Luigi Gorini

A ribosomal ambiguity mutation

Abstract The existence of a genetic determinant ram ‡ for a 30 s ribosomal component controlling translation ambiguity has been demonstrated. It is located in the strA (streptomycin sensitivity) region and is 99% cotransducible with the spc (spectinomycin sensitivity) gene. In vitro, ram mutant ribosomes misread extensively and by interchanging the subunits of ram and ram+ ribosomes it is shown that misreading is a property of the 30 s subunit. In vivo, the ram mutation confers on the cell the ability to suppress all three nonsense codons. Streptomycin induces a similar generalized ambiguity phenotypically both in vitro and in vivo. The effects of ram and of streptomycin are additive. Ambiguity caused by ram, by streptomycin and by their combination is antagonized in vivo and in vitro by additional mutations at the strA locus. Thus the strA40 (resistant competent) and strA1 (resistant incompetent) alleles increasingly restrict ribosomal (as well as tRNA) suppression and return to normal the reduced growth rate produced by ram mutation in a wild-type strA+ strain. Growth is inhibited completely in a ram1, strA+ strain by sublethal doses of streptomycin and in ram1, strA40 strains by higher doses, while strain ram1, strA1 is indifferent to streptomycin. The bactericidal action of the drug is unrelated to these effects.

Selecting Bacterial Mutants by the Penicillin Method

Certain improvements are described in the use of penicillin for isolating auxotrophic mutants of bacteria. By obtaining exponential growth before the penicillin is added, and by minimizing the duration of the treatment, cross-feeding is decreased and much denser populations can be screened. These modifications have made it possible to obtain, with regularity, mutants of Escherichia coli blocked in a desired step in arginine biosynthesis.

Ribosomal mutations affecting efficiency of amber suppression

Abstract The effect of strA mutations on the efficiency of amber and ochre suppressors, known to be mutated tRNAs, has been measured in vivo in isogenic strains. This effect is expressed in terms of translation rate of the amber codon, using bacterial and T4 amber mutants. Both translation through an adequate tRNA and through misreading are co-ordinately affected at different degrees by different strA mutations. The efficiency of different suppressor species is generally reduced by a given strA mutation at different degrees. This degree does not depend on the anticodon and probably also not on the amino acyl moiety. The efficiency of one species (suIII) is in fact enhanced when tested with T4 mutants while it is normally reduced when tested with bacterial mutations. Indirect evidence indicates that this tRNA is modified by T4 infection, probably in a structural element which determines its interaction with the ribosome. It is proposed that the strA protein influences the interaction of the ribosome (possibly binding) with the tRNA, controlling thereby the rate of chain elongation.

Streptomycin and Misreading of the Genetic Code

Historical Background In 1961 we accidentally isolated a streptomycin-resistant auxotroph whose arginine requirement could be satisfied by streptomycin (Gorini, Gundersen and Burger 1961). It was shown a few years later (Gorini and Kataja 1964a), using the same streptomycin-resistant parent and by selecting for streptomycin dependence in minimal medium but not in broth, that a class of mutants defective in a number of unrelated metabolic pathways could be easily obtained. The members of this class share the property of “conditional streptomycin dependence (CSD).” It could be shown in extracts that the enzyme normally missing in the mutant was formed during growth in the presence of streptomycin. We suggested that streptomycin might induce mistakes in the transmission of information from DNA to protein, compensating for the effect of the mistake encoded in the mutant DNA. Such a mechanism is analogous to that of “informational suppression” (Gorini 1970), by which the effect of a mutation persists at the level of the gene but is suppressed at the level of translation. However, informational suppression is found to be the consequence of a mutation in one of the molecules involved in the translation process (most commonly in a tRNA), whereas suppression in the case of CSD mutants appeared to depend on the environment, i.e., the presence or absence of streptomycin in the growth medium. We have suggested the designation of “phenotypic suppression” for this streptomycin effect. If streptomycin produces phenotypic suppression by inducing mistakes in translation, then the drug could be expected to induce mistakes...

Drug dependence reversed by a ribosomal ambiguity mutation, ram, in Escherichia coli.

Mutations in the strA ribosomal gene may produce strains dependent on the presence of drugs for growth. Three classes have been isolated from Escherichia coli B: (1) the majority are DrugD, equally dependent on streptomycin, paromomycin, or ethanol; (2) some are SmD EtD‡ (3) a few are strictly SmD, dependent on streptomycin exclusively. They also differ in their ability to yield spontaneous “revertants” to independence: resistant to low concentrations of streptomycin (20 μg/ml.) from (1); resistant to high concentration of streptomycin (500 μg/ml. at least) from (2) and no revertants from (3). It is found that mutations at a second ribosomal gene, ram, known to contrast the effects of strA mutations, may cancel the dependence phenotype. This is demonstrated in two ways: (a) by transducing a known ram allele, ram1, into a dependent recipient and (b) by isolating spontaneous revertants to independence and demonstrating that they contain a mutation at the ram locus. The ram1 recombinants and the revertants from each of the classes (1) and (2) display identical phenotypes: low and high resistance respectively. The introduction of ram1 allele into class (3) fails to yield drug-independent recombinants. It is suggested that ram mutations and addition of drug may equally overcome dependence for analogous reasons: introduction of a genetic or phenotypic agent inducing translational ambiguity.

A unitary account of the repression mechanism of arginine biosynthesis in Escherichia coli. I. The genetic evidence.

In Escherichia coli strain B growing in minimal medium, added arginine slightly stimulates, while in strain K it represses formation of the arginine biosynthetic enzymes. Nevertheless, mapping and complementation experiments indicate that the respective regulatory genes, argRB‡ and argR+, are allelic. Since all argR alleles producing an active repressor, including argRB, are trans-dominant to constitutive mutants of either strain, and since an amber mutant of argRB has been found which is constitutive, the pathway in both strains is under negative control, and the regulatory gene product requires translation for its synthesis. A single amino acid substitution can change B-type control to K-type control since correction of the amber argRB mutant by suI+ restores the argRB phenotype while correction by suIII+ leads to arginine repressibility. It is suggested that arginine, or an arginine derivative, activates the repressor product in both strains, but that the formation or function of the active repressor from the argRB product is reduced at high arginine concentration. The eight structural genes of the arginine pathway are scattered in five regions of the chromosome. A technique has been devised for selecting a mutation which partially relieves repression of argF, one of the unclustered genes. This mutation, which is specific for argF, closely linked, and cis-dominant has been designated argOF for operator mutation. The existence of similar operators for the other arginine genes all interacting with the same argR product but differing in affinity is implied by parallel but non-co-ordinate control of the pathway and by the behavior of an argRB temperature-sensitive mutant in which de-repression as a function of temperature differs depending on the arginine enzyme tested.

Restriction, de-restriction and mistranslation in missense suppression. Ribosomal discrimination of transfer RNA's

Abstract The strA ribosomal mutations known to restrict the level of translational ambiguity and the efficiency of nonsense transfer RNA-suppressors, are shown also to restrict the efficiency of missense tRNA-suppressors; the efficiency of wildtype tRNA is not noticeably affected by these strA mutations and the extent by which the suppressor tRNA is restricted is shown to be dependent upon some structural aspect of the suppressor-tRNA molecule other than either the anticodon or the amino acid-accepting specificity. Ribosomal alteration mutants (ram), known to reverse strA restriction of translational ambiguity, are shown to reverse also strA restriction of the efficiency of nonsense and missense tRNA-suppressors. Furthermore, introduction of the ram mutation into missense suppressor strains is shown greatly to increase the amount of mistranslation (a property peculiar to missense suppressors), while it has no significant effect in su− strains. The strA mutation is shown to reverse this ram effect on mistranslation. Presence of streptomycin, known to act on the ribosome, is shown to reverse the effect of strA on restriction and mistranslation in a manner similar and additive to that of the ram mutation. These observations suggest that: (a) the ribosome provides a recognition screen for tRNAs prior to, or simultaneous with, their interaction with messenger RNA; and (b) this postulated ribosomal screen discriminates normal from mutated tRNA.

Le rôle du calcium dans l'activité et la stabilité de quelques protéinases bactériennes

The production of proteinases by a whole series of bacteria (M. lysodeikticus, B. megatherium, Coccus P., Proteus sp, Ps. pyocyanea, B. mesentericus, B. subtilis, and B. cereus), grown aerobically and at pH 7–8, is the more, the lower the temperature; this work was carried out at 26°. The optimun pH of these proteinases is between 7 and 8. The activity of these proteinases, when separated from bacterial cells, is inhibited by various reagents for calcium: fluoride, citrate, oxalate, nitrilotriacetate (Complexon I), ethylenediamino-tetraacetate (Complexon II), and sodium hexametaphosphate. The inactivation of the various proteinases is not immediate and takes place more slowly as the temperature is lower. The sensitivity of these enzymes varies considerably. That from M. lysodeikticus is the most sensitive and is completely inactivated by all the reagents except the fluoride. That from B. cereus is the most resistant, even complexon II and the metaphosphate having only a weak action. The inactivation of the proteinases by the above-mentioned compounds can be completely prevented by addition of calcium, provided this is done immediately after addition of the reagent. If the calcium is added later, the original activity is regained only in exceptional cases. The inactivation by elimination of calcium is reversible in the beginning, but subsequently becomes more or less rapidly irreversible. On the other hand, the calcium protects the proteinases against inactivation by heat. The calcium thus plays a fundamental part in these bacterial proteinases, not only in their activation but also in their stability: it is a constituent of these enzymes, which must therefore be considered as metalloproteins in which, according to their origin, the bond between the protein and the calcium is more or less labile. The calcium in its role as the active substance as regards proteolysis can be replaced only by strontium and only to a slight extent. As a constituent of the proteinase-molecule, assuring the stability of the enzyme structure, calcium can be substituted partially by magnesium. However, the protein-magnesium complex so formed, although keeping the fundamental structure, has no proteolytic activity. In the course of the above-mentioned research, a new buffer solution, containing methyl- and ethylmorpholine, has been tried out. It can be used between PH 6.7 and 8.5 and includes neither metallic ions nor substances capable of forming complexes with calcium.

Role of ribosomes in streptomycin-activated suppression

devices, the mechanism of the maintenance and termination of pregnancy, and about functional myometrial disorders. The common basic regulatory mechanism of the myometrium must be thoroughly investigated before evaluating the importance of superimposed modifications. The recognition that it is fundamentally alike in the various mammalian species promises to provide order in an important field of reproductive biology where controversy and confusion have prevailed.

The Effect of Streptomycin and Other Aminoglycoside Antibiotics on Protein Synthesis

Streptomycin (Sm), the first nontoxic broad spectrum antibiotic also effective against the tubercle bacillus, was isolated from Streptomyces griseus by Schatz, Bugie, and Waksman in 1944. The structures of Sm and several of its active derivatives are shown in Fig. 1. It is composed of streptidine, an inositol substituted with two guanido groups, and streptobiosamine, a disaccharide containing a methylamino group. Streptidine can also be considered a substituted streptamine, which emphasizes a chemical moiety found in other aminoglycoside antibiotics, Fig. 3 and 4. Antibacterial activity is destroyed by cleavage of the glycosidic bond between streptidine and streptobiosamine, by replacing the guanido groups with amino groups, or by carbobenzyloxylation of the secondary amine (Polglase, 1965).