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W S Champney

Publications and source records attributed to W S Champney.

At least 37 records · Page 2Linked to original sources
PubMed1994

A temperature-sensitive mutant of Escherichia coli with an alteration in ribosomal protein L22.

A temperature-sensitive, protein synthesis-defective mutant of Escherichia coli exhibiting an altered ribosomal protein L22 has been investigated. The temperature-sensitive mutation was mapped to the rplV gene for protein L22. The genes from the wild type and mutant strains were amplified by the polymerase chain reaction and the products were sequenced. A cytosine to thymine transition at position 22 of the coding sequence was found in the mutant DNA, predicting an arginine to cysteine alteration in the protein. A single cysteine residue was found in the isolated mutant protein. This amino acid change accounts for the altered mobility of the mutant protein in two-dimensional gels and during reversed-phase HPLC. The temperature-sensitive phenotype was fully complemented by a plasmid carrying the wild type L22 gene. Ribosomes from the complemented cells showed only wild type protein L22 by two dimensional gel analysis and were as heat-resistant as control ribosomes in a translation assay. The point mutation in the L22 gene is uniquely responsible for the temperature-sensitivity of this strain.

Amino Acid Sequence↗
PubMed1994

Ribosomal protein gene sequence changes in erythromycin-resistant mutants of Escherichia coli.

The genes for ribosomal proteins L4 and L22 from two erythromycin-resistant mutants of Escherichia coli have been isolated and sequenced. In the L4 mutant, an A-to-G transition in codon 63 predicted a Lys-to-Glu change in the protein. In the L22 strain, a 9-bp deletion removed codons 82 to 84, eliminating the sequence Met-Lys-Arg from the protein. Consistent with these DNA changes, in comparison with wild-type proteins, both mutant proteins had reduced first-dimension mobilities in two-dimensional polyacrylamide gels. Complementation of each mutation by a wild-type gene on a plasmid vector resulted in increased erythromycin sensitivity in the partial-diploid strains. The fraction of ribosomes containing the mutant form of the protein was increased by growth in the presence of erythromycin. Erythromycin binding was increased by the fraction of wild-type protein present in the ribosome population. The strain with the L4 mutation was found to be cold sensitive for growth at 20 degrees C, and 50S-subunit assembly was impaired at this temperature. The mutated sequences are highly conserved in the corresponding proteins from a number of species. The results indicate the participation of these proteins in the interaction of erythromycin with the ribosome.

Amino Acid Sequence↗
PubMed1992

Ribosomes from trichomonad protozoa have prokaryotic characteristics.

1. Ribosomes from cells of the genera Trichomonas and Tritrichomonas have been isolated and characterized. The ribosomes from each organism had a sedimentation coefficient of 70S in calibrated sucrose gradients and the subunits sedimented as 50S and 30S particles under the same conditions. 2. The major ribosomal RNAs from each species were identical in size to prokaryotic ribosomal RNAs when examined by denaturing gel electrophoresis. The ribosomes contained both 5.8S and 5S RNAs. 3. The ribosomal proteins were compared by the methods of two-dimensional gel electrophoresis and reversed phase HPLC. Electrophoresis of the ribosomal proteins in two different gel systems indicated the presence of 56 proteins in T. gallinae, 40 in T. bactrachorum and 45 in the Tritrichomonas sp. The protein molecular mass range was 8.5-40 kDa. 4. The HPLC analysis confirmed the protein number established by the gel methods. 5. Both methods of analysis revealed greater similarities between the ribosomal proteins of the 2 Tritrichomonas sp. than between those of the more distantly related T. gallinae and T. bactrachorum.

Animals↗
PubMed1991

Characteristics of a leucine aminoacyl transfer RNA synthetase from Tritrichomonas augusta.

This study has investigated the characteristics of a leucine aminoacyl transfer RNA synthetase enzyme from Tritrichomonas augusta. Differential centrifugation and DEAE-cellulose column chromatography were used for partial enzyme purification. The column purification increased the synthetase activity 125-fold over the unfractionated cell extract. The conditions for maximum [3H] leucine charging were 37 degrees C for 20 min, with protein at 180 micrograms ml-1 using yeast leucine tRNA as an acceptor. The optimal reaction conditions were 14 mM-Mg acetate, 3 mM-ATP, 3 mM-spermidine and 5.5 mM-putrescine. Acceptor activity with T. augusta transfer RNA was 8-fold higher than with yeast transfer RNA and 25-fold higher than with Escherichia coli transfer RNA. The partially purified enzyme fraction had comparable changing activities for both leucine and valine.

Amino Acyl-tRNA Synthetases↗
PubMed1990

Reversed-phase chromatography of Escherichia coli ribosomal proteins. Correlation of retention time with chain length and hydrophobicity.

The elution times of 52 bacterial ribosomal proteins from a C4 reversed-phase column have been predicted. The prediction is based upon the use of the hydrophobicity coefficients for the protein amino acid content as defined by Guo et al. [J. Chromatogr., 359 (1986) 499-518]. A strong positive correlation was observed when the difference between predicted and observed protein retention time was plotted against the product of net hydrophobicity and natural log of protein chain length. Observations with this class of related proteins strengthens and extends the observations of Mant et al. [J. Chromatogr., 476 (1989) 363-375]. Observed deviations from predicted chromatographic behavior can be explained for several proteins which elute as dimers or which have modified amino acid residues.

Chemical Phenomena↗
PubMed1990

A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase.

A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated. Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min. on the bacterial chromosome. The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44 degrees C. The thermostability at 44 degrees C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme. Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a function of assay temperature but that initiation and elongation were not significantly affected by the alpha mutation. A major effect on the fidelity of transcription was observed with the mutant enzyme, with misincorporation on two different templates stimulated about 4 fold at 37 degrees C. The role of the alpha dimer in the structure and function of RNA polymerase is discussed.

Chromosome Mapping↗
PubMed1989

Reductive methods for isotopic labeling of antibiotics.

Methods for the reductive methylation of the amino groups of eight different antibiotics using 3HCOH or H14COH are presented. The reductive labeling of an additional seven antibiotics by NaB3H4 is also described. The specific activity of the methyl-labeled drugs was determined by a phosphocellulose paper binding assay. Two quantitative assays for these compounds based on the reactivity of the antibiotic amino groups with fluorescamine and of the aldehyde and ketone groups with 2,4-dinitrophenylhydrazine are also presented. Data on the cellular uptake and ribosome binding of these labeled compounds are also presented.

Aminoglycosides↗
PubMed1985

Temperature-sensitive translation of MS2 bacteriophage RNA.

A comparison was made of bacteriophage MS2 RNA translation in infected Escherichia coli cells and in a defined cell-free system. A number of temperature-sensitive mutants were used as hosts for viral RNA translation at permissive and restrictive temperatures. The amount of viral coat protein synthesis was determined after gel electrophoresis of proteins from the cell lysates. These results were compared to those obtained with cell-free translation assays conducted with ribosomes isolated from the same mutants. Compared with control cells, a reduced activity in vivo and in vitro was found for each mutant examined at elevated temperatures. A good correlation between the two types of translational assays was observed. These findings are discussed in terms of the translational defects known to be a characteristic of some of these mutant strains.

Coliphages↗
PubMed1981

Effects of (hydroxymethyl)trimethylpsoralen on structure and function of bacteriophage MS2 ribonucleic acid.

Treatment of bacteriophage MS2 with 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen and 360-nm light caused a dose-dependent decline in the infectivity of the virus. Covalent photobinding of a single psoralen molecule on the phage genome was a lethal event. Ribonucleic acid (RNA) extracted from psoralen and light-treated virus had a dose-dependent 385-nm fluorescence emission but was unaltered in its physical properties compared to control RNA samples. Phage adsorption and penetration in Escherichia coli host cells were unaffected, but in vivo replication of the treated virus was affected to the same extent as infectivity. The cell-free translational activity of the MS2 RNA was also severely reduced after psoralen and light treatment of the phage. Examination of the in vitro translation products revealed that the synthesis of the viral replicase protein was most substantially affected. Psoralen treatment of purified, protein-free MS2 RNA promoted an even greater reduction in cell-free synthesis of all viral proteins. This difference in translational function was consistent with the observation that virion-free RNA bound approximately 4 times as much psoralen as did RNA treated within the phage capsid. It was concluded that the replicase gene is the most sensitive region of the viral RNA molecule for psoralen binding.

Coliphages↗
PubMed1980

Protein synthesis defects in temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins.

The ribosomes from four temperature-sensitive mutants of Escherichia coli have been examined for defects in cell-free protein synthesis. The mutants examined had alterations in ribosomal proteins S10, S15, or L22 (two strains). Ribosomes from each mutant showed a reduced activity in the translation of phage MS2 RNA at 44 degrees C and were more rapidly inactivated by heating at this temperature compared to control ribosomes. Ribosomal subunits from three of the mutants demonstrated a partial or complete inability to reassociate at 44 degrees C. 70-S ribosomes from two strains showed a reducton in messenger RNA binding. tRNA binding to the 30 S subunit was reduced in the strains with altered 30-S proteins and binding to the 50 S subunit was affected in the mutants with a change in 50 S protein L22. The relation between ribosomal protein structure and function in protein synthesis in these mutants is discussed.

Bacterial Proteins↗
PubMed1979

Protein synthesis kinetics with ribosomes from temperature-sensitive mutants of Escherichia coli.

The kinetics of MS2 ribonucleic acid (RNA) directed protein synthesis have been investigated at seven temperatures between 30 and 47 degrees C by using ribosomes isolated from a wild type strain and seven temperature-sensitive mutants of Escherichia coli. The amount of MS2 coat protein formed at each temperature was determined by gel electrophoresis of the products formed with control ribosomes. With ribosomes from each of the mutant strains, the activation energy required to drive protein synthesis below the maximum temperature (up to 40 degrees C) was increased relative to the control (wild type) activity. Preincubation of the ribosomes at 44 degrees C revealed the kinetics of thermal inactivation, with ribosomes from each of the mutants having a half-life for inactivation less than that of the control ribosomes. A good correlation was observed between the relative activity of the different ribosomes at 44 degrees C and their relative rate of thermal inactivation. Mixing assays allowed the identification of a temperature-sensitive ribosomal subunit for each of the mutants. Defects in one or more of three specific steps in protein synthesis (messenger RNA binding, transfer RNA binding, transfer RNA binding, and subunit reassociation) were identified for the ribosomes from each mutant. The relationship between temperature sensitivity and protein synthesis in these strains is discussed.

Bacterial Proteins↗
PubMed1979

Properties of Escherichia coli 16S ribosomal ribonucleic acid treated with 4,5',8-trimethylpsoralen and light.

16S rRNA reacted with the furocoumarin 4,5',8-trimethylpsoralen (trioxsalen) and 360-nm light showed a number of chemical and physical differences from untreated RNA. After extensive irradiation, five molecules of trioxsalen were bound per molecule of RNA. The trioxsalen-treated RNA had an altered ultraviolet absorption spectrum and a distinctive fluorescence emission spectrum. The modified RNA was significantly more resistant to T1 ribonuclease digestion than was control RNA. Treated RNA, when mixed with purified ribosomal proteins, was not functional in the in vitro reconstitution of 30S subunits and yielded more slowly sedimenting particles which were inactive in protein synthesis assays. By contrast, 16S rRNA within the 30S subunit structure did not exhibit these changes when reacted with the same dose of trioxsalen and light, suggesting that the ribosomal proteins were effective in protecting the RNA from interaction with the drug.

Cell Fractionation↗
PubMed1979

Localized mutagenesis for the isolation of temperature-sensitive mutants of Escherichia coli affected in protein synthesis.

Two variations of the method of localized mutagenesis were used to introduce mutations into the 72 min region of the Escherichia coli chromosome. Twenty temperature-sensitive mutants, with linkage to markers in this region, have been examined. Each strain showed an inhibition of growth in liquid medium at 44 degrees, and 19 of the mutants lost viability upon prolonged incubation at this temperature. A reduction in the rate of in vivo RNA and protein synthesis was observed for each mutant at 44 degrees, relative to a control strain. Eleven of the mutants were altered in growth sensitivity or resistance to one or more of three ribosomal antibiotics. The incomplete assembly of ribosomal subunits was detected in nine strains grown at 44 degrees. The characteristics of these mutants suggest that many of them are altered in genes for translational or transcriptional components, consistent with the clustering of these genes at this chromosomal locus.

Bacterial Proteins↗
PubMed1977

Kinetics of ribosome synthesis during a nutritional shift-up in Escherischia coli K-12.

The rates of total protein synthesis, polyribosome formation and 70S ribosome accumulation were measured following a nutritional shift-up of Escherichia coli K-12. Changes in ribosome content and distribution during the shift-up were measured by examining the total cellular content of free and polysome-associated ribosomes using a sensitive double isotope labeling method. The kinetics of ribosomal subunit formation and the biosynthesis of subunit protein and RNA species were also defined. The results indicated that a pre-shift population of ribosomal subunits was utilized for the immediate post shift increase in both total and ribosomal-specific protein synthesis. An assembly time for new subunits of about 3 min was observed. The formation of certain ribosomal proteins during the shift suggested that new subunit assembly was limited by the rate of synthesis of particular ribosomal proteins during this growth transition.

Amino Acids↗
PubMed1977

Transcription of ribosomal protein genes carried on F' plasmids of Escherichia coli.

The transcriptional activity of the DNA sequences coding for certain ribosomal proteins has been measured in Escherichia coli. Two partial diploid strains were isolated from mating of a recA, argD, aroE recipient and an Hfr with an origin at 69.5 min. One contained an F' element carrying the genes from 69.5 to 72.7 min including the rpsL locus (72.4) and the second was diploid for the genes from 69.5 to 71.5 min but did not include any mapped ribosomal protein loci. The molecular weights of the plasmids were estimated to be 140 and 110 x 10(6) daltons, respectively. The extent of in vivo transcription of the chromosomal genes on the plasmid and the ribosomal protein mRNA fraction of the total cellular RNA weer calculated from DNA-RNA liquid hybridization experiments using both DNA and RNA excess procedures. The results indicated a high degree of transcriptional activity concentrated in the ribosomal protein sequences with 83% of the F' chromosomal sequences transcribed into mRNA products representing about 0.12% of the total cellular RNA.

Conjugation, Genetic↗
PubMed1977

Conditionally lethal ribosomal protein mutants: characterization of a locus required for modification of 50S subunit proteins.

Mutagenized P1 bacteriophage were used to transduce a marker (aroE) adjacent to the cluster or ribosomal protein genes located at 72 min on the Escherichia coli chromosome. Linked temperature-sensitive transductants were isolated and characterized. A mutant unable to grow at 44 degrees was found to be defective in protein synthesis both in vivo and in vitro. At the restrictive temperature mutant cells lost all polyribosomes. Analysis of the ribosomal proteins revealed alterations in at least four 50S subunit proteins. The mutation (called rimE1, ribosomal protein modification) mapped between rpsE and aroE. It is suggested that the rimE locus is the structural gene for an activity that modifies a selected number of ribosomal proteins.

Coliphages↗
PubMed1976

A proposal for a uniform nomenclature for the genetics of bacterial protein synthesis.

A new genetic nomenclature for the macromolecules involved in bacterial protein synthesis is proposed and explained. Genes for ribosomal proteins are designated rsp, rpl and rpm while genes for ribosomal RNAs are rrs and rrl. Protein synthesis factors and ribosome assembly and modification activities are also consistantly named.

Bacterial Proteins↗