Antigen presentation. Naturally processed peptides.
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Antigen-Presenting Cells↗
Biomedical subjects
T Elliott
Publications and source records attributed to T Elliott.
Assembly of MHC class I molecules analyzed in vitro.
Recent evidence suggests that peptide ligands take part in the assembly of class I molecules in living cells. We now describe a simple system for studying class I assembly in vitro. Detergent extracts of the mutant cells RMA-S and .174, in which class I assembly does not occur spontaneously, will support assembly in vitro when specific peptides are added. Peptides stabilize a conformational change in the class I heavy chain and association with beta 2-microglobulin, at concentrations approximately 100-fold lower than required in "peptide feeding" experiments with whole cells. We show that peptides bind class I molecules during assembly and demonstrate that the conformational change induced in the heavy chain is influenced by the concentrations of both peptide and beta 2-microglobulin.
Amino Acid Sequence↗
Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase.
Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected.
Amino Acid Sequence↗
General anesthesia in sickle cell disease.
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Adult↗
Heme-deficient mutants of Salmonella typhimurium: two genes required for ALA synthesis.
The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). We have isolated, mapped and characterized a large number of Salmonella typhimurium mutants auxotrophic for ALA. These mutants carry defects in either one of two genes, both required for ALA synthesis. The previously identified hemA gene maps at 35 min, and the hemL gene maps at 5 min on the S. typhimurium genetic map. Mutants in hemA and hemL are defective for aerobic and anaerobic respiration, and appear to be oxygen sensitive. The Hem- phenotype of hemL mutants is less severe than that of hemA mutants. Although hemA and hemL mutants are deficient in heme synthesis, genetic tests indicate that they still synthesize two minor products of the heme pathway, siroheme and cobalamin (vitamin B12), under anaerobic conditions. In contrast, hemB, hemC and cysG mutants, blocked after ALA synthesis, make neither siroheme nor vitamin B12. Double mutants defective in both hemA and hemL also make siroheme. We suggest that hemA and hemL are required for one route of ALA synthesis and that a second, minor route of ALA synthesis may operate in S. typhimurium; this second pathway would be independent of the hemA and hemL functions.
Aminolevulinic Acid↗
Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.
The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). Mutations in two genes, hemA and hemL, result in auxotrophy for ALA in Salmonella typhimurium, but the roles played by these genes and the mechanism of ALA synthesis are not understood. I have cloned and sequenced the S. typhimurium hemA gene. The predicted polypeptide sequence for the HemA protein shows no similarity to known ALA synthases, and no ALA synthase activity was detected in extracts prepared from strains carrying the cloned hemA gene. Genetic analysis, DNA sequencing of amber mutations, and maxicell studies proved that the open reading frame identified in the DNA sequence encodes HemA. Another surprising finding of this study is that hemA lies directly upstream of prfA, which encodes peptide chain release factor 1 (RF-1). A hemA::Kan insertion mutation, constructed in vitro, was transferred to the chromosome and used to show that these two genes form an operon. The hemA gene ends with an amber codon, recognized by RF-1. I suggest a model for autogenous control of prfA expression by translation reinitiation.
5-Aminolevulinate Synthetase↗
Two rare unstable beta chain variants, Hb Mozhaisk or alpha 2 beta 292(F8)His----Arg and Hb Djelfa or alpha 2 beta 298(Fg5)Val----Ala, each being observed for the second time.
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Adult↗
Characterization of Tn10d-Cam: a transposition-defective Tn10 specifying chloramphenicol resistance.
We have constructed a small, transposition-defective derivative of the transposon Tn10 that carries the chloramphenicol acetyltransferase gene of pACYC184. This new genetic element, Tn10d-Cam, transposes when Tn10 transposase is provided from a multi-copy plasmid. Transposon insertion mutagenesis of Salmonella typhimurium was performed by using a strain carrying a Tn10d-Cam insertion in an Escherichia coli F' episome as the donor in transductional crosses into recipients that carried a plasmid expressing Tn10 transposase. Tn10d-Cam insertion mutations were also generated by complementation in cis of Tn10d-Cam by a cotransducible Tn10 element that overproduces transposase. Here, transposase was provided only transiently, and the Tn10d-Cam insertion mutations were recovered in a transposase-free strain. Cis complementation was used for mutagenesis of a plasmid target. The site specificity of insertion and the effect of insertions on expression of a downstream gene were investigated, using Tn10d-Cam insertions in a plasmid carrying a segment of the histidine operon.
Chloramphenicol Resistance↗
Packaging specific segments of the Salmonella chromosome with locked-in Mud-P22 prophages.
Hybrid genetic elements, Mud-P and Mud-Q (collectively, Mud-P22s), have been constructed that carry two-thirds of the temperate Salmonella phage P22 genome sandwiched between the ends of transposon Mu. Insertions of these elements in the Salmonella chromosome generate locked-in P22 prophages that cannot excise. Upon induction (as a consequence of the inactivation of P22 c2 repressor), a locked-in prophage replicates its DNA in situ, resulting in the amplification of neighboring regions of the chromosome and the processive packaging of three contiguous headsful of adjacent DNA in one direction from the P22 packaging site, pac. Phage particles in an induced lysate of a Mud-P22 lysogen contain DNA molecules corresponding to several minutes of chromosomal DNA adjacent to the site of prophage insertion and transduce nearby genetic markers with high efficiencies. Mud-P22 prophages have been introduced into an F' episome by transposition; resident Mud insertions on the Salmonella chromosome may be converted to Mud-P22 insertions by homologous recombination in P22-mediated transductional crosses.
Chromosomes, Bacterial↗
Haemoglobin M-Hyde Park associated with polyagglutinable red blood cells in a South African family.
Twelve of 35 members tested in a large ethnically-mixed South African family were found to have both haemoglobin M type Hyde Park and persistent polyagglutinable red blood cells. The characteristics of the polyagglutination have not been recorded previously. The cells of affected family members were not agglutinated by Arachis hypogea, Dolichos biflorus or Salvia sclarea, but were agglutinated weakly by Salvia horminum and BSII (GSII) and reacted strongly with Glycine soja and Sophora japonica lectins. BSI (GSI) lectin agglutinated the group A but not the group O cells. The N and MN cells were agglutinated more strongly than normal by Vicia graminea, other anti-N lectins and human anti-N but the M and MN cells reacted as expected with human anti-M. The name 'Hyde Park' is provisionally suggested for this type of polyagglutination, although it appears unlikely that the evidently complete association between the polyagglutination and the variant haemoglobin is the result of a single genetic mutation. More likely, the connection has a post-genetic origin, perhaps showing that bonds, possibly affected adversely by precocious senescence, normally occur between the haemoglobin and alpha-sialoglycoprotein molecules in red blood cells.
Adolescent↗
The complex pattern of transcription in the segment of the bacteriophage T4 genome containing three of the head protein genes.
A detailed analysis of the temporal pattern of transcription in the gene 22-24 region of bacteriophage T4 has been made. Each of these three late genes has its own promoter, activated during the late phase of viral development. There is also late transcription of the opposite DNA strand across two of these late promoters. The 5' ends of these two sets of convergent transcripts have been mapped to sites designated Q22 and Q23. Middle (T4 gene mot product-dependent) transcription was found opposing the third late promoter, P24. An early promoter was mapped to the region between genes 23 and 24.
DNA Restriction Enzymes↗
Regulation of transcription in the segment of the bacteriophage T4 genome containing three of the head protein genes: plasmid-specific and phage chromosome-specific effects.
The activity of the phage T4 late promoters, P22, P23, and P24, is regulated by the gene 33, 45, and 55 products and, through the gene 43 product, by DNA replication. The accumulation of RNA of opposing polarity, with 5' ends mapping to Q22 and Q23, as described by T. Elliott, G. A. Kassavetis, and E. P. Geiduschek (Virology, 1984, 260-282), is subject to the same regulation. When located on a plasmid, the P23 promoter escapes the gene 43 product dependence.
Chromosomes, Bacterial↗
Defining a bacteriophage T4 late promoter: absence of a "-35" region.
We performed a deletion analysis to identify the minimal DNA sequence required for the function of the T4 late promoter, P23. A minipromoter derivative of P23 was constructed, containing 35 bp of T4 DNA from -18 to +17 with respect to the transcriptional initiation site. This derivative retains the TATAAATA homology and is competent to serve as a T4 late promoter both in vitro and in vivo. Its transcriptional activity in vivo is regulated identically to a wild-type plasmid-borne P23, requiring the function of T4 genes essential for late transcription, but not requiring T4 DNA replication. Recombination with the T4 phage chromosome is not significant for mini-P23 activity in vivo.
Base Sequence↗
Comparison of combination chemotherapy programs in advanced adenocarcinoma-large cell carcinoma of the lung: a North Central Cancer Treatment Group study.
One hundred and six patients with advanced adenocarcinoma and large cell carcinoma of the lung with no prior chemotherapy were entered in a prospectively randomized trial comparing cyclophosphamide, doxorubicin, and cisplatin versus methotrexate, doxorubicin, cyclophosphamide, and lomustine. The two regimens resulted in nearly identical regression probabilities (36% vs 34%), distributions of time to progression, and survival distributions. The major toxic effects from both regimens consisted primarily of myelosuppression and nausea and vomiting, with severe vomiting occurring more frequently in patients treated with cyclophosphamide, doxorubicin, and cisplatin (43% vs 19%).
Adenocarcinoma↗
Initiation of transcription at phage T4 late promoters with purified RNA polymerase.
We have previously identified T4 late promoters governing the in vivo expression of T4 late genes 23 and 24 (P23 and P24). T4 late transcription in vivo is known to involve the binding of at least five phage-coded proteins to the bacterial RNA polymerase and normally requires concurrent DNA replication for DNA template activation. We show here that in vitro transcription, primarily of plasmids carrying T4 genes 23 and 24, by RNA polymerase purified from Escherichia coli at late times after T4 infection allows specific initiation at P23 and P24 in the absence of DNA replication. These promoters are not utilized by E. coli RNA polymerase holoenzyme, by RNA polymerase core, or by T4-modified RNA polymerase purified from cells infected with a T4 gene 55 mutant (gene 55 codes for an RNA polymerase binding protein required for late transcription). The utilization of P23 and P24 in vitro is sharply inhibited by NaCl concentrations greater than 100 mM, and this inhibition is partly reversed by the addition of 10% DMSO. Relaxation of plasmid DNA containing P23 (with topoisomerase I) reduces P23 utilization at low salt (50 mM Na+) and nearly abolishes it at high salt (250 mM Na+). P23 utilization is discernible in linear, glucosylated hydroxymethylcytosine-containing T4 virion DNA.
Cell-Free System↗
Phosphorylation and metabolism of the transforming protein of Rous sarcoma virus.
p60src, the transforming protein of Rous sarcoma virus, was found to contain 0.5 to 0.9 mol of total phosphate per mol of polypeptide. The protein is known to be phosphorylated at two sites, a serine in the amino-terminal domain and a tyrosine in the carboxy-terminal domain. Because our indirect analysis suggests that the serine is phosphorylated to approximately twice the extent of the tyrosine, we estimate that p60src contains approximately 0.3 to 0.6 mol of phosphoserine and 0.2 to 0.3 mol of phosphotyrosine per mol of polypeptide. p60src was found to represent approximately 0.02% of the total incorporated radioactivity in Rous sarcoma virus-transformed chick cells labeled with [35S]methionine for 48 h. This corresponds to approximately 500,000 molecules of p60src per cell. Pulse-chase experiments revealed that the half-life of p60src ranged from 2 to 7 h, depending on the strain of virus examined. The P60src of the Schmidt-Ruppin strain was significantly more stable than that of the Prague strain.
Avian Sarcoma Viruses↗
Purification of mature schizonts of Plasmodium falciparum on colloidal silica gradients.
The density of human erythrocytes infected in vitro with Plasmodium falciparum has been measured by isopycnic centrifugation in colloidal silica gradients. The densities of uninfected cells, rings, trophozoites, young schizonts, and mature schizonts were approximately 1.110, 1.110, 1.106, 1.097, and 1.090 g/ml, respectively. This information has been used to design a simple procedure for the separation of schizonts from other parasite stages and uninfected erythrocytes. By using synchronized cultures it is possible to obtain essentially pure schizonts after two centrifugations using a bench centrifuge. Such preparations are an excellent source of parasite antigen for immunological studies.
Centrifugation↗
Plasmodium falciparum strains from Papua New Guinea: culture characteristics and drug sensitivity.
Seven strains of Plasmodium falciparum from Papua New Guinea have been established in continuous in vitro culture. Samples with a high initial parasitaemia were more likely to form continuous lines, possibly due to the time required for transport of infected blood samples from Papua New Guinea to laboratories in Australia. Most but not all established lines were resistant to chloroquine and all were resistant to pyrimethamine, possibly reflecting the parasite strain characteristics in that region.
Adolescent↗