Mutagenic activity of nitrosourea antitumor agents.

The relative mutagenic activities of chloroethyl-nitrosourea and methylnitrosourea antitumor agents in active clinical use were determined with the use of the Ames Salmonella typhimurium assay. The results indicated that the drugs induced base substitutions. 2-Deoxy-2-[[(methylnitrosoamino)carbonyl]amino]-D-glucopyranose (also called streptozotocin), a glucose-containing methylnitrosourea, was the most mutagenic of all compounds tested and showed at least a 250-fold increase in activity when compared to that of its chloroethyl analog, 2-[[[(2-chloroethyl) nitrosoamino]carbonyl]-amino]-2-deoxy-D-glucose (also called chlorozotocin). All nitrosoureas, with the exception of N'-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N-(2-chloroethyl)-N-nitrosourea monohydrochloride, a pyrimidine chloroethyl analog, demonstrated an increase in mutagenicity after incubation with induced Sprague-Dawley rat liver microsomes. No correlation between in vitro chemical alkylating activity and mutagenic potential was observed. Mutagenic activity was not observed to be of predictive value for antitumor activity in the L1210 leukemia model system.

Modified intraoral film holders for postmortem identification.

To date there have not been any commercial dental X-ray film holders marketed to accommodate the special needs of forensic odontologists. Modification of standard Rinn XCP film holders by the investigators produced self-supporting film holders that do not require the active participation from the examinee. The modified film holders greatly simplify the operator's technique of exposing dental radiographs on cadavers.

Efficacy of topical disinfectants.

A comparison of bactericidal activity of two surface disinfectants showed that both were highly effective. The glutaraldehyde-based disinfectant showed higher efficacy on roughed surfaces than the alcohol-based disinfectant. The study used a novel, and generally applicable, method to evaluate the surface killing potential of disinfectants.

Rich culture medium for the radiochemical labeling of proteins and nucleic acids.

Yeast extract was treated with tyrosine decarboxylase and used to prepare a rich, complex medium virtually free of tyrosine. The medium supported maximal growth rates for Escherichia coli prototrophs, as well as for defined and undefined auxotrophs. It has made possible the efficient radiochemical labeling of cells growing optimally in complex medium and the characterization of mutants with undefined requirements. Similarly prepared media may be useful for the study of fastidious organisms and organisms for which no defined medium has been described.

An amber mutation in the gene encoding the beta' subunit of Escherichia coli RNA polymerase.

An Escherichia coli strain carrying an amber mutation (UAG) in rpoC, the gene encoding the beta prime subunit of RNA polymerase, was isolated after mutagenesis with nitrosoguanidine. The mutation was moved into an unmutagenized strain carrying the supD43,74 allele, which encodes a temperature-sensitive su1 amber suppressor, and sue alleles, which enhance the efficiency of the suppressor. In this background, beta prime is not synthesized at high temperature. Suppression of the mutation by the non-temperature-sensitive amber suppressor su1+ yields a protein which is functional at all temperatures examined (30, 37, and 42 degrees C).

High efficiency temperature-sensitive amber suppressor strains of Escherichia coli K12: construction and characterization of recombinant strains with suppressor-enhancing mutations.

A set of eight strains combining the supD43,74 ts1 suppressor gene with alleles of three suppressor-enhancing (sue) genes have been constructed and characterized. The sue mutations work cooperatively to raise suppressor activity and together raise the activity of the supD43,74-encoded suppressor 40-fold. These strains further expand the utility of the ts suppressor system by providing as much as 100% suppressor activity at temperatures at or below 20 degrees C to as little as 0.015% suppressor activity at 43 degrees C.

Regulation of RNA polymerase synthesis in Escherichia coli: a mutant unable to synthesize the enzyme at 43 degrees.

We report the isolation of a mutant of E. coli in which the capacity to synthesize RNA polymerase (EC 2.7.7.6) (the beta and beta' subunits) is rapidly lost at 43 degrees. The mutation has no effect on the stability or activity of the polymerase itself. The mutation is recessive and is closely linked to the rif locus (the structural gene for the beta subunit). Using strains carrying the mutation, we have shown that polymerase is present in excess in rapidly growing E. coli cells.

Growth of bacteriophage H on male and female strains of Escherichia coli.

Phage H propagated on Yersinia pestis was reported by Molnar and Lawton to be rapidly adsorbed to female but not to male strains of Escherichia coli. In contrast, we find phage H adsorbs to all E. coli strains tested (both male and female) and forms plaques on a wide variety of male strains. Phage H appears to be related to the T3-T7 group of coliphages.

A temperature-sensitive suppressor enabling the manipulation of the level of individual proteins in intact cells.

A temperature-sensitive suppressor strain of E. coli has been isolated and characterized. The properties of the mutant indicate a strong potential for its use in biochemical and genetic work. In particular, the mutant makes possible the variation of the intracellular concentration of selected protein, permitting an evaluation of its role in cell growth and biochemistry. The mutant also permits the selective radiochemical labeling of proteins in vivo for in vitro identification and analysis. The utilization of the mutant for these and other applications is discussed.

Live attenuated bacterial vaccines: new approaches for safety and efficacy.

Problems arising from reversion to virulence in genetically attenuated bacterial vaccines can be overcome by the combination, in one strain, of multiple temperature-sensitive mutations of identical phenotype. Immunogenicity of attenuated strains may be enhanced by incorporation of mutations which permit limited replication in the vaccinee (thereby increasing antigen mass while minimising the possibility of vaccine reactions) and the expression of genes coding for antigens which are synthesised only during infection of the host.

In vivo titration of araC protein.

The requirement for araC protein in the induction of the araBAD operon was investigated. Strains of Escherichia coli carrying an araC(Am) mutation and temperature-sensitive amber suppressors were used to vary the intracellular level of araC protein. The levels of araC protein studied ranged from 0.007 to 1.8 times the normal amount. The results indicate that the normal level of araC protein is just sufficient to provide maximal expression of the araBAD operon.

Temperature-sensitive mutants of Pseudomonas aeruginosa: isolation and preliminary immunological evaluation.

The immunogenicity of two temperature-sensitive (ts) mutants of Pseudomonas aeruginosa immunotype 1, isolated and characterized for the development of a safe, live vaccine strain, was evaluated in a mouse protection model. One mutant, A/10/25, had a limited "coasting" property (i.e., continued replication for two divisions) at the nonpermissive temperature (36 degrees C), whereas the other mutant, E/9/9, continued replication for five generations after transfer to 36 degrees C. Groups of 3- to 5-week-old ICR mice were immunized intraperitoneally with various doses of the two ts mutants; at various times thereafter, the mice were challenged intraperitoneally with lethal doses of the parental wild type. The more extensive coaster, E/9/9, induced 100% protection at immunizing doses lower than those required for A/10/25 to induce the same protection (1 x 10(8) to 2 x 10(8) and 6 x 10(8) colony-forming units, respectively). Both ts strains induced significant protection for up to 5 weeks after immunization. The results of these studies suggest that the use of P. aeruginosa ts mutants might provide a novel approach to the prevention of P. aeruginosa colonization of patients with cystic fibrosis.

High efficiency temperature-sensitive amber suppressor strains of Escherichia coli K12: isolation of strains with suppressor-enhancing mutations.

Two independent high efficiency ts amber suppressor strains have been isolated as derivatives of a well-characterized supD ts suppressor strain (Oeschger and Woods, 1976). The mutations which raise suppressor activity have been shown by Hfr mapping to be distinct from each other and the supD locus. One of the isolates provides up to 100% efficient suppressor activity at low temperatures.

Ideal target organism for quantitative bactericidal assays.

We have developed a target organism which permits quantitative bactericidal assays. The organism is an Escherichia coli mutant which cannot grow at the temperature of the assay (37 degrees C), but retains full colony-forming potential for subsequent quantitation at 25 degrees C. We show that quantitative data on the bactericidal capacity of polymorphonuclear leukocytes and alveolar macrophages can be obtained when this mutant is used as a target. The procedure used to generate the strain is described in detail and should be applicable to many bacterial species. Characterization of the properties of the mutant indicates that it has a strong potential for use in other in vivo and in vitro investigations of host responses to microbial invasion.