Effect of neomycin and protein S1 on the binding of streptomycin to the ribosome

The binding of [3H]dihydrostreptomycin to the 70-S ribosome or to the 30-S subunit has been investigated in the presence of neomycin by the Millipore filtration or the equilibrium dialysis procedure. It was observed that dihydrostreptomycin binds equally well to the 30-S subunit and the 70-S ribosome, and that neomycin stimulates the binding of dihydrostreptomycin to the ribosome by increasing the association constant and not by creating new binding sites. Specific removal of protein S1 from the 30-S subunit neither affected the binding of dihydrostreptomycin to the ribosome nor the stimulation of dihydrostreptomycin binding by neomycin.     

A new class of promoter mutations in the lactose operon of Escherichia coli

The isolation and genetic characterization of a number of mutations that are located in the promoter region of the lac2 operon are described. These mutations have reduced levels of lac operon expression in a wild, type (crp⁺cya⁺) genetic background. Three of the mutations also have lower levels of lac operon expression than lacP⁺ in a crp^?cya^? genetic background, that is in the absence of the catabolite activator protein and 3′,5′-adenosine cyclic monophosphate. These three mutations are located nearest to the lac operator. They define a second essential site in the promoter region.     

Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo

Summary Mutations in seven different maternal-effect loci on the second chromosome of Drosophila melanogaster all cause alterations in the anterior-posterior pattern of the embryo. Mutations in torso (tor) and trunk (trk) delete the anterior- and posterior-most structures of the embryo. At the same time they shift cellular fates which are normally found in the subterminal regions of the embryo towards the poles. Mutations in vasa (vas), valois (vls), staufen (stau) and tudor (tud) cause two embryonic defects. For one they result in absence of polar plasm, polar granules and pole cells in all eggs produced by mutant females. Secondly, embryos developing inside such eggs show deletions of abdominal segments. In addition, embryos derived from staufen mothers lack anterior head structures, embryos derived from valois mothers frequently fail to cellularize properly. Mutations in exuperantia (exu) cause deletions of anterior head structures, similar to torso, trunk and staufen. However in exu, these head structures are replaced by an inverted posterior end which comprises posterior midgut, proctodeal region, and often malpighian tubules.The effects of all mutations can be traced back to the beginning stages of gastrulation, indicating that the alterations in cellular fates have probably taken place by that time. Analysis of embryos derived from double mutant mothers suggests that these three phenotypic groups of mutants interfere with three different, independent pathways. All three pathways seem to act additively on the system which specifies anterior-posterior cellular fates within the egg.     

Comparison of the action of streptomycin and neomycin on the structure of the bacterial ribosome

The influence of streptomycin and neomycin upon the conformation of the ribosome has been investigated using spin-labeled and fluorescent analogs of the sulfhydryl reagent, N-ethylmaleimide. Changes in the electron paramagnetic resonance spectra or in the polarization of fluorescence of labeled ribosomes reveal that streptomycin alters the mobility of labels bound to the sulfhydryl group of protein S18 while neomycin affects the mobility of labels bound to the sulfhydryl groups of proteins S1, S21 and/or L10. It is also observed that both streptomycin and neomycin interfere with changes in the mobility of labels induced by storage under inactivating conditions. From these results, it is concluded that: 1. streptomycin and neomycin distort the conformation of the ribosome at different sites, streptomycin disturbing preferentially the area around the sulfhydryl group of protein S18 while neomycin affects the environment of the sulfhydryl groups of proteins S1, S21 and/or L10; 2. streptomycin and neomycin interefere with the ability of the ribosome to undergo conformational changes.     

Potential new antibiotic sites in the ribosome revealed by deleterious mutations in RNA of the large ribosomal subunit

The ribosome is the main target for antibiotics that inhibit protein biosynthesis. Despite the chemical diversity of the known antibiotics that affect functions of the large ribosomal subunit, these drugs act on only a few sites corresponding to some of the known functional centers. We have used a genetic approach for identifying structurally and functionally critical sites in the ribosome that can be used as new antibiotic targets. By using randomly mutagenized rRNA genes, we mapped rRNA sites where nucleotide alterations impair the ribosome function or assembly and lead to a deleterious phenotype. A total of 77 single-point deleterious mutations were mapped in 23 S rRNA and ranked according to the severity of their deleterious phenotypes. Many of the mutations mapped to familiar functional sites that are targeted by known antibiotics. However, a number of mutations were located in previously unexplored regions. The distribution of the mutations in the spatial structure of the ribosome showed a strong bias, with the strongly deleterious mutations being mainly localized at the interface of the large subunit and the mild ones on the solvent side. Five sites where deleterious mutations tend to cluster within discrete rRNA elements were identified as potential new antibiotic targets. One of the sites, the conserved segment of helix 38, was studied in more detail. Although the ability of the mutant 50 S subunits to associate with 30 S subunits was impaired, the lethal effect of mutations in this rRNA element was unrelated to its function as an intersubunit bridge. Instead, mutations in this region had a profound deleterious effect on the ribosome assembly.     

Effects of mutations to streptomycin resistance on the rate of translation of mutant genetic information

Gartner, T. K. (University of California, Santa Barbara), and E. Orias. Effects of mutations to streptomycin resistance on the rate of translation of mutant genetic information. J. Bacteriol. 91:1021-1028. 1966.-The effects of mutations to streptomycin resistance of independent origin upon the translation of suppressible mutant information were studied in an isogenic series of strains of Escherichia coli. The group of suppressible mutants included 1 mutation in the z gene of the lac operon of E. coli (O(0) (2) allele), 12 mutations distributed among the two rII cistrons of T4, and 13 mutations distributed among at least five cistrons of phage T7. It was concluded that the mutations to streptomycin resistance cause a significant decrease in the rate of translation of the suppressible codons, and that this effect is limited to a few types of codons.     

Linkage relationships of mutations endowing Streptococcus pyogenes with resistance to antibiotics that affect the ribosome

Summary Several mutations conferring resistance to streptomycin, kanamycin, spectinomycin, erythromycin, and lincomycin on the group A streptococcal strain 56188 have been mapped by two- and three-point crosses using transduction with bacteriophage A25. The markers are located in two linkage regions too distant to be cotransduced. One harbors the streptomycin and kanamycin loci which are transduced jointly at 78% and the other bears loci for spectinomycin (spc), erythromycin (eryA), and lincomycin (linA) resistance, in this order. spc and linA are cotransduced at a frequency of about 27%. Analysis of three-point crosses involving spc-4, eryA300, and linA12 according to the Wu model for random general transduction shows consistency of the theoretical predictions with the experimental data and indicates that the intervals of the above sequence are about 22% and 6% of the average length of the transduced piece.     

Genetic study of plasmid-associated high-frequency mutations to streptomycin resistance in Escherichia coli]

Escherichia coli CTR1(RT1)RHfm1) carrying two H-factors and having unusually high frequency of mutation to high level streptomycin resistance is studied. The high frequency of mutation (about 10(-6) to streptomycin resistance is connected with the presence of R factor RHfm1, controlling the resistance to chloramphenicol and low level streptomacin resistance, but not with RT1, controlling the resistance to tetracycline. Spontaneous or ethidium bromide-induced loss of RHfm1 is accompanied by a decrease of the mutation frequency to 10(-9). RHfm1 is efficiently transmissible to other strains at 28 degrees C. The acquisition of RHfm1 by strains of E. coli K-12 ans S. typhimurium LT2 was followed by a 1000--10000-fold increase of the frequejcy of mutation to streptomycin resistance. Some streptomycin resistant mutants were isolated, and chromosome location of the mutations was demonstrated. The streptomycin resistant mutants were unable to transmit high level of resistance to streptomycin with R factor, but only low level one. The loss of RHfm1 by streptomycin resistant mutants was accompanied by the return to the streptomycin sensitivity of the initial R- strans (E. coli K-12 mutants) or by a decrease of the streptomycin resistance to the level, only 2-fold higher than that of R- wild type (E. coli CTR1 mutant). Thus, the mutantions had practically no effect on streptomycin resistance of R- strains, but could lead to high resistance phenotypes in the presence of RHfm1. The mutant loci in all three studied strains were found to be closely linked to the locus "fus" on the genetic map of E. coli.     

Site-directed mutations altering methyl-accepting residues of a sensory transducer protein

The Trg protein is one of a family of transducer proteins that mediate chemotactic response in Escherichia coli. Transducers are methyl-accepting proteins that gain or lose methyl esters on specific glutamyl residues during sensory adaptation. In this study, the significance of multiple sites of methylation on transducer proteins was addressed by using oligonucleotide-directed, site-specific mutagenesis to substitute an alanyl residue at each of the five methyl-accepting sites in Trg. The resulting collection of five mutations, each inactivating a single site, was analyzed for effects on covalent modification at the remaining sites on Trg and for the ability of the altered proteins to mediate sensory adaptation. Most of the alanyl substitutions had substantial biochemical effects, enhancing or reducing methyl-accepting activity of other sites, including one case of activation of a site not methylated in wild-type protein. Analysis of the altered proteins provided explanations for many features of the complex pattern of electrophoretic forms exhibited by Trg. The mutant proteins were less efficient than normal Trg in mediating adaptation. Correlation of biochemical and behavioral data indicated that reduction in the number of methyl-accepting sites on the transducer lengthened the time required to reach an adapted state.     

Systematic characterization of mutations altering protein degradation in human cancers

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A new mechanism for altering chromosome number during karyotype evolution

Summary A new mechanism for changing chromosome numbers (preserving the fundamental number of long chromosome arms) during karyotype evolution is suggested. It includes: 1) Occurrence of individuals heterozygous for two interchanges between different arms of three chromosomes (a metacentric and two acrocentric ones). 2) Formation in heterokaryotypes of multivalents during meiosis between the chromosomes involved in the interchanges and their unchanged homologues. 3) Mis-segregation of chromosomes from these multivalents resulting in hypoploid (n-1) and hyperploid (n+1) simultaneously instead of euhaploid gametes. 4) Fusion of n-1 or n+1 gametes which gives rise to (zygotes and) individuals representing homokaryotypes with changed number of chromosomes (2n+2 or 2n-2), but preserves (as compared to the parental karyotypes) the number of long chromosome arms. Under definite conditions, chromosome numbers of the progeny may be changed by this process in both directions (upwards and downwards). The mechanism is free of the difficulties associated with the explanation for such changes by direct Robertsonian interchanges (see Discussion), which are usually considered to be responsible for such alterations in chromosome number. The above-mentioned process has been experimentally documented in Vicia faba and it probably also occurred naturally within the Vicia sativa group.     

Effect of streptomycin on the response of Escherichia coli ribosomes to the dissociation factor

Ribosomes recovered from cells of Escherichia coli treated with streptomycin are impaired in their response to the dissociation factor. This effect of Str evidently depends on a direct action on the ribosome and not on stabilization of a complex with normal ligands. Thus, such Str-ribosomes lack firmly bound transfer RNA; treatment with puromycin does not remove the resistance to dissociation; and similar resistance is produced when free ribosomes (in the absence of normal ligands) are exposed to Str in buffer and then washed.The impairment of dissociation of Str-ribosomes in cells is evidently incomplete, for these ribosomes maintain a reduced polysome level by engaging in cyclic abortive reinitiation (see preceding paper, Wallace &; Davis, 1973) and this process requires formylation (and hence presumably dissociation). Str-inhibited dissociation may be the limiting step in this reinitiation, for the polysome level is much lower in Str-treated cells of strain W than in those of K12, and Str impairs dissociation much more with ribosomes of the former strain.     

Interaction of mutations affecting growth rate and resistance to streptomycin in pneumococci and streptococci

Krauss, Marjorie R. (New York University Medical Center, New York, N.Y.), James C. King, and Rody P. Cox. Interaction of mutations affecting growth rate and resistance to streptomycin in pneumococci and streptococci. J. Bacteriol. 92:1337-1344. 1966.-A strain of Streptococcus (Viridans group) was shown by transformation reactions to be the carrier of two interacting mutations. One produced resistance to streptomycin and a slow rate of growth; the only effect of the second was an increase in growth rate when it was added by transformation to streptococcal strains that had already been transformed to bear the first. Similar modifying mutations were observed in strains of streptococci and pneumococci into which the first mutation had been introduced by transformation.