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T Elliott

Publications and source records attributed to T Elliott.

At least 109 records · Page 6Linked to original sources
PubMed1993

Mutants defective in the energy-conserving NADH dehydrogenase of Salmonella typhimurium identified by a decrease in energy-dependent proteolysis after carbon starvation.

NADH dehydrogenase is the first component of the respiratory chain. It transfers electrons from NADH to ubiquinone and concomitantly establishes a proton motive force across the membrane. Salmonella typhimurium mutants defective in this enzyme were isolated in a screen for strains with increased expression of beta-galactosidase from a hemA-lacZ protein fusion. This unexpected phenotype results from stabilization of the hybrid protein during carbon starvation and is apparently due to an energy requirement for proteolytic attack. Sequence analysis of DNA fragments cloned from an insertion mutant indicates that S. typhimurium has a large cluster of genes encoding the energy-conserving NADH dehydrogenase, similar to one recently described in Paracoccus denitrificans. These findings establish the potential for genetic analysis of a complex enzyme whose function, especially in proton efflux, is poorly understood.

Base Sequence↗
PubMed1993

A method to quantify binding of unlabeled peptides to class I MHC molecules and detect their allele specificity.

A general method has been developed for measuring the stabilization of class I MHC molecules in extracts of the mutant cell lines .174/T2 and RMA-S. 35S-Met-labeled class I molecules which have been stabilized by peptides in vitro are immunoprecipitated with conformation dependent monoclonal antibodies and electrophoresed on polyacrylamide gels. The heavy and light chains are excised from the dried gel and quantified on a flat bed scintillation counter. The stabilizing effect of peptides on class I molecules in vitro correlates well with peptide binding measured by direct methods and can be therefore used to assess peptide binding affinity. We show that a peptide from HIV-1 gag (which has a high affinity for Db) is a CTL epitope restricted through Db, and also use the assay to analyse the effects of amino acid substitution on peptide affinity. In addition, the effect of a given peptide on a class I molecule within a mixture of human class I molecules can be distinguished by immunoprecipitation with the monomorphic antibody W6/32 and separation by 1-D isoelectric focussing. The technique therefore requires neither labeled peptide ligands nor allele-specific antibodies. It can be used to identify the peptide ligand of any human class I molecule, and gives a measure of peptide binding affinity. The technique should be of value in identifying epitopes recognized by CTL since we have found that these tend to bind with the highest affinities.

Alleles↗
PubMed1993

An oxygen-dependent coproporphyrinogen oxidase encoded by the hemF gene of Salmonella typhimurium.

The 8th step in the 10-step heme biosynthetic pathway of Salmonella typhimurium is the oxidation of coproporphyrinogen III to protoporphyrinogen IX. On the basis of genetic studies, we have suggested that this reaction may be catalyzed by either of two different enzymes, an oxygen-dependent one encoded by hemF or an oxygen-independent enzyme encoded by hemN. Here, we report the cloning of the S. typhimurium hemF gene and its DNA sequence. The predicted amino acid sequence of the HemF protein is 44% identical to that of the coproporphyrinogen oxidase encoded by the yeast HEM13 gene. The wild-type S. typhimurium strain LT-2 produces an oxygen-dependent coproporphyrinogen oxidase activity detectable in crude extracts, which is not found in hemF mutants and is overproduced in strains carrying the hemF gene on a multicopy plasmid. the hemF gene is the second gene in an operon with an upstream gene with an unknown function, whose amino acid sequence suggests a relation to amidases involved in cell wall synthesis or remodeling. The upstream gene and hemF are cotranscribed from a promoter which was mapped by primer extension. A weaker, hemF-specific promoter is inferred from the behavior of an omega-Cm insertion mutation in the upstream gene. Although this insertion decreases expression of beta-galactosidase about 7.5-fold when placed upstream of a hemF-lacZ operon fusion, it still allows sufficient HemF expression from an otherwise wild-type construct to confer a Hem+ phenotype. The hemF operon is transcribed clockwise with respect to the genetic map.

Amino Acid Sequence↗
PubMed1993

Transport of 5-aminolevulinic acid by the dipeptide permease in Salmonella typhimurium.

In a previous search for mutants of Salmonella typhimurium that are defective in heme synthesis, one class that is apparently defective in 5-aminolevulinic acid (ALA) uptake (alu) was found. Here, I describe the characterization of these mutations. The mutations all map to a single locus near 77.5 min on the genetic map, which is transcribed counterclockwise. Nutritional tests, genetic and physical mapping, and partial DNA sequence analysis revealed that alu mutants are defective in a periplasmic binding protein-dependent permease that also transports dipeptides, encoded by the dpp operon. The uptake of labeled ALA is defective in dpp mutants and is markedly increased in a strain that has elevated transcription of the dpp locus. Unlabeled L-leucyl-glycine competes with labeled ALA for uptake. In a strain carrying both a dpp-lac operon fusion and a functional copy of the dpp locus, the expression of beta-galactosidase is not induced by ALA, nor, in a hemL mutant, does expression of dpp change substantially during starvation for ALA. The dipeptide permease displays a relaxed substrate specificity that allows transport of the important nonpeptide nutrient ALA, whose structure is closely related to that of glycyl-glycine.

Aminolevulinic Acid↗
PubMed1993

Regulation of apolipoprotein A-I-containing lipoproteins in IDDM.

In IDDM patients, serum high-density lipoprotein cholesterol concentrations have been reported to be normal or elevated. The spectrum of high-density lipoprotein particles is highly heterogeneous, but no data are available on the subpopulations of high-density lipoprotein in IDDM. We, therefore, studied the spectrum of high-density lipoprotein particles in 86 IDDM patients (51 men and 35 women) 37 +/- 10 yr of age and in 74 sex-, age-, and body mass index-matched healthy nondiabetic subjects. The concentrations of high-density lipoprotein and HDL2 cholesterol were higher in the IDDM group than in the control subjects (P < 0.01). The apoA-I-to-apoA-II ratio was higher in the IDDM patients than in the nondiabetic subjects (P < 0.001) because of an increased concentration of LpA-I particles (61 +/- 17 vs. 53 +/- 15, P < 0.01). LpA-I particles correlated positively with high-density lipoprotein and HDL2 cholesterol in the two groups. Postheparin plasma lipoprotein lipase activity was significantly higher in the IDDM group than in the control group (P < 0.001), whereas postheparin plasma hepatic lipase activities were similar in both groups. Plasma cholesteryl ester transfer protein activity was estimated in an in vitro isotopic assay using exogenous labeled donor (low-density) and acceptor (high-density) lipoproteins in the absence of native lipoproteins. We observed no difference in cholesteryl ester transfer protein activity between the groups, and no significant correlations existed between cholesteryl ester transfer protein activity and high-density lipoprotein subpopulations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Assembly and function of the two ABC transporter proteins encoded in the human major histocompatibility complex.

Presentation of cytoplasmic antigens to class I-restricted cytotoxic T cells implied the existence of a specialized peptide transporter. For most class I heavy chains, association with peptides of the appropriate length is required for stable assembly with beta 2-microglobulin. Mutant cells RMA-S and .174/T2 neither assemble stable class I molecules nor present intracellular antigens, and we have suggested that they have lost a function required for the transport of short peptides from the cytosol to the endoplasmic reticulum. The genetic defect in .174 has been localized to a large deletion in the class II region of the major histocompatibility complex, within which two genes (RING4 and RING11) have been identified that code for 'ABC' (ATP-binding cassette) transporters. We report here that the protein products of these two genes assemble to form a complex. Defects in either protein result in the formation of unstable class I molecules and loss of presentation of intracellular antigens. The molecular defect in a new mutant, BM36.1, is shown to be in the ATP-binding domain of the RING11/PSF2 protein. This is in contrast to the mutant .134, which lacks the RING4/PSF1 protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Structural requirements for the peptide-induced conformational change of free major histocompatibility complex class I heavy chains.

In an attempt to define the structural features of peptides which are important for inducing the folding of free class I heavy chains in the absence of beta 2-microglobulin, and to determine whether they are the same as those required to form stable major histocompatibility complex (MHC): peptide adducts, we have used a panel of peptides related to the Db-binding nonamer ASNENMDAM (influenza nucleoprotein residues 366-374) with altered primary structures, and a number of other peptides which have the Db-binding "motif". In this way, we have shown that in addition to the "anchor" residues which define this motif, the alpha amino and carboxyl groups at the N and C termini also play a major role in both inducing the conformational change in free heavy chain (HC) and formation of a stable Db:peptide complex. We also show that the importance of the key residues is affected by the primary sequence "context" in which they appear. In addition, we have extended our original finding that naturally processed epitopes induce a conformational change in free HC to the H2Kb HC, and show that the effect does not require the presence of the class I alpha 3 domain.

Amino Acid Sequence↗
PubMed1992

Association of the human invariant chain with H-2 Db class I molecules.

We describe two proteins of 24 kDa and 33 kDa (p24 and p33) which associate with H-2 Kb and H-2 Db molecules, respectively, in human cells transfected with H-2 Kb and H-2 Db genes. This association is particularly clear in the mutant cell line T2, in which association of endogenous peptide with newly synthesized class I molecules may not occur (V. Cerundolo et al., Nature 1990. 345: 449). We show that p33 is the 33-kDa form of the human invariant chain which is resident in the endoplasmic reticulum of T2 cells (P. Cresswell, Cold Spring Harbor Symp. Quant. Biol. 1989. LIV:309). The stability of the invariant chain H-2 Db complex is critically dependent upon occupation of the class I binding site by peptide ligand. In the absence of peptide, the complex is stable at 4 degrees C whereas following exposure to peptide, the invariant chain dissociates rapidly from H-2 Db molecules (half-life of 30 min at 4 degrees C). Although the interaction between the human invariant chain and murine H-2 Db is unlikely to have any functional significance, the peptide-induced dissociation of the invariant chain is consistent with a conformational change in H-2 Db on peptide binding.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1992

Short peptides assist the folding of free class I heavy chains in solution.

Previous experiments have shown that short peptides coresponding to naturally processed epitopes of viral antigens can induce a conformational change in the class I heavy chain (HC) to which they bind in the fully assembled molecule. Here, we present evidence that the mechanism for this conformational change may involve binding of peptide to a partially unfolded form of free HC, followed by its subsequent folding. These results may be important for understanding the way in which class I molecules are assembled in vivo, and how certain epitopes are selected for presentation to T cells.

Amino Acid Sequence↗
PubMed1992

A comparison of the costs of ceftazidime therapy and gentamicin combinations in three UK hospitals.

This study compares the utilization costs of ceftazidime therapy with those of gentamicin in combination with other antibacterial drugs. The results show that the relatively high purchase cost of ceftazidime compared to combinations is more than counterbalanced by the additional materials used for drug administration and serum antibiotic assays, even when other drugs were combined with ceftazidime. The average drug and equipment costs were 230.13 pounds for ceftazidime regimens and 253.94 pounds for gentamicin regimens. It is also shown that ceftazidime therapy is associated with a reduction in personnel time compared to gentamicin regimens. The average times per patient for administration and assay were 1 h 43 min for ceftazidime and 4 h 57 min for gentamicin regimens. We conclude that ceftazidime regimens are cheaper than gentamicin regimens when all drug and equipment costs are quantified. Moreover, the use of ceftazidime will release staff time for other purposes.

Anti-Bacterial Agents↗
PubMed1992

A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.

This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.

Chromosome Deletion↗
PubMed1992

The genes required for heme synthesis in Salmonella typhimurium include those encoding alternative functions for aerobic and anaerobic coproporphyrinogen oxidation.

Insertion mutagenesis has been used to isolate Salmonella typhimurium strains that are blocked in the conversion of 5-aminolevulinic acid (ALA) to heme. These mutants define the steps of the heme biosynthetic pathway after ALA. Insertions were recovered at five unlinked loci: hemB, hemCD, and hemE, which have been mapped previously in S. typhimurium, and hemG and hemH, which have been described only for Escherichia coli. No other simple hem mutants were found. However, double mutants are described that are auxotrophic for heme during aerobic growth and fail to convert coproporphyrinogen III to protoporphyrinogen IX. These mutant strains are defective in two genes, hemN and hemF. Single mutants defective only in hemN require heme for anaerobic growth on glycerol plus nitrate but not for aerobic growth on glycerol. Mutants defective only in hemF have no apparent growth defect. We suggest that these two genes encode alternative forms of coproporphyrinogen oxidase. Anaerobic heme synthesis requires hemN function, while either hemN or hemF is sufficient for aerobic heme synthesis. These phenotypes are consistent with the requirement of a well-characterized class of coproporphyrinogen oxidase for molecular oxygen.

Aerobiosis↗
PubMed1991

Peptide-induced conformational change of the class I heavy chain.

There is evidence that peptide ligands take part in the assembly of class I molecules. In particular, addition of peptides to extracts of the mutant cells RMA-S and .174/T2, in which stable assembly of class I does not occur, results in a conformational change in the class I heavy chain and stable association of the heavy chain with beta 2-microglobulin (beta 2m). Thus specific peptides may stabilize or induce a conformational change in the class I heavy chain that results in a rise in the binding affinity of the heavy chain for beta 2m (Fig. 1a). Here we show that peptides have two cooperative roles in class I assembly. Specific short peptides (9-10 amino acids) can induce folding of the heavy chain in the absence of beta 2m. Both short (nine amino acids) and longer sequences (15 amino acids) can stabilize performed low-affinity complexes of heavy chain and beta 2m. To alter the conformation of free heavy chains, the peptides must be exactly the correct size, and they are found to correspond to the sequences isolated from infected cells. This property may therefore be the basis for selection of epitopes presented in vivo.

Allosteric Regulation↗
PubMed1991

A quantitative assay of peptide-dependent class I assembly.

We have developed a quantitative assay for the measurement of class I assembly induced by peptide. We have applied this assay to H-2Db, Kb and HLA-A2.1 with a panel of 49 overlapping peptides derived from HIV-1 gag protein. We find that the effects of peptide on assembly form a continuous distribution. By defining positives as those that increase the concentration of folded heavy chains more than three standard deviations from the control we show that 7/48 bind A2.1, 11/49 bind Db and 7/47 bind Kb. The assembly assay contrasts with solid-phase assays in being more discriminating (fewer peptides binding any given class I molecule), and showing less overlap in the patterns of peptides bound by the three class I molecules.

Amino Acid Sequence↗
PubMed1991

The binding affinity and dissociation rates of peptides for class I major histocompatibility complex molecules.

Peptides of various lengths derived from the influenza nucleoprotein (NP) bind to H-2Db class I molecules with affinities at 4 degrees C between approximately 3 x 10(5)- approximately 3 x 10(7) M-1. The peptide with the highest affinity corresponds to the sequence of nine amino acids (NP366-374) recently isolated from cells infected with influenza. This peptide forms stable complexes with half-lives greater than 110 h at 4 degrees C, 39 h at 22 degrees C and 3 h at 37 degrees C. Small increases in length of the peptide greatly reduce the stability of the complex (t1/2 approximately 1-10 h at 4 degrees C). These results may explain the homogeneous length of peptides isolated from class I molecules formed in vivo, and suggest that class I and II may differ in their dependence on the length of peptides for the formation of stable complexes.

Amino Acid Sequence↗
PubMed1991

Salmonella typhimurium prfA mutants defective in release factor 1.

Mutations have been characterized that map in the prfA gene of Salmonella typhimurium. These weak amber suppressors show increased readthrough of UAG but not UAA or UGA codons. Some hemA mutants exhibit a similar suppressor activity due to transcriptional polarity on prfA. All of the suppressors mapping in prfA are recessive to the wild type. Two mutant prfA genes were cloned onto plasmids, and their DNA sequences were determined. A method was devised for transferring the sequenced mutant alleles back to their original location in S. typhimurium via an Escherichia coli recD strain that carries the entire S. typhimurium hemA-prfA operon as a chromosomal insertion in trp. This reconstruction experiment showed that the mutations sequenced are sufficient to confer the suppressor phenotype.

Cloning, Molecular↗