntheses, comes to an abrupt halt after 10 minutes, but can be restored by the addition of Mg ++ to the medium. Mg ++ is not effective in promoting phage development if aerobic metabolism is blocked.

ON THE TOPOGRAPHY OF THE GENETIC FINE STRUCTURE

In an earlier paper [1], a detailed examination was made of the structure of a small portion of the genetic map of phage T4, the rII region. This region, which controls the ability of the phage to grow in Escherichia coli strain K, consists of two adjacent cistrons, or functional units. Various rII mutants, unable to grow in strain K, have mutations affecting various parts of either or both of these cistrons. The topology of the region; i.e., the manner in which its parts are interconnected, was intensively tested and it was found that the active structure can be described as a string of subelements, a mutation constituting an alteration of a point or segment of the linear array. This paper is a sequel in which inquiry is made into the topography of the structure, i.e., local differences in the properties of its parts. Specifically, are all the subelements equally mutable? If so, mutations should occur at random throughout the structure and the topography would be trivial. On the other hand, sites or regions of unusually high or low mutability would be interesting topographic features.