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PubMed1983

Isolation of conditionally lethal amber mutations affecting synthesis of the nusA protein of Escherichia coli.

Amber mutants (am3 and am4) of Escherichia coli K12 defective in the synthesis of nusA protein (Friedman 1971) were isolated from a strain harboring an amber suppressor (sup-126) that is active only at low temperatures. These mutants grew at low temperature (30 degrees C) but did not grow at temperatures above 38 degrees C. Complementation experiments with plasmids carrying the nusA+ gene and its derivatives or with plasmids carrying the nusA1 or am4 mutation indicated that the mutations am3, am4 and nusA1 affected the same gene function. Analysis of proteins produced by minicells containing a plasmid demonstrated that the plasmid pYN87, which can complement the nusA1 and amber mutations, codes for three bacterial proteins, a truncated nusA gene product (61 K), argG gene product (48 K) and a 21 K dalton protein, and that the am4 mutation affects the synthesis of only NusA protein. lambda Nam7 (and lambda Nam7Nam53) phages could grow on these amber mutants at 32 degrees C but not on the parental strain. Spontaneous temperature-resistant revertants of the amber mutants simultaneously lost the ability to permit lambda Nam7 phage development, indicating that the two phenotypes are due to a single mutation. These results suggest that the nusA gene function is essential for the growth of E. coli, and that the lambda N function is dispensable for phage development if the nusA gene is defective.

Bacterial Proteins↗

PubMed1980

Amber mutations in the structural gene for RNA polymerase sigma factor of Escherichia coli.

Amber mutants of Escherichia coli K-12 affected in the structural gene (rpoD) for the sigma subunit of RNA polymerase have been obtained from a strain harboring a temperature-sensitive amber suppressor (supF-Ts6) which is active only at low temperatures. These mutants grow normally at low temperature (30 degrees C) but do not grow at high temperature (42 degrees C) due to the inability to synthesize sigma factor. In one mutant studied in detail (rpoD40), the rate of sigma-factor synthesis at 30 degrees C is about half that of the wild type and is decreased to 10%-15% within 1 h of incubation at 42 degrees C. The synthesis of core polymerase subunits or bulk protein is virtually unaffected at least for 2 h. The defect of the mutant in sigma synthesis and growth at high temperature can be suppressed by any of the amber suppressors tested (supD. supE or supF). RNA-polymerase holoenzymes prepared from the mutant cells carrying each of the suppressors (grown at 42 degrees C) exhibit different thermostabilities attributable to alterations in the sigma factor. The reduced sigma synthesis in the mutant is accompanied by the synthesis of polypeptide tentatively identified as 'amber fragment'. These results as well as the genetic mapping data indicate that the amber mutation (rpoD40) resides within the structural gene for the sigma factor and directly affects sigma synthesis upon inactivation of the suppressor at high temperature.

Chromosome Mapping↗

PubMed1991

Adeno-associated viruses having nonsense mutations in the capsid genes: growth in mammalian cells containing an inducible amber suppressor.

When an adeno-associated virus (AAV) genome contained in a recombinant plasmid is transfected into adenovirus-infected cells, infectious AAV particles are efficiently generated. We previously described the construction of a conditional lethal mutant of AAV having an amber termination codon inserted in the rep gene. This mutant was propagated on a monkey kidney cell line (SupD12) having an inducible amber suppressor tRNAser. We now describe the construction and propagation of two additional conditional lethal mutants of AAV having amber codons affecting all three capsid proteins (AAV Capam) or only the VP1 capsid protein (AAV VP1am). Suppression of the amber mutations in the capsid proteins was demonstrated directly by immunoblot analysis. The efficiency of amber suppression on the SupD12 cell was about 6 to 10% for AAV VP1am and 4 to 5% for AAV Capam. The reversion frequency of either mutant was apparently less than 10(-5). On nonsuppressing cells AAV VP1am exhibited an Lip (Inf) phenotype, whereas AAV Capam exhibited a Cap phenotype.

Animals↗

PubMed1987

An inducible mammalian amber suppressor: propagation of a poliovirus mutant.

We describe a general protocol for controlled gene amplification, which allows conditional expression of high levels of amber suppressor activity in monkey kidney cells, and we demonstrate its use in the genetic analysis of animal viruses by the generation and propagation of the first nonsense mutant of poliovirus. A human amber suppressor tRNASer gene linked to the SV40 origin of replication and a second DNA carrying a temperature-sensitive SV40 large T antigen gene were cotransfected into monkey cells. Cell lines having stably integrated the DNAs were isolated. Shifting the cells from the nonpermissive temperature to a lower permissive temperature caused the amplification of the suppressor tRNA gene, which resulted in suppression efficiencies at amber codons of 50%-70%, as measured by suppression of an amber codon in the E. coli chloramphenicol acetyltransferase gene. A mutant of poliovirus, in which a serine codon in the replicase gene was converted to an amber codon, was efficiently propagated on the suppressor-positive cell lines. The mutant virus reverted to wild-type by a single base change to a serine codon at a frequency of approximately 2.5 x 10(-6), surprisingly low for a RNA genome.

Animals↗

PubMed1991

Advanced multiple beam equalization radiography (AMBER) in the detection of diffuse lung disease.

To evaluate the effects of scanning equalization radiography (SER) on the detection of diffuse lung disease a clinical comparison between an Advanced Multiple Beam Equalization Radiography (AMBER) unit and conventional chest radiography was performed. Even though the overall detection of focal pulmonary lesions with the AMBER unit has been shown to be significantly higher than with conventional radiography because of the improved demonstration of the costophrenic and retrocardiac regions, the utility of AMBER in the demonstration of diffuse lung disease has not been established. Twenty-one patients with diffuse lung disease (fibrosing alveolitis or sarcoidosis) and six patients with no pulmonary disease had high kVp frontal and lateral chest radiographs on both an AMBER unit and a conventional chest stand. The pooled results of five observers using Receiver Operating Characteristic (ROC) analysis indicate that there is a slight improvement but no statistically significant difference in observer performance between AMBER (Area under the ROC curve AZ = 0.934) and conventional radiography (AZ = 0.868) in the task of detecting diffuse lung disease.

Evaluation Studies as Topic↗

PubMed1990

Genetic and molecular analysis of eight tRNA(Trp) amber suppressors in Caenorhabditis elegans.

Over 100 revertants of five different amber mutants were analyzed by Southern blot hybridization using synthetic oligomers as probes to detect a single base change at the anticodon, CCA to CTA (amber), of tRNA(Trp) genes of Caenohrabditis elegans. Of the 12 members of the tRNA(Trp) gene family, a total of eight were converted to amber suppressor alleles. All eight encode identical tRNAs; three of these are new tRNA(Trp) suppressors, sup-21, sup-33 and sup-34. Previous results had suggested that individual suppressor tRNA genes were expressed differentially in a cell-type- or developmental stage-specific manner. To extend these observations to the new genes and to test the specificity of expression against additional genes, cross suppression tests of these eight amber suppressors were carried out against amber mutations in several different genes including genes likely to be expressed in the same cell-type: three nervous system-affecting genes, two muscle structure-affecting genes and two genes presumed to be expressed in hypodermis. Seven out of eight suppressors could be distinguished one from another by the spectrum of their suppression efficiencies. These results also provide further evidence of cell-type-specific patterns of expression in the nervous system, muscle and hypodermis. The suppression pattern of the suppressor against the two muscle-affecting genes, unc-15 and unc-52, suggested that either the suppressors are expressed in a developmental stage-specific manner or that the unc-52 products are expressed in cell-types other than muscle, possibly hypodermis.

Animals↗

PubMed1995

[Value of the AMBER system in interstitial lung changes].

PURPOSE: To evaluate the diagnostic value of the AMBER technique in the diagnosis of interstitial lung disease. METHODS: A prospective comparison of conventional and AMBER chest radiographs of 52 patients was performed using HR-CT as a gold standard on the same day. ROC analysis based on the results of 5 observers was carried out to quantify the diagnostic value of both chest radiograph techniques. RESULTS: The ROC area for the AMBER system (0.860) showed statistically significant superiority in comparison to the conventional technique (0.828). This was mainly due to the better sensitivity of the AMBER system (65.3%) compared to the conventional chest radiographs (39.8%) within the lung regions superimposed by the diaphragm or the mediastinum. No difference between both methods was observed within the lung regions without superimposion. CONCLUSION: AMBER technique improved conventional chest radiography in the diagnosis of interstitial lung disease due to superiority in the superimposed lung regions.

Evaluation Studies as Topic↗

PubMed2001

Import of amber and ochre suppressor tRNAs into mammalian cells: a general approach to site-specific insertion of amino acid analogues into proteins.

A general approach to site-specific insertion of amino acid analogues into proteins in vivo would be the import into cells of a suppressor tRNA aminoacylated with the analogue of choice. The analogue would be inserted at any site in the protein specified by a stop codon in the mRNA. The only requirement is that the suppressor tRNA must not be a substrate for any of the cellular aminoacyl-tRNA synthetases. Here, we describe conditions for the import of amber and ochre suppressor tRNAs derived from Escherichia coli initiator tRNA into mammalian COS1 cells, and we present evidence for their activity in the specific suppression of amber (UAG) and ochre (UAA) codons, respectively. We show that an aminoacylated amber suppressor tRNA (supF) derived from the E. coli tyrosine tRNA can be imported into COS1 cells and acts as a suppressor of amber codons, whereas the same suppressor tRNA imported without prior aminoacylation does not, suggesting that the supF tRNA is not a substrate for any mammalian aminoacyl-tRNA synthetase. These results open the possibility of using the supF tRNA aminoacylated with an amino acid analogue as a general approach for the site-specific insertion of amino acid analogues into proteins in mammalian cells. We discuss the possibility further of importing a mixture of amber and ochre suppressor tRNAs for the insertion of two different amino acid analogues into a protein and the potential use of suppressor tRNA import for treatment of some of the human genetic diseases caused by nonsense mutations.

Animals↗

PubMed1976

Isolation and characterization of amber suppressors in yeast.

Nonsense suppressors were obtained in a haploid yeast strain containing eight nutritional mutations, that are assumed to be amber or ochre, and the cyc1-179 amber mutation that has a UAG codon corresponding to position 9 in iso-1-cytochrome c. Previous studies established that the biosynthesis and function of iso-1-cytochrome c is compatible with replacements at position 9 of amino acids having widely different structures (Stewart and Sherman 1972). UV-induced revertants, selected on media requiring the reversion of one or two of the amber nutritional markers, were presumed to contain a suppressor if there was the unselected reversion of at least one other marker. The 1088 suppressors that were isolated could be divided into 78 phenotypic classes. Only 43 suppressors of three classes caused the production of more than 50% of the normal amount of iso-1-cytochrome c in the cyc1-179 strain. Genetic analyses indicated that all of these highly efficient amber suppressors are allelic to one or another of the eight suppressors which cause the insertion of tyrosine at ochre (UAA) codons (Gilmore, Stewart and Sherman 1971). Furthermore, only tyrosine has been identified at position 9 in iso-1-cytochrome c in cyc1-179 strains suppressed with these efficient amber suppressors.

Cytochrome c Group↗

PubMed1977

Search for revertants of the glutamine mischarging mutans of Escherichia coli su+3 tyrosine suppressor tRNA that are able to insert tyrosine at the site of amber mutation.

We have isolated a bacterial amber mutation (nadam) that is suppressed by the tyrosine inserting suppressor su+3 but not by the glutamine (su+2, su+3 A1, su+3 G82 and su+3 A1G82), serine (su+1) and leucine (su+6) inserting suppressors. The su+7 suppressor which inserts glutamine and tryptophan also suppresses this mutation indicating that tryptophan, in addition to tyrosine, is accepted at the site of amber mutation. We have used this amber mutation to search for revertants of the su+3 glutamine mischarging mutants su+3 A1, su+3 G82 and su+3 A1G82 that are able to insert tyrosine at the site of amber mutation. Two types of revertants were found in the case of su+3 A1. One type corresponding to the true revertant A1 leads to G, and the other to the second site revertants C81 leads to U (A1U81). The A1U81 revertant has been shown to insert both glutamine and tyrosine at the site of amber mutation. Only true revertants (G82 leads to A) were obtained when su+3 G82 was analyzed. No revertants were obtained in the case of the su+3 A1G82. These results are discussed in relation to aminoacyl-tRNA recognition.

2-Aminopurine↗

PubMed2006

Development of amber suppressor tRNAs appropriate for incorporation of nonnatural amino acids.

An amber suppression method has been used for incorporation of nonnatural amino acids into proteins. However, the incorporation efficiency of nonnatural amino acids through an amber codon has been low, which restricts the application of the proteins containing nonnatural amino acids. In this study, we screened a wide variety of amber suppressor tRNAs to discover tRNAs capable to incorporate nonnatural amino acids with high efficiency. To this purpose, synthetic amber suppressor tRNAs of E. coli and Mycoplasma capricolum were screened for the incorporation of a fluorescently labeled nonnatural amino acid in an E. coli cell-free translation system. tRNAs that showed high capability for the incorporation were then mutated not to be aminoacylated by any of endogenous aminoacyl-tRNA synthetases of E. coli and to enhance the incorporation capability. As a result of these investigations, we successfully obtained several amber suppressor tRNAs with high ability for the incorporation of nonnatural amino acids.

Amino Acids↗

PubMed1995

Genes Essential for Amber Disease in Grass Grubs Are Located on the Large Plasmid Found in Serratia entomophila and Serratia proteamaculans.

The bacteria Serratia entomophila and S. proteamaculans cause amber disease in the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Disease symptoms include rapid cessation of feeding and amber coloration of larvae. A 105-kb plasmid (designated pADAP) has consistently been found only in pathogenic isolates of both species. Investigations into the involvement of pADAP in amber disease have been hindered by the lack of both a selectable marker on the plasmid and a reliable transposon delivery system. Kanamycin-resistant transposon insertions into three cloned HindIII fragments (9.5, 9.6, and 10.6 kb) were isolated and introduced into pADAP by shuttle mutagenesis. Inserts into the 9.5-and 9.6-kb HindIII fragments on pADAP did not alter disease-causing ability. When plasmids with inserts into the 9.6-kb region were conjugated into plasmid-minus, nonpathogenic isolates of S. entomophila and S. proteamaculans, all of them became pathogenic. Transposon insertions into two regions of the 10.6-kb HindIII fragment continued to cause cessation of feeding but failed to produce amber coloration. Further analysis of a mutant from each amber-minus region (pADK-10 and pADK-13) demonstrated that the antifeeding effect was produced only at dosages higher than that of the wild-type strain. Complementation with the wild-type HindIII fragment restored full-blown disease properties for pADK-13, but not for pADK-10.

Journal Article↗

PubMed1979

Characterization of amber and ochre suppressors in Salmonella typhimurium.

Amber and ochre suppressor mutations in Salmonella typhimurium were selected. The amino acid insertions directed by the suppressors were inferred from suppression patterns of Escherichia coli lacI amber mutations. These amber mutations only respond to nonsense suppressors that direct the insertion of particular amino acids. Four Salmonella amber suppressors characterized insert serine, glutamine, tyrosine, and (probably) leucine. Of the three ochre suppressors characterized, two direct the insertion of tyrosine and one directs that of lysine. Of the three amber and two ochre suppressors which have been mapped by phage P22 cotransduction, all are located in the same relative position on the Salmonella map as the analogous E. coli suppressors are on the E. coli map.

Bacterial Proteins↗

PubMed1989

Conditionally lethal amber mutations in the leader peptidase gene of Escherichia coli.

The lep gene of Escherichia coli encodes the leader peptidase which cleaves amino-terminal leader sequences of secreted proteins. To facilitate the study of structure-function relationships of the leader peptidase, 22 amber mutations in lep were isolated by localized mutagenesis. These amber mutants grew at 32 degrees C but not at 42 degrees C in the presence of a temperature-sensitive amber suppressor. Most of them were lethal under sup0 conditions. However, one amber mutant, the lep-9 mutant, exhibited temperature-sensitive growth in the sup0 strain, indicating that the amber fragment is active at 32 degrees C but not at 42 degrees C. Protein precursors of the maltose-binding protein and OmpA accumulate strikingly in the lep-9 mutant.

Chromosome Mapping↗

PubMed2004

Conditional lethal amber mutations in essential Escherichia coli genes.

The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in Escherichia coli that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential E. coli genes. Amber stop codons were introduced as "tagalong" mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes frr, gcpE, lpxC, map, murA, ppa, and rpsA. We were unable to isolate an amber mutation in ftsZ. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in gcpE, which was bacteriostatic. The strain carrying an amber mutation in murA was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development.

Escherichia coli↗

PubMed1981

Genetic and amber fragment maps of genes 46 and 47 of bacteriophage T4D.

We constructed genetic recombinational maps of genes 46 and 47 by using five amber mutants in gene 46, nine amber mutants in gene 47, and two-factor crosses. Two different amber fragments in gene 46 and three different amber fragments in gene 47 were detected on polyacrylamide slab gels in the presence of sodium dodecyl sulfate. The genetic maps agreed with the amber fragment maps; taken together, the data oriented all of the sites in both genes with respect to each other. Given the relative map positions of genes 46 and 47 determined genetically by Epstein et al. (Cold Spring Harbor Symp. Quant. Biol. 28:375-394, 1963), our results extend and reinforce the work of Hercules and Sauerbier (J. Virol. 12: 872-881, 1973) and that of Minner and Bernstein (J. Gen. Virol. 31:277-280, 1976), which indicated that the direction of transcription and translation of these genes if counterclockwise on the T4 genetic map (i.e., from gene 47 toward gene 46).

Chromosome Mapping↗

PubMed2004

Digital slot-scan charge-coupled device radiography versus AMBER and Bucky screen-film radiography for detection of simulated nodules and interstitial disease in a chest phantom.

PURPOSE: To evaluate the diagnostic performance of full-field slot-scan charge-coupled device (CCD)-based digital radiography in the detection of simulated chest diseases in clinical conditions versus that of two screen-film techniques: advanced multiple beam equalization radiography (AMBER) and Bucky radiography. MATERIALS AND METHODS: Simulated nodules and interstitial nodular and interstitial linear lesions were attached onto an anthropomorphic chest phantom. One hundred sixty-eight lesions were distributed over 25 configurations. A posteroanterior chest radiograph of each configuration was obtained with each technique. The images were presented to six observers. Each lesion was assigned one of two outcome scores: "detected" or "not detected." False-positive readings were evaluated. Differences between the imaging methods were analyzed by using a semiparametric logistic regression model. RESULTS: For simulated nodules and interstitial linear disease, no statistically significant difference was found in diagnostic performance between CCD digital radiography and AMBER. The detection of simulated interstitial nodular disease was better with CCD digital radiography than with AMBER: Sensitivity was 71% (77 of 108 interstitial nodular lesions) with CCD digital radiography but was 56% (60 of 108 lesions) with AMBER (P =.041). Better results for the detection of all lesion types in the mediastinum were observed with CCD digital radiography than with Bucky screen-film radiography: Sensitivity was 45% (227 of 504 total simulated lesions) with CCD digital radiography but was 24% (119 of 504 lesions) with Bucky radiography (P <.001). There were fewer false-positive observations with CCD digital radiography (35 [5.7%] of 609 observations) than with Bucky radiography (47 [9.5%] of 497 observations; P =.012). CONCLUSION: Differences were in favor of the full-field slot-scan CCD digital radiographic technique. This technique provides a digital alternative to AMBER and Bucky screen-film radiography.

Dose-Response Relationship, Radiation↗

PubMed1993

Film-based chest radiography: AMBER vs asymmetric screen-film systems.

OBJECTIVE: Contrast-to-noise ratios were measured on radiographs from two types of state-of-the-art chest imaging systems (an Advanced Multiple Beam Equalization Radiography [AMBER] system and an asymmetric screen-film system) to facilitate an objective comparison of image quality. MATERIALS AND METHODS: Radiographs of a chest phantom were obtained by using the AMBER system with a medium-latitude screen-film image recorder (Kodak T-MAT L film and Lanex regular screens) and a commercially available asymmetric, zero-crossover screen-film system optimized for chest radiography (Kodak InSight and InSight HC). Conventionally acquired radiographs (T-MAT L/Lanex regular) were also evaluated as a reference. Films were digitized, radiographic contrast and noise were measured in the lung-, mediastinum-, and subdiaphragm-equivalent regions of each image, and contrast-to-noise ratios were computed. RESULTS: Radiographic contrast and contrast-to-noise values were found to be higher on AMBER images in all chest regions when compared with radiographs obtained with the asymmetric screen-film systems (InSight contrast-to-noise ratio approximately 77% of AMBER contrast-to-noise in the lung-equivalent region, 57% in the mediastinum-equivalent region, and 43% in the subdiaphragm-equivalent region). On conventional radiographs, the contrast and contrast-to-noise values were higher than on all other image types in the lung-equivalent region and lower than on all other image types in the less well penetrated chest areas. CONCLUSION: Image quality was higher, most notably in dense phantom regions, on radiographs obtained with the AMBER system than on radiographs obtained with the new asymmetric screen-film systems. Clinical studies are needed to determine whether this level of image improvement justifies the additional expense of the exposure equalization system.

Humans↗