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Previous work has indicated that ribosomes isolated from Salmonella typhimurium were highly immunogenic and afforded excellent protection against homologous challenge. Effective protection was obtained also when ribonucleic acid (RNA) extracted from these ribosomes was used as a vaccine. In this investigation ribosomes prepared by another method and washed repeatedly in 1 M NH4Cl lost much of their prophylactic potency and yielded poorly protective

In situ hybridization, using a biotinylated cDNA probe for the epidermal growth factor receptor (EGFR) gene, indicates that the amplified EGFR genes in the colon tumor cell line, DiFi, are localized in many small double minute chromosomes (dmin) of varying size and visibility. Analysis of the electrophoretic mobility of gamma-irradiated DNA from DiFi by pulsed-field gel electrophoresis and Southern blot hybridization using EGFR probe, indicates that the amplified EGFR in DiFi exists in extrachromosomal, covalently closed circular episomes, presumably equivalent to dmin. Two major and one minor species were observed which had estimated sizes of 650, 1,300, and 2,000 kb, respectively. The DiFi cell line appears to represent a unique case of extrachromosomal EGFR gene amplification in human cells.

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We have determined the nucleotide sequences of two unusual UGG-suppressing glycine tRNAs from Escherichia coli and, as a result, have discovered a new mechanism for the generation of missense suppressors. The suppressor tRNAs translate UGG but not UGA. Each arose as a consequence of spontaneous mutational alteration of glyT, the gene for the GGA/Greading glycine tRNA of E. coli In each mutant tRNA, the change in primary structure involved the insertion ofan adenylate residue on the 3' side of the anticodon and the loss ofa modification of the uridylate residue at the 5' end of the anticodon. A "shift" of the effective anticodon by one nucleotide in the 3' direction can account for the new coding specificity of these tRNAs.(19). Nevertheless, both mutant glyT tRNAs suppressed UGG mutations occurring at the same positions in trpA (19). The conversion of a GGA/G-reading glycine tRNA to a UGG-specific suppressor alters its recognition of two positions of the codon (first and third). But suppressors of this type occur frequently, suggesting that members of this class of UGG suppressor can arise from a single mutational event. We determined the nucleotide sequence ofboth of these tRNAs and discovered a new type of missense suppressor tRNA. MATERIALS AND METHODSThe original hypothesis oftranslational suppression of missense mutations (1-3) proposed the general nature of tRNA involvement. To date, a variety of specific molecular alterations have been found to result in tRNA-mediated suppression. For example, the coding specificity of a tRNA can be altered by a change in the anticodon, either by base substitution (for review, see refs. 4-6) or by loss ofa base modification (7-9). In addition to anticodon changes, nucleotide substitutions in other parts of a tRNA molecule can result in suppression by causing attachment of an incorrect amino acid (5, 10). Moreover, a particular anticodon change was found to alter a tRNA so that it accepts a new amino acid and reads a different codon (11). An especially interesting suppressor tRNA is a mutant form of tRNA rP that can translate the termination codon UGA and the tryptophan codon, UGG (12). This mutant tRNA retains the original anticodon, and its suppressor activity is due to a single base change in the dihydrouracil arm of the molecule. How this change, distant from the anticodon, alters the codon response has not been completely resolved, but a consequent change in the tertiary structure of the molecule has been implicated (13)(14)(15). Other mutational studies (7-9, 16-19) also have indicated the importance oftRNA conformation in determining the specificity of codon response.In a recent report, we described the isolation and initial characterization of translational suppressors of a UGG missense mutation in Eschertichia coli (20). Eleven UGG suppressors were shown by genetic mapping and reversed-phase column chromatography to have alterations in glyT, the structural gene for a species of glycine tRNA. Nine suppressed both UGA and UGG mutations. This result was expe...