Vernon C. Bode

Effects of ENU dosage on mouse strains.

Abstract. The germline supermutagen, N-ethyl-N-nitrosourea (ENU), has a variety of effects on mice. ENU is a toxin and carcinogen as well as a mutagen, and strains differ in their susceptibility to its effects. Therefore, it is necessary to determine an appropriate mutagenic, non-toxic dose of ENU for strains that are to be used in experiments. In order to provide some guidance, we have compiled data from a number of laboratories that have exposed male mice from inbred and non-inbred strains or their F1 hybrids to ENU. The results show that most F1 hybrid animals tolerate ENU well, but that inbred strains of mice vary in their longevity and in their ability to recover fertility after treatment with ENU.

Three ENU-induced alleles of the murine quaking locus are recessive embryonic lethal mutations

Trois nouveaux alleles du phenotype «quaking» ont ete selectionnes par mutagenese par ENU. Ces alleles sont des mutations letales embryonnaires, qui ne se complement pas entre elles

Biochemical Defect of the hph‐1 Mouse Mutant Is a Deficiency in GTP‐Cyclohydrolase Activity

A hyperphenylalaninemic mouse mutant, hph‐1, has been identified in the progeny of mice treated with the mutagen ethylnitrosourea. Phenylalanine hydroxylase activity levels in mutant liver lysates are reduced relative to normal, but correction for the amount of enzyme protein present demonstrates that the specific activity of this enzyme is normal in mutant mice. Quinonoid‐dihydropteridine reductase activity is also normal. GTP‐cyclohydrolase activity levels are essentially absent early in life and greatly diminished later in life. This finding has significant implications for the study of catecholamine neurotransmitter synthesis because GTP‐cyclohydrolase catalyzes an important step in the de novo synthesis of tetrahydrobiopterin, an enzyme cofactor required for the synthesis of 3,4‐dihydroxyphenylalanine (DOPA) and serotonin.

Induction of new mutations in a mouse t-haplotype using ethylnitrosourea mutagenesis.

N-ethyl-N-nitrosourea (ENU) was used to induce mutations within the r-haplotype of the mouse to make possible a further study of gene arrangement in /-mutants and to provide potential landmarks for cloning and sequence studies in the region. Two independent mutants were isolated for each of three loci in the /-region, brachyury (T), quaking {qk), and tufted (//). The new T alleles produce tailless mice when a tct mutation is present in trans. The new qk alleles are recessive and homozygous lethal. They are viable, male fertile, and cause seizures and quaking when paired with the qk mutation which previously defined the locus. The tf mutations are recessive and phenotypically similar to the mutant alleles available in non-/ chromosomes. The mutations were induced in the /-haplotype at an average per locus frequency of 1 in 1500. Their isolation demonstrates the power of this technique for obtaining the specific mouse mutants that are needed to genetically dissect a complex mammalian system.

Events in lambda injection between phage adsorption and DNA entry

Injection kinetics indicate that, in the time period from irreversible adsorption through stable uptake of λ DNA, several reactions must occur in or between the phage and the bacterium. The events are grouped in three steps: lag, triggering, and uptake. The results further suggest that an exchange of information occurs between the distal and the proximal ends of the phage tail. The triggering of DNA injection from λ preadsorbed to Escherichia coli is essentially immediate at 37° but occurs only after a lag of 6 min at 25° or 10 min at 23°. At 15°, injection does not occur. When phage-cell complexes are incubated at 37° in 10 mM putrescine-10−5 M Mg2+, the lag functions are completed but injection is blocked. After resuspension in normal buffer (10 mM Mg2+, 10 mM phosphate, pH 7.1), DNA injection is triggered without a lag, even at 12.5°. It still does not occur at 4°. Events during the lag involve more than a simple interaction between the tail tip and the outer membrane of the bacterial cell wall. When tails alone are attached to cells, incubated at 37 or at 4°, and then joined with heads in vitro at 4°, the resulting phage particles inject at 25° with the same kinetics, including a lag, regardless of whether the tail-bacterial complexes initially were incubated at 37 or at 4°. When phage-cell complexes in which the λ DNA is 3H-labeled are treated with CHCl3 and then incubated at 37°, 90% of the labeled DNA is ejected from the phage but it is found in the medium rather than inside the host cell. If cells are exposed to chloroform prior to phage attachment of after the phage-cell complex has completed the lag reaction, the pattern of triggering and of uptake is very different. This reflects, in another way, the existence of several different possible states during injection.

The lambda head-tail joining reaction: purification, properties and structure of biologically active heads and tails.

Abstract Procedures were developed to obtain biologically active lambda heads and tails at high purity with 20 to 40% recovery. Free heads, free tails and phage particles differ markedly in stability. Phage are stable in solutions containing Mg 2+ but tails are not. The protein subunits which form the shaft of the tail dissociate in the presence of Mg 2+ and form multisubunit spherical structures. EDTA protects free tails against inactivation but disrupts heads and phage particles. The four carbon diamine, putrescine, stabilizes heads against inactivation; the three and five carbon diamines are less effective. Electron micrographs reveal a new “knob” structure at the distal end of the tail fiber of phage and of free tails. Tails released from EDTA-disrupted phage possess a “head-tail connector”, a structure not present on the tail before its joining with a head.

Putrescine and certain polyamines can inhibit DNA injection from bacteriophage lambda.

Abstract The injection of λDNA from attached phage into a host bacterium can be reversibly inhibited by putrescine. The concentration of di- or polyamine required to inhibit injection varies with the Mg2+ concentration and the amount of DNA in the phage head. In a series of n-alkyl diamines, those with more than five or fewer than three CH2 groups between amino groups were ineffective.

Arrangement of DNA in lambda bacteriophage heads: III. Location and number of nucleotides cleaved from λ DNA by micrococcal nuclease attack on heads☆☆☆

If lambda phage heads are exposed to micrococcal nuclease before phage tails are attached, the cohesive ends of the packaged λ DNA are damaged. The altered ends were used as templates for repair synthesis catalyzed by Escherichia coli DNA polymerase I. The set of nucleotides added to the 3′-termini of DNA from micrococcal nuclease treated lambda heads was compared with that added to the DNA from control heads not exposed to this nuclease. The comparison indicates that micrococcal nuclease treatment of heads removes four terminal bases from the right-hand cohesive end of the packaged DNA, but leaves the left-hand cohesive end intact.

The arrangement of DNA in lambda phage heads: I. Biological consequences of micrococcal nuclease attack on a portion of the chromosome exposed in tailless heads☆

Abstract If they lack tails, lambda heads are sensitive to inactivation by micrococcal DNase. Nuclease-treated heads still join with tails, producing phage which adsorb to host cells and inject their DNA normally. The injected chromosomes are defective since both lytic phage production and lysogeny are decreased 85 to 95%. The intracellular circularization of λ DNA is the first step in phage development altered by nuclease damage. When extracted from an infected cell, most parental molecules from nuclease-treated heads are of normal size but linear. The failure to cyclize in vivo suggests the cohesive ends are damaged.

Binding to isolated phage receptors and λ DNA release in vitro

Abstract Lambda DNA injection was investigated using partially purified λ-receptors isolated from the membrane of Escherichia coli K12. Binding of receptor and triggering of DNA ejection are distinct events. The binding occurs at 4° and may require the presence of a chloroform phase. The subsequent triggering of DNA ejection from phage-receptor complexes requires temperatures above 15°, begins without a lag period, and can occur in the absence of chloroform. The rate of stable binding in the absence of chloroform can be enhanced or diminished by mutations that alter either the phage tail or the host receptor.