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S A Ellis

Publications and source records attributed to S A Ellis.

At least 37 records · Page 2Linked to original sources
PubMed1998

DNA typing for BoLA class I using sequence-specific primers (PCR-SSP).

The analysis of cattle MHC (BoLA) class I gene expression is an essential component of studies on immune responses and susceptibility to disease. International BoLA workshops have generated data and reagents that allow discrimination of class I molecules at the haplotype level, but progress has been limited by difficulties encountered in defining single alleles. Our aim in this study was to develop a DNA-based system for improved identification of expressed class I alleles, utilizing available cDNA sequences derived from cattle carrying a series of serologically defined class I specificities. This method has allowed more accurate typing of animals for expression of the class I genes present within a small number of haplotypes. The method has also reliably differentiated between allelic variants (identified by prior sequence analysis) and has split existing serological specificities. The data show that MHC class I genes in cattle are more polymorphic than demonstrated by serology and biochemical analysis.

Alleles↗
PubMed1996

Identification of potential CTL epitopes of bovine RSV using allele-specific peptide motifs from bovine MHC class I molecules.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in young infants and housed calves. Depletion of CD8+ lymphocytes from calves inhibited their ability to clear the virus from the nasopharynx and lungs. To study these cells further, a cytotoxic T lymphocyte (CTL) assay was established. CTL could be demonstrated in the peripheral blood of gnotobiotic calves 7-10 days post infection (p.i.) with RSV and in lungs 10 days p.i. This response was both MHC-restricted and virus-specific. Following separation of the lung lymphocytes by magnetic activated cell sorting, it was shown that the cytolytic activity was mediated by cells of the CD8+ phenotype. To identify epitopes recognised by bovine CTL, the consensus motifs from MHC class I alleles found in the herd at Compton were identified. cDNA libraries were constructed and screened for full length class I sequences. The isolated cDNA clones were then transfected into mouse P815 cells and the expressed product immunoprecipitated and matched with a serological specificity. The bovine MHC class I molecules were isolated from lysed transfected cells by affinity chromatography, using a monoclonal antibody specific for bovine MHC class I, and bound peptides were separated by reverse-phase HPLC. Analysis of the protein sequences of bovine RSV for the defined motifs has identified potential CTL epitopes.

Alleles↗
PubMed1995

Methylation status and transcriptional expression of the MHC class I loci in human trophoblast cells from term placenta.

Of the various molecular regulatory mechanisms that may be used by human trophoblast cells to down-regulate expression of HLA class I genes, we chose to investigate the methylation of DNA, generally associated with inhibition of transcription. We analyzed the methylation status of different HLA class I loci in villous and extravillous cytotrophoblast cells and in vitro-differentiated syncytiotrophoblast, purified from human term placenta, as well as in the human trophoblast-derived JAR and JEG-3 cell lines. We then compared methylation status and transcriptional activity. An inverse relationship was established between JAR and JEG-3: HLA-A, -B, and -G are methylated and repressed in JAR, whereas in JEG-3, HLA-A is methylated and repressed but HLA-B and -G are partially methylated and transcribed. HLA-E is unmethylated and transcribed in both cell lines. Apart from HLA-E, which is always unmethylated and transcribed, no such relationship exists for the other class I loci in trophoblast cells. Whereas nonclassical HLA-G and classical HLA-A and -B class I genes are undermethylated in both cytotrophoblast and syncytiotrophoblast, they are clearly transcribed in the former but minimally transcribed in the latter subpopulation. Thus, the down-regulation of class I gene expression in the in vitro-differentiated syncytiotrophoblast is unlikely to be caused by DNA methylation. Furthermore, there is no detectable expression of any class I molecule at the cell surface of either trophoblast cell subpopulation, suggesting a negative control on translation and/or on the secretory pathway to the plasma membrane.

Base Sequence↗
PubMed1995

At least four MHC class I genes are transcribed in the horse: phylogenetic analysis suggests an unusual evolutionary history for the MHC in this species.

Nineteen horse MHC class I specificities have been serologically identified previously at a single locus (ELA-A), and two other specificities appear to be coded at other loci. Biochemical studies indicate that there are at least two expressed loci. In order to establish the number of transcribed horse MHC class I genes, we made a cDNA library from a heterozygous animal (ELA-A3/A7), and screened for positive clones using a bovine class I probe. More than 200 class I clones were isolated in this way, and so far seven unique full length sequences have been identified. All of the sequences are predicted to code for surface expressed, functional molecules. The number of different sequences identified demonstrate that at least four genes are transcribed, although variations in transmembrane length (which is generally conserved in class I loci) suggest that five genes could be represented. Evolutionary analysis of these sequences (and two additional sequences known to represent different horse class I loci) reveals no firm relationships, such that the division between the different loci cannot be discerned. These results suggest an unusual evolutionary history for the horse MHC, the precise nature of which may be revealed only following further cross-species comparisons.

Amino Acid Sequence↗
PubMed1992

Transmembrane and cytoplasmic domain sequences demonstrate at least two expressed bovine MHC class I loci.

We have used the polymerase chain reaction to amplify cDNA from expressed bovine major histocompatibility complex class I genes. Sequences obtained from transmembrane and cytoplasmic domains were used to identify the number of expressed alleles. Data from three animals suggest that there are four major expressed alleles, representing the products of two (or more) loci. We have also demonstrated the presence of an alternatively spliced mRNA, which has been observed in five animals. The alternative splicing removes exon 7 (the major site of class I phosphorylation), which predicts a truncated molecule with a cytoplasmic portion 16 amino acids shorter than usual. This phenomenon was detected for only a single class I allele within each individual.

Alternative Splicing↗
PubMed1990

Human trophoblast and the choriocarcinoma cell line BeWo express a truncated HLA Class I molecule.

Extravillous trophoblast from normal human placenta has been shown previously to express an unusual form of HLA class I molecule, as does a choriocarcinoma cell line, BeWo. This molecule has a H chain of approximately 40 kDa and appears to be nonpolymorphic. We have isolated and sequenced a HLA class I cDNA clone, which probably corresponds to this molecule, from a library derived from BeWo. The nucleotide sequence shows a high degree of homology with the published sequence of a genomic clone, HLA 6.0, which is the product of a class I locus other than A, B, or C, (provisionally designated "HLA G"). The expressed product of this locus has not previously been localized. We have used the polymerase chain reaction to demonstrate the presence of similar HLA class I sequences in cDNA from normal extravillous trophoblast. Although there is some nucleotide sequence polymorphism the amino acid sequence of this molecule is conserved. It is therefore unlikely to provoke immune responses even though it is found at the fetal-maternal interface.

Amino Acid Sequence↗
PubMed1990

Structural homologies between two HLA B27-restricted peptides suggest residues important for interaction with HLA B27.

Recently we described an HLA B27-restricted peptide derived from HIV gag p24 protein. In this study we have isolated an HLA B27-restricted peptide from the nucleoprotein (NP) of influenza A virus. The shortest fragment recognized by cytotoxic T lymphocyte (CTL) is eight amino acids long, residues 384-391. Comparison of the sequence of these two HLA B27 restricted peptides reveals homologies which can be aligned from one peptide to the other. Of the eight residues, two are identical: tryptophan and isoleucine. Both peptides have a positively charged residue at the N terminus, lysine at position 265 of gag and arginine at position 384 of NP. Using modified peptides we have shown that lysine or arginine is crucial for the interaction with HLA B27. The wild-type gag peptide blocked CTL recognition of NP peptide by influenza-specific CTL, but removal of the lysine prevented inhibition of NP peptide recognition. The importance of these charged residues was confirmed by the observation that truncated NP and gag peptides where the lysine or arginine was removed were not recognized by specific CTL. Further studies showed that the tryptophan residue influenced the association of the gag peptide with HLA B27, because the affinity of the gag peptide for B27 was strongly increased after replacing this residue with a leucine or a tyrosine. However, these peptides were not recognized by gag-specific CTL, suggesting that the tryptophan may interact with both HLA B27 and T cell receptor. These observations should help in the identification of HLA B27-restricted peptides from other viruses or organisms.

Amino Acid Sequence↗
PubMed1989

Complete nucleotide sequence of a unique HLA class I C locus product expressed on the human choriocarcinoma cell line BeWo.

We have used Northern blot analysis to detect mRNA from class I HLA genes in the human choriocarcinoma cell line BeWo, which has been previously shown to express an atypical HLA class I molecule, in the absence of HLA A and B. Hybridization was seen with three class I DNA probes, the strongest being seen with the probe pC800, which corresponds to an 800-bp section of the Cw3 gene. We have made cDNA libraries from BeWo cells and screened for positive clones by using class I DNA probes. Of the clones isolated, we determined the complete sequence of one and partial sequence of five shorter clones. They all code for an identical C locus-related product, which does not correspond to published C locus sequences.

Amino Acid Sequence↗