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PubMed2003

Development of polyphosphate parameters for use with the AMBER force field.

Accurate force fields are essential for reproducing the conformational and dynamic behavior of condensed-phase systems. The popular AMBER force field has parameters for monophosphates, but they do not extend well to polyphorylated molecules such as ADP and ATP. This work presents parameters for the partial charges, atom types, bond angles, and torsions in simple polyphosphorylated compounds. The parameters are based on molecular orbital calculations of methyldiphosphate and methyltriphosphate at the RHF/6-31+G* level. The new parameters were fit to the entire potential energy surface (not just minima) with an RMSD of 0.62 kcal/mol. This is exceptional agreement and a significant improvement over the current parameters that produce a potential surface with an RMSD of 7.8 kcal/mol to that of the ab initio calculations. Testing has shown that the parameters are transferable and capable of reproducing the gas-phase conformations of inorganic diphosphate and triphosphate. Also, the parameters are an improvement over existing parameters in the condensed phase as shown by minimizations of ATP bound in several proteins. These parameters are intended for use with the existing AMBER 94/99 force field, and they will permit users to apply AMBER to a wider variety of important enzymatic systems.

Adenosine Triphosphate↗
PubMed2006

Comparison of multiple Amber force fields and development of improved protein backbone parameters.

The ff94 force field that is commonly associated with the Amber simulation package is one of the most widely used parameter sets for biomolecular simulation. After a decade of extensive use and testing, limitations in this force field, such as over-stabilization of alpha-helices, were reported by us and other researchers. This led to a number of attempts to improve these parameters, resulting in a variety of "Amber" force fields and significant difficulty in determining which should be used for a particular application. We show that several of these continue to suffer from inadequate balance between different secondary structure elements. In addition, the approach used in most of these studies neglected to account for the existence in Amber of two sets of backbone phi/psi dihedral terms. This led to parameter sets that provide unreasonable conformational preferences for glycine. We report here an effort to improve the phi/psi dihedral terms in the ff99 energy function. Dihedral term parameters are based on fitting the energies of multiple conformations of glycine and alanine tetrapeptides from high level ab initio quantum mechanical calculations. The new parameters for backbone dihedrals replace those in the existing ff99 force field. This parameter set, which we denote ff99SB, achieves a better balance of secondary structure elements as judged by improved distribution of backbone dihedrals for glycine and alanine with respect to PDB survey data. It also accomplishes improved agreement with published experimental data for conformational preferences of short alanine peptides and better accord with experimental NMR relaxation data of test protein systems.

Alanine↗
PubMed2001

Structural characterisation of Baltic amber and its solvent extracts by several mass spectrometric methods.

A sample of Baltic amber ( approximately 40 million yrs old) has been extracted using pentane, toluene and 1-methyl-2-pyrrolidinone (NMP). The relationship between solubility characteristics of the extracts in relation to molecular mass and chemical makeup has been investigated. The extracts were first characterised by (13)C-NMR spectrometry, size exclusion chromatography (SEC) and UV-fluorescence spectroscopy. The fractions differed less in terms of chemical structural features than they did in terms of molecular mass. This contrasts markedly with data on fractions of coal-derived liquids, but parallels results from petroleum-derived vacuum residues. In SEC, the toluene soluble/pentane insoluble fraction gave a peak for high mass material at about 67 000 u. Material excluded from the column porosity in this fraction and in NMP solubles eluted between 8 and 11 min, corresponding to polystyrene masses between 200 000 and several million u. A column with a larger pore size distribution was calibrated using polystyrene and polymethylmethacrylate standards with detection by a light-scattering evaporative analyser. The largest polystyrene standard (15.4 million u) eluted at 13.4 min, similar to that of the earliest eluting amber-derived material in the NMP solubles fraction. Results from probe-MS and pyrolysis-GC/MS have been used to confirm the similarity of chemical structures of the three solubility fractions. Broadly, low mass ions appear to correspond to the various monomeric units of structures present in the amber, the higher mass ions to dimer units and the molecular ions to the different combinations of three or more monomeric units. The main monomer groups have been identified in detail, showing a situation very different from that of coal-derived materials, where the sizes of aromatic ring systems increase with molecular size.

Journal Article↗
PubMed1994

Modulation of the suppression efficiency and amino acid identity of an artificial yeast amber isoleucine transfer RNA in Escherichia coli by a G-U pair in the anticodon stem.

The artificial amber suppressor corresponding to the major isoleucine tRNA from yeast (pVBt5), when expressed in E. coli, is a poor suppressor of the amber mutation lacIam181-Z. By analysing mutant forms, we could show that this was due to the presence of a U30-G40 wobble pair in the anticodon stem of the yeast tRNA and not to the level of the heterologously expressed tRNA. Efficient suppressors were obtained by restoring a normal U30-A40 or G30-C40 Watson-Crick pair. In vivo the mutant forms are exclusively charged by the bacterial lysyl-tRNA synthetase (LysRS), whereas the original yeast amber tRNA is charged at a low level by E. coli glutaminyl-tRNA synthetase (GlnRS) and LysRS. The inversion of the U30-G40 pair also induces a loss of the Gln identity. We conclude from these experiments that the U30-G40 base pair constitutes a negative determinant for LysRS interaction which operates either at the level of complex formation or at the catalytic step. As no direct contacts are seen between GlnRS and positions 30-40 of the complexed homologous tRNA, the U30-G40 pair of pVBt5 is believed to influence aminoacylation by GlnRS indirectly, probably at the level of the anticodon loop conformation by favouring an optimal apposition of the anticodon nucleotides with the protein.

Amino Acyl-tRNA Synthetases↗
PubMed2001

Gene function analysis by amber stop codon suppression: CMBF is a nuclear protein that supports growth and development of Dictyostelium amoebae.

The C-module-binding factor, CMBF, is a nuclear DNA-binding protein which was originally identified through its specific binding to a promoter element within the retrotransposable element TRE5-A of Dictyostelium discoideum AX2 cells. In order to analyse putative physiological functions of CMBF for the TRE5-A-hosting D. discoideum cells, we used a novel strategy to create mutant cell lines which stably underexpressed functional CMBF. An amber (UAG) translation stop codon was introduced into the chromosomal copy of the CMBF-encoding gene (cbfA), and an amber suppressor tRNA gene was expressed in the same mutant cells. Due to the low efficiency of translation stop codon suppression in this system all recovered cell lines expressed <20 % of wild-type CMBF levels. The mutant cell lines displayed strong growth phenotypes when plated on their natural food source, bacteria. We show evidence that growth reduction was due to impaired phagocytosis of bacteria in the mutants. All obtained mutants showed a strong developmental defect which was defined by the formation of very small fruiting bodies. The strength of the developmental phenotype appeared to depend upon the residual CMBF levels maintained in the mutants. We propose that CMBF is a general transcription regulator which supports the normal expression of several genes required for the maintenance of high proliferation rates of D. discoideum amoebae as well as proper aggregation and development. Our results demonstrate that amber stop codon suppression may be a useful strategy to stably underexpress proteins whose coding genes cannot be successfully disrupted by homologous recombination.

Active Transport, Cell Nucleus↗
PubMed1980

Amber-mutants of plasmid mini-F defective in replication.

Amber mutants of the mini-F plasmid pML31 were isolated with the mutagen hydroxylamine. Under non-permissive conditions amber mutants segregate and show no incorporation of label into supercoiled plasmid DNA in double-label experiments. Wild-type and one mutant of mini-F were integrated by recombinant DNA techniques into the single EcoRI site of plasmid vector pBR322. Plasmid specific proteins were labeled in minicells and analysed by SDS-PAGE. A 34,000 dalton molecular weight protein was identified to be missing in the amber mutant of plasmid mini-F.

Bacterial Proteins↗
PubMed1977

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

A method to obtain amber mutations in ribosomal protein genes is described. tit 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 lambda prophage which kills suppressor hosts at 42 degrees 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.

Bacteriological Techniques↗
PubMed1993

Expression of a Drosophila melanogaster amber suppressor tRNA(Ser) in Caenorhabditis elegans.

The purpose of this study was to test a cloned amber-suppressing tRNA(Ser) gene derived from Drosophila melanogaster for its ability to produce amber suppression in the nematode Caenorhabditis elegans. To date, all characterized nonsense suppressors in C. elegans have been derived from tRNA(Trp) genes. Suppression was assayed by monitoring the reversal of a mutant tra-3 phenotype among individuals transformed with the cloned Drosophila suppressor gene. An amber allele of tra-3 results in masculinization of XX animals with accompanying sterility. Complete suppression was observed among the transformants. The presence of the heterologous transgene, in both suppressed experimental animals and controls injected with a non-suppressing wild-type Drosophila tRNA(Ser) gene, was verified by PCR amplification of DNA from single worms using primers flanking the tRNA(Ser) gene. Suppression by the heterologous transgene was comparable in quality to that produced by endogenous C. elegans suppressors, and, in frequency as well as quality, to that produced by a transgenic C. elegans tRNA(Trp)-derived suppressors. Thus, a heterologous suppressor gene will function in C. elegans, and it need not be based on tRNA(Trp).

Animals↗
PubMed1982

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. II. Evidence that large N-terminal amber fragments of the beta subunit interfere with RNA polymerase function.

A collection of 95 independent, spontaneously-occurring mutants carrying amber lesions that affect expression of the beta gene, rpoB, has been isolated (see accompanying paper (Nene and Glass 1982)). Certain rpoB amber mutations act in trans, preventing a functional allele present on an F' plasmid from acting at high temperature. Two such temperature-sensitive rpoB(Am) strains are shown to produce large, N-terminal amber fragments. The possibility that these truncated polypeptides are the cause of this transdominant conditional-lethal phenotype is supported by analysis of fragment levels in thermoresistant survivors: the nonsense fragments are degraded at a significantly faster rate (half-lives 1.4- to 2.6-fold reduced) in Ts+ derivatives likely to carry second-site mutations within rpoB. We suggest that the beta fragments interfere with RNA polymerase function by interacting with one or more of the polymerase subunits.

DNA-Directed RNA Polymerases↗
PubMed1985

Safety evaluation of flashing amber operation at signalized intersections.

The safety of a flashing amber signal for all directions at off-peak hours as a replacement for regular traffic signal operations was evaluated. The proposed control strategy was motivated by the need for energy conservation through reduced amounts of acceleration and of idling time of vehicles. The methodology of the study employed as a measure a broadened definition of "conflict" that freed observers from the need to detect only emergency evasive manoeuvres and decreased their subjective interpretations. Observations were carried out by trained observers at a sample of intersections, using two control strategies: full signal operation and flashing amber phase. Stationed at each leg of an intersection, the observers noted the travel direction of any two vehicles involved in a conflict. The results showed that the most frequent type of conflict under full signal operation was of the rear-end type; during the flashing amber operation, crossing and merging conflicts were dominant. It was concluded that up to a volume of 600 vehicles per hour, flashing operation does not increase the number of conflicts.

Accidents, Traffic↗
PubMed1984

Yeast amber suppressors corresponding to tRNA3Leu genes.

Amber suppressors previously isolated from the yeast Saccharomyces cerevisiae and belonging to the same phenotypic class (Liebman et al., 1976) were assigned to nine different linkage groups named SUP52 through SUP60. One of these suppressors, SUP52, had been shown to cause the insertion of leucine and had been genetically mapped (Liebman et al., 1977). The following additional amber suppressors were mapped: SUP53 maps near the centromere of chromosome III closely linked to leu2; SUP54 maps on chromosome VII, 6 cM distal to trp5; SUP56 maps on chromosome I, 5.4 cM distal to ade1; SUP57 maps on chromosome VI, closely linked to met10; and SUP58 maps on the left arm of chromosome XI, loosely linked to met14. We show by protein analysis that like SUP52, the suppressors SUP53 through SUP56 are leucine-inserters. Furthermore, by hybridization with a cloned tRNA3Leu probe we demonstrate that at least SUP53, SUP54, SUP55 and SUP56 contain mutations in redundant tRNA3Leu genes because they each generate a new XbaI site in a DNA fragment encompassing a tRNA3Leu gene. These new XbaI sites are predicted by the known sequences of tRNA3Leu genes if the CAA anticodon mutates to the amber suppressing anticodon CTA. It is likely that each of the nine suppressors in this phenotypic class contain similar mutations in different tRNA3Leu genes since we find that there are approximately nine unlinked redundant copies of tRNA3Leu genes in haploid strains.

Autoradiography↗
PubMed1986

Mutation in the D arm enables a suppressor with a CUA anticodon to read both amber and ochre codons in Escherichia coli.

Su9 of Escherichia coli differs from tRNATrp by only a G to A transition in the D arm, yet has an enhanced ability to translate UGA by an unusual C X A wobble pairing. In order to examine the effects of this mutation on translation of the complementary and wobble codons in vivo, we constructed the gene for an amber (UAG) suppressing variant of Su9, trpT179, by making the additional nucleotide change required for an amber suppressor anticodon. The resultant suppressor tRNA, Su79, is a very strong amber suppressor. Furthermore, the D arm mutation enables Su79 to suppress ochre (UAA) codons by C X A wobble pairing. These data demonstrate that the effect of the D arm mutation on wobble pairing is not restricted to a CCA anticodon. The effect extends to the CUA anticodon of Su79, thereby creating a new type of ochre suppressor. The new coding activity of Su79 cannot be explained by alterations in the level of aminoacylation, steady-state tRNA concentration, or nucleotide modification. The A24 mutation could permit unorthodox wobble pairings by generally enhancing tRNA efficiency at all codons or by altering codon specificity.

Anticodon↗
PubMed1987

Temperature sensitivity of the multiplication of bacteriophage T4 amber mutants on nonpermissive host: characterization of the phenomenon.

The existence of temperature sensitivity of the multiplication of amber mutants on a nonpermissive host has been established for a considerable number of mutants in tail and head genes and for mutants in some other T4 genes as well. Temperature sensitivity of multiplication appears to be gene specific, and is typical of amber mutants in genes the products of which are not numerous per phage or which play the role of catalytic factor. Moreover, in most cases temperature sensitivity is characteristic of amber mutants in definite gene clusters.

Escherichia coli↗
PubMed1983

The genes sup-7 X and sup-5 III of C. elegans suppress amber nonsense mutations via altered transfer RNA.

The sup-5 III and sup-7 X suppressors in C. elegans have previously been shown to have many genetic properties in common with tRNA nonsense suppressors of microorganisms. We report here the results of two lines of investigation into the molecular basis of these suppressors. In one, which sought to determine the nature of suppressible alleles, we demonstrate through DNA sequencing studies that a suppressible allele, unc-54(e 1300) I, of the myosin heavy chain gene contains a C leads to T substitution, which changes a glutamine codon to amber terminator at residue 1903. In the other approach, which sought to define the nature of the suppressing activity, we show through in vitro translation studies that tRNA fractions from the suppressor strains, but not wild-type, promote the specific readthrough of amber terminators of three different messenger RNAs. We conclude that sup-5 and sup-7 result in readthrough of amber terminators in vivo through an altered tRNA.

Alleles↗
PubMedDate not supplied

Isolation of a DNA fragment that is expressed as an amber suppressor when present in high copy number in yeast.

A plasmid carrying a DNA sequence conferring amber suppression has been isolated from yeast pool 35 DNA. The plasmid YEp13-SUP suppresses the amber mutations rad50-1 and trp1-289 as well as other known amber mutations. This sequence is located on the 2.5-kb insert and is expressed only when the plasmid bearing it is present in high copy number. The suppressor sequence was shown to integrate close to the MAL4 gene located on the right arm of chromosome XI.

Base Sequence↗
PubMed1995

A ROC study of AMBER and conventional chest imaging in the detection of simulated interstitial lung disease.

PURPOSE: The aim of this study was to compare the detection of simulated interstitial lung disease with Advanced Multiple Beam Equalization Radiography (AMBER) and conventional wide latitude screen-film radiography. MATERIALS AND METHODS: Interstitial disease of varying severity was simulated with overlays on an anthropomorphic chest phantom. A total of 60 images per modality was used in a Receiver Operating Characteristic (ROC) study. RESULTS: AMBER performed significantly better than conventional radiography for all readers (P < 0.02). The difference was even more significant for the radiologist readers (P = 0.001). In each case the difference in ROC areas was between 5% and 9%. CONCLUSION: AMBER is superior to conventional wide latitude screen-film imaging in detecting the subtle patterns used to simulate interstitial lung disease.

Humans↗
PubMedDate not supplied

Amber suppressors of Erwinia chrysanthemi.

Mutations trp1 and thyA1, both of a polyauxotrophic derivative of the Erwinia chrysanthemi strain B374, were characterized as amber mutations with an Escherichia coli suppressor, supA1P2, which inserts a glutamine in response to UAG. Simultaneous reversion of both mutations allowed us to isolate amber suppressor mutants of E. chrysanthemi. These suppressors were tested with a set of amber mutants of bacteriophage Mu which had been previously characterized on E. coli. The two independently isolated suppressors behaved as supD and supE mutants, respectively, of E. coli.

Bacteriophage mu↗
PubMed2006

A fast mutagenesis procedure to recover soluble and functional scFvs containing amber stop codons from synthetic and semisynthetic antibody libraries.

The selection and production of scFvs from phage display synthetic antibody libraries are frequently delayed by the presence of amber (TAG) stop codons within the sequences corresponding to the variable CDRs. This is due to the use of randomised oligonucleotides for library design and amber mutations for joining the scFv to the phage protein pIII. The screening of such libraries may lead to the selection of scFvs containing stop codons. Then, multiple site-directed mutagenesis is required for their removal or, alternatively, the proteins must be expressed as scFv-pIII fusions, which are not suitable for many functional assays. We describe here an alternative procedure to express soluble scFvs, despite the presence of TAG stop codons, in the currently used Escherichia coli suppressor strain TG1. It is based on a simple mutagenesis protocol that replaces the amber codon between the scFv and the pIII gene by a different stop codon (TAA), functional in E. coli TG1. The expression of soluble scFvs in the suppressor strain TG1 permits their fully functional characterization including the determination of affinity constants, which are critical for selecting the right scFvs for further studies.

Amino Acid Sequence↗