Teresa Cabezón

Alteration of ribosomal protein S17 by mutation linked to neamine resistance in Escherichia coli: I. General properties of neaA mutants

A mutant of Escherichia coli strain K12S isolated after selection for resistance to the amino glycoside antibiotic neamine shows severe restriction of amber suppressors in vivo . Ribosomes from the mutant exhibit low neamine-induced misreading in vitro and a decreased affinity for the related antibiotic streptomycin. The S17 ribosomal protein from the mutant strain shows altered electrophoretic mobility on two-dimensional polyacrylamide gels and also differs in chromatographical behaviour from the S17 protein from the parental strain. When ribosomes from the mutant strain are reconstituted in the presence of a molar excess of the S17 protein from the wild type species, the particles exhibit normal druginduced misreading properties as well as a normal capacity for binding streptomycin. The gene involved in the alteration of protein S17 ( rpxQ ) is located inside the ribosomal protein gene cluster at 64 minutes on the E. coli genetic map and cotransduces at 96% with rpxE (S5).

Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli

SummaryThe effect on translational fidelity of a particular mutation in the gene coding for protein S5 (rpxE) has been investigated. This mutation has the opposite effect of a restrictive strA mutation; in vivo, it relieves the restriction imposed by strA on the suppression of T4 nonsense mutants and results in hypersensitivity to streptomycin; in vitro, the presence of the altered S5 protein in 30S ribosomes results in increased intrinsic misreading. It is concluded that this mutation, ramC319, acts as a ribosomal ambiguity mutation similar to certain mutations of protein S4 (ramA).

Simultaneous synthesis and assembly of various hepatitis B surface proteins in Saccharomyces cerevisiae

Yeast transposon of class-1-based vectors, allowing integration at a series of chromosomal loci by homologous recombination with resident transposons, were constructed. Using such vectors, we have introduced several copies of an expression cassette encoding the major hepatitis B surface protein as well as expression cassettes encoding the middle (M) or/and the large (L) surface protein into Saccharomyces cerevisiae. In extracts of such strains, coassembly of the different proteins into a single lipoprotein structure is observed. This was demonstrated by immunoprecipitation of the major protein using monoclonal antibodies directed specifically against epitopes that are present only on the M or the L protein. These results show that hepatitis B surface antigen envelope proteins synthesized in yeast are able to assemble into structures composed of different polypeptides. This opens the possibility of producing in yeast a variety of particles carrying well-defined amounts of preS epitopes on their surface. Also, one can envisage the production of mixed particles containing different foreign epitopes on their surface, in defined relative abundance, which could be useful for vaccine applications.

Expression of ribosomal protein genes in Escherichia coli.

SummaryStreptomycin or spectinomycin treatment of an E. coli strain, carrying the strR and spcR alleles on the chromosome and the wild-type (sensitive) alleles on the episome, selects for inactivation of the relevant sensitive allele. After Mu induced mutagenesis, in the absence of selection against extended deletions upon the episome, a large proportion of stro mutants are also spco, and vice versa. However, when markers flanking the strA and spcA gene cluster are simultaneously selected, effectively eliminating long deletions, the majority of stro mutants continue to express the spcs allele, and vice versa.Insofar as inactivation after Mu treatment is due to prophage insertion within or proximal to the genes in question, this result indicates that the genes strA and spcA are not parts of a single operon. In virtue of the high frequency of extended deletions observed in the absence of suitable counter-selection, we must place a word of caution upon the use of phage Mu-1 as a means for isolating polar mutations and defining transcriptional units.

A missense mutation in the gene coding for ribosomal protein S17 (rpsQ) leading to ribosomal assembly defectivity in Escherichia coli

SummaryThe conditionally lethal mutation, 286lmis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and was found to cotransduce at 97% with rpsE (S5). The 2861mis mutation leads to thermosensitivity and impaired assembly in vivo of 30S ribosomal particles at 42°C. The strain carrying the mutation has an altered S17 ribosomal protein; the mutational alteration involves a replacement of serine by phenylalanine in protein S17. Spontaneous reversion to temperature independence can restore the normal assembly in vivo of 30S ribosomal subunits at 42°C and the normal chromatographical sehaviour of the S17 ribosomal protein in vitro. We conclude therefore that the 2861mis mutation affects the structural gene for protein S17 (rpsQ).

Alteration of ribosomal protein S17 by mutation linked to neamine resistance in Escherichia coli: II. Localization of the amino acid replacement in protein S17 from a neaA mutant

Abstract A mutant of Escherichia coli strain K12S, nea R 301, resistant to the antibiotic neamine was found to have an altered 30 S ribosomal protein S17. The modification involves a change in the electrophoretic mobility of this protein. S17 proteins wore purified from the mutant and the parental strain, respectively, and the amino acid compositions of all tryptic peptides were compared. The results show that the mutational alteration involves a replacement of histidine by proline in peptide T8 from mutant nea R 301. The amino acid replacement is located at position 30 of the S17 protein chain. We conclude, therefore, that the mutation nea R 301 affects the structural gene for protein S17 ( rps Q).

Mapping of neamine resistance: Identification of two genetic loci, nea A and nea B

SummaryMutants of Escherichia coli resistant to the antibiotic neamine have been isolated. The mutants have been mapped and two separate loci for neamine resistance, nea A and nea B, have been identified on the genome. Nea A maps very closely to spc A and nea B is near to the str A locus. Both neamine resistance markers are situated, therefore, in the socalled ribosomal operon.

Apports de la génétique à la connaissance du ribosome bactérien

Summary In this review, we have tried to describe the role of the ribosome, and particularly of the 30S subunit, in the translation of the genetic message ; we present the current status of the genetic knowledge in this field. For more extensive reviews, we refer the interested reader to the recent publication Ribosomes (1974) [1] .

Amber mutations in Escherichia coli essential genes: isolation of mutants affected in the ribosomes.

SummaryA method to obtain amber mutations in ribosomal protein genes is described. It relies on the P1-mediated localized mutagenesis (Hong and Ames, 1971) and on the fact that the recipient strain contains (a) an efficient but genetically unstable suppressor, (b) a particular thermoinducible λ prophage which kills suppressor hosts at 42° C. Exposure of these bacteria to the high temperature yields frequent suppressor-free derivatives while none will be found if the strain carries an amber mutation in an essential gene. Eleven mutants have been isolated by this method, of which at least six appear to carry amber mutations. All of them map close to, and to the right of spcA, in a region which codes mostly for ribosomal proteins. Three mutants were studied biochemically; all three show defective ribosomal assembly in vivo upon loss of suppression.

Translational fidelity in Escherichia coli: contrasting role of neaA and ramA gene products in the ribosome functioning.

SummaryStrains carrying both the ramA1 and the neaA301 mutations do not exhibit the restriction of informational suppressors normally associated with resistance to neamine. Furthermore, ribosomes from such strains exhibit increased misreading in vitro with respect to particles from the neaA strain. These properties suggest that translational fidelity may be cooperatively controlled by ribosomal proteins S4 and S17, coded by ramA (rpsD) and neaA (rpsQ) genes respectively.