There is reason to believe' that host-cell enzymes catalyze several of the steps in the replication of the single-stranded DNA of the virus 4bX174, particularly the conversion of the single-stranded DNA to the double-stranded replicative form (RF)l and, perhaps, the subsequent replication of the RF. Hence one could expect to find bacterial mutants in which the 4X DNA cannot be replicated. Such mutants would more clearly define host-controlled steps in the viral life cycle. In addition, one might be able to use the viral DNA as a probe of the fate of nucleic acids in the mutant cell; that is, it should be possible to understand any concomitantly occurring deficiency in cellular nucleic acid metabolism in terms of the experimentally analyzable defect in 4X DNA replication.Because of the potentially informative nature of mutants with altered patterns of DNA metabolism, and because of our interest in 4X replication, we sought mutants of a 4IX-sensitive strain that would adsorb but not replicate the virus. We report here the initial characterization of a bacterial mutant (called REP-) that does not allow the replication of 4X RF and additionally has a reduced capacity for bacterial recombination. It is representative of a heretofore undescribed class of recombination-deficient mutants.Materials and Methods.-Solutions and media: TKCaB contains 10 gm Bacto-Tryptone (Difco), 5 gm KCl, and 0.015 M CaC12 in 1 liter of distilled H20. When growing thyminerequiring cells, 10 Ag/ml thymidine is added. Tryptone broth contains, per liter: 10 gm tryptone, 5 gm NaCl, 0.01 M MgSO4, and 20 mg thymidine. m3XD contains 3 gm NH4Cl, 0.3 gm MgS04 7H20, 15 gm Difco Casamino acids, 24 ml glycerol, 1 mg gelatin, 0.9 gm KH2P04, 2.1 gm
A simple and practical method is presented for demonstrating the presence of the Australia/SH antigen and its corresponding antibody in serum specimens, both qualitatively and quantitatively. The method is based on the electronmicroscopic visualization of characteristic aggregates of antigen–antibody complexes formed in the mixture of a serum specimen and the appropriate Australia/SH detector reagent. It involves the use of a microtechnique requiring minute amounts of reagents and provides, as a result of diffusion and filtration through agar gel, partially purified and concentrated preparations, ready for electronmicroscopic examination in less than an hour. The method is highly specific and yields reproducible results. Its sensitivity was found to be greater than that of the crossover electrophoresis test and closely approximates that of the complement fixation test, with the added advantage of not being affected by the "prozone phenomenon." The method can be recommended for use in laboratories equipped with electronmicroscopic facilities to establish a differential diagnosis of viral hepatitis cases, perform rapid screening of blood samples (blood products) for the presence of Australia/SH antigen, and clarify equivocal results obtained by other methods. It is expected that the agar–diffusion–filtration technique will also prove useful, in general, for enhancing the chances of detecting virus particles in suspensions of relatively low virus concentrations.
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