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At least 217 records · Page 12Linked to original sources
PubMed1980

The selection of amber mutations in genes required for completion of start, the controlling event of the cell division cycle of S. cerevisiae.

Using a modification of a procedure developed for the isolation of temperature-sensitive mutants defective in the start event of cell division, amber mutations were obtained for two Class-I start genes, cdc28 and cdc37. Genetic analysis demonstrated that co-segregation of an amber suppressor with such alleles was required for viability of spores subsequent to meiosis. These mutations are expected to be useful in the identification of the molecular products of the genes cdc28 and cdc37.

Alleles↗
PubMed1990

In vitro suppression of an amber mutation by a chemically aminoacylated transfer RNA prepared by runoff transcription.

An amber suppressor tRNA was prepared in vitro by runoff transcription with T7 RNA polymerase. Both full-length tRNA and truncated tRNA lacking the 3' terminal pCpA from the acceptor stem could be synthesized from the same DNA template. Truncated runoff suppressor tRNA could be enzymatically ligated to phenylalanyl-pCpA to generate aminoacylated full-length suppressor tRNA (Phe-tRNA(CUA)). Phe-tRNA(CUA) is capable of suppressing an amber (UAG) mutation in vitro with equivalent efficiency as suppressor prepared by anticodon-loop replacement of a naturally-isolated tRNA. The ease of suppressor tRNA preparation using this method, compared to anticodon-loop replacement, greatly facilitates the use of chemically acylated suppressor tRNA's for site-specifically incorporating unnatural amino acids into proteins.

Acylation↗
PubMed1995

Genetic selection for active E.coli amber tRNA(Asn) exclusively led to glutamine inserting suppressors.

Suppressor tRNAs are useful tools for determining identity elements which define recognition of tRNAs in vivo by their cognate aminoacyl-tRNA synthetases. This study was aimed at the isolation of active amber tRNA(Asn). Nineteen mutated tRNA(Asn)CUA having amber suppressor activity were selected by an in vivo genetic screen, and all exclusively inserted glutamine. From analysis of the different mutations it is concluded that glutamine accepting activity was obtained upon reducing the interaction strength between the first base pair of the tRNA(Asn)CUA by direct or indirect effects. Failure to isolate tRNA(Asn)CUA suppressors charged with asparagine as well as other evolutionary related amino acids is discussed.

Base Sequence↗
PubMed1987

Isolation and characterization of conditional lethal amber nonsense mutants of vesicular stomatitis virus.

We describe the isolation and characterization of conditional lethal amber nonsense mutants of vesicular stomatitis virus (VSV), Indiana serotype. The mutants were isolated from a chemically mutagenized stock of wild-type virus by their ability to grow on genetically engineered cells which express a Xenopus laevis amber suppressor tyrosine tRNA gene (su+ cells) but not on the non-suppressor parental cells (su- cells). Five mutations were assigned to complementation group I (the L gene) and one to complementation group V (the G gene) by complementation analysis using temperature-sensitive mutants representing each of the five VSV cistrons. Four of the group I mutants were observed to synthesize a novel polypeptide species in su+ cells. Immunoprecipitation and immunoblotting studies using monospecific antisera directed against the N and C termini of the VSV L protein showed that the novel polypeptide species contain N terminal- but not C terminal-specific sequences and can thus be considered to be truncated versions of the L protein. In addition a protein which again contained N terminal- but not C terminal-specific sequences could be identified for the fifth group I mutant. Revertants of four of the group I mutants were isolated on the su- cells. The revertants all synthesized normal L protein but not the putative truncated version.

DNA Mutational Analysis↗
PubMed1991

Further characterization of conditional lethal amber nonsense mutants of vesicular stomatitis virus: nucleotide sequence analysis.

Conditional lethal amber nonsense mutants of vesicular stomatitis virus, Indiana serotype, classified in complementation group I (the L gene), synthesize truncated versions of the L protein. This paper reports further characterization of mutants AmbL1, AmbL2 and AmbL3 by nucleic acid sequence analysis, which was achieved by sequencing L mRNA directly using appropriate synthetic oligonucleotides. In each case a single point mutation altered a glutamine-specifying codon to an amber stop codon. The L mRNA from wild-type and revertant viruses was sequenced for comparison. Of the revertants sequenced, each had reverted by back mutation within the same codon as the original mutation. A revertant of AmbL2 reverted by a second site mutation, also within the same codon as the original mutation. These mutants may be useful for assigning functions to different parts of the L polypeptide chain.

Base Sequence↗
PubMed1996

Suppression of amber nonsense mutations of herpes simplex virus type 1 in a tissue culture system.

We have investigated the ability of monkey kidney cell lines (SupD3 and SupD12) inducibly expressing an amber suppressor tRNA(ser) to suppress amber nonsense mutations in three genes of herpes simplex virus type 1 (HSV-1). HSV-1 mutant TK4, which contains a nonsense mutation in the non-essential viral thymidine kinase (TK) gene, synthesized a full-length TK polypeptide at about 30% of the wild-type (wt) level in induced SupD3 cells but not in the parental non-suppressor (Sup0) cells. Using complementing cells, we constructed HSV-1 mutants carrying nonsense mutations in an essential gene, UL8, encoding a protein essential for viral DNA replication (ambUL8) or in a partially dispensable gene, UL12, encoding alkaline nuclease (ambUL12). The growth of the mutants in Vero or Sup0 cells was either totally (ambUL8) or severely (ambUL12) impaired, whereas in cells expressing suppressor tRNA the mutants produced infectious virus. However, the yields were much lower than obtained with wt HSV-1. In Vero or Sup0 cells the mutants ambUL8 and ambUL12 failed to synthesize full-length UL8 and UL12 protein products, respectively. Western immunoblotting showed that the virus ambUL12 produced full-length UL12 protein in SupD12 cells which yielded a level of 25.9% of the alkaline nuclease activity of the wt HSV-1 control. Our results show that the levels of suppression of the nonsense mutations in ambUL8 and ambUL12 are insufficient to allow their continuing propagation in the available Sup+ cells. Possible reasons are discussed.

Animals↗
PubMed1980

Characterization of lambda cysB and of two derivatives carrying cysB amber mutations.

The specialized transducing phage lambda cysB (Borck et al., 1976) was found to carry about 5 kilobases of Escherichia coli DNA. It was shown to have an intact cysB gene but none of the known neighbouring genetic loci. The phage (which is known to be deficient in its site-specific recombination functions) was shown to integrate into the chromosome of bacterial recipients at the cysB locus. Excision from this site occasionally generated recombinant phages that had exchanged their cysB allele for the one originally present in the host. In this way lambda cysB derivatives were prepared from lysogens of two strains carrying the amber mutations cysB242 and cysB257; these phases were proved by several tests to contain the expected cysB amber mutations.

Alleles↗
PubMed2005

Acquisition of a stable mutation in metY allows efficient initiation from an amber codon in Escherichia coli.

Escherichia coli strains harbouring elongator tRNAs that insert amino acids in response to a termination codon during elongation have been generated for various applications. Additionally, it was shown that expression of an initiator tRNA containing a CUA anticodon from a multicopy plasmid in E. coli resulted in initiation from an amber codon. Even though the initiation-based system remedies toxicity-related drawbacks, its usefulness has remained limited for want of a strain with a chromosomally encoded initiator tRNA 'suppressor'. E. coli K strains possess four initiator tRNA genes: the metZ, metW and metV genes, located at a single locus, encode tRNA(1)(fMet), and a distantly located metY gene encodes a variant, tRNA(2)(fMet). In this study, a stable strain of E. coli K-12 that affords efficient initiation from an amber initiation codon was isolated. Genetic analysis revealed that the metY gene in this strain acquired mutations to encode tRNA(2)(fMet) with a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended on the presence of a plasmid-borne copy of the mutant metY and recA(+) host background. The mutations were observed when the plasmid-borne gene encoded tRNA(2)(fMet) (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/psi39G40A41). The usefulness of this strain, and a possible role for multiple tRNA(1)(fMet) genes in E. coli in safeguarding their intactness, are discussed.

Anticodon↗
PubMed1988

Effect of calcium (II) and magnesium (II) ions on the 18-23 gamma-carboxyglutamic acid containing cyclic peptide loop of bovine prothrombin. An AMBER molecular mechanics study.

The effect of calcium (II) and magnesium (II) ions on the conformation of the 18-23 cyclic peptide loop of bovine prothrombin are investigated by the molecular mechanics program AMBER (Assisted Model Building with Energy Refinement). The work is an extension of an earlier paper (Eastman et al., Int. J. Peptide Protein Res. 27, 1986, 530-553) that employed the program ECEPP (Empirical Conformational Energy Program for Peptides). In the absence of either metal ion, or in the presence of either one Ca(II) or one Mg(II) ion, the lowest-energy forms found by AMBER have the Gla21-Pro22 peptide bond in a trans conformation. In the presence of two Ca(II) or Mg(II) ions, the loop form of lowest energy is decidedly cis. The coordination about the Ca(II) and Mg(II) ions is different in both the single and double metal cases. In addition, the peptide chains that emerge from the loop are oriented parallel to each other in the lowest-energy complex with two Ca(II) ions, but are not parallel in the lowest-energy complex with two Mg(II) ions.

1-Carboxyglutamic Acid↗
PubMed1974

Proteins of the inner membrane of Escherichia coli: identification of succinate dehydrogenase by polyacrylamide gel electrophoresis with sdh amber mutants.

The inner or cytoplasmic membrane fraction of the cell envelope of Escherichia coli was isolated by isopycnic centrifugation on sucrose gradients. The membrane proteins were analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels (8.5%), and up to 56 bands were resolved. Different preparations gave very similar patterns of proteins. Succinate dehydrogenase mutants (sdh) were isolated which could not grow on succinate minimal medium, although growth on fumarate was unimpaired. The protein patterns of inner membrane preparations from sdh amber mutants were compared with the wild type, and one major band was greatly reduced in the mutants. This component, which represented approximately 5% of the inner membrane protein, was restored by introducing an amber suppressor gene (supU), which also restored the Sdh(+) phenotype. The band corresponded to a protein with a molecular weight of 67,000 daltons, which is close to that for the large subunits of the succinate dehydrogenases of Rhodospirillum rubrum and beef heart mitochondria.

Bacterial Proteins↗
PubMed1975

Genetic analyses of an amber mutation in Escherichia coli K-12, affecting deoxyribonucleic acid ligase and viability.

Genetic analyses of an Escherichia coli K-12 mutant possessing the amber mutation lig-321 were carried out. This mutant is defective in deoxyribonucleic acid (DNA) ligase and conditionally lethal. We constructed strains harboring an F'lig+ or F'lig-321 plasmid. Genetic complementation analyses were done by using these plasmids and by constructing a lig-4/F'lig-321 merodiploid. It was shown that lig-321 does not complement lig-4, unless the former is suppressed by an amber suppressor. The same was found to be the case between lig-321 and lig-ts7. Transductional mapping of lig-321, by a four-factor cross, revealed that lig-321 is very closely linked to lig-4. The frequency of recombinants between the two alleles was not unreasonable for assuming that they arose by intragenic recombination. The lig-4 and lig-ts7 alleles are known to reside in the structural gene for DNA ligase, in which lig-321 may also be located.

Alleles↗
PubMed1988

Accumulation of trehalose by Escherichia coli K-12 at high osmotic pressure depends on the presence of amber suppressors.

When grown at high osmotic pressure, some strains of Escherichia coli K-12 synthesized substantial levels of free sugar and accumulated proline if it was present in the growth medium. The sugar was identified as trehalose by chemical reactivity, gas-liquid chromatography, and nuclear magnetic resonance spectroscopy. Strains of E. coli K-12 could be divided into two major classes with respect to osmoregulation. Those of class A showed a large increase in trehalose levels with increasing medium osmolarity and also accumulated proline from the medium, whereas those in class B showed no accumulation of trehalose or proline. Most class A strains carried suppressor mutations which arose during their derivation from the wild type, whereas the osmodefective strains of class B were suppressor free. When amber suppressor mutations at the supD, supE, or supF loci were introduced into such sup0 osmodefective strains, they became osmotolerant and gained the ability to accumulate trehalose in response to elevated medium osmolarity. It appears that the original K-12 strain of E. coli carries an amber mutation in a gene affecting osmoregulation. Mutants lacking ADP-glucose synthetase (glgC) accumulated trehalose normally, whereas mutants lacking UDP-glucose synthetase (galU) did not make trehalose and grew poorly in medium of high osmolarity. Trehalose synthesis was repressed by exogenous glycine betaine but not by proline.

Betaine↗
PubMed1991

Synthesis, accumulation, and excretion of trehalose in osmotically stressed Escherichia coli K-12 strains: influence of amber suppressors and function of the periplasmic trehalase.

It has been reported previously that Escherichia coli K-12 carries an amber mutation that prevents osmotic stress-dependent accumulation of trehalose (M. L. Rod, K. Y. Alam, P. R. Cunningham, and D. P. Clark, J. Bacteriol. 170:3601-3610, 1988). We report that E. coli K-12 and W1485 (sup0) accumulated trehalose but that they required a higher osmotic strength in the growth medium than that required by their sup+ derivatives. Furthermore, the sup+ derivatives displayed both strongly increased trehalose-6-phosphate synthase activity and expression of otsA-lacZ and otsB-lacZ operon fusions compared with their parental strains. It is suggested that the amber mutation in question may be in a gene system encoding a transcriptional activator of the ots genes which govern the synthase. The much-used sup0 strain MC4100 behaved like the sup+ derivatives of W1485 with respect to trehalose synthesis. treA mutants with a defective periplasmic trehalase accumulated trehalose extracellularly under osmotic stress. The amount of trehalose excreted correlated with their synthase activity. Strains with an intact trehalase did not display extracellular trehalose accumulation. Thus, stressed E. coli cells regulate the cytoplasmic level of trehalose by a futile cycle involving overproduction, excretion, and degradation to glucose, which is reutilized.

Escherichia coli↗
PubMed1992

Identification of a Serratia entomophila genetic locus encoding amber disease in New Zealand grass grub (Costelytra zealandica).

Serratia entomophila UC9 (A1MO2), which causes amber disease in the New Zealand grass grub Costelytra zealandica, was subjected to transposon (TnphoA)-induced mutagenesis. A mutant (UC21) was found to be nonpathogenic (Path-) to grass grub larvae in bioassays and was shown, by Southern hybridization, to contain a single TnphoA insertion. This mutant failed to adhere to the gut wall (Adn-) of the larvae and also failed to produce pili (Pil-). A comparative study of the total protein profiles of wild-type S. entomophila UC9 and mutant UC21 revealed that the mutant lacked an approximately 44-kDa protein and overexpressed an approximately 20-kDa protein. Transfer of cosmids containing homologous wild-type sequences into mutant strain UC21 restored wild-type phenotypes (Path+, Pil+, and Adn+). One of the complementing cosmids (pSER107) conferred piliation on Pil- Escherichia coli HB101. The TnphoA insertion in UC21 was mapped within an 8.6-kb BamHI fragment common to the complementing cosmids, and we designated this gene locus amb-1. Six gene products with molecular masses of 44, 36, 34, 33, 20, and 18 kDa were detected in E. coli minicells exclusive to the cloned 8.6-kb fragment (pSER201A). The 44-kDa gene product was not detected in E. coli minicells containing the cloned mutant fragment. Saturation mutagenesis of this fragment produced four unlinked insertional mutations with active fusions to TnphoA. These active fusions disrupted the expression of one or more gene products encoded by amb-1. The 8.6-kb fragment cloned in the opposite orientation (pSER201B) expressed only a 20-kDa protein. We propose that these are the products of structural and/or regulatory genes involved in adhesion and/or piliation which are prerequisites in the S. entomophila-grass grub interaction leading to amber disease.

Animals↗
PubMed1994

Inducible amber suppressor for Bacillus subtilis.

An amber suppressor variant of Bacillus subtilis tyrosyl-tRNA was constructed and placed under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter. Addition of IPTG resulted in a 50-fold increase in the expression of an rpsD-lacZ fusion containing a UAG amber codon. This system permitted isolation of a conditional lethal mutant which required IPTG for growth.

Bacillus subtilis↗
PubMed1974

Biochemistry of DNA-defective amber mutants of bacteriophage T4. IV. DNA synthesis in plasmolyzed cells.

Requirements for bacteriophage T4 DNA synthesis have been investigated in situ by use of plasmolyzed infected cells. When such cells are incubated with dATP, dGTP, dTTP, hydroxymethyldeoxycytidine triphosphate, and rATP, significant semiconservative synthesis of DNA occurs. This DNA hybridizes preferentially to T4 DNA. T4 amber mutants defective in genes 44 and 45, which display a DNA-negative phenotype in vivo, are unable to synthesize DNA in situ. By contrast, T4 amber mutants bearing lesions in genes 41 and 62, which also display a DNA-negative phenotype in vivo, do allow DNA synthesis in situ, the extent of synthesis being 80 to 90% that of the wild-type synthesis under the same conditions. Cells infected with gene 42 mutants (dCMP hydroxymethylase) are unable to synthesize DNA in situ even though exogenous nucleotides are provided. Also one gene 1 mutant (deoxynucleotide kinase) was found to synthesize DNA in situ, but two other gene 1 mutants did not. These results point to possible roles of hydroxymethylase and kinase in DNA metabolism, in addition to provision of essential DNA precursors, as has recently been suggested by Wovcha et al. (1973).

Adenosine Triphosphate↗
PubMed1969

Phenotypic suppression by streptomycin of amber mutants in the ribonucleic Acid bacteriophage coat protein cistron.

After mutagenesis with nitrosoguanidine or ultraviolet light, 298 streptomycin high-resistant and 98 streptomycin high-dependent mutants were isolated from HfrC Su(-). They were tested for their ability to phenotypically suppress five different amber ribonucleic acid (RNA) bacteriophage mutants in the presence of streptomycin. The phage mutants are all in the coat protein, which is 129 amino acids long; the uracil-adenine-guanine codons were at the following positions: sus3 and amB2, 6; amB11, 50; amB21, 54; sus11, 70. Only sus3 and amB2 could be phenotypically suppressed by streptomycin; this was clearly demonstrated in nine mutant strains, seven str-HR and two str-HD. The suppression was always dependent upon added streptomycin and was dose-dependent in all cases. None of the mutants showed measurable suppression in absence of the drug. Among revertants to streptomycin independence from streptomycin-dependent strains that could show phenotypic suppression, most of those that were still resistant to streptomycin (10 mug or more) retained the capacity to show phenotypic suppression; whereas among those revertants sensitive to 10 mug of streptomycin or less, none retained the capacity. Eight different amber polar mutants (strong and weak) in gene 34 of phage T4 were also tested for pleiotypic suppression by streptomycin in all the streptomycin-resistant and -dependent strains isolated. No suppression was found in any of the 396 strains tested.

Journal Article↗