Genetic and biochemical distinction among Chinese hamster cell emtA, emtB, and emtC mutants.

Genetic and biochemical experiments have enabled us to more clearly distinguish three genetic loci, emtA, emtB, and emtC, all of which can be altered to give rise to resistance to the protein synthesis inhibitor, emetine, in cultured Chinese hamster cells. Genetic experiments have demonstrated that, unlike the emtB locus, neither the emtA locus nor the emtC locus is linked to chromosome 2 in Chinese hamster cells, clearly distinguishing the latter two genes from emtB. emtA mutants can also be distinguished, biochemically, from emtB and emtC mutants based upon different degrees of cross-resistance to another inhibitor of protein synthesis, cryptopleurine. Two-dimensional gel electrophoretic analysis of ribosomal proteins failed to detect any electrophoretic alterations in ribosomal proteins from emtA or emtC mutants that could be correlated with emetine resistance. However, a distinct electrophoretic alteration in ribosomal protein S14 was observed in an emtB mutant. In addition, the parental Chinese hamster peritoneal cell line of an emtC mutant, and the emtC mutant itself, are apparently heterozygous for an electrophoretic alteration in ribosomal protein L9.     

Cotransduction of strA and Ribosomal Protein Cistrons in Escherichia coli-Salmonella typhimurium Hybrids

Genetic mapping of ribosomal protein cistrons of Salmonella typhimurium and Escherichia coli was performed by phage P1 mediated, generalized transduction. From an E. coli hybrid strain which carried a S. typhimuirum F' factor, an E. coli strain was constructed which had integrated S. typhimurium genetic material including the region of the strA locus. Salmonella genetic material from this hybrid was transduced into E. coli recipients. The ribosomal protein electrophoretic patterns of these hybrid transductants were correlated with the presence of markers contributed by each parent.The results of these studies indicate that cistrons for at least three characteristic S. typhimurium and two E. coli 30S ribosomal proteins are closely linked to the strA locus on the genetic maps of both organisms. At least one cistron coding for a 50S ribosomal protein is also closely linked to this locus on both maps. These findings support the concept that cistrons coding for the ribosomal proteins are clustered in one area of the genome. Mutations to spectinomycin and streptomycin resistance are closely linked in S. typhimurium and are located at strA.     

Mitochondrial Genetics VII. Allelism and Mapping Studies of Ribosomal Mutants Resistant to Chloramphenicol, Erythromycin and Spiramycin in S. CEREVISIAE

We have isolated 15 spontaneous mutants resistant to one or several antibiotics like chloramphenicol, erythromycin and spiramycin. We have shown by several criteria that all of them result from mutations localized in the mitochondrial DNA. The mutations have been mapped by allelism tests and by two- and three-factor crosses involving various configurations of resistant and sensitive alleles associated in cis or in trans with the mitochondrial locus omega which governs the polarity of genetic recombination. A general mapping procedure based on results of heterosexual (omega(+)x omega(-)) crosses and applicable to mutations localized in the polar segment is described and shown to be more resolving than that based on results of homosexual crosses. Mutations fall into three loci which are all linked and map in the following order: omega-R(I)-R(II)-R(III). The first locus is very tightly linked with omega while the second is less linked to the first. Mutations of similar resistance phenotype can belong to different loci and different phenotypes to the same locus. Mutations confer antibiotic resistance on isolated mitochondrial ribosomes and delineate a ribosomal segment of the mitochondrial DNA. Homo- and hetero-sexual crosses between mutants of the ribosomal segment and those belonging to the genetically unlinked ATPase locus, O(I), have been performed in various allele configurations. The polarity of recombination between R(I), R(II), R(III) and O(I) decreases as a function of the distance of the R locus from the omega locus rather than as a function of the distance of the R locus from the O(I) locus.     

Genetic studies of he ribosoml proteis in Escherichaoli. V. Mapp-ing of erythromycin resistance mutations which lea to alteration of a 50s ribosomal protein component

Summary It was previously reported that in Escherichia coli the mutation from erythromycin sensitive to resistance accompanied the alteration of one 50s ribosomal protein component, 50-8, and that the chemical nature of this alteration differed in one mutant to another. In the present paper, it has been demonstrated that the character of the altered 50-8 component was always cotransduced with the erythromycin resistance from the donor to the recipient bacteria by Plkc phage. The geneic locus of these mutations has been shown to be in the str region by transduction and conjugation experiments.     

Erythromycin and spiramycin resistance mutations of yeast mitochondria: nature of the rib2 locus in the large ribosomal RNA gene.

Two linked genetic loci, rib 2 and rib 3, of yeast mitochondrial genome are the sites of mutations that confer resistance to erythromycin and/or spiramycin. We have examined two mutations at the rib 2 locus. Mutation ER354 was found at the nucleotide position 3993 of the large ribosomal RNA gene; it corresponded to a C to G transversion leading to a double resistance to erythromycin and spiramycin. Mutation SR551 was found also at the same position, but the C was replaced by a T, conferring resistance to spiramycin only. Rib 2 and rib 3 are 836 base pairs apart on the gene sequence, but are very close to each other in the secondary structure of ribosomal RNA.     

A Third Kasugamycin Resistance Locus, ksgC, Affecting Ribosomal Protein S2 in Escherichia coli K-12

A third kasugamycin-resistant mutant affecting ribosomal protein S2 has been isolated from Escherichia coli K-12. Mating and transduction revealed that this newly recognized kasugamycin resistance locus, designated as ksgC, is located at 0.1 to 0.2 min from purE.     

Mapping of chloroplast genes involved in chloroplast ribosome biogenesis in Chlamydomonas reinhardtii

Summary Chloroplast gene mutations which confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii have been tested for allelism and mapped by recombination analysis of progeny from biparental zygote clones. Thirty-one independently isolated streptomycin resistant mutants have chloroplast ribosomes which are resistant to this drug in an assay based on misreading of isoleucine in response to a poly U template, and comprise one nuclear and four chloroplast gene loci. Four mutants resistant to spectinomycin, and three mutants resistant to neamine and kanamycin, which have chloroplast ribosomes resistant to their respective antibiotics in poly U directed phenylalanine incorporation, appear to map in a single chloroplast gene locus. Representative alleles of this nr/spr locus, the four streptomycin resistance loci, and two chloroplast gene loci for erythromycin resistance, have been analyzed in a series of parallel crosses to establish the following map order for these seven genes in the chloroplast genome: er-u-la-er-u-37-nr-u-2-1/spr-u-1-H-4-sr-u-2-23-sr-u-2-60-sr-u-sm3-sr-u-sm2. These seven genes may constitute a ribosomal region within the chloroplast genome of Chlamydomonas comparable to the ribosomal gene clusters in bacteria.     

Macrolide and aminoglycoside antibiotic resistance mutations in the Bacillus subtilis ribosome resulting in temperature-sensitive sporulation

Summary Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins). Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance. These mutants are all temperature-sensitive for sporulation (Spo^ts). Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17. These mutations do not cause a defective sporulation phenotype. All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B. subtilis chromosome. Hence, changes in a single ribosomal protein can result in different sporulation phenotypes.Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated. Approximately 5% of these are Spo^ts. Representative mutations, neo 162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map as single-site lesions in the Str-Spc region of the chromosome. Strains bearing neo162 or kan25 are equally cross-resistant to several aminoglycoside antibiotics but show no resistance to streptomycin or spectinomycin. These mutations define a new B. subtilis drug resistance locus at which mutation can cause defective sporulation.     

Mutations determining generalized resistance to aminoglycoside antibiotics in Escherichia coli.

Summary Mutations conferring resistance to low levels of kanamycin in Escherichia coli have been mapped at 3 locations: the unc locus (min. 83), a locus we have designated, kanA (min. 72), close to strA (rpsL), and a locus at min. 86.5 previously discovered by Plate (1976) that we have designated ecfB. The unc and ecfB mutations are associated with defects in energy metabolism, while mutations at kanA may be in the gene coding for ribosomal protein S12 (rpsL). The three types of mutations cause cross resistance to a number of different aminoglycoside antibiotics and the effects of the mutations are cumulative in combination.     

Analysis of lincomycin resistance mutations in Escherichia coli.

Summary High level lincomycin resistant strains of Escherichia coli were isolated and screened for altered ribosomal proteins and functions. Amongst 58 strains investigated by electrophoresis one had an altered ribosomal protein S7, another one a mutated L14 and two showed altered L15 proteins.A correlation between these alterations and lincomycin resistant growth could not be demonstrated by genetic analysis for any of the mutants. In vitro, however, extracts from the two L15 mutants were less sensitive to inhibition by the drug. A gene locus (lin ^R) responsible for the lincomycin resistance phenotype was mapped at min 30 of the Escherichia coli chromosome near tyrR; it seems to be identical to the previously described linB locus (Apirion, 1967); however, in contrast to these reports it does not seem to alter any ribosomal function.     

Mapping of chloroplast mutations conferring resistance to antibiotics in Chlamydomonas: Evidence for a novel site of streptomycin resistance in the small subunit rRNA

Summary A major obstacle to out understanding of the mechanisms governing the inheritance, recombination and segregation of chloroplast genes in Chlamydomonas is that the majority of antibiotic resistance mutations that have been used to gain insights into such mechanisms have not been physically localized on the chloroplast genome. We report here the physical mapping of two chloroplast antibiotic resistance mutations: one conferring cross-resistance to erythromycin and spiramycin in Chlamydomonas moewusii (er-nM1) and the other conferring resistance to streptomycin in the interfertile species C. eugametos (sr-2). The er-nM1 mutation results from a C to G transversion at a well-known site of macrolide resistance within the peptidyl transferase loop region of the large subunit rRNA gene. This locus, designated rib-2 in yeast mitochondrial DNA, corresponds to residue C-2611 in the 23 S rRNA of Escherichia coli. The sr-2 locus maps within the small subunit (SSU) rRNA gene at a site that has not been described previously. The mutation results from an A to C transversion at a position equivalent to residue A-523 in the E. coli 16 S rRNA. Although this region of the E. coli SSU rRNA has no binding affinity for streptomycin, it binds to ribosomal protein S4, a protein that has long been associated with the response of bacterial cells to this antibiotic. We propose that the sr-2 mutation indirectly affects the nearest streptomycin binding site through an altered interaction between a ribosomal protein and the SSU rRNA.     

Chloramphenicol resistance mutation in Escherichia coli which maps in the major ribosomal protein gene cluster.

Localized mutagenesis and selection for streptomycin resistance were utilized to isolate a chloramphenicol resistance mutation in Escherichia coli K-12 linked to the strA (rpsL) locus. Bacteriophage P1 transduction verified the map position of the new resistance mutation at 72 min, placing it within a dense cluster of ribosomal protein genes. The map position differs from that of known cmlA and cmlB mutations, which map at 18 and 21 min, respectively. Ribosomes prepared from chloramphenicol-resistant and -sensitive isogenic transductants were analyzed in vitro for activity in formation of N-formylmethionyl-puromycin, polyphenylalanine, and polylysine in the presence of inhibitory concentrations of chloramphenicol. Comparisons were also made of 14C-chloramphenicol binding to 70S ribosomes and of the two-dimensional polyacrylamide gel electrophoresis pattern of ribosomal proteins from each strain. There was no detectable difference between ribosomes from sensitive and resistant strains as measured by these assays. Enzymatic modification by chloramphenicol acetyltransferase is not responsible for the observed phenotype.     

New antibiotic resistance Loci in the ribosomal region of yeast mitochondrial DNA

A large number of mitochondrial antibiotic-resistant mutants have been isolated following mutagenesis with manganese. These include several different phenotypic classes of mutants, as distinguished by cross-resistance patterns, that have been found to be allelic at cap1 or ery1; some have been found to be heteroallelic.--Seven chloramphenicol-resistant mutants have been identified that are nonallelic by recombination tests with the three loci (cap1, spi1 and ery1) previously identified in the ribosomal region. Four of these are allelic with each other and define a new locus, cap3; two others are allelic and define another new locus, cap2; the seventh maps at yet a different locus, cap4. One new spiramycin-resistant mutant has been identified that defines still another new locus, spi2. A variety of genetic techniques have been used to map these loci within the ribosomal region of the mitochondrial genome.-Manganese has been shown to be effective in inducing the mutation from omega(-) to omega(n) in many mutants that experience a simultaneous mutation at the closely linked cap1 locus. The omega(n) mutation has also been described in the cap4 mutant, and this locus has been shown to be more closely linked to omega than cap1 is to omega.     

Chromosomal mutations causing resistance to tetracycline in Bacillus subtilis.

Summary We have isolated, after ethylmethanesulfonate mutagenesis, several chromosomal mutations causing resistance to tetracycline in Bacillus subtilis. These mutations fall into two classes, tetA and tetB. 30 S ribosomal protein S10 shows an altered mobility on two-dimensional acrylamide gels in cells bearing the former type of mutation. Ribosomes from these cells show elevated levels of resistance to tetracycline in vitro as measured by polyuridine dependent polyphenylalanine synthesis. The tetA locus maps adjacent to the tuf gene in the B. subtilis ribosomal protein gene cluster. Cells with the tetB mutation do not show any altered ribosomal protein, and their ribosomes are as sensitive, in vitro, to tetracycline as ribosomes isolated from wild type cells. The tetB mutation has been mapped proximal to cysA14.In partial fulfillment of the requirements for the doctoral degree by G.W. in the Department of Biology at the New York University Graduate School of Arts and Sciences     

Cloning the gyrA gene of Bacillus subtilis.

We have isolated an eight kilobase fragment of Bacillus subtilis DNA by specific integration and excision of a plasmid containing a sequence adjacent to ribosomal operon rrn O. The genetic locus of the cloned fragment was verified by linkage of the integrated vector to nearby genetic markers using both transduction and transformation. Functional gyrA activity encoded by this fragment complements E. coli gyrA mutants. Recombination between the Bacillus sequences and the E. coli chromosome did not occur. The Bacillus wild type gyrA gene, which confers sensitivity to nalidixic acid, is dominant in E. coli as is the E. coli gene. The cloned DNA precisely defines the physical location of the gyrA mutation on the B. subtilis chromosome. Since an analogous fragment from a nalidixic acid resistant strain has also been isolated, and shown to transform B. subtilis to nalidixic acid resistance, both alleles have been cloned.     

A ribosomal orphon sequence from Xenopus laevis flanked by novel low copy number repetitive elements

We have used a differential cloning approach to isolate ribosomal/non-ribosomal frontier sequences from Xenopus laevis. A ribosomal intergenic spacer sequence (IGS) was cloned and shown not to be physically linked with the ribosomal locus. This ribosomal orphon contained the IGS sequences found immediately downstream of the 28S gene and included an array of enhancer repetitions and a non-functional spacer promoter. The orphon sequence was flanked by a member of the novel 'Frt' low copy repetitive element family. Three individual Frt repeats were sequenced and all members of this family were shown to lie clustered at two chromosomal sites, one of which contained the ribosomal orphon. One of the Frt elements contained an insertion of 297 bp that showed extensive homology to sequences within at least three other Xenopus genes. Each homology region was flanked by members of the T2 family of short interspersed repetitive elements, (SINEs), and by its target insertion sequence, suggesting multiple translocation events. The data are discussed in terms of the evolution of the ribosomal gene locus.     

Chromosomal location of the 28S ribosomal RNA gene of channel catfish by in situ polymerase chain reaction

This study describes the use of the polymerase chain reaction for physical mapping of fish genes. A 287–base pair (bp) fragment of the 28S ribosomal RNA gene (28S rDNA) of channel catfish Ictalurus punctatus was isolated and sequenced with human-derived primers. The nucleotide (nt) sequence of this fragment was 20 bp shorter than that of the corresponding region of the human 28S rDNA. The gene was mapped to chromosomes of channel catfish by fluorescence in situ hybridization (FISH) and in situ polymerase chain reaction (ISPCR). A major locus and a minor locus of 28S rDNA were found on chromosomes of channel catfish. The major locus was associated with the active nucleolus organizer region (NOR) sites. The minor locus was highly resolved and not detectable by silver staining, suggesting that this locus was not involved in synthesis of ribosomal RNA and possessed fewer copies of 28S rDNA. Both loci contained GC-rich DNA elements that could be components of 28S rDNA repeated units. In this study, a potential method of comparative mapping of the channel catfish genome has been presented by using human-derived oligonucleotide sequences. These data demonstrate that ISPCR is highly specific and will be useful in physical mapping of fish genomes.     

Ribosomal protein S14 is not responsible for the Minute phenotype associated with the M(1)7C locus in Drosophila melanogaster.

Summary A locus associated with a severe Minute effect has been mapped at 7C on the X chromosome of Drosophila melanogaster. Previous work has suggested that this Minute encodes ribosomal proteins S14A and S141B. We have made a chromosomal deficiency that removes the S14 ribosomal protein genes, yet does not display the Minute phenotype. These data suggest that the S14 genes do not actually correspond to the Minute locus.     

PerR controls Mn-dependent resistance to oxidative stress in Neisseria gonorrhoeae

In previous studies it has been established that resistance to superoxide by Neisseria gonorrhoeae is dependent on the accumulation of Mn(II) ions involving the ABC transporter, MntABC. A mutant strain lacking the periplasmic binding protein component (MntC) of this transport system is hypersensitive to killing by superoxide anion. In this study the mntC mutant was found to be more sensitive to H2O2 killing than the wild-type. Analysis of regulation of MntC expression revealed that it was de-repressed under low Mn(II) conditions. The N. gonorrhoeae mntABC locus lacks the mntR repressor typically found associated with this locus in other organisms. A search for a candidate regulator of mntABC expression revealed a homologue of PerR, a Mn-dependent peroxide-responsive regulator found in Gram-positive organisms. A perR mutant expressed more MntC protein than wild-type, and expression was independent of Mn(II), consistent with a role for PerR as a repressor of mntABC expression. The PerR regulon of N. gonorrhoeae was defined by microarray analysis and includes ribosomal proteins, TonB-dependent receptors and an alcohol dehydrogenase. Both the mntC and perR mutants had reduced intracellular survival in a human cervical epithelial cell model.